Methods for improving adeno-associated virus (AAV) delivery

Abstract

Provided herein is a method for improving delivery of a pharmaceutical composition to the central nervous system in a subject in need thereof, the method comprising administering to the subject, in combination with the pharmaceutical composition, an agent that enhances glymphatic influx.

Description

[Technical field]

[0001] The present disclosure is directed to improving the delivery of pharmaceutical compositions to target tissues, such as the central nervous system, by modulating glymphatic influx. [Background technology]

[0002] A variety of therapeutic agents have been developed to treat central nervous system (CNS) diseases. However, therapeutic delivery to the brain is largely limited by the nearly impermeable blood-brain barrier (BBB) ​​and poor penetration of therapeutic agents into the brain. AAV vectors have emerged as a promising approach to treat a variety of genetically determined diseases, but delivery to and transgene expression in specific tissues and cell types is required to achieve efficacy and avoid unwanted toxicity. This process requires first exposure of the intended tissue to the vector, followed by vector tropism to the intended target cell type. For diseases requiring transduction of the CNS, several delivery routes have been attempted to achieve sufficient tissue exposure, including systemic intravascular administration and direct injection into the intrathecal and intraventricular spaces. Following intravascular administration, the vector must cross the blood-brain barrier, which appears to limit exposure to the brain parenchyma for many AAV serotypes. Direct injection into the intrathecal and intraventricular space bypasses the blood-brain barrier, but the mechanism by which the vector distributes from the cerebrospinal fluid to the brain parenchyma remains unclear.

[0003] To further clarify the distribution mechanism after intrathecal administration of AAV vectors, a non-human primate study in cynomolgus monkeys was performed. The results suggest that there is a low level of AAV transduction in the brain. The glymphatic system, a network of perivascular spaces that facilitates fluid exchange between the CSF and the interstitial space, may be exploited to enhance drug delivery from the CSF to the parenchyma. Glymphatic flow is maximal during anesthetic regimens that induce sleep and slow-wave sleep-like states. (Lilius TO,et al.Dexmedetomidine enhances glymphatic brain delivery of intrathecally administered drugs.J Control Release.2019 Jun 28;304:29-38). Therefore, there is a need to improve AAV delivery to the brain by modulating glymphatic flow. Summary of the Invention

[0004] The present disclosure provides a method for improving the delivery of pharmaceutical compositions to target tissues, such as the central nervous system, by modulating glymphatic influx. Glymphatic is a recently recognized system in which CSF is drawn into deeper regions of the brain along periarterial spaces formed by blood vessel-adjacent astrocytes, where CSF may interact with interstitial fluid before exiting the brain in corresponding perivenular spaces. This system is believed to play a major role in fluid movement and removal of macromolecules from the brain parenchyma. Larger particles, such as lipoproteins, which are comparable in size to AAV vectors, move through the glymphatic system. It has been discovered that AAV distribution patterns in the brain are consistent with limited vector diffusion across membranes lining the brain surface and vector entry occurring primarily through glymphatic influx. It has been unexpectedly discovered that AAV delivery to the brain can be improved by modulating glymphatic influx. Enhanced glymphatic influx may also reduce variability in brain distribution of viral vectors among patient populations treated with pharmaceutical compositions containing the viral vector, and may reduce liver and / or DRG toxicity associated with AAV gene therapy.

[0005] In one aspect, the disclosure provides a method of improving delivery of a pharmaceutical composition to the central nervous system in a subject in need thereof, the method comprising administering to the subject an agent that enhances glymphatic influx in combination with the pharmaceutical composition.

[0006] In some embodiments, the agent is administered simultaneously or sequentially with the pharmaceutical composition. In some embodiments, the agent is administered prior to administration of the pharmaceutical composition. In some embodiments, the agent is administered after administration of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is administered by intrathecal (IT), intracisternal (ICM) and / or intracerebroventricular (ICV) administration. In some embodiments, the agent is administered by intravenous infusion, intravenous injection, inhalation, intraperitoneal, oral, subcutaneous or intramuscular routes.

[0007] In some embodiments, the agent promotes interstitial fluid circulation within the blood-brain barrier, for example, the agent comprises an aquaporin 4 (AQP4) promoter, such as TGN-073. In some embodiments, the agent comprises a compound that upregulates AQP4 expression (e.g., sevoflurane) or a compound that alters the intracellular localization of AQP4.

[0008] In some embodiments, the agent comprises an alpha-2 adrenergic agonist, such as clonidine, cizanidine, or dexmedetomidine (e.g., Precedex or Dexdomitol). In some embodiments, the agent comprises one or more FDA approved anesthetic agents that enhance glymphatic influx. In some embodiments, the anesthetic agent is ketamine, dexmedetomidine, or xylazine, or a combination thereof. In a preferred embodiment, the agent comprises a combination of ketamine and dexmedetomidine.

[0009] In some embodiments, the subject is first administered ketamine, followed by the pharmaceutical composition, and followed by dexmedetomidine.

[0010] In some embodiments, the subject is first administered ketamine, followed by the pharmaceutical composition, followed by dexmedetomidine, and followed by sevoflurane.

[0011] In some embodiments, ketamine is administered about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, preferably about 10-15 minutes, prior to administration of the pharmaceutical composition.

[0012] In some embodiments, ketamine is administered at about 100 mg / kg, about 90 mg / kg, about 80 mg / kg, about 70 mg / kg, about 60 mg / kg, about 50 mg / kg, about 40 mg / kg, about 30 mg / kg, about 20 mg / kg, about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, preferably about 10 mg / kg.

[0013] In some embodiments, dexmedetomidine is administered at about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.09 mg / kg, about 0.08 mg / kg, about 0.07 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, about 0.009 mg / kg, about 0.008 mg / kg, about 0.007 mg / kg, about 0.006 mg / kg, about 0.005 mg / kg, preferably about 0.02 mg / kg.

[0014] In some embodiments, the subject is further administered sevorlan, followed by dexmedetomidine.

[0015] In some embodiments, sevorlan is administered as an inhalant.

[0016] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg). The subject is then maintained in a hindlimb elevated Trendelenburg position for 10-15 minutes after completion of ketamine and dexmedetomidine administration. The subject is further administered atipamezole (0.2 mg / kg IM), with dosing occurring between 8-10 AM.

[0017] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg), followed by sevoflurane inhalation anesthesia.

[0018] In some embodiments, the agent induces plasma hyperosmolarity. In some embodiments, the agent comprises hypertonic saline (e.g., sodium chloride with or without sodium acetate) or mannitol. In preferred embodiments, the agent comprises hypertonic saline with or without sodium acetate. In some embodiments, the hypertonic saline is 2% NaCl, 3% NaCl, 5% NaCl, 7% NaCl, or 23% NaCl, preferably 3% NaCl. In some embodiments, 3% NaCl is administered at about 2-3.5 ml / kg.

[0019] In some embodiments, the agent is administered intravenously or by infusion injection about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 minutes, preferably about 5 minutes, before or after administration of the pharmaceutical composition, which administration may optionally be repeated.

[0020] In some embodiments, the agent enhances glymphatic influx by increasing slow wave sleep, hi some embodiments, the agent is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine, and trazodone, or combinations thereof.

[0021] In some embodiments, the agent comprises a VEGF, such as VEGF-C. In some embodiments, the VEGF-C comprises (i) an amino acid sequence of any one of the sequences provided in Table 1, or a sequence having at least 95% sequence identity thereto (optionally with or without a linker (e.g., a glycine-serine linker) and / or a his-tag); and / or (ii) an amino acid substitution of C137A, numbered according to SEQ ID NO:1.

[0022] In some embodiments, the subject is maintained in a hind limbs elevated position, eg, Trendelenburg position, for about 1-2 hours after administration of the pharmaceutical composition.

[0023] In some embodiments, the pharmaceutical composition comprises a viral vector, an antibody, an antisense oligonucleotide, or a nanoparticle.

[0024] In some embodiments, the pharmaceutical composition comprises an adeno-associated virus (AAV) viral vector.

[0025] In some embodiments, AAV virus vector is AAV1、AAV2、AAV2G9、AAV3、AAV3a、AAV3b、AAV3-3、AAV4、AAV4-4、AAV5、AAV AV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV 9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27. 3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV 42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20 、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223 .4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-3 / rh.61 AV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r 11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu .10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127. 2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV A3.3、AAV A3.4、AAV A3.5、AAV A3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2 、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAV hu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.2 5、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu. u.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu. AVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20 、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R 2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、 AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R A586R、AAVrh8R R533A、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAV hEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, A AV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-L K16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 Capsid proteins from AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPEN AAV10 and / or Japanese AAV10 serotypes and variants thereof.

[0026] In some embodiments, the AAV viral vector comprises a capsid protein from AAV9.

[0027] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein.

[0028] In some embodiments, the AAV viral vector comprises a polynucleotide encoding the methyl-CpG binding protein 2 (MECP2) protein.

[0029] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a small hairpin RNA (shRNA) that targets superoxide dismutase 1 (SOD1).

