Compositions and methods for improved treatment of disorders affecting the central nervous system - Patents.com

Abstract

Described herein are methods for treating a subject having or at risk of developing a disorder affecting the central nervous system (CNS), such as a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders, or a lysosomal storage disorder. The disclosed methods can include administering an adeno-associated virus (AAV) vector expressing a therapeutic protein (e.g., the deficiency or lack of activity of which is associated with the disorder or whose supplementation is likely to benefit the patient). The disclosed AAV vectors can be administered in specific amounts and by specific routes of administration that achieve gene expression in the CNS, while avoiding, for example, transduction in peripheral tissues (e.g., liver, lung, and spleen).

Description

[Technical field]

[0001] The present disclosure relates to compositions and methods for treating diseases affecting the central nervous system of a subject (e.g., a human subject). [Background technology]

[0002] Progranulin (PGRN) is a 68.5 kD glycoprotein that has long been implicated in tumorigenesis, inflammation, and repair, including growth factor signaling pathways. PGRN is primarily expressed in microglia and neurons in brain tissues, where it may perform growth factor-like functions. Recent findings suggest that PGRN is involved in neurodegenerative disorders, particularly frontotemporal dementia (FTD), and autosomal dominant mutations in the GRN gene have been described as the underlying FTD phenotype. PGRN has also been found associated with beta-amyloid plaques in Alzheimer's disease and other amyloid-related diseases (e.g., dementia with Lewy bodies), and with transactivation response element TAR DNA-binding protein 43 (TDP-43), the main disease-associated protein, in patients with amyotrophic lateral sclerosis (ALS). Existing treatments for such neurodegenerative diseases, such as FTD, strive to ameliorate the symptoms of the disease. However, therapies targeting the underlying neurodegeneration are lacking, thus highlighting the need for new therapeutic avenues. Summary of the Invention

[0003] The present disclosure provides compositions and methods that can be used to treat disorders of the central nervous system, such as neurocognitive or neuromuscular disorders (e.g., neurodegenerative diseases) or lysosomal storage disorders, among other disorders that adversely affect the central nervous system. Exemplary disorders that can be treated using the compositions and methods of the present disclosure include frontotemporal dementia (FTD), Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS), and related neurocognitive and motor neuron disorders. Using the compositions and methods of the present disclosure, a patient (e.g., a mammalian patient, such as a human patient) with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders) or lysosomal storage disorder, among other conditions described herein, can be administered an adeno-associated virus (AAV) containing a transgene encoding a therapeutic protein (e.g., a deficiency or lack of activity of which is associated with the disorder or whose supplementation is likely to benefit the patient). Exemplary transgenes useful in combination with the compositions and methods of the present disclosure include progranulin (PGRN), among other therapeutic proteins described herein, which in some embodiments may be delivered in the form of a codon-optimized transgene to further increase protein expression. The methods of use described herein are particularly beneficial because they avoid significant transgene expression in peripheral tissues, including, but not limited to, the liver, lung, and spleen.

[0004] In a first aspect, the disclosure provides a method of achieving expression of a therapeutic transgene (e.g., PGRN, among various other therapeutic transgenes described herein) in the central nervous system (CNS) of a patient while minimizing or completely avoiding expression of the transgene in peripheral tissues (e.g., liver, lung, and / or spleen) of the patient. The patient may be a patient with a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders), or a lysosomal storage disorder), and the method comprises administering to the patient an AAV vector comprising the therapeutic transgene. In some embodiments, the AAV vector is administered to the patient in an amount of about 1×10 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., about 5 × 10 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, approx. 1×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 5 × 10 10 vg / hemisphere ~ approx. 1×10 11 The drug is administered intrathalamally to patients in a dose of 100 mg / hemisphere (vg / hemisphere).

[0005] In another aspect, the disclosure provides a method of improving cognition, reducing neurodegeneration, and / or improving neuromuscular function in a patient in need thereof by introducing a therapeutic transgene (e.g., PGRN, among other therapeutic transgenes described herein) into the CNS of the patient. The method may minimize or completely avoid expression of the transgene in peripheral tissues (e.g., liver, lung, and / or spleen) of the patient. The patient may be a patient with a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders), or a lysosomal storage disorder), and the method includes administering to the patient an AAV vector comprising the therapeutic transgene. In some embodiments, the AAV vector is administered to the patient in an amount of about 1×10 9 vg / hemisphere ~ approx. 9×1012 vg / hemisphere (e.g., about 5 × 10 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, approx. 1×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 5 × 10 10 vg / hemisphere ~ approx. 1×10 11 The drug is administered intrathalamally to patients in a dose of 100 mg / hemisphere (vg / hemisphere).

[0006] In another aspect, the disclosure provides a method of treating a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders), or a lysosomal storage disorder) in a human patient in need thereof, the method comprising administering to the patient an AAV vector containing a transgene encoding a therapeutic protein (e.g., the deficiency or lack of activity of which is associated with the disorder or whose supplementation is likely to benefit the patient), such as PGRN, among various other therapeutic proteins described herein. In some embodiments, the AAV vector is administered in an amount of about 1×10 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., about 5 × 10 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, approx. 1×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 5 × 10 10 vg / hemisphere ~ approx. 1×10 11 The drug is administered intrathalamally to patients in a dose of 100 mg / hemisphere (vg / hemisphere).

[0007] In a further aspect, the disclosure provides a method of improving cognitive function in a human patient diagnosed with a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders), or a lysosomal storage disorder), the method comprising administering to the patient an AAV vector comprising a transgene encoding a therapeutic protein (e.g., a deficiency or lack of activity of which is associated with the disorder or whose supplementation is likely to benefit the patient, such as PGRN, among various other therapeutic proteins described herein). In some embodiments, the AAV vector is administered in an amount of about 1×10 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., about 5 × 10 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, approx. 1×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 5 × 10 10 vg / hemisphere ~ approx. 1×10 11 The drug is administered intrathalamally to patients in a dose of 100 mg / hemisphere (vg / hemisphere).

[0008] In another aspect, the disclosure provides a method of expressing or restoring expression of a therapeutic protein (e.g., PGRN, or another therapeutic protein described herein) in the brain (e.g., frontal cortex) of a human patient diagnosed with a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders, or a lysosomal storage disorder), the method comprising administering to the patient an AAV vector comprising a transgene encoding a therapeutic protein (e.g., PGRN, among various other therapeutic proteins described herein, the deficiency or lack of activity of which is associated with the disorder or whose supplementation is likely to benefit the patient). In some embodiments, the AAV vector is administered to the patient in an amount of about 1×10 9 vg / hemisphere ~ approx. 9×1012 vg / hemisphere (e.g., about 5 × 10 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, approx. 1×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 5 × 10 10 vg / hemisphere ~ approx. 1×10 11 The drug is administered intrathalamally to patients in a dose of 100 mg / hemisphere (vg / hemisphere).

[0009] In some embodiments of any of the aforementioned aspects, the AAV vector is about 1×10 10 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., 5×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 1×10 11 For example, in some embodiments, the AAV vector is administered to a patient in an amount of about 1×10 10 vg / hemisphere, 2×10 10 vg / hemisphere, 3×10 10 vg / hemisphere, 4×10 10 vg / hemisphere, 5×10 10 vg / hemisphere, 6×10 10 vg / hemisphere, 7×10 10 vg / hemisphere, 8×10 10 vg / hemisphere, 9×10 10 vg / hemisphere, 1×10 11 vg / hemisphere, 2×10 11 vg / hemisphere, 3×10 11 vg / hemisphere, 4×10 11 vg / hemisphere, 5×10 11 vg / hemisphere, 6×10 11 vg / hemisphere, 7×10 11 vg / hemisphere, 8×10 11 vg / hemisphere, 9×10 11 vg / hemisphere, 1×10 12 vg / hemisphere, 2×10 12 vg / hemisphere, 3×10 12 vg / hemisphere, 4×10 12 vg / hemisphere, 5×10 12 vg / hemisphere, 6×10 12 vg / hemisphere, 7×10 12 vg / hemisphere, 8×1012 vg / hemisphere, or 9×10 12 In some embodiments, the AAV vector is administered to a patient in an amount of about 5×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 1×10 11 In some embodiments, the AAV vector is administered to a patient in an amount of about 1×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered to a patient in an amount of about 5×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered to a patient in an amount of about 1×10 vg / hemisphere. 11 vg / hemisphere is administered to the patient.

[0010] In some embodiments of any of the foregoing aspects, the therapeutic protein is a secreted protein. In some embodiments, the therapeutic protein is a protein listed in Table 5 herein. In some embodiments, the therapeutic protein is PGRN.

[0011] In some embodiments of any of the above aspects, the disorder is a neurocognitive disorder, a neuromuscular disorder, a neurodegenerative disorder, or a lysosomal storage disorder. In some embodiments, the disorder is frontotemporal dementia (FTD), Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS), or related neurocognitive or motor neuron disorders.

[0012] In some embodiments of any of the aforementioned aspects, the AAV vector is administered to the patient in a single dose per hemisphere comprising that amount.

[0013] In some embodiments, the AAV vector is administered to the patient in multiple doses per hemisphere (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses), all of which contain that amount.

[0014] In some embodiments of any of the foregoing aspects, the transgene (e.g., a transgene encoding PGRN, among other therapeutic proteins described herein) is operably linked to a promoter active in neuronal and / or glial cells. For example, in some embodiments, the promoter is a synapsin promoter, a tetracycline-controlled transactivator protein (tTA) promoter, a reverse tetracycline-controlled transactivator protein (rTA) promoter, a U1 promoter, a U6 promoter, a U7 promoter, a prion promoter, a phosphoglycerate kinase (PGK) promoter, a CB7 promoter, a H1 promoter, a cytomegalovirus (CMV) promoter, a CMV-chicken β-actin (CBA) promoter, a glial fibrillary acidic protein (GFAP) promoter, a calcium / calmodulin-dependent protein kinase III promoter, a tubulin alpha I promoter, a microtubulin-associated protein IB (MAP IB) promoter, a neuron-specific enolase promoter, a platelet-derived growth factor beta chain promoter, a neurofilament light chain promoter, a neuron-specific VGF gene promoter, a neuronal nucleus (NeuN) promoter, an adenomatous polyposis coli (APC) promoter, an ionized calcium-binding adaptor molecule 1 (Iba-1) promoter, or a homeobox protein 9 (HB9) promoter. In some embodiments, the promoter is a synapsin promoter.

[0015] In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO: 1. For example, in some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 1, and optionally, the synapsin promoter has a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the synapsin promoter has the nucleic acid sequence of SEQ ID NO: 1.

[0016] In some embodiments of any of the aforementioned aspects, the transgene encodes PGRN. PGRN may have an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:2, for example. For example, in some embodiments, PGRN has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, and optionally, PGRN has an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has the amino acid sequence of SEQ ID NO:2.

[0017] In some embodiments, the transgene (e.g., encoding PGRN) is codon-optimized. For example, in some embodiments, the transgene encoding PGRN has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the transgene encoding PGRN has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:3, and optionally, the transgene encoding PGRN has a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the transgene encoding PGRN has a nucleic acid sequence of SEQ ID NO:3.

[0018] In some embodiments, the transgene (e.g., the transgene encoding PGRN) is operably linked to a human growth hormone (hGH) intron. For example, in some embodiments, the hGH intron is hGH intron 3. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:4. For example, in some embodiments, the hGH intron has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:4, and optionally, the hGH intron has a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence of SEQ ID NO:4.

[0019] In some embodiments, the transgene (e.g., encoding PGRN) is operably linked to a 3' enhancer element. In some embodiments, the 3' enhancer element has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:5. For example, in some embodiments, the 3' enhancer element has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:5, and optionally, the 3' enhancer element has a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the 3' enhancer element has a nucleic acid sequence of SEQ ID NO:5.

[0020] In some embodiments of any of the above aspects, the AAV has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:6. For example, in some embodiments, the AAV has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:6, and optionally, the AAV has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence of SEQ ID NO:6.

[0021] In some embodiments of any of the aforementioned aspects, prior to administration of the AAV vector, the patient exhibits a level of expression of an endogenous therapeutic protein (e.g., PGRN) that is about 1% to about 40% of the level of endogenous therapeutic protein expression (e.g., endogenous PGRN expression) observed in a human subject of the same age, sex, and / or body mass index who does not have a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorder) or a lysosomal storage disorder).

[0022] In some embodiments, following administration of the AAV vector, the patient exhibits increased expression of a therapeutic protein (e.g., PGRN, among other therapeutic proteins described herein) relative to a measurement of the patient's therapeutic protein expression levels obtained prior to administration of the AAV vector. In some embodiments, increased therapeutic protein (e.g., PGRN) expression is observed in the patient's thalamus, frontal cortex, basal ganglia, parietal cortex, temporal cortex, parietal and temporal cortex, and / or cerebrospinal fluid (CSF).

[0023] In some embodiments, following administration of the AAV vector, the patient exhibits a level of therapeutic protein expression (e.g., PGRN expression) in the frontal cortex of about 2 ng / mg to about 100 ng / mg (e.g., 3 ng / mg to about 99 ng / mg, 4 ng / mg to about 98 ng / mg, 5 ng / mg to about 97 ng / mg, 10 ng / mg to about 90 ng / mg, 20 ng / mg to about 80 ng / mg, 30 ng / mg to about 70 ng / mg, 40 ng / mg to about 60 ng / mg, or about 50 ng / mg).

[0024] In some embodiments of any of the aforementioned aspects, the AAV vector is administered to the patient in a convectively assisted manner.

[0025] In another aspect, the disclosure provides a method of treating a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders, or a lysosomal storage disorder) in a human patient in need thereof, the method comprising administering to the patient an AAV vector comprising a transgene encoding a therapeutic protein (e.g., PGRN), wherein the AAV vector is administered to the patient in an amount sufficient to achieve a level of therapeutic protein (e.g., PGRN) expression in the patient's brain (e.g., frontal cortex) that is greater than or equal to about 1×10 expression of an AAV vector described herein (e.g., an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6). 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., 5×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 1×10 11 vg / hemisphere), is equivalent to the level of expression of a therapeutic protein (e.g., PGRN) observed in a human subject having a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder, such as FTD, AD, PD, dementia with Lewy bodies, ALS, or a related neurocognitive or motor neuron disorder, or a lysosomal storage disorder).

[0026] In another aspect, the disclosure provides a method of improving cognitive function in a human patient diagnosed with a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or a related neurocognitive or motor neuron disorder), or a lysosomal storage disorder), the method comprising administering to the patient an AAV vector comprising a transgene encoding a therapeutic protein (e.g., PGRN), wherein the AAV vector is administered to the patient in an amount sufficient to achieve a level of expression of the therapeutic protein (e.g., PGRN) in the patient's brain (e.g., frontal cortex) that is greater than or equal to about 1 x 10 of an AAV vector described herein (e.g., an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO: 6). 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., 5×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 1×10 11 vg / hemisphere), which is equivalent to the level of therapeutic protein (e.g., PGRN) expression observed in a human subject having a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder, such as FTD, AD, PD, dementia with Lewy bodies, ALS, or a related neurocognitive or motor neuron disorder, or a lysosomal storage disorder).

[0027] In another aspect, the disclosure provides a method of expressing or restoring expression of a therapeutic protein (e.g., PGRN) in the brain (e.g., frontal cortex) of a human patient diagnosed with a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders, or a lysosomal storage disorder), the method comprising administering to the patient an AAV vector comprising a transgene encoding a therapeutic protein (e.g., PGRN), wherein the AAV vector is administered to the patient in an amount sufficient to achieve a level of therapeutic protein expression (e.g., PGRN expression) in the brain (e.g., frontal cortex) of the patient that is greater than or equal to about 1×10 of an AAV vector described herein (e.g., an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6). 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., 5×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 1×10 11 vg / hemisphere), which is equivalent to the level of therapeutic protein (e.g., PGRN) expression observed in a human subject having a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder, such as FTD, AD, PD, dementia with Lewy bodies, ALS, or a related neurocognitive or motor neuron disorder, or a lysosomal storage disorder).

[0028] In another aspect, the disclosure provides a method of treating a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders), or a lysosomal storage disorder) in a human patient in need thereof, the method comprising administering to the patient an AAV vector comprising a transgene encoding a therapeutic protein (e.g., a deficiency or lack of activity of which is associated with the disorder or whose supplementation is likely to benefit the patient, such as PGRN, among other therapeutic proteins described herein). In some embodiments, the AAV vector is administered to a patient in an amount sufficient to achieve a level of therapeutic protein (e.g., PGRN) expression in the patient's brain (e.g., frontal cortex) of about 2 ng / mg to about 8 ng / mg (e.g., 3 ng / mg to about 7 ng / mg, 4 ng / mg to about 6 ng / mg, or about 5 ng / mg), or more (e.g., about 9 ng / mg, about 10 ng / mg, about 15 ng / mg, about 20 ng / mg, about 30 ng / mg, about 40 ng / mg, about 50 ng / mg, about 60 ng / mg, about 70 ng / mg, about 80 ng / mg, about 90 ng / mg, or about 100 ng / mg).

[0029] In another aspect, the disclosure provides a method of improving cognitive function in a human patient diagnosed with a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders), or a lysosomal storage disorder), the method comprising administering to the patient an AAV vector comprising a transgene encoding a therapeutic protein (e.g., a protein whose deficiency or lack of activity is associated with the disorder or whose supplementation is likely to benefit the patient, such as PGRN), Methods are provided in which the AV vector is administered to the patient in an amount sufficient to achieve a level of therapeutic protein (e.g., PGRN) expression in the patient's brain (e.g., frontal cortex) of about 2 ng / mg to about 8 ng / mg (e.g., 3 ng / mg to about 7 ng / mg, 4 ng / mg to about 6 ng / mg, or about 5 ng / mg), or more (e.g., about 9 ng / mg, about 10 ng / mg, about 15 ng / mg, about 20 ng / mg, about 30 ng / mg, about 40 ng / mg, about 50 ng / mg, about 60 ng / mg, about 70 ng / mg, about 80 ng / mg, about 90 ng / mg, or about 100 ng / mg).