[0030] In some embodiments, the AAV viral vector comprises two ITRs (e.g., a modified AAV2 ITR and an unmodified AAV2 ITR), a promoter (e.g., the chicken beta actin (CB) promoter), an enhancer (e.g., the cytomegalovirus (CMV) immediate early / early enhancer), an intro (e.g., a modified SV40 late 16s intron), and a polyadenylation signal (e.g., the bovine growth hormone (BGH) polyadenylation signal).

[0031] In some embodiments, the pharmaceutical composition comprises 1×10 10 ~1×10 15 viral vector genomes, e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 The vector comprises a viral vector genome.

[0032] In some embodiments, the composition is at least 1×10 10 ~1×10 15 vector genomes / milliliter (vg / ml), e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 Contains vector genomes / milliliter (vg / ml).

[0033] In another aspect, the disclosure provides a method of treating a neurological disease comprising administering to a subject in need thereof a pharmaceutical composition according to any one of the preceding embodiments, wherein the pharmaceutical composition comprises an AAV encoding a gene associated with the neurological disease, and wherein administration of the pharmaceutical composition is coincident with CSF influx during the sleep cycle.

[0034] In some embodiments, the pharmaceutical composition is administered when the subject falls asleep, as indicated, for example, by electroencephalogram (EEG) monitoring.

[0035] In some embodiments, the subject is administered a sleep-promoting drug in combination with the pharmaceutical combination.

[0036] In some embodiments, the sleep-promoting agent is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine, and trazodone, or combinations thereof.

[0037] In another aspect, the disclosure provides a method of improving transduction efficiency and / or distribution of a neurodegenerative therapeutic agent to the brain, the method comprising administering the neurodegenerative therapeutic agent in a subject in need thereof in combination with a second agent that enhances glymphatic influx, thereby improving the transduction efficiency of the neurodegenerative therapeutic agent in the subject.

[0038] In some embodiments, the neurodegenerative therapeutic is a viral vector, an antibody, an antisense oligonucleotide, or a CNS-targeted nanoparticle.

[0039] In some embodiments, the second agent is administered simultaneously or sequentially with the neurodegenerative therapeutic agent. In some embodiments, the second agent is administered before the neurodegenerative therapeutic agent. In some embodiments, the second agent is administered after the neurodegenerative therapeutic agent.

[0040] In some embodiments, the neurodegenerative therapeutic agent is administered intrathecally (IT), intracisternomagna (ICM) and / or ICV by bolus, slow bolus and / or infusion through an implanted intrathecal or intraventricular catheter.

[0041] In some embodiments, the second agent is administered by intravenous infusion, intravenous injection, and / or inhalation.

[0042] In some embodiments, the second agent comprises an AQP4 promoter, such as TGN-073. In some embodiments, the second agent comprises a compound that upregulates AQP4, such as sevoflurane.

[0043] In some embodiments, the second agent comprises an alpha-2 adrenergic agonist, such as clonidine, cizanidine, or dexmedetomidine (eg, Precedex or Dexdomitol).

[0044] In some embodiments, the second agent comprises one or more FDA approved anesthetic agents that enhance glymphatic inflow, hi some embodiments, the anesthetic agent is ketamine, dexmedetomidine, or xylazine, or a combination thereof.

[0045] In a preferred embodiment, the second agent comprises a combination of ketamine and dexmedetomidine.

[0046] In some embodiments, ketamine is administered about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, preferably about 10-15 minutes, prior to administration of the neurodegenerative therapeutic agent.

[0047] In some embodiments, ketamine is administered at about 100 mg / kg, about 90 mg / kg, about 80 mg / kg, about 70 mg / kg, about 60 mg / kg, about 50 mg / kg, about 40 mg / kg, about 30 mg / kg, about 20 mg / kg, about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, preferably about 10 mg / kg.

[0048] In some embodiments, dexmedetomidine is administered at about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.09 mg / kg, about 0.08 mg / kg, about 0.07 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, about 0.009 mg / kg, about 0.008 mg / kg, about 0.007 mg / kg, about 0.006 mg / kg, about 0.005 mg / kg, preferably about 0.02 mg / kg.

[0049] In some embodiments, the subject is further administered sevorlan followed by dexmedetomidine. In some embodiments, the sevorlan is administered as an inhalant.

[0050] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg). The subject is then maintained in a hindlimb elevated Trendelenburg position for 10-15 minutes after completion of ketamine and dexmedetomidine administration. The subject is further administered atipamezole (0.2 mg / kg IM), with dosing occurring between 8-10 AM standard time.

[0051] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg), followed by sevoflurane inhalation anesthesia.

[0052] In some embodiments, the second agent induces plasma hyperosmolarity. In some embodiments, the second agent comprises hypertonic saline (e.g., sodium chloride with or without sodium acetate) or mannitol. In some embodiments, the second agent comprises hypertonic saline with or without sodium acetate. In some embodiments, the hypertonic saline is 2% NaCl, 3% NaCl, 5% NaCl, 7% NaCl, or 23% NaCl, preferably 3% NaCl. In some embodiments, the 3% NaCl is administered at about 2-3.5 ml / kg.

[0053] In some embodiments, the agent is administered intravenously or by infusion injection about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 minutes, preferably about 5 minutes, before or after administration of the pharmaceutical composition, which administration may optionally be repeated.

[0054] In some embodiments, the second agent enhances glymphatic influx by increasing slow wave sleep, hi some embodiments, the second agent is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine, and trazodone, or combinations thereof.

[0055] In some embodiments, the second agent comprises a VEGF, such as VEGF-C. In some embodiments, the VEGF-C comprises (i) an amino acid sequence of any one of the sequences provided in Table 1, or a sequence having at least 95% sequence identity thereto (optionally, the sequence includes or does not include a linker (e.g., a glycine-serine linker) and / or a his tag); and / or (ii) an amino acid substitution of C137A, numbered according to SEQ ID NO:1.

[0056] In some embodiments, the subject is maintained in a hind limbs elevated position, eg, Trendelenburg position, for about 1-2 hours after administration of the pharmaceutical composition.

[0057] In some embodiments, the neurodegenerative therapeutic is an adeno-associated virus (AAV) viral vector.

[0058] In some embodiments, AAV virus vector is AAV1、AAV2、AAV2G9、AAV3、AAV3a、AAV3b、AAV3-3、AAV4、AAV4-4、AAV5、AAV AV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV 9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27. 3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV 42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20 、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223 .4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-3 / rh.61 AV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r 11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu .10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127. 2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV A3.3、AAV A3.4、AAV A3.5、AAV A3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2 、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAV hu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.2 5、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu. u.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu. AVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20 、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R 2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、 AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R A586R、AAVrh8R R533A、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAV hEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, A AV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-L K16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 Capsid proteins from AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPEN AAV10 and / or Japanese AAV10 serotypes and variants thereof.

[0059] In some embodiments, the AAV viral vector comprises a capsid protein from AAV9.

[0060] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein.

[0061] In some embodiments, the AAV viral vector comprises a polynucleotide encoding the methyl-CpG binding protein 2 (MECP2) protein.

[0062] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a small hairpin RNA (shRNA) that targets superoxide dismutase 1 (SOD1).

[0063] In some embodiments, the AAV viral vector comprises two ITRs (e.g., a modified AAV2 ITR and an unmodified AAV2 ITR), a promoter (e.g., the chicken beta actin (CB) promoter), an enhancer (e.g., the cytomegalovirus (CMV) immediate early / early enhancer), an intro (e.g., a modified SV40 late 16s intron), and a polyadenylation signal (e.g., the bovine growth hormone (BGH) polyadenylation signal).

[0064] In some embodiments, the pharmaceutical composition comprises 1×10 10 ~1×10 15 viral vector genomes, e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 The vector comprises a viral vector genome.

[0065] In some embodiments, the composition is at least 1×10 10 ~1×10 15 viral vector genomes / milliliter (vg / ml), e.g., 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 Contains viral vector genomes / milliliter (vg / ml).

[0066] In another aspect, the disclosure provides a method of enhancing the efficacy of a pharmaceutical composition delivered intrathecally, comprising administering to a subject in need thereof the pharmaceutical composition in combination with an agent that enhances glymphatic inflow.

[0067] In some embodiments, the agent is administered simultaneously or sequentially with the composition. In some embodiments, the agent is administered prior to administration of the composition. In some embodiments, the agent is administered after administration of the composition. In some embodiments, the agent is administered by intravenous infusion, intravenous injection, and / or inhalation.

[0068] In some embodiments, the agent comprises an AQP4 promoter, such as TGN-073.

[0069] In some embodiments, the agent comprises a compound that upregulates AQP4, such as sevoflurane.

[0070] In some embodiments, the agent comprises an alpha-2 adrenergic agonist, such as clonidine, cizanidine, or dexmedetomidine (eg, Precedex or Dexdomitol).

[0071] In some embodiments, the agent comprises one or more FDA approved anesthetic agents that enhance glymphatic inflow, hi some embodiments, the anesthetic agent is ketamine, dexmedetomidine, or xylazine, or a combination thereof.

[0072] In preferred embodiments, the medicament comprises a combination of ketamine and dexmedetomidine. In some embodiments, the subject is administered ketamine first, followed by the pharmaceutical composition, followed by dexmedetomidine. In some embodiments, the subject is administered ketamine first, followed by the pharmaceutical composition, followed by dexmedetomidine, and followed by sevoflurane.

[0073] In some embodiments, ketamine is administered at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, preferably about 10 to 15 minutes, prior to administration of the pharmaceutical composition. In some embodiments, ketamine is administered at about 100 mg / kg, about 90 mg / kg, about 80 mg / kg, about 70 mg / kg, about 60 mg / kg, about 50 mg / kg, about 40 mg / kg, about 30 mg / kg, about 20 mg / kg, about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, preferably about 10 mg / kg. In some embodiments, the method of any one of claims 90-92, wherein dexmedetomidine is administered at about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.09 mg / kg, about 0.08 mg / kg, about 0.07 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, about 0.009 mg / kg, about 0.008 mg / kg, about 0.007 mg / kg, about 0.006 mg / kg, about 0.005 mg / kg, preferably about 0.02 mg / kg. In some embodiments, the subject is further administered sevorlan followed by dexmedetomidine. In some embodiments, the sevorlan is administered as an inhalant.

[0074] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg). The subject is then maintained in a hindlimb elevated Trendelenburg position for 10-15 minutes after completion of ketamine and dexmedetomidine administration. The subject is further administered atipamezole (0.2 mg / kg IM), with dosing occurring between 8-10 AM standard time.

[0075] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg), followed by sevoflurane inhalation anesthesia.

[0076] In some embodiments, the agent induces plasma hyperosmolarity. In some embodiments, the agent comprises hypertonic saline or mannitol. In some embodiments, the agent comprises hypertonic saline. In some embodiments, the hypertonic saline is 3% NaCl. In some embodiments, the 3% NaCl is administered at about 2-3.5 ml / kg.

[0077] In some embodiments, the agent is administered intravenously or by infusion injection about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 minutes, preferably about 5 minutes, before or after administration of the pharmaceutical composition, which administration may optionally be repeated.

[0078] In some embodiments, the agent enhances glymphatic influx by increasing slow wave sleep, hi some embodiments, the agent is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine, and trazodone, or combinations thereof.

[0079] In some embodiments, the agent comprises a VEGF, such as VEGF-C. In some embodiments, the VEGF-C comprises (i) an amino acid sequence of any one of the sequences provided in Table 1, or a sequence having at least 95% sequence identity thereto (optionally with or without a linker (e.g., a glycine-serine linker) and / or a his-tag); and / or (ii) an amino acid substitution of C137A, numbered according to SEQ ID NO:1.

[0080] In some embodiments, the subject is maintained in a hind limbs elevated position, eg, Trendelenburg position, for about 1-2 hours after administration of the pharmaceutical composition.

[0081] In some embodiments, the pharmaceutical composition comprises a viral vector, an antibody, an antisense oligonucleotide, or a nanoparticle.

[0082] In some embodiments, the pharmaceutical composition comprises an adeno-associated virus (AAV) viral vector.

[0083] In some embodiments, AAV virus vectors are AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV7. V8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV2 7.3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-1 3、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AA V223.4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、 AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / rh.58、AAV16. 8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV A3.3、AAV A3.4、AAV A3.5、AAV A3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2 、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAV hu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.2 5、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu. u.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu. AVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh .17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.3 6、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52 AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R R533A、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAV hEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, A AV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-L K16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 Capsid proteins from AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPEN AAV10 and / or Japanese AAV10 serotypes and variants thereof.

[0084] In some embodiments, the AAV viral vector comprises a capsid protein from AAV9.

[0085] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein.

[0086] In some embodiments, the AAV viral vector comprises a polynucleotide encoding the methyl-CpG binding protein 2 (MECP2) protein.

[0087] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a small hairpin RNA (shRNA) that targets superoxide dismutase 1 (SOD1).

[0088] In some embodiments, the AAV viral vector comprises two ITRs (e.g., a modified AAV2 ITR and an unmodified AAV2 ITR), a promoter (e.g., the chicken beta actin (CB) promoter), an enhancer (e.g., the cytomegalovirus (CMV) immediate early / early enhancer), an intro (e.g., a modified SV40 late 16s intron), and a polyadenylation signal (e.g., the bovine growth hormone (BGH) polyadenylation signal).

[0089] In some embodiments, the pharmaceutical composition comprises 1×10 10 ~1×10 15 viral vector genomes, e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 The vector comprises a viral vector genome.

[0090] In some embodiments, the composition is at least 1×10 10 ~1×10 15 viral vector genomes / milliliter (vg / ml), e.g., 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 Contains vector genomes / milliliter (vg / ml).

[0091] In another aspect, the disclosure provides a method of reducing variability in brain distribution of a viral vector among a population of patients treated with a pharmaceutical composition comprising the viral vector, the method comprising administering to a subject an agent that enhances glymphatic influx in combination with the pharmaceutical composition.

[0092] In some embodiments, the agent is administered simultaneously or sequentially with the composition. In some embodiments, the agent is administered prior to administration of the composition. In some embodiments, the agent is administered after administration of the composition.

[0093] In some embodiments, the pharmaceutical composition is administered intrathecally (IT) and / or intracisternally (ICM). In some embodiments, the agent is administered by intravenous infusion, intravenous injection and / or inhalation.

[0094] In some embodiments, the agent comprises an AQP4 promoter, such as TGN-073.

[0095] In some embodiments, the agent comprises a compound that upregulates AQP4, such as sevoflurane.

[0096] In some embodiments, the agent comprises an alpha-2 adrenergic agonist, such as clonidine, cizanidine, or dexmedetomidine (eg, Precedex or Dexdomitol).

[0097] In some embodiments, the agent comprises one or more FDA approved anesthetic agents that enhance glymphatic inflow, hi some embodiments, the anesthetic agent is ketamine, dexmedetomidine, or xylazine, or a combination thereof.

[0098] In preferred embodiments, the medicament comprises a combination of ketamine and dexmedetomidine. In some embodiments, the subject is administered ketamine first, followed by the pharmaceutical composition, followed by dexmedetomidine. In some embodiments, the subject is administered ketamine first, followed by the pharmaceutical composition, followed by dexmedetomidine, and followed by sevoflurane.

[0099] In some embodiments, ketamine is administered at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, preferably about 10 to 15 minutes, prior to administration of the pharmaceutical composition. In some embodiments, ketamine is administered at about 100 mg / kg, about 90 mg / kg, about 80 mg / kg, about 70 mg / kg, about 60 mg / kg, about 50 mg / kg, about 40 mg / kg, about 30 mg / kg, about 20 mg / kg, about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, preferably about 10 mg / kg. In some embodiments, dexmedetomidine is administered at about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.09 mg / kg, about 0.08 mg / kg, about 0.07 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, about 0.009 mg / kg, about 0.008 mg / kg, about 0.007 mg / kg, about 0.006 mg / kg, about 0.005 mg / kg, preferably about 0.02 mg / kg. In some embodiments, the subject is further administered sevorlan followed by dexmedetomidine. In some embodiments, the sevorlan is administered as an inhalant.

[0100] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg). The subject is then maintained in a hindlimb elevated Trendelenburg position for 10-15 minutes after completion of ketamine and dexmedetomidine administration. The subject is further administered atipamezole (0.2 mg / kg IM), with dosing occurring between 8-10 AM standard time.

[0101] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg), followed by sevoflurane inhalation anesthesia.

[0102] In some embodiments, the agent induces plasma hyperosmolarity. In some embodiments, the agent comprises hypertonic saline or mannitol. In some embodiments, the agent comprises hypertonic saline. In some embodiments, the hypertonic saline is 2% NaCl, 3% NaCl, 5% NaCl, 7% NaCl or 23% NaCl, preferably 3% NaCl. In some embodiments, the 3% NaCl is administered at about 2-3.5 ml / kg.

[0103] In some embodiments, the agent is administered intravenously or by infusion injection about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 minutes, preferably about 5 minutes, before or after administration of the pharmaceutical composition, which administration may optionally be repeated.

[0104] In some embodiments, the agent enhances glymphatic influx by increasing slow wave sleep, hi some embodiments, the agent is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine, and trazodone, or combinations thereof.

[0105] In some embodiments, the agent comprises a VEGF, such as VEGF-C. In some embodiments, the VEGF-C comprises (i) an amino acid sequence of any one of the sequences provided in Table 1, or a sequence having at least 95% sequence identity thereto (optionally with or without a linker (e.g., a glycine-serine linker) and / or a his-tag); and / or (ii) an amino acid substitution of C137A, numbered according to SEQ ID NO:1.

[0106] In some embodiments, the subject is maintained in a hind limbs elevated position, eg, Trendelenburg position, for about 1-2 hours after administration of the pharmaceutical composition.