[0030] In another aspect, the disclosure provides a method of expressing or restoring the level of expression of a therapeutic protein (e.g., PGRN) in the brain (e.g., frontal cortex) of a human patient diagnosed with a disorder affecting the central nervous system (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders, or a lysosomal storage disease), the method comprising administering to the patient a transgene encoding a therapeutic protein (e.g., PGRN, the deficiency or lack of activity of which is associated with the disorder or whose supplementation is likely to benefit the patient), such as PGRN. The method includes administering to the patient an AAV vector comprising administering to the patient an AAV vector comprising a genomic DNA sequence comprising: a) a genomic DNA sequence comprising ...

[0031] In some embodiments of any of the aforementioned aspects, the AAV vector comprises a capsid protein derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, AAVrh.8, and AAVrh.10.

[0032] In some embodiments, the AAV is an anterograde transport AAV or a retrograde transport AAV.

[0033] In some embodiments of any of the foregoing aspects, the AAV vector comprises a 5' inverted terminal repeat (ITR) and / or a 3' ITR from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, AAVrh.8, or AAVrh.10, and optionally the AAV vector comprises a 5' ITR and a 3' ITR from AAV2. For example, in some embodiments, the AAV vector comprises a 5' ITR and a 3' ITR from one AAV serotype and a capsid protein from a different AAV serotype.

[0034] In some embodiments, the AAV vector is an AAV2 / 9 vector.

[0035] In some embodiments of any of the aforementioned aspects, the human patient is diagnosed with FTD due to a mutation in the GRN gene.

[0036] In some embodiments of any of the foregoing aspects, upon administration of the AAV vector, there is no significant increase in therapeutic protein (e.g., PGRN) expression in peripheral tissues, or any such increase in therapeutic protein (e.g., PGRN) expression is 10% or less (e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%). In some embodiments, peripheral tissues include, but are not limited to, liver, lung, and / or spleen. In some embodiments, PGRN transgene expression is calculated relative to GAPDH expression.

[0037] In another aspect, the disclosure provides a kit comprising an AAV vector comprising a transgene encoding a therapeutic protein (e.g., a therapeutic protein described herein, such as PGRN), the kit further comprising a package insert instructing a user of the kit to administer the AAV vector to a patient according to a method of any of the preceding aspects. [Brief description of the drawings]

[0038] [Figure 1]1 is a map of an adeno-associated virus (AAV) encoding a codon-optimized human progranulin (hPGRN) gene (abbreviated herein as "AAV9-SYN-PGRN"). The shaded arrow and rectangle represent a nucleic acid molecule comprising the first AAV2 inverted terminal repeat (ITR) from 5' to 3', the human synapsin (hSyn) promoter, the human growth hormone intron (hGHi3), the codon-optimized hGRN gene (PGRN-GS), the hPGRN 3' untranslated region (hPGRN-3'UTR), the bovine growth hormone polyadenylation (poly(A)) signal, the phage-derived origin of replication (f1 ori), the citrobacter freundii ampC β-lactamase (AmpR) promoter, the kanamycin selection gene (KanR), and the second origin of replication (ori). [Diagram 2] Photomicrographs of progranulin (PGRN) expression across six layers of the cortex in sheep injected intrathalamically (ITM) with a low dose of 1x1010 vg / hemisphere of AAV9-SYN-PGRN as described in Figure 1. Abbreviations: NeuN, neuronal nuclei; DAPI, 4',6-diamidino-2-phenylindole. [Diagram 3] 1 is a set of graphs showing vector genomes per μg (vg) across brain regions of biopsy tissue from sheep ITM-injected with low dose (1×10 vg / hemisphere), medium dose (5×10 vg / hemisphere), or high dose (1×10 vg / hemisphere) of AAV9-SYN-PGRN as described in FIG 1. Abbreviations: CD / PT, caudate-putamen / parieto-temporal. [Figure 4] A set of photomicrographs of hPGRN, NeuN, and IBA1 (e.g., a marker of microglial activation) expression in the prefrontal (PF) cortex of sheep (e.g., KCL9-KCL13) ITM-injected with low dose (1 x 1010 vg / hemisphere), medium dose (5 x 1010 vg / hemisphere), or high dose (1 x 1011 vg / hemisphere) of AAV9-SYN-PGRN described in Figure 1. [Diagram 5]1 is a set of graphs showing hPGRN protein levels across brain regions in sheep ITM-injected with low (1×10 vg / hemisphere), medium (5×10 vg / hemisphere), or high (1×10 vg / hemisphere) doses of AAV9-SYN-PGRN described in Figure 1. Abbreviations: CD / PT, caudate-putamen / parieto-temporal. [Figure 6] 1 is a set of graphs showing hPGRN protein levels across the frontal A cortex, frontal B cortex, and thalamus, respectively, in sheep ITM-injected with 1×10 vg / hemisphere, 5×10 vg / hemisphere, 1×10 vg / hemisphere, 5×10 vg / hemisphere, or 5×10 vg / hemisphere of AAV9-SYN-PGRN as described in FIG. [Figure 7] 1 is a set of graphs showing hPGRN protein levels across brain regions in sheep ITM-injected with low dose (1x1010vg / hemisphere), medium dose (5x1010vg / hemisphere), or high dose (1x1011vg / hemisphere) of AAV9-SYN-PGRN described in Figure 1, normalized to percent (%) of hPGRN protein level expression in the thalamus. Abbreviations: CD / PT, caudate-putamen / parieto-temporal. [Figure 8] (A and B) are a set of graphs showing hPGRN protein levels in cerebrospinal fluid (CSF). (A) is a graph showing hPGRN levels in CSF of patients diagnosed with frontotemporal dementia (FTD) before and after the onset of symptoms (pre-symptomatic and post-symptomatic, respectively) compared to healthy control subjects. (B) is a graph showing hPGRN levels in CSF of sheep 4 weeks after transduction with low dose (1×1010vg / hemisphere), medium dose (5×1010vg / hemisphere), or high dose (1×1011vg / hemisphere) AAV9-SYN-PGRN as described in FIG. 1 administered ITM. [Figure 9](A and B) are a set of graphs showing hPGRN protein levels in serum. (A) is a graph showing hPGRN levels in serum of patients diagnosed with FTD before and after the onset of symptoms (pre-symptomatic and post-symptomatic, respectively) compared to healthy control subjects. (B) is a graph showing hPGRN levels in serum of sheep 1 week before or 4 weeks after transduction with low (1×1010vg / hemisphere), medium (5×1010vg / hemisphere), or high dose (1×1011vg / hemisphere) AAV9-SYN-PGRN as described in FIG. 1 administered ITM. [Figure 10] A set of photomicrographs of PF cortex and thalamus from hematoxylin stained sections of sheep (e.g., KCL9-KCL13) injected with AAV9-SYN-PGRN as described in Figure 1 at doses of 1 x 1010 vg / hemisphere, 5 x 1010 vg / hemisphere, or 1 x 1011 vg / hemisphere. [Figure 11] 2 is a set of graphs showing hPGRN protein levels normalized to vg / μg across brain regions of sheep ITM-transduced with AAV9-SYN-PGRN as described in FIG. 1 at 1×10 vg / hemisphere, 5×10 vg / hemisphere, or 1×10 vg / hemisphere. [Figure 12] Set of photomicrographs of hPGRN expression in sheep cerebellum transduced intracisternally (ICM) with 1x1013 AAV9-SYN-PGRN described in Figure 1, or AAV9 or AAV1 vectors expressing a transgene encoding PGRN (AAV9-CB7-PGRN and AAV1-CB7-PGRN), respectively. Panels A-C are merged images of PGRN, NeuN, and DAPI. Panels D-F show tissue stained with anti-hPGRN antibody alone. Panels G-I show tissue stained with anti-NeuN antibody alone. Abbreviations: SYN: synapsin; CB7, chicken β-actin promoter with cytomegalovirus enhancer. [Figure 13]Set of photomicrographs of PGRN expression in the PF cortex and thalamus, respectively, of sheep ITM- or ICM-injected with 1x1010 vg / hemisphere or 1x1013 AAV9-SYN-PGRN as described in Figure 1, or AAV9 or AAV1 vectors expressing a transgene encoding hPGRN, respectively (AAV9-CB7-PGRN and AAV1-CB7-PGRN). Abbreviations: CB7, chicken β-actin promoter with cytomegalovirus enhancer. [Figure 14] 13 is a set of graphs showing vg / μg across regions of biopsy brain tissue (e.g., KCL-9 to KCL-13) from sheep ITM-transduced with AAV9-SYN-PGRN, AAV1-CB7-PGRN, or AAV9-CB7-PGRN as described in FIG. 12 at 1×10 vg / hemisphere, 5×10 vg / hemisphere, or 1×10 vg / hemisphere, or ICM-transduced with AAV9-SYN-PGRN, AAV1-CB7-PGRN, or AAV9-CB7-PGRN at 1×10 vg / animal. Shading intensity indicates vg expression levels. [Figure 15] 13 is a set of graphs showing hPGRN expression across brain regions across sheep (e.g., KCL-9 to KCL-13) ITM-transduced with 1×1010 vg / hemisphere, 5×1010 vg / hemisphere, 1×1011 AAV1-CB7-PGRN, AAV9-CB7-PGRN, or AAV9-SYN-PGRN as described in FIG. 12, or ICM-transduced with 1×1013 AAV1-CB7-PGRN, AAV9-CB7-PGRN, or AAV9-SYN-PGRN. Shading intensity indicates hPGRN expression levels. [Figure 16] FIG. 13 is a graph showing hPGRN expression across cortical brain regions in sheep ITM-transduced with 5×10 vg / hemisphere AAV9-SYN-PGRN, or ICM-transduced with 1×10 vg / animal AAV9-SYN-PGRN, AAV1-CB7-PGRN, or AAV9-CB7-PGRN as described in FIG. [Figure 17]13 is a set of graphs showing hPGRN expression in the frontal A cortex of sheep ITM-transduced with 1×10 vg / hemisphere, 5×10 vg / hemisphere, or 1×10 vg / hemisphere AAV9-SYN-PGRN, or ICM-transduced with AAV9-SYN-PGRN, AAV1-CB7-PGRN, or AAV9-CB7-PGRN at 1×10 vg / animal as described in FIG. [Figure 18] 13 is a set of graphs showing hPGRN expression in the frontal B cortex of sheep ITM or ICM transduced with 1×10 vg / hemisphere, 5×10 vg / hemisphere, or 1×10 vg / hemisphere AAV9-SYN-PGRN, or ICM transduced with 1×10 vg / animal AAV9-SYN-PGRN, AAV1-CB7-PGRN, or AAV9-CB7-PGRN as described in FIG. [Figure 19] FIG. 11 is a set of photomicrographs of PGRN and lipofuscinosis levels, as measured by the lipofuscinosis marker subunit C mitochondrial ATP synthase (SCMAS), in the thalamus of GRN− / − mice injected ITM with very low dose (2.3×107 vg / hemisphere, e.g., equivalent to a sheep dose of 1×1010 vg / hemisphere), low dose (1.1×108 vg / hemisphere, e.g., equivalent to a sheep dose of 5×1010 vg / hemisphere), medium dose (2.3×108 vg / hemisphere, e.g., equivalent to a sheep dose of 1×1011 vg / hemisphere), or high dose (2.3×109 vg / hemisphere, e.g., equivalent to a sheep dose of 1×1012 vg / hemisphere). PBS injection was used as a control for SCMAS. Panels A-E are merged images of PGRN and SCMAS, Panels F-J show tissue stained with anti-hPGRN antibody alone, and Panels K-O show tissue stained with anti-SCMAS antibody alone. [Figure 20]13 is a graph showing that AAV-SYN-PGRN administration significantly reduced lipofuscinosis in the brains of GRN− / − mice. Lipofuscinosis was quantified by SCMAS-positive granules in GRN− / − mice ITM-injected with AAV-SYN-PGRN at very low doses (2.3×107vg / hemisphere, e.g., equivalent to a sheep dose of 1×1010vg / hemisphere), low doses (1.1×108vg / hemisphere, e.g., equivalent to a sheep dose of 5×1010vg / hemisphere), medium doses (2.3×108vg / hemisphere, e.g., equivalent to a sheep dose of 1×1011vg / hemisphere), or high doses (2.3×109vg / hemisphere, e.g., equivalent to a sheep dose of 1×1012vg / hemisphere) compared to wild-type (WT) controls or GRN− / − home cage controls ("20w Hom", e.g., untreated mice that spent 20 weeks in their home cages). Abbreviations: ns, not significant. [Figure 21]

[0023] Figure 1 shows a graph depicting vector biodistribution in sheep liver 4 weeks after ITM administration of AAV9-SYN-PGRN vector in sheep brain. Six sites per sheep liver were biopsied and two sheep were administered vector per vector dose. [Figure 22] Graph demonstrating that minimal to no significant increase in PGRN expression levels is observed in the bloodstream following ITM administration of AAV9-PGRN vector. Data were obtained from a single bilateral ITM injection of AAV9.PGRN in cynomolgus monkeys using convection-enhanced delivery (low dose: 2.5×1010 vg / hemisphere, high dose: 2.5×1011 vg / hemisphere). Blood samples were collected at day 0, 2, 4, 8, and 12 weeks and analyzed for PGRN protein by ELISA. (Note: 2.5×1011 vg / hemisphere in non-human primates (e.g., cynomolgus monkeys) is equivalent to 4×1012 vg / hemisphere in humans.) [Figure 23]1 is a table demonstrating that no significant expression of PGRN is observed in non-neural tissues following ITM administration of AAV9-PGRN vectors. Data are from a single bilateral ITM injection of AAV9.PGRN in cynomolgus monkeys using convection-enhanced delivery (low dose: 2.5×1010 vg / hemisphere, high dose: 2.5×1011 vg / hemisphere). Terminal biopsies at 12 weeks were taken from major non-neural organs and assayed for levels of hPGRN RNA by qPCR. (Note: 2.5×1011 vg / hemisphere in non-human primates (e.g., cynomolgus monkeys) is equivalent to 4×1012 vg / hemisphere in humans.)

[0039] definition As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0040] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, and any other AAV now known or later discovered. See, e.g., Fields et al. Virology, 4 thed. Lippincott-Raven Publishers, Philadelphia, 1996. Additional AAV serotypes and clades have been recently identified. (See, e.g., Gao et al. J. Virol. 78:6381 (2004); Morris et al. Virol. 33:375 (2004). The genomic sequences of the various serotypes of AAV, as well as the sequences of the native ITRs, Rep proteins, and capsid subunits, are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. NC-002077, NC-001401, NC-001729, NC-001863, NC-001829, NC-001862, NC-000883, NC-001701, NC-001510, NC-001601, NC-001610, NC-001621, NC-001632, NC-001641, NC-001642, NC-001651, NC-001661, NC-001671, NC-001681, NC-001691, NC-001692, NC-001693, NC-001694, NC-001695, NC-001696, NC-001697, NC-001698, NC-001699, NC-001610, NC-001623, NC-001624, NC-001636, NC-001638, NC-001646, NC See, e.g., 06152, NC-006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC-001358, NC-001540, AF513851, AF513852, and AY530579, the disclosures of which are incorporated by reference herein for their teaching of AAV nucleic acid and amino acid sequences.Also, for example, Bantel-Schaal et al.J.Virol.73:939(1999), Chiorini et al.J.Virol.71:6823(1997), Chiorini et al.J.Virol.73:1309(1999), Gao et al.Proc.Nat.Acad.Sci.USA 99:11854(2002), Morris et al.Virol.33:375(2004), Muramatsu et al.Virol.221:208(1996), Ruffing et al.J.Gen.Virol.75:3385(1994), Rutledge et al.J.Virol.72:309(1998), Schmidt et al. J. Virol. 82:8911 (2008), Shade et al. See, e.g., Srivastava et al. J. Virol. 58:921 (1986), Srivastava et al. J. Virol. 45:555 (1983), Xiao et al. J. Virol. 73:3994 (1999), WO00 / 28061, WO99 / 61601, WO98 / 11244, and US 6,156,303, the disclosures of which are incorporated herein by reference for their teaching of AAV nucleic acid and amino acid sequences. As used herein, the term "AAV" encompasses anterograde transport AAV and / or retrograde transport AAV.

[0041] As used herein, the terms "amyotrophic lateral sclerosis" and "ALS", also known as Lou Gehrig's disease, refer to a fatal disease that affects motor neurons in the cortex, brainstem, and spinal cord. In the context of the present invention, the term "ALS" includes a range of neurodegenerative disorders known as classical (Charcot) ALS, Lou Gehrig's disease, motor neuron disease (MND), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), bulbar onset ALS, spinal onset ALS, and ALS with multisystem involvement (Wijesekera LC and Leigh P N. Amyotrophic lateral sclerosis. Orphanet J. Rare Dis. 2009, 4:3).

[0042] As used herein, "Alzheimer's disease" and "AD" refer to a late-onset neurodegenerative disorder that presents as cognitive decline, short-term and long-term memory loss, attention deficits, language-specific problems, disorientation, impulse control, social withdrawal, anhedonia, and other symptoms. Brain tissue from AD patients exhibits neuropathological features such as extracellular aggregates of amyloid beta protein and neurofibrillary tangles of hyperphosphorylated microtubule-associated tau protein. The accumulation of these aggregates is associated with neuronal loss and atrophy in many brain regions, including the frontal, temporal, and parietal lobes of the cerebral cortex, as well as subcortical structures such as the basal forebrain cholinergic system and the locus coeruleus in the brainstem. AD ​​is also associated with increased neuroinflammation, characterized by reactive gliosis and elevated levels of proinflammatory cytokines.