[0107] In some embodiments, the pharmaceutical composition comprises an adeno-associated virus (AAV) viral vector.

[0108] In some embodiments, AAV virus vector is AAV1、AAV2、AAV2G9、AAV3、AAV3a、AAV3b、AAV3-3、AAV4、AAV4-4、AAV5、AAV AV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV 9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27. 3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV 42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20 、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223 .4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-3 / rh.61 AV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r 11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu .10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127. 2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV A3.3、AAV A3.4、AAV A3.5、AAV A3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2 、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAV hu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.2 5、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu. u.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu. AVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh .17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.3 6、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52 AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R R533A、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAV hEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, A AV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-L K16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 Capsid proteins from AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPEN AAV10 and / or Japanese AAV10 serotypes and variants thereof.

[0109] In some embodiments, the AAV viral vector comprises a capsid protein from AAV9.

[0110] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein.

[0111] In some embodiments, the AAV viral vector comprises a polynucleotide encoding the methyl-CpG binding protein 2 (MECP2) protein.

[0112] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a small hairpin RNA (shRNA) that targets superoxide dismutase 1 (SOD1).

[0113] In some embodiments, the AAV viral vector comprises two ITRs (e.g., a modified AAV2 ITR and an unmodified AAV2 ITR), a promoter (e.g., the chicken beta actin (CB) promoter), an enhancer (e.g., the cytomegalovirus (CMV) immediate early / early enhancer), an intro (e.g., a modified SV40 late 16s intron), and a polyadenylation signal (e.g., the bovine growth hormone (BGH) polyadenylation signal).

[0114] In some embodiments, the pharmaceutical composition comprises 1×10 10 ~1×10 15 viral vector genomes, e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 The vector comprises a viral vector genome.

[0115] In some embodiments, the composition is at least 1×10 10 ~1×10 15 vector genomes / milliliter (vg / ml), e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 Contains vector genomes / milliliter (vg / ml).

[0116] In another aspect, the present disclosure provides a method of reducing systemic exposure of a CNS-targeted pharmaceutical composition in a subject in need thereof to reduce liver and / or DRG toxicity in the subject, the method comprising administering to the subject an agent that enhances glymphatic influx in combination with the pharmaceutical composition.

[0117] In some embodiments, the agent is administered simultaneously or sequentially with the composition. In some embodiments, the agent is administered prior to administration of the composition. In some embodiments, the agent is administered after administration of the composition.

[0118] In some embodiments, the pharmaceutical compositions are administered by intrathecal (IT) administration and / or intracisternal (ICM) administration.

[0119] In some embodiments, the agent is administered by intravenous infusion, intravenous injection, and / or inhalation.

[0120] In some embodiments, the agent comprises an AQP4 promoter, such as TGN-073.

[0121] In some embodiments, the agent comprises a compound that upregulates AQP4, such as sevoflurane.

[0122] In some embodiments, the agent comprises an alpha-2 adrenergic agonist, such as clonidine, cizanidine, or dexmedetomidine (eg, Precedex or Dexdomitol).

[0123] In some embodiments, the agent comprises one or more FDA approved anesthetic agents that enhance glymphatic inflow, hi some embodiments, the anesthetic agent is ketamine, dexmedetomidine, or xylazine, or a combination thereof.

[0124] In preferred embodiments, the medicament comprises a combination of ketamine and dexmedetomidine. In some embodiments, the subject is administered ketamine first, followed by the pharmaceutical composition, followed by dexmedetomidine. In some embodiments, the subject is administered ketamine first, followed by the pharmaceutical composition, followed by dexmedetomidine, and followed by sevoflurane.

[0125] In some embodiments, ketamine is administered at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, preferably about 10 to 15 minutes, prior to administration of the pharmaceutical composition. In some embodiments, ketamine is administered at about 100 mg / kg, about 90 mg / kg, about 80 mg / kg, about 70 mg / kg, about 60 mg / kg, about 50 mg / kg, about 40 mg / kg, about 30 mg / kg, about 20 mg / kg, about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, preferably about 10 mg / kg. In some embodiments, dexmedetomidine is administered at about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.09 mg / kg, about 0.08 mg / kg, about 0.07 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, about 0.009 mg / kg, about 0.008 mg / kg, about 0.007 mg / kg, about 0.006 mg / kg, about 0.005 mg / kg, preferably about 0.02 mg / kg. In some embodiments, the subject is further administered sevorlan followed by dexmedetomidine. In some embodiments, the sevorlan is administered as an inhalant.

[0126] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg). The subject is then maintained in a hindlimb elevated Trendelenburg position for 10-15 minutes after completion of ketamine and dexmedetomidine administration. The subject is further administered atipamezole (0.2 mg / kg IM), with dosing occurring between 8-10 AM standard time.

[0127] In some preferred embodiments, subjects are administered ketamine (10 mg / kg) 10-15 minutes prior to dosing, followed by dexmedetomidine (0.02 mg / kg), followed by sevoflurane inhalation anesthesia.

[0128] In some embodiments, the agent induces plasma hyperosmolarity. In some embodiments, the agent comprises hypertonic saline (e.g., sodium chloride with or without sodium acetate) or mannitol. In some embodiments, the agent comprises hypertonic saline with or without sodium acetate. In some embodiments, the hypertonic saline is 2% NaCl, 3% NaCl, 5% NaCl, 7% NaCl, or 23% NaCl, preferably 3% NaCl. In some embodiments, 3% NaCl is administered at about 2-3.5 ml / kg.

[0129] In some embodiments, the agent is administered intravenously or by infusion injection about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 minutes, preferably about 5 minutes, before or after administration of the pharmaceutical composition, which administration may optionally be repeated.

[0130] In some embodiments, the agent enhances glymphatic influx by increasing slow wave sleep, hi some embodiments, the agent is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine, and trazodone, or combinations thereof.

[0131] In some embodiments, the agent comprises a VEGF, such as VEGF-C. In some embodiments, the VEGF-C comprises (i) an amino acid sequence of any one of the sequences provided in Table 1, or a sequence having at least 95% sequence identity thereto (optionally with or without a linker (e.g., a glycine-serine linker) and / or a his-tag); and / or (ii) an amino acid substitution of C137A, numbered according to SEQ ID NO:1.

[0132] In some embodiments, the subject is maintained in a hind limbs elevated position, eg, Trendelenburg position, for about 1-2 hours after administration of the pharmaceutical composition.

[0133] In some embodiments, the pharmaceutical composition comprises a viral vector, an antibody, an antisense oligonucleotide, or a nanoparticle.

[0134] In some embodiments, the pharmaceutical composition comprises an adeno-associated virus (AAV) viral vector.

[0135] In some embodiments, AAV virus vector is AAV1、AAV2、AAV2G9、AAV3、AAV3a、AAV3b、AAV3-3、AAV4、AAV4-4、AAV5、AAV AV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV 9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27. 3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV 42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20 、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223 .4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-3 / rh.61 AV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r 11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu .10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127. 2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV A3.3、AAV A3.4、AAV A3.5、AAV A3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2 、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAV hu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.2 5、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu. u.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu. AVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20 、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R 2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、 AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R A586R、AAVrh8R R533A、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAV hEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, A AV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-L K16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 Capsid proteins from AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPEN AAV10 and / or Japanese AAV10 serotypes and variants thereof.

[0136] In some embodiments, the AAV viral vector comprises a capsid protein from AAV9.

[0137] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein.

[0138] In some embodiments, the AAV viral vector comprises a polynucleotide encoding the methyl-CpG binding protein 2 (MECP2) protein.

[0139] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a small hairpin RNA (shRNA) that targets superoxide dismutase 1 (SOD1).

[0140] In some embodiments, the vector comprises two ITRs (e.g., a modified AAV2 ITR and an unmodified AAV2 ITR), a promoter (e.g., chicken beta actin (CB) promoter), an enhancer (e.g., the cytomegalovirus (CMV) immediate early / early enhancer), an intro (e.g., a modified SV40 late 16s intron), a polyadenylation signal (e.g., the bovine growth hormone (BGH) polyadenylation signal).

[0141] In some embodiments, the pharmaceutical composition comprises 1×10 10 ~1×10 15 viral vector genomes, e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 The vector comprises a viral vector genome.

[0142] In some embodiments, the composition is at least 1×10 10 ~1×10 15 vector genomes / milliliter (vg / ml), e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 Contains vector genomes / milliliter (vg / ml).