[0043] As used herein, "capsid protein" refers to any of the AAV capsid proteins that are components of the AAV viral particle, including AAV8 and AAV9.

[0044] The term "codon" as used herein refers to any group of three consecutive nucleotide bases in a given messenger RNA molecule or coding strand of DNA that specifies a particular amino acid or a start or stop signal for translation. The term codon also refers to a base triplet in a DNA strand.

[0045] As used herein, "codon optimization" refers to the process of modifying a nucleic acid sequence according to the principle that the frequency of occurrence of synonymous codons (e.g., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Such modified sequences are referred to herein as "codon optimization." This process can be performed on any of the sequences described herein to enhance expression or stability. Codon optimization can be performed by any method known in the art, such as, for example, as described in U.S. Pat. Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. The sequence surrounding the translation start site can be converted to a consensus Kozak sequence according to known methods. See, for example, Kozak et al, Nucleic Acids Res. 15(20): 8125-8148, incorporated herein by reference in its entirety. Multiple stop codons may be incorporated.

[0046] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0047] As used herein, the terms "conservative mutation," "conservative substitution," and "conservative amino acid substitution" refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk. These properties are summarized for each of the 20 naturally occurring amino acids in Table 1 below. [Table 1]

[0048] From this table, it is understood that conservative amino acid families include, for example, (i) G, A, V, L, I, P, and M, (ii) D and E, (iii) C, S, and T, (iv) H, K, and R, (v) N and Q, and (vi) F, Y, and W, and thus a conservative mutation or substitution is the replacement of one amino acid with a member of the same amino acid family (e.g., replacement of Ser with Thr or replacement of Lys with Arg).

[0049] By "CpG site" is meant a region of DNA in which a cytosine nucleotide occurs next to a guanine nucleotide in a linear nucleic acid sequence of nucleotides along its length, e.g., -C-phosphate-G-, the cytosine and guanine are separated by only one phosphate, or a cytosine 5' to a guanine nucleotide.

[0050] As used herein, the terms "dementia with Lewy bodies" and "Lewy body dementia" are used interchangeably to refer to a disorder that includes fluctuating cognitive impairment, recurrent hallucinations of specific detailed events, and / or parkinsonian dementia symptoms.

[0051] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of an AAV vector described herein refer to an amount sufficient to achieve a beneficial or desired result, including a clinical result, when administered to a subject, including a mammal, e.g., a human. Thus, "effective amount," or synonyms thereof, depend on the context in which it is applied. For example, in the context of treating a neurocognitive or neuromuscular disorder, it is an amount of an AAV vector sufficient to achieve a therapeutic response compared to the response obtained without administration of the AAV vector. The amount of a given AAV vector described herein that corresponds to such an amount will vary depending on various factors, such as, for example, the pharmaceutical formulation, subject attributes (e.g., age, sex, weight), but may nevertheless be routinely determined by one of skill in the art. Also, as used herein, a "therapeutically effective amount" of an AAV vector of the present disclosure is an amount that results in a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of an AAV vector of the present disclosure is an amount that is approximately 1×10 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere amount.

[0052] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., humans) or in a particular location within an organism (e.g., an organ, tissue, or cell, e.g., a human cell).

[0053] As used herein, the terms "frontotemporal dementia" and "FTD" refer to disorders caused by degeneration of the frontal lobe of the brain and degeneration that may extend to the temporal lobe. FTD is one of three syndromes caused by frontotemporal lobe degeneration and is the second most common cause of early dementia after AD. Diagnostic criteria according to the Lund-Manchester criteria include onset and gradual progression, early decline in social and interpersonal behavior, early impairment in control of personal behavior, early emotional blunting, and early loss of insight. Symptoms of FTD may appear between the ages of about 45 to about 65 years (e.g., 50 to about 60 years). As used herein, "FTD" is intended to include all stages (e.g., preclinical stages) and subtypes of the disease.

[0054] As used herein, the term "GC content" refers to the amount of nucleosides in a particular nucleic acid molecule, such as a DNA or RNA polynucleotide, that is, either guanosine (G) or cytidine (C), relative to the total amount of nucleosides present in the nucleic acid molecule. GC content can be expressed as a percentage, for example, according to the following formula: GC content = ((total amount of guanosine nucleosides) + (total amount of cytidine nucleosides) / (total amount of nucleosides)) x 100

[0055] As used herein, a patient suffering from "GRN mutation" or "GRN-associated FTD" is a patient who has been diagnosed with FTD and contains a deleterious mutation in the GRN gene. More than 70 pathogenic mutations have been reported in the GRN gene, the majority of which result in premature stop codons and the nonsense intervening decay of truncated GRN mRNA. GRN mutations are described in Gijselinck et al., Hum.Mutat.29(12), 1373-1386, (2012) and Pottier et al., J.Neurochem.138(Suppl.1), 32:53, (2016), the disclosures of which are incorporated herein by reference as they relate to human GRN mutations.

[0056] As used herein, the term "intron" refers to a region in the coding region of a gene whose nucleotide sequence is not translated into the amino acid sequence of the corresponding protein. The term intron also refers to the corresponding region of the RNA transcribed from the gene. In some embodiments, a gene may contain, for example, at least two introns, each of which forms an intervening sequence between two exons. Introns are transcribed into pre-mRNA but are removed during processing and are not included in mature mRNA.

[0057] An "ITR" is a palindromic nucleic acid, e.g., an inverted terminal repeat, about 120 nucleotides to about 250 nucleotides in length, capable of forming a hairpin. The term "ITR" includes a site of viral genome replication that can be recognized and bound by a parvoviral protein (e.g., Rep78 / 68). The ITR can be from any adeno-associated virus (AAV), with serotype 2 being preferred. The ITR includes a replication protein-binding element (RBE) and a terminal resolution sequence (TRS). The term "ITR" does not require a wild-type parvoviral ITR (e.g., the wild-type nucleic acid sequence may be altered by insertion, deletion, truncation, or missense mutation), so long as the ITR functions to mediate viral packaging, replication, integration, and / or proviral rescue, etc. The term "5'ITR" is intended to mean a parvoviral ITR located at the 5' border of a nucleic acid molecule, and the term "3'ITR" is intended to mean a parvoviral ITR located at the 3' border of a nucleic acid molecule.

[0058] As used herein, the term "modified nucleotide" refers to a nucleotide or portion thereof (e.g., adenosine, guanosine, thymidine, cytidine, or uridine) that has been altered by one or more enzymatic or synthetic chemical transformations. Exemplary changes observed in modified nucleotides described herein or known in the art include the introduction of chemical substituents, such as halo, thio, amino, azido, alkyl, acyl, or other functional groups, at one or more positions (e.g., 2', 3', and / or 5' positions) of 2-deoxyribonucleotides or ribonucleotides.

[0059] As used herein, the terms "motor neuron disorder" and "motor neuron disease" refer interchangeably to a group of progressive neurological disorders that destroy motor neurons, the cells that control skeletal muscle activities such as walking, breathing, speaking, and swallowing. Exemplary non-limiting motor neuron disorders include ALS, progressive bulbar palsy, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy, Kennedy's disease, and post-polio syndrome. It should be understood that the above list is not exhaustive.

[0060] As used herein, the term "mutation" refers to a change in the nucleotide sequence of a gene (e.g., GRN) or a change in the polypeptide sequence of a protein (e.g., PGRN). Mutations in genes or proteins can occur naturally, for example, as a result of errors in DNA replication, DNA repair, irradiation, and exposure to carcinogens, or mutations can be induced as a result of administration of a transgene expressing a mutant gene. Mutations can result from the insertion, deletion, or substitution of single or multiple nucleotides.

[0061] As used herein, the term "neurocognitive disorder" (NCD) refers to a set of clinical disorders or syndromes in which the primary clinical deficit is cognitive function, such as, for example, deficits in complex attention, executive function, learning and memory, language, sensorimotor function, and social cognition. NCDs are characterized as acquired rather than developmental pathologies. For example, NCDs are conditions in which cognitive impairment was not evident at birth or from a very early age, thus requiring that cognitive function in NCDs has declined from a previously acquired level. NCDs are distinguished from other disorders in which patients present with cognitive impairment in that NCDs include only disorders in which the core deficit is cognitive function. NCDs can be "major NCDs" or "mild NCDs." Major NCDs are characterized by significant cognitive decline that interferes with an individual's independence and normal daily functioning, and are not due to delirium or other psychiatric disorders. Mild NCDs are characterized by moderate cognitive decline that does not interfere with an individual's independence and normal daily functioning, and are not due to delirium or other psychiatric disorders. Severe and mild NCDs may also be distinguished based on quantitative cognitive testing across any one of the above specific cognitive functions. For example, severe NCDs may be characterized by scores obtained on cognitive tests by subjects identified as having or at risk of developing NCDs that are more than two standard deviations away from the mean score of a reference population (e.g., the mean score of the general population), or scores that are in the third percentile of the distribution of scores of the reference population. Mild NCDs may be characterized by scores obtained on cognitive tests by subjects identified as having or at risk of developing NCDs that are one to two standard deviations away from the mean score of the reference population, or scores that are between the third and sixteenth percentiles of the distribution of scores of the reference population. Non-limiting examples of cognitive tests that may be used to classify NCD patients as having either severe or mild NCDs include AD8, AWV, GPCOG, HRA, MIS, MMSE, MoCA, SLUMS, and short IQCODE. Additionally, NCDs include syndromic subtypes that indicate a specific etiological origin of the NCD, such as, for example, FTD, AD, or dementia with Lewy bodies.

[0062] As used herein, the terms "neurodegenerative disorder" and "neurodegenerative disease" refer interchangeably to disorders characterized by progressive loss of number (e.g., by cell death), structure, and / or function of neurons. In some cases, neurocognitive or neuromuscular disorders (e.g., neurodegenerative diseases) may be associated with genetic defects (e.g., mutations in the GRN gene), protein misfolding, defects in protein degradation, programmed cell death, membrane damage, or other processes. Exemplary non-limiting neurodegenerative disorders include FTD, AD, PD, dementia with Lewy bodies, ALS, Lou Gehrig's disease, MND, PBP, PMA, PLS, bulbar onset ALS, spinal onset ALS, and ALS with multisystem involvement, as well as associated motor neuron disorders.

[0063] As used herein, the term "neuromuscular disorder" refers to a disease that impairs the ability of one or more neurons to control the activity of the associated muscle. Examples of neuromuscular disorders include, among others, Parkinson's disease (PD), ALS, congenital myasthenic syndromes, congenital myopathies, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal-bulbar muscular atrophy, spinal muscular atrophy, stiff-person syndrome, Troyer syndrome, and Guillain-Barre syndrome.

[0064] It should be understood that the above list is not exhaustive, and disorders or diseases can be classified into various categories.For example, AD can be considered as a neurocognitive disorder and a neurodegenerative disease.Similarly, PD can be considered as a neuromuscular disorder and a neurodegenerative disease.

[0065] "Nucleic acid" or "polynucleotide", as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.

[0066] As used herein, the term "operably linked" refers to a first molecule bound to a second molecule, where the molecules are positioned such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter regulates the transcription of a transcribable polynucleotide molecule of interest in a cell. Furthermore, two portions of a transcriptional regulatory element are operably linked to each other if they are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to each other without the presence of any intervening nucleotides.

[0067] "Percentage of sequence identity" to a reference polynucleotide sequence or a reference polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in a reference polynucleotide sequence or a reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining percentage of nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. For example, percentage of sequence identity values ​​can be generated using the sequence comparison computer program BLAST. As an example, the percentage of sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which can alternatively be translated as a given nucleic acid or amino acid sequence A having a certain percentage of sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in a programmatic alignment of A and B, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0068] The terms "polyadenylation signal," "polyadenylation site," and "pA" are used interchangeably herein to refer to a nucleic acid sequence sufficient to direct the addition of polyadenosine ribonucleic acid to an RNA molecule expressed in a cell.

[0069] As used herein, the term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule to which additional DNA segments can be ligated. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. Certain plasmids can replicate autonomously in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain plasmids can direct the expression of genes to which they are operatively linked.

[0070] As used herein, the terms "Progranulin" and "PGRN" refer to the secreted trophic factor and precursor peptide of granulin. The gene is located on chromosome 17q21.31 and is known as GRN. The terms "Progranulin" and "PGRN" also refer to variant proteins having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or more) to the amino acid sequence of the wild-type PGRN peptide (e.g., SEQ ID NO: 2), or the nucleic acid sequence of the wild-type GRN gene (e.g., SEQ ID NO: 7). "PGRN" refers to variants of wild-type Progranulin peptides and the nucleic acids encoding them, such as polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or more) with the wild-type PGRN peptide, provided that the encoded PGRN analog retains the therapeutic function of wild-type PGRN. The terms "Progranulin" and "PGRN" may also refer to the PGRN protein in which the natural secretory signal peptide is present. As used herein, as will be understood by those skilled in the art, "PGRN" refers to a peptide, while "GRN" refers to the gene encoding this protein.

[0071] As used herein, the terms "Parkinson's disease" and "PD" refer to neurodegenerative disorders characterized by motor and non-motor symptoms. Motor symptoms primarily include dyskinesia, hypotonia, rigidity, and progression, and motor dyskinesia includes exercise-induced relaxation and even anemia. Non-motor symptoms include pain, constipation, delayed gastric emptying, depression, and sleep disorders.

[0072] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives transcription of the transgene. Exemplary promoters suitable for use with the compositions and methods described herein are described, for example, in Sandelin et al., Nat Rev. Genet. 8:424 (2007), the disclosure of which is incorporated herein by reference as it relates to nucleic acid regulatory elements.

[0073] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response and other significant complications, commensurate with a reasonable benefit / risk ratio.

[0074] As used herein, the term "secretory signal peptide" refers to a short (usually 16-60 amino acids) peptide region within a precursor protein that directs secretion of the precursor protein from the host cytoplasm to the periplasmic space or extracellular space. Such secretory signal peptides are generally located at the amino terminus of the precursor protein. In some embodiments, the secretory signal peptide is linked to the amino terminus. Typically, the secretory signal peptide is cleaved during transport through the cellular secretory pathway. Cleavage is not essential as long as the secreted protein retains the desired activity. Exemplary secretory signal peptides include the PGRN secretory signal peptide.

[0075] As used herein, the term "therapeutic protein" refers to (i) a protein whose deficiency or lack of activity is associated with a disorder (e.g., a neurological disorder as described herein), as well as (ii) a protein that is not necessarily deficient in a patient, but whose replacement would nevertheless have a beneficial effect on the patient. Exemplary therapeutic proteins useful in conjunction with the compositions and methods of the present disclosure are set forth in Table 5 herein.

[0076] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, sonoporation, phototransfection, magnetofection, imparefection, and the like.

[0077] As used herein, the term "transgene" refers to a recombinant nucleic acid (e.g., DNA or cDNA) that encodes a gene product (e.g., PGRN). The gene product may be an RNA, a peptide, or a protein. In addition to the coding region for the gene product, the transgene may include or be operably linked to one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), destabilization domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements. Embodiments of the present disclosure may utilize any known suitable promoter, enhancer(s), destabilization domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0078] As used herein, the terms "subject" and "patient" refer to an animal (e.g., a mammal, such as a human). The subject treated according to the methods described herein may be a subject diagnosed with FTD or GRN-related neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder, such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorder, ALS, or related motor neuron disorder), or a subject at risk of developing one or more of these conditions. Diagnosis may be performed by any method or technique known in the art. Those skilled in the art will understand that the subject treated according to the present disclosure may have been subjected to standard testing, or may have been identified as a subject at risk due to the presence of one or more risk factors associated with a disease or condition without testing.

[0079] As used herein, the terms "transduction" and "transducing" refer to the method of introducing a viral vector construct, or a portion thereof, into a cell and the subsequent expression of a transgene encoded by the vector construct, or a portion thereof, within the cell.

[0080] As used herein, "treatment" and "treating" refer to an approach to obtain a beneficial or desired result, e.g., a clinical result. Beneficial or desired results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable; reduction in the extent of the disease or condition; stabilization (i.e., not worsening) of the state of the disease, disorder, or condition; prevention of the spread of the disease or condition; delaying or slowing the progression of the disease or condition; amelioration or alleviation of the disease or condition; and remission (partial or complete). "Ameliorating" or "alleviating" a disease or condition means that the extent and / or undesirable clinical symptoms of the disease, disorder, or condition are reduced and / or the time course of progression is slowed or prolonged compared to the extent or time course in the absence of treatment. "Treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in need of preventing the condition or disorder.

[0081] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors such as plasmids, RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed to deliver polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO1994 / 011026, which is incorporated herein by reference as it relates to vectors suitable for expressing genes of interest. Expression vectors suitable for use with the compositions and methods described herein contain polynucleotide sequences and additional sequence elements that are used, for example, for protein expression and / or for integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express PGRN as described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for the expression of PGRN contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, IRES, and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also include a polynucleotide encoding a marker for the selection of cells containing such a vector. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] Detailed Description Described herein are compositions and methods for the treatment of a disorder affecting the central nervous system (CNS) in a subject (e.g., a mammalian subject, e.g., a human) (e.g., a neurocognitive, neuromuscular, or neurodegenerative disorder (FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders), or a lysosomal storage disorder). The compositions and methods described herein are useful for stimulating expression of a therapeutic protein, such as a protein whose deficiency or lack of activity is associated with the disorder of the subject, or a protein that is not necessarily deficient in the patient, but whose supplementation is likely to have a beneficial effect on the patient. An exemplary therapeutic protein of the disclosure is the human progranulin (PGRN) protein, which is particularly useful for treating disorders associated with mutations in the progranulin gene (GRN), such as FTD. The compositions and methods described herein are also useful for stimulating expression of a variety of other therapeutic proteins that may improve or otherwise benefit patients suffering from neurocognitive and / or neuromuscular disorders, as well as lysosomal storage disorders. Exemplary therapeutic proteins useful in combination with the compositions and methods of the disclosure are listed below in Table 5.

[0083] The compositions described herein include adeno-associated viruses (AAV) that code for therapeutic proteins. Therapeutic transgenes that can be used to code for such proteins include, for example, human GRN or codon-optimized human GRN, which are useful for expressing PGRN protein in cells. The AAV described herein can be used to express approximately 1×10 9 vg / hemisphere ~ approx. 9×10 12vg / hemisphere to the patient. Without being limited by mechanism, the compositions described herein can improve pathology associated with disorders affecting the central nervous system (e.g., neurocognitive, neuromuscular, or neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders, or lysosomal storage disorders) by effectively stimulating expression of a therapeutic protein (e.g., PGRN, among others described herein). The compositions and methods described herein can be used to treat one or more of the above disorders by administering the AAV described herein.

[0084] The present invention is based, at least in part, on the discovery that intrathalamic delivery of AAV containing a transgene encoding a therapeutic protein (e.g., PGRN) results in a surprisingly good ability to transduce the cortex (e.g., by anterograde and / or retrograde transport of AAV) and induce expression of the therapeutic protein (e.g., PGRN) in the cortex. In the context of PGRN gene therapy, this property is particularly beneficial considering the prevalence of mutations in the GRN gene in mammalian genomes, such as those of human patients with FTD, a disorder characterized by neurodegeneration in the frontal and temporal lobes of the cerebral cortex. The present invention is also based, at least in part, on the identification of an optimal dosage range for achieving said expression of a therapeutic protein (e.g., PGRN) in the cortex after intrathalamic delivery. The optimal dosage range in combination with the delivery method results in highly specific transduction and subsequent transduced gene expression in the CNS relative to peripheral tissues (e.g., liver, lung, and spleen). This specificity is highly desirable, because the increase in PGRN expression in peripheral tissues is associated with certain adverse effects, including but not limited to cancer growth and inflammation-related adverse reactions.Therefore, a delivery method that ensures little or no transgene expression in peripheral tissues is highly advantageous.Using the compositions and methods described herein, for example, the expression of vital and healthy GRNs, or their codon-optimized variants, and their encoded PGRN protein products can be effectively enhanced in the cortex.

[0085] The following sections provide descriptions of exemplary codon optimizations and methods for producing codon-optimized therapeutic transgenes (e.g., human GRN) that can be used in combination with dosing regimens and AAV vectors encoding such constructs to provide methods that can be used to treat disorders affecting the CNS (e.g., neurocognitive, neuromuscular, or neurodegenerative disorders (FTD, AD, PD, dementia with Lewy bodies, ALS, or related neurocognitive or motor neuron disorders) or lysosomal storage disorders).

[0086] Neurocognitive and neuromuscular disorders Neurocognitive disorders are defined as a set of disorders that are characterized by cognitive impairment as a core symptom, and are not developmental disorders, but rather show cognitive decline (e.g., acquired disorders) against a previous higher level of cognition, while neuromuscular disorders are characterized by progressive muscle weakness. Neurocognitive disorders can be classified based on their etiological origin. For example, non-limiting examples of neurocognitive disorders can include neurocognitive disorders due to AD, neurocognitive disorders with Lewy bodies (e.g., Lewy body dementia), neurocognitive disorders due to PD, frontotemporal neurocognitive disorders (e.g., FTD), neurocognitive disorders due to leukodystrophy (e.g., PLOSL), vascular neurocognitive disorders, neurocognitive disorders due to traumatic brain injury, neurocognitive disorders due to HIV infection, substance / drug-induced neurocognitive disorders, neurocognitive disorders due to Huntington's disease, neurocognitive disorders due to prion disease, neurocognitive disorders due to another medical condition, neurocognitive disorders due to multiple etiologies, and unspecified neurocognitive disorders. Non-limiting examples of neuromuscular disorders include PD, ALS, congenital myasthenic syndromes, congenital myopathies, fasciculations, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophies, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal-bulbar muscular atrophy, spinal muscular atrophy, stiff-person syndrome, Troyer syndrome, and Guillain-Barre syndrome, and related motor neuron disorders. The compositions and methods disclosed herein are useful for treating neurocognitive disorders and / or neuromuscular disorders.

[0087] Neurocognitive and neuromuscular disorders associated with GRN mutations FTD is a clinical syndrome characterized by progressive neurodegeneration in the frontal and temporal lobes of the cerebral cortex. The clinical symptoms of FTD are complex and heterogeneous, but may manifest as progressive aphasia, cognitive decline (e.g., reduced working memory and executive function), decreased impulse control, emergence of persistent behaviors, apraxia, apathy, and / or social withdrawal. Neuronal loss in the brains of FTD patients is associated with distinct neuropathologies, including mutations in the GRN gene, the presence of tau-positive neuronal and glial inclusions, or ubiquitin (ub)-positive and TAR DNA-binding protein 43 (TDP43)-positive, but tau-negative inclusions. These neuropathologies are believed to be important in the pathogenesis of FTD and highlight some of the other proteins with which PGRN is thought to interact in order to also play a role in other neurodegenerative diseases such as AD, dementia with Lewy bodies, related neurocognitive disorders, ALS, and related motor neuron disorders. For example, there is a known relationship between the reduction of PGRN and the accumulation of TDP-43, a protein that has been shown to be a primary component in cytoplasmic aggregates in postmortem tissue from patients with ALS. Furthermore, nearly half of FTD patients have first-degree family members with dementia, ALS, or PD, suggesting a strong genetic link to the etiology of the disease, and several mutations in chromosome 17q21 have been associated with the presentation of FTD.

[0088] Studies investigating the association between chromosome 17q21 and FTD have found many FTD-associated mutations in the PGRN gene, GRN. These mutations often result in the aggregation and accumulation of ub-positive, TDP43-positive, tau-negative neuropathological inclusions in the brains of FTD patients. PGRN is a secreted precursor peptide to several mature granulin proteins and is thought to function primarily as a neurotrophic growth factor, promoting neuronal differentiation and survival. PGRN has also been demonstrated to perform anti-inflammatory and neuroprotective functions. PGRN is expressed ubiquitously, but as a result of its association with FTD, much attention has been directed to the central nervous system (CNS), where PGRN is expressed in multiple cell types, including neurons, glial cells, and endothelial cells. Over 70 loss-of-function mutations in the GRN gene have been identified in FTD, the majority of which result in haploinsufficiency and a greater than 50% reduction in serum PGRN levels. GRN mutations are described in Gijselinck et al., Hum. Mutat. 29(12), 1373-86 (2008), the disclosure of which is incorporated herein by reference as it relates to human GRN mutations. The effects of GRN mutations are dose-dependent, as homozygous patients who completely lack functional PGRN protein develop a lysosomal storage disease known as CLN11 neuronal ceroid lipofuscinosis (NCL), suggesting an additional role for this protein in normal lysosomal function. Neurodegeneration, dementia, and early cognitive decline are also characteristic of NCL symptoms.

[0089] Clinical management of neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorders, ALS, or associated motor neuron disorders) is primarily used to ameliorate disease symptoms. For example, in FTD, current approaches to clinical management often use selective serotonin reuptake inhibitors and antipsychotics to manage the emotional and behavioral changes associated with FTD. However, this strategy aims to ameliorate disease symptoms without addressing its development and progression. Unlike these treatments, the compositions and methods described herein offer the benefit of treating a different biochemical phenomenon that may underlie the development of PGRN-associated pathology. Thus, the compositions and methods described herein target the physiological cause of the disease and represent a potential curative treatment. The compositions and methods described herein may be used to treat neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorders, ALS, or associated motor neuron disorders) by intrathalamic administration of an AAV vector containing a transgene encoding PGRN. The compositions and methods described herein may be used to treat neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorders, ALS, or associated motor neuron disorders) having any etiology, e.g., genetic mutation, environmental toxin, or sporadic. These compositions and methods may also be used to treat patients with GRN-associated FTD. The compositions and methods described herein may be used to treat patients with reduced PGRN activity and / or expression (e.g., a level of expression of endogenous PGRN that is about 1% to about 40% of the level of endogenous PGRN activity and / or expression observed in a human subject of the same age, sex, and / or body mass index without a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorders, ALS, or associated motor neuron disorders), or patients whose GRN mutation status and / or PGRN activity levels are unknown.Healthy physiological levels of 2-10 ng / mL of PGRN are observed in the CNS of human subjects without neurocognitive or neuromuscular disorders. The compositions and methods described herein may also be administered as a prophylactic treatment to patients at risk of developing a neurocognitive or neuromuscular disorder, to patients with reduced PGRN activity and / or expression (e.g., levels of expression of endogenous PGRN that are about 1% to about 40% of the levels of endogenous PGRN activity and / or expression observed in human subjects of the same age, sex, and / or body mass index without a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders), or to patients with a mutation in the GRN gene.

[0090] According to the methods described herein, a patient may be administered a transgene encoding the amino acid sequence of SEQ ID NO:2 below, or a polynucleotide encoding a polypeptide having at least 90% sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:2, or a polynucleotide encoding a polypeptide containing one or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more conservative amino acid substitutions) to SEQ ID NO:2, such as an AAV vector expressing SEQ ID NO:3 below, provided that the encoded PGRN analog retains the therapeutic function of wild-type PGRN. In some embodiments, the polynucleotide encoding wild-type PGRN may be a codon-optimized polynucleotide, as described in detail below.

[0091] Wild-type human PGRN (GenBank accession number: NP_002078.1) has the following amino acid sequence: MWTLVSWVALTAGLVAGTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWG CCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTC CRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGC DQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL (SEQ ID NO:2)

[0092] Wild-type human GRN (GenBank Accession Number: NM_002087.3) has the following nucleic acid sequence: (SEQ ID NO:10)

[0093] Treatment method The present disclosure is based, at least in part, on the discovery that an AAV vector encoding PGRN delivered to a patient by the routes of administration (e.g., intrathalamic administration) and dosages described herein can achieve PGRN expression levels in the patient's CNS on the order of double-digit ng / mL (see, e.g., Examples described below). This discovery is significant because physiological levels of PGRN protein in the CNS of a healthy human subject are on the order of 2-6 ng / mL (as measured in the patient's cerebrospinal fluid). Thus, the PGRN-encoding compositions described herein can produce physiological PGRN expression levels in the CNS of a human subject. Guided by this discovery, the inventors have found that a wide variety of other therapeutic proteins also have healthy concentration levels in the CNS on the order of up to double-digit ng / mL, and therefore the compositions and methods described herein can be used to deliver other therapeutic proteins in physiologically relevant amounts. The following table provides a list of exemplary therapeutic proteins of the present disclosure and their corresponding physiological expression levels in the CNS. [Table 2]

[0094] The exemplary subject that can be treated as described herein is the subject that has or is at risk of developing neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders), such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders.The type of FTD can be GRN-related FTD, sporadic FTD, FTD caused by environmental toxins, such as herbicides or pesticides, or FTD associated with non-GRN mutation, such as mutation in one or more of the genes associated with FTD. The compositions and methods described herein may be used to treat patients with reduced PGRN activity and / or expression (e.g., a level of endogenous PGRN expression that is about 1% to about 40% of the level of endogenous PGRN expression observed in a human subject of the same age, sex, and / or body mass index who does not have a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders)) or patients whose GRN mutation status and / or PGRN activity levels are unknown.The compositions and methods described herein also include methods for evaluating the efficacy and safety of a GRN mutation in patients at risk of developing a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders), e.g., patients with a GRN mutation, reduced PGRN activity and / or expression (e.g., intrinsic to the GRN mutation observed in human subjects of the same age, sex, and / or body mass index who do not have a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders), and / or in patients with a GRN mutation, who have a GRN mutation in the GRN mutation. The present invention may be administered as a preventative treatment to patients with a neurodegenerative disorder, such as a neurocognitive disorder (e.g., a level of expression of endogenous PGRN that is about 1% to about 40% of the level of endogenous PGRN expression), a patient with a mutation in one or more of the genes associated with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders), or a patient who has been exposed to an environmental toxin associated with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). A patient at risk for a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders) may be showing early symptoms of a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders) or may not yet be symptomatic when treatment is administered.

[0095] In some embodiments, the methods and compositions described herein are used to detect and treat a variety of cancers, including, for example, frameshift mutations (e.g., p.C31LfsX35, p.C31LfsX35, p.S82VfsX174, p.L271LfsX174, and / or p.T382NfsX32 mutations), missense mutations (p.C521Y, p.A9D, p.P248L, p.R432C, p.C139R, p.C521Y, and / or p.C139R mutations), nonsense mutations (e.g., p.C31LfsX35, p.C31LfsX35, p.S82VfsX174, p.L271LfsX174, and / or p.T382NfsX32 mutations), The present invention may be administered to patients with GRN mutations, including GRN mutations (e.g., p.Q125X mutation), insertion mutations (e.g., c.1145insA mutation), and / or transversion mutations (e.g., p.0(IVS1+5G>C mutation). In some embodiments, the methods and compositions described herein may be administered to patients carrying any other pathogenic mutation in the GRN gene. For example, the pathogenic mutation in the GRN gene may be any of the mutations discussed in Gijselinck et al., Human Mutation 29(12), 1373-1386, (2012), the disclosure of which is incorporated herein by reference as it relates to human GRN mutations.

[0096] In some embodiments, the disclosure provides methods of treating a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders) in a human patient in need thereof.

[0097] In some embodiments, the disclosure provides methods of improving cognitive function in a human patient diagnosed with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders).

[0098] In some embodiments, the disclosure provides methods of expressing or restoring expression of PGRN in the brain (e.g., frontal cortex) of a human patient diagnosed with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders).

[0099] Polynucleotide encoding PGRN PGRN activity is reduced in patients with FTD. The compositions and methods described herein target this dysfunction by administering an AAV vector expressing a transgene encoding PGRN. Such constructs can be produced using methods well known to those skilled in the art.

[0100] The recognition and binding of the polynucleotide encoding PGRN by mammalian RNA polymerase is important for gene expression. Therefore, sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and promote the assembly of a transcription complex at the transcription initiation site can be included in the polynucleotide. Such sequence elements include, for example, mammalian promoters, whose sequences can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters are described in Smith et al., Mol. Sys. Biol., 3:73, online publication, the disclosure of which is incorporated herein by reference.

[0101] Polynucleotides suitable for use with the compositions and methods described herein also include those encoding PGRN downstream of a mammalian promoter. Promoters useful for expression of GRN in mammalian cells include, for example, the synapsin promoter, the tetracycline-controlled transactivator protein (tTA) promoter, the cytomegalovirus (CMV) promoter, the reverse tetracycline-controlled transactivator protein (rTA) promoter, the U1 promoter, the U6 promoter, the U7 promoter, the prion promoter, the phosphoglycerate kinase (PGK) promoter, the CB7 promoter, the H1 promoter, the CMV-chicken β-actin (CBA) promoter, the glial fibrillary acidic protein (GFAP) promoter, the calcium / calmodulin-dependent protein kinase III promoter, the tubulin alpha I promoter, the microtubulin-associated protein IB (MAP IB) promoter, neuron-specific enolase promoter, platelet-derived growth factor beta chain promoter, neurofilament light chain promoter, neuron-specific VGF gene promoter, neuron nucleus (NeuN) promoter, adenomatous polyposis coli (APC) promoter, ionized calcium-binding adaptor molecule 1 (Iba-1) promoter, or homeobox protein 9 (HB9) promoter, elongation factor 1 alpha (EF1α) promoter, CD68 molecule (CD68) promoter (Dahl et al. al., Mol. Ther. 23:835 (2015), incorporated herein as it relates to the use of PGK and CD68 promoters to express GRN), C-X3-C motif chemokine receptor 1 (CX3CR1) promoter, integrin subunit alpha M (ITGAM) promoter, allograft inflammatory factor 1 (AIF1) promoter, purinergic receptor P2Y12 (P2Y12) promoter, transmembrane protein 119 (TMEM119) promoter, and colony stimulating factor 1 receptor (CSF1R) promoter. In some embodiments, the promoter is a synapsin promoter.

[0102] In some embodiments, the transgene encoding PGRN is operably linked to a promoter that is active in neuronal cells (eg, synapsin) and / or glial cells.

[0103] Other DNA sequence elements that may be included in polynucleotides for use in the compositions and methods described herein are enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in polynucleotides containing genes of interest such that the DNA adopts a three-dimensional orientation that favors the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode PGRN, as well as mammalian enhancer sequences. Many enhancer sequences are currently known from mammalian genes, examples being enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include enhancers derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples are the SV40 enhancer (100-270 bp) on the late side of the replication origin, the CMV early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences directing activation of the transcription of eukaryotic genes are disclosed in Yaniv et al., Nature 297:17 (1982).

[0104] Example PGRN In one approach, the invention provides PGRN having an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:2. For example, in some embodiments, PGRN has an amino acid sequence that is at least 86% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 87% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 88% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 89% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 91% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 93% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 94% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN encodes a protein that is identical to the amino acid sequence of SEQ ID NO:2.