[0143] Further embodiments of the present invention are provided in the following sections. [Brief description of the drawings]

[0144] [Figure 1-1] Figure 1: Figures 1A-G are images showing immunohistochemistry for GFP protein expression in the brain after intrathecal dosing by lumbar puncture of 3.0x1013 vg of scAAV9-CB-GFP at 1 month. Figure 1A is an image of animal P0304 block 44. Figure 1B is an image of animal P0303 block 46. Figure 1C is an image of animal P0304 block 44. Figure 1D is an image of animal P0302 block 47. Figure 1E is an image of animal P0503 block 47. Figure 1F is an image of animal P0303 block 47. Figure 1G is an image of animal P0301 block 51. Representations a, b, c, d and e correspond to enlargements of the boxed areas in the low power micrographs. [Figure 1-2] (As stated above.) [Figure 1-3] (As stated above.) [Figure 1-4] (As stated above.) [Figure 1-5] (As stated above.) [Figure 1-6] (As stated above.) [Figure 1-7] (As stated above.) [Diagram 2] Images showing immunohistochemistry for co-localized glial fibrillary acidic protein (GFAP) and GFP. GFP positive cells are morphologically consistent with astrocytes (GFP-DAB, left) and co-localize with GFAP (GFAP-blue and GFP-yellow, right). [Figure 3-1] Figure 3: Figures 3A and 3B are images and scatter plots, respectively, showing quantitative image analysis of GFP expression in spinal cord, dorsal root ganglion and brain regions, expressed as percent DAB positive pixels. Minimal and variable expression is detected in multiple regions of the brain, while moderate to high expression is detected in lower motor neurons of the spinal cord and neurons of the dorsal root ganglion. [Figure 3-2] (As stated above.) [Figure 4] 1 shows images showing immunohistochemistry for GFP protein on sections of cerebellum and brainstem. Minimal transduction of Purkinje cell neurons and neurons within the deep cerebellar nuclei is observed. GFP signal is primarily present with Bergmann glia. [Diagram 5] Immunohistochemistry for GFP protein. Periventricular GFP protein expression is observed in astrocytes. This periventricular expression was variable between individual animals and was restricted to the immediate 500-1000 um of the surrounding neuropil. [Figure 6] Images showing immunohistochemistry for GFP protein on sections of occipital cortex. Multifocal protein expression is present in perivascular astrocytes. Labels in a, b, and c correspond to magnifications of boxed areas in low-power photomicrographs. [Figure 7] Immunohistochemistry for GFP protein in the occipital cortex shows expression in perivascular astrocytes adjacent to perforating arteries in the occipital cortex, consistent with exposure to vector through glymphatic influx. [Figure 8] 11 shows detection of GFP protein in the occipital cortex using immunohistochemistry and quantitative image analysis. A linear pattern of astrocyte expression is observed adjacent to the arterial vascular supply, consistent with exposure to vector through glymphatic influx. [Figure 9] Image showing model of vector distribution to the central nervous system and systemic tissues after intrathecal administration of AAV in cynomolgus macaques. Due to rapid turnover of CSF, most of the vector leaves the intrathecal space through the arachnoid granulations and nerve roots and is distributed to systemic tissues. Limited vector reaches brain tissue through glymphatic influx and periventricular diffusion. Approximately 0.01% of the intrathecally administered dose is present in brain tissue at 30 days compared to 1.3% in the liver. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0145] Throughout the detailed description, certain terms are defined so that the present disclosure may be more readily understood. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0146] The disclosed compositions and methods may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure.

[0147] Throughout this specification, the description refers to compositions and methods of using the compositions. When the disclosure discloses or claims a feature or embodiment related to a composition, such feature or embodiment is equally applicable to a method or use of using the composition. Similarly, when the disclosure discloses or claims a feature or embodiment related to a method of using the composition, such feature or embodiment is equally applicable to the composition. When a range of values ​​is expressed, it includes embodiments using any specific value within the range. Furthermore, reference to values ​​stated in a range includes every single value within that range. All ranges are inclusive of their endpoints and can be combined. When values ​​are expressed as approximations, it will be understood that the particular value forms another embodiment by using the antecedent "about." Reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. The use of "or" means "and / or" unless the particular context of its use dictates otherwise. All references cited herein are incorporated by reference for any purpose. In the event of a conflict between a reference and this specification, this specification will control. It should be understood that certain features of the disclosed compositions and methods that are, for clarity, disclosed herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods that are, for brevity, disclosed in the context of a single embodiment, can also be provided separately or in any subcombination.

[0148] definition Unless otherwise stated, the following terms and phrases as used herein shall have the following meanings:

[0149] As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. When used in the context of numerical values ​​and ranges, the term "about" or "approximately" refers to a value or range that is close to or near the recited value or range such that the embodiment can function as intended, as would be apparent to one of ordinary skill in the art from the teachings contained herein. In some embodiments, about means plus or minus 10% of the numerical value.

[0150] As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value that is similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise stated or otherwise evident from the context (except that such number exceeds 100% of possible values).

[0151] As used herein, the terms "glymphatic" or "glialymphatic", "glymphatic system" or "glymphatic pathway" refer to brain waste clearance pathways for the central nervous system ("CNS") via perivascular pathways of cerebrospinal fluid (CSF) flow. The glymphatic system relies on the reciprocal exchange of CSF and interstitial fluid (ISF) to allow waste products to be transferred to the CSF and transported out of the brain.

[0152] As used herein, the term "pharmaceutical composition" refers to a composition in which the biological activity of the active ingredient has a therapeutic effect, and thus the composition can be administered in a subject, e.g., a human, for therapeutic purposes. The phrase "pharmaceutical acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0153] As used herein, the term "therapeutic agent" refers to any pharmacologically active substance that can be administered to achieve a desired effect.

[0154] As used herein, "neurodegenerative disorder" or "neurodegenerative disease" refers to a central nervous system (CNS) disorder characterized by neuronal death in one or more regions of the nervous system and subsequent functional impairment of the affected population. In some embodiments, the neurological disorder may be a neurodegenerative disorder, including, but not limited to, Alzheimer's disease (AD); amyotrophic lateral sclerosis (ALS); Creutzfeldt-Jakob disease; Huntington's disease (HD); Friedreich's ataxia (FA); Parkinson's disease (PD); multiple system atrophy (MSA); spinal muscular atrophy (SMA), multifocal sclerosis (MS); primary progressive aphasia; progressive supranuclear palsy; dementia; brain cancer, degenerative neurological diseases, encephalitis, epilepsy, inherited brain disorders causing neurodegeneration, retinitis pigmentosa (RP), head and brain malformations, hydrocephalus, stroke, prion diseases, infantile neuronal ceroid lipofuscinosis (INCL), a pediatric neurodegenerative disease caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1), and the like.

[0155] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in attempting to alter the natural course of the individual being treated, and may be performed either prophylactically or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or remitigating the disease state, and improving the prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the onset of disease or slow the progression of the disease.

[0156] An "effective amount" of an agent, eg, a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0157] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0158] As used herein, the term "intrathecal (IT) administration" or "intrathecal (IT) injection" refers to an injection into the spinal canal (the intrathecal space surrounding the spinal cord). A variety of techniques may be used, including but not limited to lateral ventricular injection through a burr hole or cisterna magna or lumbar puncture, etc. In some embodiments, "intrathecal administration" or "intrathecal delivery" according to the present disclosure refers to IT administration or delivery via the lumbar or lumbar region, i.e., lumbar IT administration or delivery. As used herein, the term "lumbar region" or "lumbar" refers to the vertebrae in the region between the third and fourth lumbar vertebrae (lower back), more generally the L2-S region of the spine.

[0159] As used herein, the term "intracisternal (ICM) administration" or "intracisternal (ICM) injection" refers to injection into the space around and below the cerebellum via the opening between the skull and the top of the spine.

[0160] As used herein, the term "intracerebroventricular (ICV) administration" or "intracerebroventricular (ICV) injection" refers to injection into a cavity in the brain that is continuous with the central canal of the spinal cord.

[0161] As used herein, the term "AQP4" or "aquaporin 4" refers to a membrane protein that functions as a water transporter in the central nervous system. It is concentrated in the perivascular endfeet of astroglial cells that surround blood vessels and maintain the integrity of the blood-brain barrier, and has a key role in controlling cerebral water balance.

[0162] As used herein, the term "alpha-2 adrenergic agonist" refers to a chemical entity, such as a compound, ion, complex, etc., that acts at or binds to an alpha-2 adrenergic receptor and is effective to produce a therapeutic effect.

[0163] As used herein, "hyperosmolality" and "hypotonicity" are relative terms, for example, in the context of physiological osmolality, but may differ from this, as long as the ultimate goal of an osmolality difference or gradient between two compartments (such as plasma and central nervous system interstitium) is achieved to enhance the influx of glymphatic flow into the central nervous system interstitium, brain interstitium and / or spinal cord interstitium. Thus, "hyperosmolality solution" refers to any physiologically and / or pharmacologic acceptable solution that is hyperosmolar with respect to physiological osmolality, including hypertonic saline or sugar solutions. As mentioned herein, the preferred hyperosmolality solution of the present disclosure preferably does not cause BBB disruption.

[0164] As used herein, the term "slow wave sleep" refers to phase 3 sleep or deep sleep, which is the deepest phase of non-rapid eye movement (NREM) sleep and is characterized by delta waves (measured by EEG).