[0105] Secretory signal peptide A polynucleotide encoding PGRN may include one or more polynucleotides encoding secretory signal peptides. Secretory signal peptides may have an amino acid sequence between 5 and 30 residues long and may be located upstream (i.e., 5' from) the polynucleotide encoding PGRN. These secretory signal peptides allow recognition of the nascent polypeptide during synthesis by a signal recognition particle resulting in translocation to the ER, packaging into transport vesicles, and eventual secretion. Exemplary secretory signal peptides for protein secretion are from PGRN, IGF-II, alpha 1 antitrypsin, IL-2, IL-6, CD5, immunoglobulins, trypsinogen, serum albumin, prolactin, elastin, tissue plasminogen activator signal peptide (tPA-SP), and insulin. In some embodiments, pluripotent cells (e.g., ESCs, iPSCs, or CD34+ cells) expressing a secreted form of PGRN may be utilized as a therapeutic strategy to correct a protein defect (e.g., PGRN) by injecting the missing protein into the bloodstream. As blood perfuses the patient's tissues, PGRN is taken up by cells and transported to its site of action.

[0106] Codon optimization The compositions and methods described herein can be used to optimize the nucleic acid sequence of GRN or its RNA equivalent encoding PGRN, for example, to achieve enhanced expression of PGRN in a particular cell type. For example, the compositions and methods described herein can be used to optimize genes and their RNA equivalents for tissue-specific expression of encoded proteins, such as PGRN. Genes and their RNA equivalents optimized using the compositions and methods described herein can be synthesized by chemical synthesis techniques and amplified, for example, using polymerase chain reaction (PCR)-based amplification methods, or by transfection of the genes into cells, such as bacterial or mammalian cells, that can replicate exogenous nucleic acids.

[0107] The gene and RNA equivalents described herein may have important clinical utility. For example, FTD is a manifestation of the deficiency of native PGRN protein. With the advent of gene therapy, a wide range of vectors and gene delivery techniques have been developed for the introduction of exogenous protein-encoding nucleic acid into target cells (e.g., human cells). However, there remains a need for a unified set of guidelines that can be followed to optimize the sequence of the exogenous transgene encoding PGRN to achieve robust and stable expression of the protein in the cells of interest.

[0108] Single nucleotide variations that preserve the amino acid sequence of the encoded protein can be informed, for example, by the standard genetic code, as represented in Table 3 below, compiled by the National Center for Biotechnology Information, Bethesda, Maryland, USA. [Table 3]

[0109] The codon optimization process can be carried out iteratively.For example, one skilled in the art can start with a wild-type gene sequence (e.g., excluding intron DNA) and introduce substitutions into this sequence that reduce the sequence identity of genes to genes that are expressed in target cells (e.g., genes whose expression levels are in the top 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the gene expression levels in the intended target cells).This process can be repeated until all codons in the gene of interest are evaluated for the opportunity to introduce single nucleotide substitutions that can reduce sequence identity to genes that are expressed at high levels in target cells. Alternatively, one can start with a gene sequence that has been previously modified relative to the wild-type sequence of the gene, for example by incorporating codon substitutions that increase the GC content of the gene relative to the wild-type sequence and / or reduce the CpG content of the gene. The resulting gene sequence can then be aligned to the coding strand of a gene that is expressed in the desired target cell (e.g., a gene whose expression level is within the top 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more of gene expression levels in the intended target cell), and repeated codon substitutions can be introduced throughout the gene to minimize the sequence identity of the previously modified gene with respect to genes that are expressed at high levels in the target cell.

[0110] Preparation of codon-optimized genes Once designed, the final codon-optimized gene can be prepared, for example, by solid-phase nucleic acid procedures known in the art. For example, a solid-phase synthesis process using the phosphoramidite method can be used to perform chemical synthesis of nucleic acid molecules such as DNA, RNA, etc. According to this procedure, nucleic acid is generally synthesized by the following steps:

[0111] First, the 5-OH protected nucleoside occurring at the 3'-end of the nucleic acid to be synthesized is esterified to a solid support via the 3'-OH function by attaching the nucleoside to a cleavable linker.Then, the support for solid-phase synthesis on which the nucleoside is fixed can be placed in a reaction column, which is then set into an automated nucleic acid synthesizer.

[0112] Then, an iterative synthesis process including the following steps can be carried out in the reaction column according to the synthesis program of the automated nucleic acid synthesizer: (1) a step of deprotection of the 5'-OH moiety of the protected immobilized nucleoside (e.g., using an acid such as trichloroacetic acid in dichloromethane solution to remove the acid-labile hydroxyl protecting group), (2) coupling a 5-OH protected nucleoside phosphoramidite with the deprotected 5'-OH group of the immobilized nucleoside in the presence of an activating agent (e.g., tetrazole, etc.); (3) capping the unreacted 5'-OH group of the 3'-terminal nucleoside (e.g., with acetic anhydride, etc.); and (4) oxidizing the immobilized phosphite substituent (e.g., with aqueous iodine).

[0113] The above process can be repeated to extend the nucleic acid in the 3' to 5' direction as necessary, and the 5' end direction is promoted to synthesize a nucleic acid having a desired sequence.

[0114] Finally, the cleavable linker is hydrolyzed (e.g., with aqueous ammonia, methylamine solution, etc.) to cleave the synthesized nucleic acid from the solid support. Procedures such as those described above for chemical synthesis of nucleic acids are known in the art and are described, for example, in U.S. Patent No. 8,835,656, the disclosure of which is incorporated herein by reference where it relates to protocols for the synthesis of nucleic acid molecules.

[0115] Additionally, the prepared genes can be amplified, for example, using PCR-based techniques described herein or known in the art, and / or by transformation of DH5α E. coli with a plasmid containing the designed gene. The bacteria can then be cultured to amplify the DNA therein, and the gene can be isolated by plasmid purification techniques known in the art, optionally followed by restriction digestion and / or sequencing of the plasmid to verify the identity of the codon-optimized gene.

[0116] Exemplary codon-optimized PGRN In one approach, the present invention provides a codon-optimized PGRN having a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:3. For example, in some embodiments, the PGRN has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN encodes a protein that is identical to the nucleic acid sequence of SEQ ID NO:3.

[0117] AAV for delivering PGRN to target cells Viral genomes provide a rich source of vectors that can be used to efficiently deliver genes of interest to the genome of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for gene delivery, because the polynucleotides contained within such genomes are typically integrated into the genome of target cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require additional proteins or reagents to induce gene integration. Examples of viral vectors include AAV.

[0118] The nucleic acids of the compositions and methods described herein can be incorporated into recombinant AAV (rAAV) vectors and / or virions to facilitate their introduction into cells. AAV vectors can be used in the central nervous system, and suitable promoters and serotypes are discussed in Pignataro et al., J Neural Transm (2017), epub ahead of print, the disclosure of which is incorporated herein by reference as it relates to promoters and AAV serotypes useful in CNS gene therapy. rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that contain (1) the heterologous sequence to be expressed (e.g., a polynucleotide encoding PGRN) and (2) viral sequences that facilitate integration and expression of the heterologous gene. The viral sequences can include sequences of AAV required in cis for DNA replication and packaging into virions (e.g., functional ITRs). Such rAAV vectors can also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part, but retain functional adjacent ITR sequences.AAV ITRs can be of any serotype suitable for a particular application.Methods for using rAAV vectors are described, for example, in Tai et al., J.Biomed.Sci.7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0119] The nucleic acids and vectors described herein can be incorporated into rAAV viral particles to facilitate the introduction of the nucleic acid or vector into cells. The capsid protein of AAV constitutes the outer non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins VP1, VP2, and VP3, which are required for virion assembly. The construction of rAAV virions is described, for example, in US 5,173,414, US 5,139,941, US 5,863,541, US 5,869,305, US 6,057,152, and US 6,376,237, as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0120] rAAV virions useful in combination with the compositions and methods described herein include those derived from various AAV serotypes, including AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and rh74. For targeting cells located in or delivered to the central nervous system, AAV2, AAV9, and AAV10 may be particularly useful. The construction and use of AAV vectors of different serotypes and AAV proteins are described, for example, in Chao et al., Mol. Ther. 2:619 (2000), Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000), Xiao et al., J. Virol. 72:2224 (1998), Halbert et al., J. Virol. 74:1524 (2000), Halbert et al., J. Virol. 75:6615 (2001), and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated by reference herein as they relate to AAV vectors for gene delivery.

[0121] Also useful in combination with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). For example, a representative pseudotyped vector is an AAV8 or AAV9 vector encoding a therapeutic protein (e.g., frataxin) pseudotyped with a capsid gene from AAV serotype 2. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001), Halbert et al., J. Virol. 74:1524 (2000), Zolotukhin et al., Methods, 28:158 (2002), and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0122] AAV virions with mutations in the virion capsid can be used to infect specific cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations to facilitate targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in the methods described herein include capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0123] Exemplary AAV Vectors As described herein, exemplary AAV vector components can include a promoter, an intron, a polynucleotide encoding PGRN or a codon-optimized version thereof, a 3' enhancer element, and / or a bovine growth hormone (bGH) polyadenylation site (pA).

[0124] In some embodiments, the AAV may comprise a synapsin promoter, a tetracycline-regulated transactivator protein (tTA) promoter, a reverse tetracycline-regulated transactivator protein (rTA) promoter, a U1 promoter, a U6 promoter, a U7 promoter, a prion promoter, a phosphoglycerate kinase (PGK) promoter, a CB7 promoter, an H1 promoter, a cytomegalovirus (CMV) promoter, a CMV-chicken β-actin (CBA) promoter, a glial fibrillary acidic protein (GFAP) promoter, a calcium / calmodulin-dependent protein kinase III promoter, a tubulin alpha I promoter, a microtubulin-associated protein IB (MAP IB) promoter, a neuron-specific enolase promoter, a platelet-derived growth factor beta chain promoter, a neurofilament light chain promoter, a neuron-specific VGF gene promoter, a neuronal nucleus (NeuN) promoter, an adenomatous polyposis coli (APC) promoter, an ionized calcium-binding adaptor molecule 1 (Iba-1) promoter, or a homeobox protein 9 (HB9) promoter. For example, in some embodiments, the promoter is a synapsin promoter. In some embodiments, the PGRN is operably linked to a promoter active in neuronal and / or glial cells.

[0125] In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:1. For example, in some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of synapsin ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the synapsin promoter has a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:1.

[0126] In some embodiments, the PGRN is operably linked to a human growth hormone (hGH) intron. For example, in some embodiments, the hGH intron is hGH intron 3.

[0127] In some embodiments, the hGH intron has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:4. For example, in some embodiments, the hGH intron has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of ID NO:1. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the hGH intron has a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:4.

[0128] In some embodiments, the AAV may comprise a PGRN having an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:2. For example, in some embodiments, the PGRN has an amino acid sequence that is at least 86% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the PGRN has an amino acid sequence that is at least 87% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the PGRN has an amino acid sequence that is at least 88% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the PGRN has an amino acid sequence that is at least 89% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the PGRN has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the PGRN has an amino acid sequence that is at least 91% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 93% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 94% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN has an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, PGRN encodes a protein that is identical to the amino acid sequence of SEQ ID NO:2.

[0129] In some embodiments, the AAV may comprise a codon-optimized PGRN having a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:3. For example, in some embodiments, the PGRN has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the PGRN has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, PGRN encodes a protein that is identical to the nucleic acid sequence of SEQ ID NO:3.

[0130] In some embodiments, the PGRN is operably linked to a 3' enhancer element. In some embodiments, the 3' enhancer element is a human PGRN 3' untranslated region (UTR).

[0131] In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:5. For example, in some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:5.In some embodiments, the human PGRN 3'UTR has a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:5.

[0132] In some embodiments, the PGRN is operably linked to bGH pA.

[0133] In some embodiments, bGH pA has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:8. For example, in some embodiments, bGH pA has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, bGH pA has a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:8.

[0134] Exemplary nucleic acids that can be incorporated into an AAV are set forth in Table 4 below.

Table 4

[0135] In some embodiments, the AAV has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:6. For example, in some embodiments, the AAV has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the AAV has a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:6.

[0136] As described herein, an exemplary AAV having the nucleic acid sequence of SEQ ID NO:6 is shown below: TGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCAGGCCTACATGT

[0137] Additional therapeutic transgenes and proteins The AAV described herein may comprise a polynucleotide encoding a therapeutic protein useful for treating a disorder affecting the CNS, such as a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disease) or a lysosomal storage disorder, among other disorders that adversely affect the CNS. Exemplary therapeutic proteins useful in combination with the compositions and methods of the present disclosure are shown in Table 5, below. [Table 5] TIFF2024530050000010.tif208165TIFF2024530050000011.tif226165TIFF2024530050 000012.tif206165TIFF2024530050000013.tif70165TIFF2024530050000014.tif237165

[0138] Exemplary AAVs encoding transgenes encoding proteins listed in Table 5 are useful for expressing or restoring healthy physiological concentrations of the proteins in the CNS of healthy human subjects without neurocognitive or neuromuscular disorders. For example, the healthy physiological concentration of GDNF is less than 10 ng / mL in the CNS. The healthy physiological concentration of BDNF is less than 50 pg / mL in the cerebrospinal fluid (CSF) and up to about 25 pg / mL in the prefrontal cortex. The healthy physiological concentration of ApoE is 4.5 ng / mL in the CSF. The healthy physiological concentration of GCase is 0.02-0.14 ng / mL in the CSF.

[0139] The AAV described herein may comprise a polynucleotide encoding a CNS protein associated with a lysosomal storage disorder. Exemplary proteins include α-galactosidase a, α-1-iduronidase, iduroate sulfatase, lysosomal acid α-glucosidase, sphingomyelinase, hexosaminidase A (HexA), hexosaminidase B (HexB), arylsulfatase A (ARSA), lysosomal acid lipase, acid ceramidase, galactosylceramidase, α-fucosidase, α-, β-mannosidosis, aspartylglucosidase, α-galactosylceram ... Examples of AAVs that can be used to treat lysosomal storage disorders include, but are not limited to, saminidase, neuramidase, heparan-N-sulfatase, N-acetyl-α-glucosaminidase, acetyl-CoA:α-glucosamide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, arylsulfatase B (ARSB), and β-glucuronidase. Exemplary AAVs encoding transgenes encoding proteins associated with lysosomal storage disorders are useful for expressing or restoring healthy physiological concentrations of the proteins in the CNS of healthy human subjects without neurocognitive or neuromuscular disorders. For example, the healthy physiological concentration of ARSA is 100 ng / mg in the CNS. The healthy physiological concentration of HexA is 20 ng / mL in plasma, and the healthy physiological concentration of HexB is 40 ng / mL in plasma.

[0140] Route of Administration The AAV described herein can be administered intrathalamically to a subject with FTD. In some embodiments, the AAV vector is administered to the patient in a convectively supported manner.

[0141] In some embodiments, administration may include convectively assisted administration, such as that described in Bobo et al. PNAS. 91:6 (1994): 2076-2080, the disclosure of which is incorporated by reference herein as it relates to convectively assisted administration.

[0142] Dosing regimen Using the compositions and methods of the present disclosure, patients with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders) can receive approximately 1×10 9 vg / hemisphere ~ approx. 9×10 12 An AAV vector containing a transgene encoding PGRN (eg, codon-optimized PGRN) can be administered intrathalamically in a dose of vg / hemisphere.

[0143] In some embodiments, the AAV vector is about 1 x 10 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., 1×10 9 vg / hemisphere ~ approx. 5×10 12 vg / hemisphere, 2×10 9 vg / hemisphere ~ approx. 4×10 12 vg / hemisphere, 3×10 9 vg / hemisphere ~ approx. 3×10 12 vg / hemisphere, 4×10 9 vg / hemisphere ~ approx. 2×10 12 vg / hemisphere, 5×10 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, 1×10 10 vg / hemisphere ~ approx. 9×10 11 vg / hemisphere, 2×10 10 vg / hemisphere~approx. 8×10 11 vg / hemisphere, 3×10 10 vg / hemisphere ~ approx. 7×10 11 vg / hemisphere, 4×10 10 vg / hemisphere ~ approx. 6×10 11 vg / hemisphere, 5×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 1×10 11 vg / hemisphere) to the patient. For example, in some embodiments, the AAV vector is administered intrathalamally to the patient at a dose of about 2×10 9 vg / hemisphere ~ approx. 4×10 12 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 3×10 vg / hemisphere. 9 vg / hemisphere ~ approx. 3×10 12In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 4×10 vg / hemisphere. 9 vg / hemisphere ~ approx. 2×10 12 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 5×10 vg / hemisphere. 9 vg / hemisphere ~ approx. 1×10 12 In some embodiments, the AAV vector is administered intrathalamically to a patient in an amount of about 1×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 9×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 2×10 vg / hemisphere. 10 vg / hemisphere~approx. 8×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 3×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 7×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 4×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 6×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 5×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 5×10 11 In some embodiments, the AAV vector is administered intrathalamically to a patient in an amount of about 1×10 vg / hemisphere. 11 vg / hemisphere is administered intrathalamic to patients.

[0144] In some embodiments, the AAV vector is about 1 x 10 10 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere (e.g., 1×10 10 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, 2×10 10 vg / hemisphere ~ approx. 9×10 11 vg / hemisphere, 3×10 10 vg / hemisphere~approx. 8×10 11 vg / hemisphere, 4×10 10 vg / hemisphere ~ approx. 7×10 11 vg / hemisphere, 5×10 10 vg / hemisphere ~ approx. 6×10 11vg / hemisphere, 6×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, 7×10 10 vg / hemisphere ~ approx. 4×10 11 vg / hemisphere, 8×10 10 vg / hemisphere ~ approx. 3×10 11 vg / hemisphere, 9×10 10 vg / hemisphere ~ approx. 2×10 11 vg / hemisphere, or approximately 1×10 11 vg / hemisphere) to the patient. For example, in some embodiments, the AAV vector is administered intrathalamally to the patient at a dose of about 2×10 10 vg / hemisphere ~ approx. 9×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 3×10 vg / hemisphere. 10 vg / hemisphere~approx. 8×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 4×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 7×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 5×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 6×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 6×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 5×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 7×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 4×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 8×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 3×10 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 9×10 vg / hemisphere. 10 vg / hemisphere ~ approx. 2×10 11 In some embodiments, the AAV vector is administered intrathalamically to a patient in an amount of about 1×10 vg / hemisphere. 11 vg / hemisphere is administered intrathalamic to patients.

[0145] In some embodiments, the AAV vector is about 5×10 10 vg / hemisphere ~ approx. 1×10 11 vg / hemisphere (e.g., 5×10 10 vg / hemisphere ~ approx. 1×10 11 vg / hemisphere, 6×10 10 vg / hemisphere ~ approx. 9×10 10 vg / hemisphere, or 7×10 10 vg / hemisphere~approx. 8×10 10 For example, in some embodiments, the AAV vector is administered intrathalamally to a patient at a dose of about 6×10 10 vg / hemisphere ~ approx. 9×10 10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 7×10 vg / hemisphere. 10 vg / hemisphere~approx. 8×10 10 vg / hemisphere is administered intrathalamic to patients.

[0146] In some embodiments, the AAV vector is about 1 x 10 9 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 2×10 vg / hemisphere. 9 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 3×10 vg / hemisphere. 9 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 4×10 vg / hemisphere. 9 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 5×10 vg / hemisphere. 9 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 6×10 vg / hemisphere. 9 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 7×10 vg / hemisphere. 9 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 8×10 vg / hemisphere. 9 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 9×10 vg / hemisphere. 9 In some embodiments, the AAV vector is administered intrathalamically to a patient in an amount of about 1×10 vg / hemisphere.10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 2×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 3×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 4×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 5×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 6×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 7×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 8×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 9×10 vg / hemisphere. 10 In some embodiments, the AAV vector is administered intrathalamically to a patient in an amount of about 1×10 vg / hemisphere. 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 2×10 vg / hemisphere. 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 3×10 vg / hemisphere. 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 4×10 vg / hemisphere. 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 5×10 vg / hemisphere. 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 6×10 vg / hemisphere. 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 7×10 vg / hemisphere. 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 8×10 vg / hemisphere. 11 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 9×10 vg / hemisphere.11 In some embodiments, the AAV vector is administered intrathalamically to a patient in an amount of about 1×10 vg / hemisphere. 12 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 2×10 vg / hemisphere. 12 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 3×10 vg / hemisphere. 12 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 4×10 vg / hemisphere. 12 In some embodiments, the AAV vector is administered intrathalamally to the patient in an amount of about 5×10 vg / hemisphere. 12 vg / hemisphere is administered intrathalamic to patients.

[0147] For example, in some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is about 1×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., 1×10 9 vg / hemisphere ~ approx. 5×10 12 vg / hemisphere, 2×10 9 vg / hemisphere ~ approx. 4×10 12 vg / hemisphere, 3×10 9 vg / hemisphere ~ approx. 3×10 12 vg / hemisphere, 4×10 9 vg / hemisphere ~ approx. 2×10 12 vg / hemisphere, 5×10 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, 1×10 10 vg / hemisphere ~ approx. 9×10 11 vg / hemisphere, 2×10 10 vg / hemisphere~approx. 8×10 11 vg / hemisphere, 3×10 10 vg / hemisphere ~ approx. 7×10 11 vg / hemisphere, 4×10 10 vg / hemisphere ~ approx. 6×10 11 vg / hemisphere, 5×10 10 vg / hemisphere ~ approx. 5×10 11vg / hemisphere, or approximately 1×10 11 vg / hemisphere) is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). For example, in some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is approximately 2×10 6 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 9 vg / hemisphere ~ approx. 4×10 12 vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is approximately 3×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 9 vg / hemisphere ~ approx. 3×10 12 vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is approximately 4×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 9 vg / hemisphere ~ approx. 2×10 12vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is approximately 5×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex, which is approximately 1×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 10 vg / hemisphere ~ approx. 9×10 11 vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is approximately 2×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 10 vg / hemisphere~approx. 8×10 11vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is approximately 3×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 10 vg / hemisphere ~ approx. 7×10 11 vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is approximately 4×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 10 vg / hemisphere ~ approx. 6×10 11 vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex that is approximately 5×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 10 vg / hemisphere ~ approx. 5×10 11vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex, which is approximately 1×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 11 Following intrathalamic administration at a dose of vg / hemisphere, the levels of PGRN expression are equivalent to those observed in human subjects with neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders).

[0148] In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 2 ng / mg to about 8 ng / mg (e.g., 3 ng / mg to about 7 ng / mg, 4 ng / mg to about 6 ng / mg, or about 5 ng / mg), or more (e.g., about 9 ng / mg, about 10 ng / mg, about 15 ng / mg, about 20 ng / mg, about 30 ng / mg, about 40 ng / mg, about 50 ng / mg, about 60 ng / mg, about 70 ng / mg, about 80 ng / mg, about 90 ng / mg, or about 100 ng / mg). For example, in some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 2 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 3 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 4 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 5 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 6 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 7 ng / mg in the frontal cortex. In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 8 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 9 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 10 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 11 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 12 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 13 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 14 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 15 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 16 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 17 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 18 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 19 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 20 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 21 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 22 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 23 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 24 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 25 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 26 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 27 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 28 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 29 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 30 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 31 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 32 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 33 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 34 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 35 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 36 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 37 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 38 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 39 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 40 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 41 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 42 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 43 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 44 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 45 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 46 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 47 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 48 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 49 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 50 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 51 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 52 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 53 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 54 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 55 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 56 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 57 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 58 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 59 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 60 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 61 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 62 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 63 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 64 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 65 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 66 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 67 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 68 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 69 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 70 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 71 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 72 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 73 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 74 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 75 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 76 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 77 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 78 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 79 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 80 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 81 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 82 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 83 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 84 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 85 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 86 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 87 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 88 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 89 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 90 ng / mg in the frontal cortex.In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 91 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 92 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 93 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 94 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 95 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 96 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 97 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 98 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 99 ng / mg in the frontal cortex. In some embodiments, the AAV vector comprising a transgene encoding PGRN is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 98 ng / mg in the frontal cortex.In some embodiments, an AAV vector containing a transgene encoding PGRN is administered to a patient in an amount sufficient to achieve a level of PGRN expression in the patient's frontal cortex of about 100 ng / mg in the frontal cortex.

[0149] In some embodiments, the AAV vector is administered to the patient in a single dose per hemisphere comprising that amount.

[0150] In some embodiments, the AAV vector is administered to the patient in multiple doses per hemisphere (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses), all of which contain that amount.

[0151] In some embodiments, the AAV vector is administered to the patient in two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) doses per hemisphere, each individually comprising that amount.

[0152] In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) doses per hemisphere are separated by one year or more (e.g., 1 year, 1 year and 1 day, 1 year and 1 month, 1 year and 6 months, 2 years, 3 years, 4 years, or 5 years).

[0153] In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) doses per hemisphere are administered to the patient within about 12 months of each other (e.g., about 12 months, about 11 months, about 10 months, about 9 months, about 8 months, about 7 months, about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, or about 1 month).

[0154] In some embodiments, the AAV vector comprises a transgene encoding a protein associated with a neurocognitive disorder, neuromuscular disorder, or lysosomal storage disorder, such as a protein listed in Table 5. In some embodiments, the AAV vector comprises a transgene encoding a protein associated with a neurocognitive disorder, neuromuscular disorder, or lysosomal storage disorder, such as a protein listed in Table 5. In some embodiments, the AAV vector comprises a transgene encoding a protein associated with a neurocognitive disorder, neuromuscular disorder, or lysosomal storage disorder, such as a protein listed in Table 5. In some embodiments, the AAV vector comprises a transgene encoding a protein associated with a neurocognitive disorder, neuromuscular disorder, or lysosomal storage disorder, such as a protein listed in Table 5. ramidase, heparan-N-sulfatase, N-acetylglucosaminidase, acetyl-CoA:α-glucosamide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, arylsulfatase A, arylsulfatase B, and β-glucuronidase.

[0155] The AAV described herein may be administered in an amount sufficient to ameliorate one or more pathological features in a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). Administration of an AAV as described herein may improve cognitive ability in a subject, restore expression of a protein encoded by the transgene in the frontal cortex (e.g., about 2 ng / mg to about 8 ng / mg (e.g., 3 ng / mg to about 7 ng / mg, 4 ng / mg to about 6 ng / mg, or about 5 ng / mg), or more (e.g., about 9 ng / mg, about 10 ng / mg, about 15 ng / mg, about 20 ng / mg, about 30 ng / mg, about 40 ng / mg, about 50 ng / mg, about 60 ng / mg, about 70 ng / mg, about 80 ng / mg, about 90 ng / mg, or about 100 ng / mg)), improve motor function in a subject, and reduce alpha-synuclein protein levels, tau-positive neuronal inclusion levels, and / or TAR DNA-binding protein 43 (TDP-43)-positive inclusion levels in brain tissue of a subject. Cognitive and motor function can be assessed using standard neurological tests before and after treatment, and protein levels (e.g., PGRN) can be detected in plasma and cerebrospinal fluid (CSF) using ELISA. Neurodegeneration can be assessed using F18-fluorodeoxyglucose PET scans or MRI scans. Patients can be evaluated 1, 2, 3, 4, 5, 6 or more months after administration of AAV. Depending on the results of the evaluation, patients may receive additional treatment.

[0156] Pharmaceutical Compositions The AAV described herein can be formulated into a pharmaceutical composition for administration to a patient, such as a human patient, exhibiting or at risk of a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder, such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders) in a biologically compatible form suitable for administration in vivo. For example, a pharmaceutical composition containing an AAV comprising one or more transgenes encoding PGRN described herein typically comprises a pharma- ceutically acceptable diluent or carrier. The pharmaceutical composition can comprise (e.g., consist of), for example, a sterile saline solution and a nucleic acid. The sterile saline is typically a pharmaceutical grade saline. The pharmaceutical composition can comprise (e.g., consist of), for example, sterile water and a nucleic acid. The sterile water is typically a pharmaceutical grade water. The pharmaceutical composition can comprise (e.g., consist of), for example, phosphate buffered saline (PBS) and a nucleic acid. The sterile PBS is typically a pharmaceutical grade PBS.

[0157] In certain embodiments, a pharmaceutical composition comprises one or more compositions or nucleic acid molecules and one or more excipients, hi certain embodiments, the excipients are selected from water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0158] In certain embodiments, the nucleic acid molecules can be mixed with pharma- ceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations. The methods for formulating the compositions and pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0159] In certain embodiments, the pharmaceutical composition comprising the nucleic acid molecule includes any pharma- ceutically acceptable salt of the inhibitor, an ester of the inhibitor, or a salt of such an ester. In certain embodiments, the pharmaceutical composition comprising the nucleic acid molecule can produce (directly or indirectly) a biologically active metabolite or residue thereof when administered to a subject (e.g., a human). Thus, for example, the present disclosure is also drawn to pharma- ceutically acceptable salts of the inhibitor, prodrugs, pharma- ceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharma- ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, the prodrugs include one or more conjugate groups attached to the nucleic acid molecule, and the conjugate groups are cleaved by endogenous nucleases in the body.

[0160] Lipid moieties have been used in a variety of ways in nucleic acid therapy. In certain such methods, nucleic acids are introduced into preformed liposomes or lipoplexes made with a mixture of cationic and neutral lipids. In certain methods, DNA complexes with mono- or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to a particular cell or tissue. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to adipose tissue. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to muscle tissue.

[0161] In certain embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions, including those that comprise hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethylsulfoxide, are used.

[0162] In certain embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents of the present invention to a specific tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises a liposome coated with a tissue-specific antibody.

[0163] In certain embodiments, the pharmaceutical composition includes a co-solvent system. The composition of such a co-solvent system includes, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such a co-solvent system is used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of 3% w / v benzyl alcohol, 8% w / v of the non-polar surfactant Polysorbate 80™, and 65% w / v of polyethylene glycol 300 in absolute ethanol. The proportions of such co-solvent systems can be varied significantly without significantly altering their solubility and toxicity properties. Furthermore, the identity of the co-solvent components can be changed, for example, other surfactants can be used in place of Polysorbate 80™, the fractional amount of polyethylene glycol can be changed, other biocompatible polymers, such as polyvinylpyrrolidone, can be substituted for polyethylene glycol, and other sugars or polysaccharides can be substituted for dextrose.

[0164] In certain embodiments, the pharmaceutical composition is prepared for intrathalamic administration. In such embodiments, the pharmaceutical composition may include a carrier and is formulated in an aqueous solution, such as water or a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. In some embodiments, other ingredients (e.g., ingredients that aid solubility or function as preservatives) are included. In some embodiments, an injectable suspension is prepared using appropriate liquid carriers, suspending agents, and the like. Certain injectable pharmaceutical compositions are provided in unit dosage form, for example, in ampoules or in multi-dose containers. Certain injectable pharmaceutical compositions are suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulating agents, such as suspending agents, stabilizing agents, and / or dispersing agents. Certain solvents suitable for use in injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents, such as sesame oil, and fatty oils, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

[0165] Efficacy monitoring Clinical efficacy can be monitored using biomarkers, among other methods.The measurable biomarkers for monitoring efficacy include, but are not limited to, monitoring one or more of the physical symptoms of neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). These may include tremors, muscle spasms or weakness (e.g., affecting the arms, legs, neck, or diaphragm), stiffness (e.g., stiff muscles), poor coordination and / or balance, difficulty chewing or swallowing, weight gain from dramatic overeating, stiff muscles, shuffling and dragging of the feet when walking, problems getting up or sitting down in a chair, fatigue, problems controlling the bladder, seizures, uncontrollable contractions (e.g., fasciculations of the arms, legs, shoulders, or tongue), bradykinesia, poor posture, loss of motility, changes in speech, changes in writing, poor regulation of bodily functions (e.g., autonomic nervous system), sleep disturbances, clumsiness, stumbling, slurred speech, or muscle wasting. Observation of stabilization, improvement, and / or reversal of one or more symptoms indicates that the treatment or prevention regime is effective. Observation of progression, increase, or worsening of one or more symptoms indicates that the treatment or prevention regime is not effective. A preferred biomarker for evaluating the treatment of neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders) is the level of PGRN.This marker is preferably evaluated at protein level, but the measurement of the mRNA encoding PGRN can also be used as a surrogate measurement of PGRN expression.Such level can be measured in blood samples.Such levels are reduced in subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorder, ALS, or associated motor neuron disorder) relative to a control population of disease-free individuals (e.g., patients exhibit levels of expression of endogenous PGRN that are about 1% to about 40% of the levels of endogenous PGRN expression observed in human subjects of the same age, sex, and / or body mass index without a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorder, ALS, or associated motor neuron disorder). Thus, increased levels provide an indication of a favorable therapeutic response, whereas unchanged or decreased levels provide an indication of an unfavorable, or at least suboptimal, therapeutic response.

[0166] In certain embodiments, monitoring methods may involve determining a baseline value of a measurable biomarker or disease parameter in a subject prior to administration of a dosage of AAV described herein, and comparing it to the value of the same measurable biomarker or parameter after a course of treatment.

[0167] In other methods, control values ​​(i.e., mean and standard deviation) of a measurable biomarker or parameter are determined for a control population. For example, in some embodiments, prior to administration of the AAV vector, the patient exhibits a level of expression of endogenous PGRN that is about 1% to about 40% of the level of endogenous PGRN expression observed in a human subject of the same age, sex, and / or body mass index who does not have a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorders, ALS, or associated motor neuron disorders). In certain embodiments, the individuals in the control population have not received prior treatment and do not have a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorders, ALS, or associated motor neuron disorders), and are not at risk of developing a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, associated neurocognitive disorders, ALS, or associated motor neuron disorders). In such a case, if the value of measurable biomarker or clinical parameter approaches control value, then the treatment is considered to be effective.In other embodiments, the individuals of the control population have not received prior treatment and have been diagnosed with neurocognitive or neuromuscular disorder (e.g., neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorder, ALS, or related motor neuron disorder).In such a case, if the value of measurable biomarker or clinical parameter approaches control value, then the treatment is considered to be ineffective.

[0168] In other methods, subjects who are not currently undergoing treatment but have undergone a previous course of treatment are monitored for one or more of the biomarkers or clinical parameters to determine whether treatment needs to be resumed. One or more measurements of the biomarkers or clinical parameters in the subject can be compared to the values ​​previously achieved in the subject after a previous course of treatment. Alternatively, the measurements in the subject can be compared to a control value (mean plus standard deviation) determined in a population of subjects after undergoing a course of treatment. Alternatively, the measurements in the subject can be compared to a control value in a population of prophylactically treated subjects who remain free of disease symptoms, or in a population of therapeutically treated subjects who show improvement in disease characteristics. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then the treatment is considered to be effective and does not need to be resumed. In all of these cases, a significant difference (i.e., more than a standard deviation) from the control level is an indication that treatment needs to be resumed in the subject.

[0169] In some embodiments, after administration of the AAV vector, the patient exhibits increased PGRN expression relative to a measurement of the patient's PGRN expression levels obtained prior to administration of the AAV vector, hi some embodiments, the increased PGRN expression is observed in the patient's thalamus, frontal cortex, basal ganglia, parietal cortex, temporal cortex, parietal and temporal cortex, and / or CSF.