[0165] As used herein, the term "VEGF-C" refers to vascular endothelial growth factor C, which is a member of the platelet-derived growth factor / blood endothelial growth factor family. VEGF-C is described in detail in WO 98 / 33917; Joukov et al., J. Biol. Chem., 273(12):6599-6602 (1998); and Joukov et al., EMBO J., 16(13):3898-3911 (1997), all of which are incorporated by reference in their entireties.

[0166] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that is capable of non-covalently, reversibly and specifically binding to a corresponding antigen. For example, a natural IgG antibody is a tetramer that includes at least two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0167] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to an antibody of the present disclosure). Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0168] The term "antisense oligonucleotide" refers to a nucleic acid sequence that is complementary to the coding strand or mRNA of a nucleic acid sequence, regardless of length.Antisense RNA can be introduced into individual cells, tissues or organoids.Antisense nucleic acid can contain modified backbones, such as phosphorothioate, phosphorodithioate or other modified backbones known in the art, or can contain non-natural internucleoside linkages.

[0169] As used herein, the term "siRNA" refers to a double-stranded RNA molecule that interferes with the expression of a specific gene or genes after transcription. In some embodiments, siRNA functions to interfere with or inhibit gene expression using the RNA interference pathway. Similar interference or inhibition effects can be achieved using one or more of short hairpin RNA (shRNA), microRNA (mRNA) and / or nucleic acids (such as siRNA, shRNA or miRNA) that contain one or more modified nucleic acid residues, such as peptide nucleic acid (PNA), locked nucleic acid (LNA), unlocked nucleic acid (UNA) or triazole-linked DNA. Optimally, siRNAs are 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and have a 2-base overhang at their 3' end. These dsRNAs can be introduced into individual cells or culture systems. Such siRNAs are used to downregulate mRNA levels or promoter activity.

[0170] As used herein, the term "nanoparticle" refers to any particle with a diameter of less than 1000 nanometers (nm). In some embodiments, the diameter of a nanoparticle is less than 300 nm as defined by the National Science Foundation. In some embodiments, the diameter of a nanoparticle is less than 100 nm as defined by the National Institutes of Health. The term "nanoparticle" further includes liposomes and lipid particles of nanoparticle size.

[0171] The terms "polyadenylation (polyA) signal sequence" and "polyadenylation sequence" refer to a control element that provides a signal for transcription termination and refers to the addition of a homopolymeric chain of adenosines to the 3' end of an RNA transcript. A polyadenylation signal may include a termination signal (e.g., an AAUAAA sequence or other non-canonical sequence) and optionally flanking auxiliary elements (e.g., GU-rich elements) and / or other elements associated with efficient cleavage and polyadenylation. A polyadenylation sequence may include a series of adenosines linked by polyadenylation to the 3' end of an mRNA. A specific polyA signal sequence may include the poly(A) signal of SEQ ID NO: 22 or SEQ ID NO: 89. In some embodiments, the DNA regulatory sequence or control element is a tissue-specific regulatory sequence.

[0172] The term "post-transcriptional regulatory element" ("PRE") refers to one or more regulatory elements that, when transcribed into mRNA, regulate gene expression at the level of the mRNA transcript. Examples of such post-transcriptional regulatory elements may include sequences encoding microRNA binding sites, RNA-binding protein binding sites, and the like. Examples of post-transcriptional regulatory elements that may be used with the nucleic acid molecules and vectors disclosed herein include woodchuck hepatitis post-transcriptional regulatory element (WPRE), hepatitis post-transcriptional regulatory element (HPRE).

[0173] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length. They may contain one or more ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases, such as locked nucleic acid (LNA), peptide nucleic acid (PNA).

[0174] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide typically contains at least two amino acids or amino acid variants, with no limit to the maximum number of amino acids that may make up the sequence of a protein or peptide. A polypeptide includes any peptide or protein that contains two or more amino acids or variants linked together by peptide bonds. These terms include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc., among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0175] The terms "sequence identity" and "sequence homology" are used interchangeably herein and, when used in reference to a polynucleotide or polypeptide, refer to the percentage of bases or amino acids that are identical and in the same relative positions when comparing or aligning two sequences of a polynucleotide of a polypeptide. Sequence identity can be determined in several different ways. For example, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.). See, e.g., Altschul et al., (1990) J. Mol. Bioi., 215:403-10.

[0176] The term "isolated" with respect to a nucleic acid or protein discussed herein refers to a nucleic acid or protein that has been separated from one or more components that are normally found in association with it in its natural environment. Separation can include removal from a larger nucleic acid (e.g., from a gene or chromosome) or from other proteins or molecules that normally contact the nucleic acid or protein. The term encompasses, but does not require, complete isolation.

[0177] As used herein, an isolated nucleic acid that includes a "heterologous nucleic acid sequence" refers to an isolated nucleic acid that includes a portion (i.e., a heterologous nucleic acid portion) that is not normally found in a natural context operably linked to one or more other components of the isolated nucleic acid. For example, a heterologous nucleic acid can include a nucleic acid sequence that was not originally found in a cell, bacterial cell, virus, or organism where the other components of the isolated nucleic acid (e.g., a promoter) are naturally derived, or where the other components of the isolated nucleic acid (e.g., a promoter) are not naturally found in operably linked to a heterologous nucleic acid in the cell, bacterial cell, virus, or organism. In some embodiments, a heterologous nucleic acid includes a transgene. As used herein, a "transgene" is a nucleic acid sequence that encodes a molecule of interest (e.g., a therapeutic protein, a reporter protein, or a therapeutic RNA molecule) that is not naturally associated with one or more components of the nucleic acid molecule. In some embodiments, the heterologous nucleic acid sequence encodes a human protein. In some embodiments, the heterologous nucleic acid sequence encodes an RNA sequence, such as an shRNA.

[0178] A DNA sequence or DNA polynucleotide sequence that "encodes" a particular RNA is a sequence of DNA that can be transcribed into RNA. A DNA polynucleotide may code for an RNA (mRNA) that is translated into a protein, or a DNA polynucleotide may code for an RNA that is not translated into a protein (e.g., tRNA, rRNA, or guide RNA; also called "non-coding" RNA or "ncRNA"). A DNA sequence or DNA polynucleotide sequence may also "encode" a particular polypeptide or protein sequence, e.g., a DNA directly codes for an mRNA that can be translated into a polypeptide or protein sequence. A "protein coding sequence" or a sequence that codes for a particular protein or polypeptide is a nucleic acid sequence that can be transcribed into mRNA (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence may be determined by a start codon at the 5'-terminus (N-terminus) and a translation stop nonsense codon at the 3'-terminus (C-terminus). A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence is usually located 3' to the coding sequence.

[0179] As used herein, the term "promoter" or "promoter sequence" refers to a DNA regulatory sequence that can promote transcription (e.g., can cause detectable levels of transcription and / or can increase detectable levels of transcription above levels provided in the absence of the promoter), e.g., of a downstream (3' direction) coding or non-coding sequence, e.g., via binding of RNA polymerase, of an operably linked coding or non-coding sequence. In some embodiments, a promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to include a minimum number of bases or elements to initiate transcription at a level detectable above background. In some embodiments, a promoter sequence may include a transcription initiation site as well as a protein binding domain involved in binding of RNA polymerase. In addition to sequences sufficient to initiate transcription, a promoter may also include sequences of other regulatory elements involved in regulating transcription (e.g., enhancers, Kozak sequences, and introns). A variety of promoters, including inducible and constitutive promoters, may be used to drive the vectors disclosed herein. Examples of promoters known in the art that can be used in some embodiments, such as the viral vectors disclosed herein, include CMV promoter, CBA promoter, smCBA promoter, and promoters derived from immunoglobulin genes, SV40, or other tissue-specific genes (e.g., RLBP1, RPE, VMD2). Additionally, standard techniques are known in the art for creating functional promoters by mixing and matching known regulatory elements. Fragments of promoters, such as those that retain at least a minimum number of bases or elements to initiate transcription at a level detectable above background, can also be used.

[0180] The terms "DNA regulatory sequence," "control element," and "regulatory element," as used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, silencers, polyadenylation signals, terminators, protein degradation signals, etc., that provide and / or regulate the transcription of a non-coding sequence (e.g., a short hairpin RNA) or a coding sequence (e.g., a PGRN) and / or regulate the translation of an encoded polypeptide.

[0181] As used herein, a process performed "in vitro" refers to a process performed outside of a normal biological environment, e.g., research performed in a test tube, flask, petri dish, artificial medium. A process performed "in vivo" refers to a process performed within an organism or cell, e.g., research performed in cell culture or mice. Research performed "ex vivo" refers to research performed in or on tissues from an organism in an external environment, e.g., minimizing alteration of natural conditions and allowing the manipulation of an organism's cells or tissues under more controlled conditions than may be possible in an in vivo experiment.

[0182] As used herein, the term "naturally-occurring" or "unmodified," as applied to, for example, a nucleic acid, polypeptide, cell, or organism, is one that is found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (such as a virus) is naturally-occurring, whether present in the organism or isolated from one or more components of the organism.