[0170] In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 2 ng / mg to about 100 ng / mg (e.g., 3 ng / mg to about 99 ng / mg, 4 ng / mg to about 98 ng / mg, 5 ng / mg to about 97 ng / mg, 10 ng / mg to about 90 ng / mg, 20 ng / mg to about 80 ng / mg, 30 ng / mg to about 70 ng / mg, 40 ng / mg to about 60 ng / mg, or about 50 ng / mg). For example, in some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 3 ng / mg to about 99 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 4 ng / mg to about 98 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 5 ng / mg to about 97 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 10 ng / mg to about 90 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 20 ng / mg to about 80 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 30 ng / mg to about 70 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 40 ng / mg to about 60 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 50 ng / mg.

[0171] In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 2 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 3 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 4 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 5 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 6 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 7 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 8 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 9 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 10 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 11 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 12 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 13 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 14 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 15 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 16 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 17 ng / mg.In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 18 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 19 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 20 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 21 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 22 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 23 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 24 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 25 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 26 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 27 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 28 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 29 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 30 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 31 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 32 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 33 ng / mg.In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 34 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 35 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 36 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 37 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 38 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 39 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 40 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 41 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 42 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 43 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 44 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 45 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 46 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 47 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 48 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 49 ng / mg.In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 50 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 51 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 52 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 53 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 54 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 55 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 56 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 57 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 58 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 59 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 60 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 61 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 62 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 63 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 64 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 65 ng / mg.In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 66 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 67 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 68 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 69 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 70 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 71 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 72 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 73 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 74 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 75 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 76 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 77 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 78 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 79 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 80 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 81 ng / mg.In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 82 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 83 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 84 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 85 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 86 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 87 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 88 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 89 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 90 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 91 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 92 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 93 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 94 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 95 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 96 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 97 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 98 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 99 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 98 ng / mg. In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 100 ng / mg.

[0172] In some embodiments, after administration of the AAV vector, the patient exhibits a PGRN expression level in the frontal cortex that is greater than or equal to about 1×109 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., about 5 × 10 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere, approx. 1×10 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, or approximately 5 × 10 10 vg / hemisphere ~ approx. 1×10 11 vg / hemisphere) is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). For example, in some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex that is about 5×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 9 vg / hemisphere ~ approx. 1×10 12 vg / hemisphere is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex that is about 1×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 10 vg / hemisphere ~ approx. 5×10 11 vg / hemisphere, is equivalent to the level of PGRN expression observed in human subjects with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex that is about 5×10 of an AAV2 / 9 vector having the nucleic acid sequence of SEQ ID NO:6. 10 vg / hemisphere ~ approx. 1×10 11Following intrathalamic administration at a dose of vg / hemisphere, the levels of PGRN expression are equivalent to those observed in human subjects with neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders).

[0173] In some embodiments, after ITM administration of the AAV vector, the patient does not show a significant increase in expression of PGRN, such as an increase of less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%, 0%, etc.) in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen). For example, in some embodiments, after administration of the AAV vector, the patient shows an increase in expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 9%. In some embodiments, after administration of the AAV vector, the patient shows an increase in expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 8%. In some embodiments, after administration of the AAV vector, the patient exhibits an increase in expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 7%. In some embodiments, after administration of the AAV vector, the patient exhibits an increase in expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 6%. In some embodiments, after administration of the AAV vector, the patient exhibits an increase in expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 5%. In some embodiments, after administration of the AAV vector, the patient exhibits an increase in expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 4%. In some embodiments, after administration of the AAV vector, the patient exhibits an increase in expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 3%. In some embodiments, after administration of the AAV vector, the patient exhibits an increase in expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 2%.In some embodiments, after administration of the AAV vector, the patient exhibits increased expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of less than about 1%. In some embodiments, after administration of the AAV vector, the patient exhibits increased expression of PGRN in one or more (e.g., 2, 3, 4, or more) peripheral tissues (e.g., liver, lung, and spleen) of 0%.

[0174] In some embodiments, peripheral tissues include, but are not limited to, the liver, lung, and spleen.

[0175] In some embodiments, expression of PGRN is measured relative to expression of GAPDH.

[0176] kit The compositions described herein may be provided in a kit for use in treating neurocognitive or neuromuscular disorders (e.g., neurodegenerative disorders such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders). In some embodiments, the kit may include one or more AAVs described herein. The kit may include a package insert that instructs the user of the kit, such as a physician of skill in the art, to perform any one of the methods described herein. The kit may optionally include a syringe or other device for administering the composition. In some embodiments, the kit may include one or more additional therapeutic agents. EXAMPLES

[0177] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein may be used and evaluated, are intended to be purely illustrative of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0178] Example 1. Development of a codon-optimized human Progranulin construct for efficient cortical expression of Progranulin Codon optimization of human progranulin (hPGRN) gene for efficient protein expression The human GRN gene sequence, excluding intron DNA, is as follows:

[0179] The human PGRN amino acid sequence is as follows: MWTLVSWVALTAGLVAGTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWG CCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTC CRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGC DQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL (Sequence number 2).

[0180] Analysis of SEQ ID NO: 10 reveals the preference of certain codons for various amino acids throughout the gene. Examination of codon frequency reveals that for certain amino acids, certain codons predominate, while other codons are used less frequently or not at all. Those skilled in the art can perform codon optimization in any manner known in the art, such as, for example, those described in U.S. Patent Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety.

[0181] The final codon-optimized gene may exhibit at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 3. For example, the final codon-optimized gene may exhibit at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 3. In another example, the final codon-optimized gene may have a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:3.

[0182] Once designed, the final codon-optimized gene can be prepared, for example, by solid-phase nucleic acid procedures known in the art. Solid-phase synthesis techniques for polynucleotides are known in the art and are described, for example, in U.S. Pat. No. 5,541,307, the disclosure of which is incorporated herein by reference as it relates to solid-phase polynucleotide synthesis and purification. Furthermore, the prepared gene can be amplified, for example, using PCR-based techniques known in the art and / or by transformation of DH5α E. coli with a plasmid containing the designed gene. The bacteria can then be cultured to amplify the DNA therein, and the gene can be isolated by plasmid purification techniques known in the art, optionally followed by restriction digestion and / or sequencing of the plasmid to verify the identity of the codon-optimized gene.

[0183] Codon-optimized human PGRN construct PCR primers were designed to bind to the GRN nucleic acid sequence from genomic DNA isolated from cultured cells and amplify it by polymerase chain reaction. Using the codon optimization method described above, the isolated GRN was modified by site-directed mutagenesis. The resulting modified amplification product was gel purified and sequenced by methods known in the art.

[0184] A pseudotyped adeno-associated virus (AAV) 2 / 9 (AAV2 / 9) parent vector was used as the destination vector for codon-optimized human PGRN (hPGRN). The parent vector contains a nucleic acid molecule with the following components: a first AAV2 inverted terminal repeat, a human synapsin (hSyn) promoter, a human growth hormone intron (hGHi3), a bovine growth hormone (bGH) polyadenylation site (pA), a second AAV2 ITR (ITR2), a phage-derived origin of replication (f1 ori), a citrobacter freundii ampC β-lactamase (AmpR) promoter, a kanamycin selection gene (KanR), and a second origin of replication (ori). The codon-optimized hPGRN was cloned into the parent vector (Figure 1, referred to herein as "AAV9-SYN-PGRN").

[0185] Example 2. Establishing therapeutic expression of hPGRN in the cortex while avoiding toxic side effects the purpose The objective of this study was to evaluate anterograde and / or retrograde transport for rescued expression of PGRN in the cortex, along with systemic toxicity and / or expression of AAV9-SYN-PGRN in adult sheep as described in Example 1. Additionally, this study was designed to refine the effective dosing range of AAV9-SYN-PGRN as determined by supraphysiological levels of PGRN in cortical brain tissue.

[0186] Materials and Methods Adult (approximately 2 years old) wild-type sheep (n=2 / dose) were administered 1 × 10 10 vg / hemisphere (referred to herein as "low dose"), 5×10 10 vg / hemisphere (referred to herein as "mid-dose"), or 1 x 10 11AAV9-SYN-PGRN (250 μL / thalamus) was intrathalamic (ITM) injected (e.g., convectively supported) with 1000 μg / hemisphere (herein referred to as "high dose") of hPGRN protein expression and vg levels, respectively, over approximately 90 minutes. Twenty-eight days after ITM administration, animals were sacrificed and brain biopsies were collected. Brains, cerebrospinal fluid (CSF), and serum were analyzed for hPGRN protein expression and vg levels, while immunofluorescence imaging of the brain was performed to assess hPGRN expression and potential inflammatory responses (e.g., expression of inflammatory markers) to the viral vector.

[0187] result As a result, it was observed that the vector preparations described herein injected into the thalamus effectively transduced the cortex with normal to supraphysiological levels of hPGRN in the cortex even at low doses (Figure 2). Furthermore, a dose-dependent increase in hPGRN levels and high animal-to-animal consistency in all brain regions was observed (Figure 3). This effect was observed throughout the prefrontal (PF) cortex of all sheep, with no evidence of reactive microglia as measured by expression levels of IBA1 (Figure 4). In assessing dose-response across cortical regions (e.g., frontal A cortex (coronal slices taken from the anterior part of the cortex), frontal B cortex (coronal slices taken from the posterior part of the cortex), caudate-putamen / parietotemporal (CD / PT) cortex), basal ganglia (e.g., caudate and putamen), thalamus, and hippocampus, it was observed that even the lowest dose tested elevated hPGRN protein expression above basal levels (Figures 5 and 6). Furthermore, when this data was normalized to the percentage of hPGRN expression in the thalamus, it was observed that the low dose tended to have a greater relative hPGRN delivery to the cortex (Figure 7). In the CSF, the medium and high doses elevated hPGRN levels, achieving supraphysiological human CSF PGRN levels in the range of 10-30 ng / ml, indicating a restorative effect (Figure 8). No detectable expression of hPGRN was observed in the serum of the respective sheep (Figure 9). Studies addressing histopathology revealed that hematoxylin and eosin staining revealed that all animals exhibited unilateral or bilateral focal inflammatory lesions in the cerebral white matter and / or thalamus, consistent with damage caused during dose administration. There was no evidence of histopathological changes specific to the site of hPGRN expression (Figure 10). Gliosis was observed in some animals, but was considered to be unrelated to hPGRN expression given its minimal focal nature, and sporadic occurrence in both white and gray matter in different regions. This was likely related to dosing and / or spontaneous background variations as well. Collectively, these data demonstrated that at all dose levels, AAV9-SYN-PGRN effectively transduced the cortex and mediated elevated cortical hPGRN expression levels without eliciting an immunological response to the AAV vector.

[0188] Example 3. Establishing the administration efficiency of hPGRN in the cortex the purpose The aim of this study was to investigate the efficiency of cortical transduction by ITM administration of AAV9-SYN-PGRN in adult sheep.

[0189] Materials and Methods Materials and methods are described in Examples 1 and 2.

[0190] result Figure 11 shows the expression levels of hPGRN normalized by quantified vg / μg of AAV9-SYN-PGRN across brain regions in wild-type sheep administered ITM at low, medium, or high doses, respectively (Figure 3). It was observed that the low dose was generally more efficient in delivering hPGRN to the cortex.

[0191] Example 4. Comparison of the efficiency of hPGRN transduction in the cortex across central administration routes the purpose The aim of this study was to compare the efficiency of cortical transduction by ITM or intracisternal magna (ICM) administration of a viral vector encoding the hPGRN transgene in adult sheep.

[0192] Materials and Methods Adult (approximately 2 years old) wild-type sheep (n=2 / dose or 2 / vector, respectively) were administered 1 × 10 10 (250 µL / thalamus over approximately 90 min) vg / hemisphere of AAV9-SYN-PGRN or AAV1 or AAV9 encoding a hPGRN transgene operably linked to a chicken β-actin promoter with a cytomegalovirus enhancer (CB7, AAV1-CB7-PGRN and AAV9-CB7-PGRN, respectively) or control omnipaque was ITM injected (e.g., convectionally assisted administration) or 1 × 10 13vg (2 mL over 1 min with CSF exchange) of AAV9-SYN-PGRN or AAV1 or AAV9 or control omnipaque was injected into the ICM. Regions from the brain were biopsied and analyzed for hPGRN protein expression and vg levels, while immunofluorescence imaging of the brain was performed to assess hPGRN expression.

[0193] result Initial evaluation of cerebellar sections for hPGRN confirms that ICM administration was successful (Figure 12). All animals showed hPGRN in the cerebellum, but differences were observed. AAV1-CB7-PGRN showed strong, superficial, and almost exclusively glial staining. Both AAV9-SYN-PGRN and AAV9-CB7-PGRN showed better penetration of hPGRN into cerebellar tissue, with AAV9-CB7-PGRN being more widespread in glial cells and AAV9-SYN-PGRN being more widespread in neurons. In the PF cortex and thalamus, ICM administration mediated little expression, regardless of which AAV vector was used (Figure 13). In contrast, this comparative study highlighted that ITM administration mediated strong expression of hPGRN in the PF cortex. Similar results were obtained across brain regions in that ITM administration resulted in enhanced transduction compared to ICM administration (Figure 14).

[0194] A similar pattern of results was observed when evaluating the levels of hPGRN protein expression. Specifically, levels detected in the cortex and subcortex of ICM-treated sheep were within background levels detected in controls (untreated sheep), but were 5×10 10 Sheep that were ITM injected with AAV9-SYN-PGRN vg / hemisphere showed higher cortical levels of hPGRN compared to sheep that were ICM administered AAV9-SYN-PGRN or AAV1-CB7-PGRN or AAV9-CB7-PGRN vectors (Figures 15-18). In the cortex of ICM-injected animals, we observed negligible hPGRN levels, with small pockets of hPGRN detected in the thalamus and parietal / temporal cortex of ICM-treated sheep.

[0195] Taken together, these results indicate that ICM administration results in poor transduction of the cortex, whereas ITM administration achieves superior cortical transduction.

[0196] Example 5. Evaluation of tissue expression of hPGRN in cortical tissues compared to peripheral tissues following AAV delivery via ITM administration of a viral vector encoding an hPGRN transgene in adult sheep the purpose The objective of this study was to evaluate hPGRN expression in the central nervous system (CNS) compared with peripheral tissues (e.g., liver, spleen, and lung) following ITM administration of a viral vector encoding the hPGRN transgene in adult sheep.

[0197] Materials and Methods Adult (approximately 2 years old) wild-type sheep (n=2 / dose or 2 / vector, and 1 control sheep) were administered 1×10 10 , 5×10 10 , or 1 × 10 11 (250 μL / thalamus over approximately 90 min) vg / hemisphere of AAV9-SYN-PGRN, or AAV1 or AAV9 encoding a hPGRN transgene operably linked to a chicken β-actin promoter with a cytomegalovirus enhancer (CB7, AAV1-CB7-PGRN and AAV9-CB7-PGRN, respectively), or control omnipaque were ITM injected (e.g., convectionally assisted). Regions from brain, liver, spleen, and lung were biopsied and analyzed for hPGRN protein expression and vg levels, while immunofluorescence imaging of brain, liver, spleen, and lung was performed to assess hPGRN expression.

[0198] result As discussed in Example 4, ITM administration of AAV9 vectors encoding the hPGRN transgene mediated robust expression of hPGRN in the brain, including the thalamus, frontal cortex, parietal / temporal cortex, occipital cortex, putamen, caudate nucleus, hippocampus, and serum from both left and right hemispheres. Upon evaluation of the level of hPGRN protein expression, no significant expression of hPGRN (as assessed by viral DNA) was observed in peripheral tissues, including the liver (Figure 21), lung, and spleen.

[0199] Notably, similar results have been obtained in cynomolgus monkeys (see, eg, Figures 22 and 23).

[0200] Taken together, these results indicate that ITM administration of this vector achieves CNS-specific expression of hPGRN.

[0201] Example 6. Efficacy of codon-optimized human PGRN against lipofuscinosis in a frontotemporal dementia model the purpose Frontotemporal dementia (FTD) is a clinical syndrome characterized by progressive neurodegeneration in the frontal and temporal lobes of the cerebral cortex. More than 70 loss-of-function mutations in the GRN gene have been identified in FTD, the majority of which result in haploinsufficiency and reduced serum PGRN levels.

[0202] The goal of this study was to demonstrate the efficacy of AAV9-SYN-PGRN in a mouse model of early stage disease (e.g., FTD).

[0203] Materials and Methods Mouse model of frontotemporal dementia Mice homozygous for deletion of GRN (e.g., GRN - / - Mice (Fig. 1A, B, C, D) were used as a model of FTD. At 6-8 weeks of age, mice were injected with AAV9-SYN-PGRN as described in Example 1 and allowed 12 weeks for viral expression.

[0204] Immunofluorescence Biopsied brain tissue was fixed overnight in 10% formalin and then stored in 70% ethanol. The tissue was embedded in paraffin and cut into 5 μm sections.

[0205] For immunofluorescence analysis, tissues were permeabilized, slides were washed, and blocked with 5% rabbit serum for 30 min. Sections were incubated with primary antibodies, anti-hPGRN, anti-Iba1, anti-CD68, and anti-subunit C mitochondrial ATP synthase (SCMAS), respectively, for 1 h at room temperature. Slides were incubated with secondary antibodies for 30 min. Evaluation included detailed quantification of SCMAS immunoreactivity.