[0183] In some embodiments, a "vector" is any genetic element (e.g., DNA, RNA, or mixtures thereof) that contains a nucleic acid of interest (e.g., a transgene) that can be expressed in a host cell, e.g., a cell, tissue, and / or organism, within a larger nucleic acid sequence or structure suitable for delivery to the cell, tissue, and / or organism, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. For example, a vector can contain an insert (e.g., a heterologous nucleic acid containing a transgene encoding the gene to be expressed or the open reading frame of that gene) and one or more additional elements and / or elements suitable for delivering the insert or controlling its expression. A vector can be capable of replication and / or expression, e.g., when associated with appropriate control elements, and it can transfer genetic information between cells. In some embodiments, a vector can be a vector suitable for expression in a host cell, e.g., an AAV vector. In some embodiments, a vector can be a plasmid suitable for expression and / or replication, e.g., in a cell or bioreactor. In some embodiments, vectors specifically designed for the expression of heterologous nucleic acid sequences, such as transgenes encoding proteins of interest, shRNAs, etc., in target cells can be called expression vectors, and generally have a promoter sequence that drives the expression of transgenes. In other embodiments, vectors, such as transcription vectors, can be transcribed but not translated. They can be replicated in target cells but not expressed. Transcription vectors can be used to amplify their inserts.

[0184] The term "plasmid" refers to a non-chromosomal (and typically double-stranded) DNA sequence that contains an intact "replicon" that allows the plasmid to replicate in a host cell. A plasmid can be a circular nucleic acid. When a plasmid is placed into a unicellular organism, the characteristics of that organism are changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying a gene for tetracycline resistance (TcR) transforms a cell that was previously sensitive to tetracycline into one that is resistant to tetracycline.

[0185] As used herein, the term "recombinant virus" refers to a non-wild type and / or artificially produced recombinant virus (such as, for example, parvovirus, adenovirus, lentivirus, or adeno-associated virus) that includes a transgene or other heterologous nucleic acid. A recombinant virus may include a recombinant viral genome packaged within a viral (e.g., AAV) capsid. A particular type of recombinant virus may be a "recombinant adeno-associated virus" or "rAAV". The recombinant viral genome packaged within the viral capsid may be a viral vector. In some embodiments, the recombinant virus disclosed herein includes a viral vector. Examples of viral vectors include, but are not limited to, an adeno-associated viral (AAV) vector, a chimeric AAV vector, an adenoviral vector, a retroviral vector, a lentiviral vector, a DNA viral vector, a herpes simplex viral vector, a baculoviral vector, or a mutant or derivative thereof.

[0186] In another embodiment, the term "transfection" is used to refer to the uptake of foreign DNA by a cell, such that the cell is "transfected" when the exogenous DNA is introduced into the cell membrane. See, e.g., Graham et al., (1973) Virology, 52:456; Sambrook et al., (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al., (1986) Basic Methods in Molecular Biology, Elsevier; Chu et al., (1981) Gene, 13:197. Using such techniques, one or more exogenous DNA moieties can be introduced into a suitable host cell. In some embodiments, the term "transduction" is used to refer to the uptake of foreign DNA by a cell, the foreign DNA being provided by a virus or viral vector. As a result, the cell is "transformed" when the exogenous DNA is introduced into the cell membrane. In some embodiments, the term "transformation" is used to refer to the uptake of foreign DNA by a bacterial cell.

[0187] As used herein, the term "cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro. In certain circumstances, spontaneous or induced changes in karyotype may occur during storage or transfer of such clonal populations. Thus, cells derived from the referenced cell line may not be exactly identical to the ancestral cells or cultures, and the referenced cell line includes such variants.

[0188] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, the term refers to the functional relationship between a transcriptional regulatory sequence and a sequence to be transcribed. For example, a promoter or enhancer sequence is operably linked to a coding sequence if, for example, it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a sequence are adjacent to the sequence or separated by a short spacer sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent or located in close proximity to the coding sequence whose transcription is enhanced.

[0189] As used herein, the term "AAV vector" refers to a vector derived from or containing one or more nucleic acid sequences derived from an adeno-associated virus serotype, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, or AAV-9 viral vector. An AAV vector may have one or more AAV wild-type genes, e.g., deleted in whole or in part, e.g., the rep and / or cap genes, while retaining functional flanking inverted terminal repeat ("ITR") sequences. In some embodiments, an AAV vector may be packaged into a protein shell or capsid, e.g., containing one or more AAV capsid proteins, which may provide a vehicle for delivery of vector nucleic acid to the nucleus of a target cell. In some embodiments, an AAV vector contains one or more AAV ITR sequences (e.g., AAV2 ITR sequences). In some embodiments, an AAV vector contains one or more AAV ITR sequences (e.g., AAV2 ITR sequences) but does not contain additional viral nucleic acid sequences. In some embodiments, the AAV vector components (e.g., ITRs) are derived from a different serotype virus than the rAAV capsid (e.g., the AAV vector may contain ITRs from AAV2, and the AAV vector may be packaged into an AAV9 capsid).Embodiments of these vector constructs are provided, for example, in International Publication No. WO 2019 / 094253 (PCT / US2018 / 058744), which is incorporated herein by reference in its entirety.

[0190] In some embodiments, "scAAV" is a self-complementary adeno-associated virus (scAAV). scAAV is called "self-complementary" because at least a portion of the vector of scAAV (e.g., at least a portion of the coding region) forms intramolecular double-stranded DNA. In some embodiments, rAAV is a scAAV. In some embodiments, viral vectors are engineered from naturally occurring adeno-associated viruses (AAV) to provide scAAV for use in gene therapy. Embodiments of these vector constructs and methods for preparing and purifying them are provided, for example, in PCT / US2019 / 094253 (PCT / US2018 / 058744), which is incorporated herein by reference in its entirety.

[0191] In some embodiments, "ssAAV" is a single-stranded adeno-associated virus (ssAAV). ssAAV is referred to as "single-stranded" because at least a portion of the vector of ssAAV (e.g., at least a portion of the coding region) is single-stranded DNA. In some embodiments, rAAV is a ssAAV. In some embodiments, the viral vector is engineered from a naturally occurring adeno-associated virus (AAV) to provide ssAAV for use in gene therapy.

[0192] As used herein, "virus" or "virion" refers to a viral particle that includes a viral vector, e.g., alone or in combination with one or more additional components, such as one or more viral capsids. For example, an AAV virus can include, for example, a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat.

[0193] In some embodiments, the terms "virus", "virion", "AAV virus", "recombinant AAV virion", "rAAV virion", "AAV vector particle", "complete capsid", "complete particle", and the like refer to an infectious, replication-defective virus, e.g., one that includes an AAV protein shell that encapsidates a heterologous nucleotide sequence of interest, e.g., in a viral vector flanked on one or both sides by AAV ITRs. rAAV virions can be produced in suitable host cells that include sequences, e.g., one or more plasmids that specify an AAV vector, alone or in combination (e.g., on the same or additional plasmids) with nucleic acids encoding AAV helper and accessory functions (such as cap genes). In some embodiments, the host cell is rendered capable of encoding AAV polypeptides that provide for packaging of the AAV vector (including the recombinant nucleotide sequence of interest) into an infectious recombinant virion particle for subsequent gene delivery.

[0194] The term "inverted terminal repeat" or "ITR" refers to a set of nucleotide sequences capable of forming a T-shaped palindrome structure, for example in adeno-associated viruses (AAV) and / or recombinant adeno-associated virus vectors (rAAV). Muzyczka et al., (2001) Fields Virology, Chapter 29, Lippincott Williams & Wilkins. In recombinant AAV vectors, these sequences may play a functional role in genome packaging and second strand synthesis.

[0195] The term "host cell" refers to a cell that contains an exogenous nucleic acid of interest, such as one or more microorganisms, yeast cells, insect cells, or mammalian cells. For example, a host cell may contain an AAV helper construct, an AAV vector plasmid, an accessory function vector, and / or other transfer DNA. The term includes the progeny of the original transfected cell. The progeny of a single parent cell may not necessarily be completely identical in morphology or genome or total DNA complement to the original parent due to natural, accidental, or deliberate mutations.

[0196] The term "AAV helper functions" refers to coding sequences from AAV that can be expressed to provide AAV gene products (e.g., those that function in trans for productive AAV replication). For example, AAV helper functions can include both major AAV open reading frames (ORFs), rep and cap. The Rep expression product has been shown to have many functions, including, among others, recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The Cap expression product provides the necessary packaging functions. As used herein, AAV helper functions can be used to complement AAV functions in trans that are missing from an AAV vector.