[0206] result As a result, reactive microglial (e.g., CD68 and IBA1 positive, amoeboid cells) labeling was observed to correlate with hPGRN levels. FIG. 19 shows that SCMAS labeling of lipofuscin is reduced even in areas of low levels of hPGRN expression. This trend was evident across all doses of AAV9-SYN-PGRN and correlated with regional expression (e.g., hPGRN positive expression in the thalamus). Quantification revealed that low doses of AAV9-SYN-PGRN significantly ameliorated lipofuscinosis, as detected by SCMAS immunoreactivity (FIG. 20). Taken together, these results demonstrate the efficacy of an exemplary AAV2 / 9 plasmid encoding codon-optimized hPGRN to ameliorate lipofuscinosis in the PF cortex, hippocampus, and thalamus of a mouse model of FTD.

[0207] Example 7. Use of codon-optimized GRN genes for the treatment of neurocognitive or neuromuscular disorders The gene encoding PGRN can be codon-optimized using the procedures described herein (e.g., as described in Example 1 above). For example, the final codon-optimized GRN gene can exhibit at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:3. For example, the final codon-optimized GRN gene can exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:3. In another example, the final codon-optimized gene can have a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:3.

[0208] The gene can then be incorporated into a plasmid, such as an AAV2 / 9 vector, and administered to a patient suffering from a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disease, such as FTD, Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies, associated neurocognitive disorders, amyotrophic lateral sclerosis (ALS), or associated motor neuron disorders). For example, a patient suffering from FTD, a disorder associated with a mutation in the GRN gene, or AD, PD, dementia with Lewy bodies, associated neurocognitive disorders, ALS, or associated motor neuron disorders, can be administered an AAV2 / 9 vector containing a codon-optimized GRN gene under the control of a suitable promoter for expression in human cells, such as neurons. For example, an AAV vector, such as an AAV2 / 9 vector, can be generated that incorporates a codon-optimized GRN gene between the 5' and 3' inverted terminal repeats of the vector, and the gene can be placed under the control of a neuron-specific promoter, such as the synapsin (Syn) promoter. The AAV vector can be administered ITM to the subject.

[0209] Those skilled in the art may monitor the expression of the codon-optimized GRN gene by various methods. For example, those skilled in the art may transfect cultured neurons with the codon-optimized gene to model the expression of the codon-optimized gene in the patient's neurons. The expression of the encoded protein may then be monitored using expression assays described herein, such as, for example, qPCR, RNA-Seq, ELISA, or immunoblot procedures. Based on the data obtained from the gene expression assay, further iterations of the codon optimization procedure may be performed, for example, to further reduce the CpG content and homopolymer content in the mRNA transcript. The candidate gene sequence with the optimal expression pattern in vitro may then be prepared for incorporation into a suitable AAV vector and administration to a mammalian subject, such as an animal model of a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders), or a human patient.

[0210] Example 8. Treatment of neurocognitive or neuromuscular disorders in human patients by intrathalamic administration of human PGRN Using the compositions and methods of the present disclosure, a patient with a neurocognitive or neuromuscular disorder (e.g., a neurodegenerative disorder such as FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders) can be ITM administered (e.g., convectionally assisted) an AAV (e.g., pseudotyped AAV2 / 9) vector containing a nucleic acid sequence encoding human PGRN. The human PGRN sequence can be codon optimized and / or operably linked to an AAV vector having a Syn promoter, e.g., the nucleic acid of SEQ ID NO: 6. The AAV can be administered, for example, at a concentration of about 1×10 9 vg / hemisphere ~ approx. 9×10 12 vg / hemisphere (e.g., 5×10 9 vg / hemisphere ~ approx. 5×10 12 vg / hemisphere, 1×10 10 vg / hemisphere ~ approx. 5×10 12 vg / hemisphere, 1×10 11 vg / hemisphere ~ approx. 5×10 12vg / hemisphere, 1×10 12 vg / hemisphere ~ approx. 5×10 12 vg / hemisphere, or 1×10 13 vg / hemisphere ~ approx. 5×10 12 For example, the AAV vector can be administered in an amount of about 1×10 10 vg / hemisphere, approximately 5×10 10 vg / hemisphere, or approximately 1×10 11 vg / hemisphere is administered to the patient.

[0211] When an AAV vector containing a transgene encoding hPGRN is administered to a patient, the patient exhibits a change in PGRN levels. For example, the patient exhibits restoration of PGRN expression in the frontal cortex after administration of an AAV vector containing a nucleic acid sequence encoding hPGRN to the patient. For example, the patient exhibits a level of PGRN expression in the frontal cortex of about 2 ng / mg to about 8 ng / mg (e.g., 3 ng / mg to about 7 ng / mg, 4 ng / mg to about 6 ng / mg, or about 5 ng / mg), or more (e.g., about 9 ng / mg, about 10 ng / mg, about 15 ng / mg, about 20 ng / mg, about 30 ng / mg, about 40 ng / mg, about 50 ng / mg, about 60 ng / mg, about 70 ng / mg, about 80 ng / mg, about 90 ng / mg, or about 100 ng / mg). Additionally or alternatively, for example, when an AAV vector containing a transgene encoding hPGRN is administered to the patient, the patient exhibits improvement in cognitive function.

[0212] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0213] While the invention has been described with reference to specific embodiments, it will be understood that the invention is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including departures from the invention which come within known or customary practice in the art to which this invention pertains and which may be applicable to the essential characteristics as hereinbefore described, and which comply with the scope of the appended claims.

[0214] Other embodiments are within the scope of the claims.

Claims

1. (i) a method for treating a disorder affecting the central nervous system (CNS) in a patient; or (ii) a method for improving cognitive function in a patient diagnosed with a disorder affecting that patient's CNS.

1. A pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising a transgene encoding a therapeutic protein for use in a patient, the method comprising administering the AAV vector to the patient, the AAV vector delivering about 1 x 10 ９ vg / hemisphere ~ approx. 9 x 10 １２ The pharmaceutical composition is administered intrathalamically to the patient in an amount of 100 mg / hemisphere.

2. 1. A pharmaceutical composition comprising an AAV vector comprising a transgene encoding a therapeutic protein for use in a method for expressing the therapeutic protein in the brain (e.g., the frontal cortex) of a patient diagnosed with a disorder affecting the CNS, the method comprising administering the AAV vector to the patient, the AAV vector comprising at least about 1 x 10 ９ vg / hemisphere ~ approx. 9 x 10 １２ The pharmaceutical composition is administered intrathalamally to the patient in an amount of 0.05 mg / hemisphere.

3. (i) the AAV vector is about 1 x 10 １０ vg / hemisphere ~ approx. 5 x 10 １２ vg / hemisphere, and optionally the AAV vector is administered to the patient in an amount of about 1 x 10 １０ vg / hemisphere, 2×10 １０ vg / hemisphere, 3×10 １０ vg / hemisphere, 4×10 １０ vg / hemisphere, 5×10 １０ vg / hemisphere, 6×10 １０ vg / hemisphere, 7×10 １０ vg / hemisphere, 8×10 １０ vg / hemisphere, 9×10 １０ vg / hemisphere, 1×10 １１ vg / hemisphere, 2×10 １１ vg / hemisphere, 3×10 １１ vg / hemisphere, 4×10 １１ vg / hemisphere, 5×10 １１ vg / hemisphere, 6×10 １１ vg / hemisphere, 7×10 １１ vg / hemisphere, 8×10 １１ vg / hemisphere, 9×10 １１ vg / hemisphere, 1×10 １２ vg / hemisphere, 2×10 １２ vg / hemisphere, 3×10 １２ vg / hemisphere, 4×10 １２ vg / hemisphere, or 5 x 10 １２ vg / hemisphere to the patient, (ii) the AAV vector is administered to the patient in a single dose per hemisphere comprising said amount; (iii) the AAV vector is administered to the patient in multiple doses per hemisphere that together comprise the amount; (iv) the therapeutic protein is a protein whose deficiency or lack of activity is associated with the disorder; (v) the therapeutic protein is a secreted protein or a protein listed in Table 5 herein; (vi) the therapeutic protein is PGRN; (vii) the disorder is a neurocognitive disorder, a neuromuscular disorder, a neurodegenerative disorder, or a lysosomal storage disorder; (viii) the disorder is a lysosomal storage disorder. (ix) the disorder is frontotemporal dementia (FTD), Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS), or a related neurocognitive or motor neuron disorder; (x) the transgene is operably linked to a promoter active in neuronal and / or glial cells, and optionally the transgene encodes PGRN; (xi) the therapeutic protein is PGRN, and the PGRN has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

2. (xii) the transgene encodes PGRN, and the transgene encoding PGRN is codon-optimized. (xiii) the transgene is operably linked to a human growth hormone (hGH) intron, and optionally (i) the hGH intron is hGH intron 3, and / or (ii) the transgene encodes PGRN. (xiv) the transgene is operably linked to a 3′ enhancer element, and optionally, the transgene encodes PGRN. (xv) the AAV vector comprises a capsid protein derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, AAVrh.8, and AAVrh.10; (xvi) The AAV is an anterograde transport AAV or a retrograde transport AAV; (xvii) the AAV vector comprises a 5' inverted terminal repeat (ITR) and / or a 3' ITR from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, AAVrh.8, or AAVrh.10, and optionally, the AAV vector comprises a 5' ITR and a 3' ITR from AAV2; (xviii) the AAV vector comprises 5′ ITR and 3′ ITR from one AAV serotype and capsid proteins from a different AAV serotype; (xix) The AAV vector is an AAV2 / 9 vector. (xx) the AAV has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 6; (xxi) the therapeutic protein is PGRN, and prior to administration of the AAV vector, the patient exhibits a level of endogenous PGRN expression that is about 1% to about 40% of the level of endogenous PGRN expression observed in a human subject of the same age, sex, and / or body mass index who does not have FTD, AD, PD, dementia with Lewy bodies, ALS, or a related motor neuron disorder. (xxii) the therapeutic protein is PGRN, and after administration of the AAV vector, the patient exhibits increased PGRN expression relative to a measurement of the patient's PGRN expression level obtained before administration of the AAV vector. (xxiii) the therapeutic protein is PGRN, and after administration of the AAV vector, the patient exhibits a level of PGRN expression in the frontal cortex of about 2 ng / mg to about 100 ng / mg. (xxiv) the AAV vector is administered to the patient in a convection-assisted manner. (xxv) the patient is diagnosed with FTD due to a mutation in the GRN gene; (xxvi) Upon administration of the AAV vector, an increase in transgene expression in less than 10% of peripheral tissues is observed. (xxvii) the patient is a mammal, or (xxviii) the patient is a human. A pharmaceutical composition for use according to claim 1.

4. (i) the AAV vector is about 5 x 10 １０ vg / hemisphere ~ approx. 9 x 10 １１ vg / hemisphere to the patient, (ii) the AAV vector is about 1 × 10 １０ vg / hemisphere to the patient, (iii) The promoter is selected from the group consisting of a synapsin promoter, a tetracycline-controlled transactivator protein (tTA) promoter, a reverse tetracycline-controlled transactivator protein (rTA) promoter, a U1 promoter, a U6 promoter, a U7 promoter, a prion promoter, a phosphoglycerate kinase (PGK) promoter, a CB7 promoter, an H1 promoter, a cytomegalovirus (CMV) promoter, a CMV-chicken β-actin (CBA) promoter, a glial fibrillary acidic protein (GFAP) promoter, a calcium / calmodulin-dependent protein kinase III promoter, a tubulin alpha I promoter, and a microtubulin-associated protein IB (MAP) promoter. IB) promoter, a neuron-specific enolase promoter, a platelet-derived growth factor beta chain promoter, a neurofilament light chain promoter, a neuron-specific VGF gene promoter, a neuronal nucleus (NeuN) promoter, an adenomatous polyposis coli (APC) promoter, an ionized calcium-binding adaptor molecule 1 (Iba-1) promoter, or a homeobox protein 9 (HB9) promoter; (iv) the PGRN has an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2, and optionally, the PGRN has an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

2. (v) the transgene encoding PGRN has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 3; (vi) the hGH intron has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO:4; (vii) the 3' enhancer element has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO:5; (viii) the AAV has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 6; (ix) increased PGRN expression is observed in the thalamus, frontal cortex, basal ganglia, parietal cortex, temporal cortex, parietal and temporal cortex, and / or cerebrospinal fluid (CSF) of the patient; (x) the peripheral tissue comprises the liver, lung, and / or spleen; or (xi) the transgene expression is calculated relative to GAPDH expression; A pharmaceutical composition for use according to claim 3.

5. (i) the AAV vector is about 5 x 10 １０ vg / hemisphere to the patient, (ii) the AAV vector is about 1 × 10 １１ vg / hemisphere to the patient, (iii) the promoter is a synapsin promoter (iv) the PGRN has the amino acid sequence of SEQ ID NO: 2; (v) the transgene encoding PGRN has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:3, and optionally, the transgene encoding PGRN has a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:3; (vi) the hGH intron has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:4, and optionally, the hGH intron has a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:4; (vii) the 3' enhancer element has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:5, optionally the 3' enhancer element has a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:5; or (viii) the AAV has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:6, and optionally, the AAV has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:

6. A pharmaceutical composition for use according to claim 4.

6. (i) the synapsin promoter has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 1; (ii) the transgene encoding PGRN has the nucleic acid sequence of SEQ ID NO: 3; (iii) the hGH intron has the nucleic acid sequence of SEQ ID NO: 4; (iv) the 3' enhancer element has the nucleic acid sequence of SEQ ID NO: 5, or (v) the AAV has the nucleic acid sequence of SEQ ID NO: 6; A pharmaceutical composition for use according to claim 5.

7. 7. The pharmaceutical composition for use of claim 6, wherein the synapsin promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:1, and optionally, the synapsin promoter has a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:

1.

8. 8. The pharmaceutical composition for use according to claim 7, wherein the synapsin promoter has the nucleic acid sequence of SEQ ID NO:

1.

9. (i) a method for treating a disorder affecting the CNS in a patient; or (ii) A method for improving cognitive function in a human patient diagnosed with a disorder affecting the patient's CNS.

1. A pharmaceutical composition comprising an AAV vector comprising a transgene encoding a therapeutic protein for use in a patient, the method comprising administering the AAV vector to the patient, wherein the AAV vector is administered to the patient in an amount sufficient to achieve a level of therapeutic protein expression in the brain (e.g., frontal cortex) of the patient, the level being greater than or equal to about 1 x 10 of an AAV2 / 9 vector comprising a transgene encoding the therapeutic protein. ９ vg / hemisphere ~ approx. 9 x 10 １２ The pharmaceutical composition has a therapeutic protein expression level equivalent to that observed in human subjects with the disorder after intrathalamic administration in an amount of 1000 mg / hemisphere.

10. 1. A pharmaceutical composition comprising an AAV vector comprising a transgene encoding a therapeutic protein for use in a method for expressing a therapeutic protein in the brain (e.g., frontal cortex) of a human patient diagnosed with a disorder affecting the patient's CNS, the method comprising administering the AAV vector to the patient, wherein the AAV vector is administered to the patient in an amount sufficient to achieve a level of therapeutic protein expression in the brain (e.g., frontal cortex) of the patient, the level being greater than or equal to about 1 x 10 of an AAV2 / 9 vector comprising a transgene encoding the therapeutic protein. ９ vg / hemisphere ~ approx. 9 x 10 １２ The pharmaceutical composition has a therapeutic protein expression level equivalent to that observed in human subjects with the disorder after intrathalamic administration in an amount of 1000 mg / hemisphere.

11. (i) a method of treating FTD, AD, PD, dementia with Lewy bodies, related neurocognitive disorders, ALS, or related motor neuron disorders in a human patient in need thereof; (ii) a method of improving cognitive function in a human patient diagnosed with FTD, AD, PD, dementia with Lewy bodies, a related neurocognitive disorder, ALS, or a related motor neuron disorder; or (iii) A method for expressing PGRN in the brain (e.g., the frontal cortex) of a human patient diagnosed with FTD, AD, PD, dementia with Lewy bodies, a related neurocognitive disorder, ALS, or a related motor neuron disorder.

1. A pharmaceutical composition comprising an AAV vector comprising a transgene encoding PGRN for use in a method comprising administering to the patient the AAV vector, wherein the AAV vector is administered to the patient in an amount sufficient to achieve a level of PGRN expression in the brain (e.g., frontal cortex) of the patient of about 2 ng / mg to about 8 ng / mg or more.

12. (i) the therapeutic protein is a protein whose deficiency or lack of activity is associated with the disorder; (ii) the therapeutic protein is a secreted protein or a protein listed in Table 5 herein; (iii) the therapeutic protein is PGRN; (iv) the disorder is a neurocognitive disorder, a neuromuscular disorder, a neurodegenerative disorder, or a lysosomal storage disorder; (v) the disorder is a lysosomal storage disorder; (vi) the disorder is frontotemporal dementia (FTD), Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS), or a related neurocognitive or motor neuron disorder; (vii) the AAV2 / 9 vector containing a transgene encoding the therapeutic protein has the nucleic acid sequence of SEQ ID NO: 6; (viii) the AAV vector administered to the patient is an anterograde transport AAV or a retrograde transport AAV; (ix) the AAV vector administered to the patient comprises a capsid protein derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, AAVrh.8, and AAVrh.10; (x) the AAV vector administered to the patient comprises a 5′ ITR and / or a 3′ ITR from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, AAVrh.8, or AAVrh.10, and optionally the AAV vector administered to the patient comprises a 5′ ITR and a 3′ ITR from AAV2; (xi) the AAV vector administered to the patient comprises a 5' ITR and a 3' ITR from one AAV serotype and capsid proteins from a different AAV serotype; or (xii) the AAV vector administered to the patient is an AAV2 / 9 vector; A pharmaceutical composition for use according to claim 9.

13. 10. A kit comprising an AAV vector comprising a transgene encoding PGRN, the kit further comprising a package insert instructing a user of the kit to administer the AAV vector to the patient according to the pharmaceutical composition for use of claim 1.

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