[0197] The term "AAV helper construct" generally refers to a nucleic acid molecule that includes a nucleotide sequence that provides or encodes a protein or nucleic acid that provides an AAV function that is deleted from an AAV vector, such as a vector for delivery of a nucleotide sequence of interest to a target cell or tissue. AAV helper constructs are commonly used to provide transient expression of AAV rep and / or cap genes to complement missing AAV functions for AAV replication. Typically, the helper construct lacks AAV ITRs and cannot replicate or package itself. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus or virion. Many AAV helper constructs have been disclosed, such as the commonly used plasmids pAAV / Ad and plM29+45, which code for both Rep and Cap expression products. See, for example, Samulski et al., (1989) J. Virol., 63:3822-3828; McCarty et al., (1991) J. Virol., 65:2936-2945. Many other vectors encoding Rep and / or Cap expression products have been disclosed. See, e.g., U.S. Patent Nos. 5,139,941 and 6,376,237. Embodiments of these vector constructs and methods for preparing and purifying them are provided, for example, in International Publication No. WO 2019 / 094253 (PCT / US2018 / 058744), which is incorporated herein by reference in its entirety.

[0198] Glymphatic Style In one aspect, the present disclosure provides a method for improving AAV delivery of drugs to target tissues, such as the brain, by modulating glymphatic influx. Glymphatic is a recently recognized system in which CSF is drawn into deeper regions of the brain along periarterial spaces formed by astrocytes adjacent to blood vessels, where CSF may interact with interstitial fluid before exiting the brain in corresponding perivenular spaces. This system is believed to play a major role in fluid movement and macromolecule removal from the brain parenchyma. Larger particles, such as lipoproteins, of comparable size to AAV vectors, move through the glymphatic system. It has been found that AAV distribution patterns are consistent with limited vector diffusion across the membrane lining the brain surface and vector entry occurs primarily through glymphatic influx. It has been unexpectedly discovered that AAV delivery to the central nervous system interstitium, brain interstitium and / or spinal cord interstitium can be achieved by enhancing glymphatic influx. Specifically, the disclosure provides that promotion of glymphatic influx may be used for i) improving delivery of a pharmaceutical composition to the central nervous system of a subject in need thereof; ii) a method of treating a neurological disease in a subject in need thereof, wherein the pharmaceutical composition comprises an AAV encoding a gene associated with the neurological disease; iii) a method of improving transduction efficiency and / or distribution of a neurodegenerative therapeutic agent in the brain; iv) a method of improving efficacy of an intrathecally delivered pharmaceutical composition; v) a method of reducing variability in brain distribution of a viral vector among a population of patients treated with a pharmaceutical composition comprising the viral vector; and vi) a method of reducing systemic exposure of a CNS-targeted pharmaceutical composition in a subject in need thereof to reduce liver and / or DRG toxicity in the subject.

[0199] Glymphatic influx can be enhanced through a number of methods. In some embodiments, glymphatic influx can be promoted by timing of vector administration to coincide with CSF influx during the sleep cycle. In some embodiments, glymphatic influx can be enhanced by administering an AQP4 promoter, such as TGN-073. In some embodiments, glymphatic influx can be enhanced by AQP4 upregulation, such as by sevoflurane anesthesia. In some embodiments, glymphatic influx can be enhanced by alpha-2 adrenergic agonists, such as dexmedetomidine (Precedex or Dexdomitol). In some embodiments, glymphatic influx can be enhanced by a combination of ketamine and xylazine. In some embodiments, glymphatic influx can be enhanced by induction of plasma hyperosmolality with hypertonic saline or mannitol.

[0200] AQP4 Aquaporin 4 (AQP4), a water channel subtype, is highly expressed in the brain. CSF and ISF replacement depends on Aquaporin 4 (AQP4) water channels in astrocytic endfeet that encase the brain vasculature. Changes in AQP4 expression or polarization, indicating a different distribution of AQP4 in endfeet compared to the rest of the cells, are associated with disturbances in glymphatic function. Consistent with the observation that the glymphatic system can clear amyloid-b, reduced glymphatic function caused by deletion of the Aqp4 gene in animal models of Alzheimer's disease leads to enhanced accumulation of amyloid-b2 and tau. 16 AQP4 polarization abnormalities are also found in Alzheimer's patients, providing some evidence that glymphatic function may also play an important role in Alzheimer's disease in humans. Kylkilahti et al., Journal of Cerebral Blood Flow & Metabolism, 0(0):1-13(2021).

[0201] In some embodiments, glymphatic influx is enhanced by an agent that enhances interstitial fluid circulation in the blood-brain barrier, for example, the agent comprises an aquaporin 4 (AQP4) promoter, such as TGN-073 (N-(3-benzyloxypyridin-2-yl)-benzene-sulfonamide). In some embodiments, the agent comprises a compound that upregulates AQP4 expression (e.g., sevoflurane) or a compound that alters the intracellular localization of AQP4. In another embodiment, the agent can be an agent that prevents AQP4 depolarization or loss of AQP4 polarity, such as JNJ-17299425 or JNJ-17306861.

[0202] Alpha-2 adrenergic agonists and anesthetics Glymphatic pathways are primarily active during sleep or anesthesia, which promotes slow wave oscillations. Decreased CNS noradrenergic tone, a key characteristic of deep NREM sleep, is associated with high glymphatic inflow because it reduces resistance to interstitial fluid flow by expanding interstitial space volume. α2-adrenergic agonists are known sedatives that induce a state of sedation similar to stage II-III NREM sleep associated with elevated slow wave delta oscillations in the electroencephalogram (EEG) and dramatically reduce noradrenergic tone. Studies have shown that dexmedetomidine, a selective α2-adrenergic agonist, enhances EEG slow wave activity and increases brain and spinal drug exposure of intrathecally administered drugs in mice and rats through modulation of glymphatic flow. TOLilius et al., Journal of Controlled Release,304:29-38(2019).Glymphatic system activity increases during sleep or ketamine / xylazine(K / X)anesthesia in mice, and is correlated with high EEG delta power and low heart rate.Hablitz et al.,Sci Adv.5(2):eaav5447(2019).

[0203] In one embodiment, the agent is an alpha2-adrenergic receptor (α2-AR) agonist. In one embodiment, the α2-AR agonist is dexmedetomidine. See, for example, Lilius TO, et al. Dexmedetomidine enhances glymphatic brain delivery of intrathecally administered drugs. J Control Release. 2019 Jun 28;304:29-38, which is incorporated by reference in its entirety. In some embodiments, the agent comprises an α-2 adrenergic agonist selected from the group consisting of clonidine, cizanidine, and dexmedetomidine (e.g., Precedex or Dexdomitol).

[0204] In one embodiment, the agent enhances glymphatic flow. In one embodiment, the agent enhances glymphatic inflow. In one embodiment, the agent is an anesthetic, such as a general anesthetic. In one embodiment, the anesthesia is selected from the group consisting of propofol, fospropofol, ketamine, barbiturates (e.g., thiopental, thiopentone, and methohexital), benzodiazepines (e.g., midazolam), etomidate, isoflurane, desflurane, and sevoflurane. See, for example, Hablitz LM, et al. Increased glymphatic influx is correlated with high EEG delta power and low heart rate in mice under anesthesia. Sci Adv. 2019 Feb 27;5(2):eaav5447, which is incorporated by reference in its entirety.

[0205] In some embodiments, the agent comprises one or more FDA approved anesthetic agents that enhance glymphatic inflow. In some embodiments, the anesthetic agent is ketamine, dexmedetomidine, or xylazine, or a combination thereof. In a preferred embodiment, the agent comprises a combination of ketamine and dexmedetomidine.

[0206] In some embodiments, the subject is first administered ketamine, followed by the pharmaceutical composition, and followed by dexmedetomidine.

[0207] In some embodiments, ketamine is administered about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, and preferably about 10-15 minutes, prior to administration of the pharmaceutical composition.

[0208] In some embodiments, ketamine is administered at about 100 mg / kg, about 90 mg / kg, about 80 mg / kg, about 70 mg / kg, about 60 mg / kg, about 50 mg / kg, about 40 mg / kg, about 30 mg / kg, about 20 mg / kg, about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, preferably about 10 mg / kg.

[0209] In some embodiments, dexmedetomidine is administered at about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.09 mg / kg, about 0.08 mg / kg, about 0.07 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, about 0.009 mg / kg, about 0.008 mg / kg, about 0.007 mg / kg, about 0.006 mg / kg, about 0.005 mg / kg, preferably about 0.02 mg / kg.

[0210] In some embodiments, the subject is further administered sevorlan, followed by dexmedetomidine.

[0211] In some embodiments, sevorlan is administered as an inhalant.

[0212] Induction of plasma hyperosmolality In another aspect, enhancement of glymphatic influx can be achieved by inducing plasma hyperosmolality. As used herein, "hyperosmolality" and "hyposmolality" are relative terms, for example, in the context of physiological osmolality, but can be different, as long as the ultimate goal of osmolality difference or gradient between two compartments (plasma and central nervous system interstitium) is achieved to promote glymphatic influx into central nervous system interstitium, brain interstitium and / or spinal cord interstitium. Thus, "hyperosmolar solution" refers to any physiologically and / or pharmacologic acceptable solution that is hyperosmolar with respect to physiological osmolality, including hypertonic saline or sugar solutions. As mentioned herein, the preferred hyperosmolar solution in the present disclosure does not cause BBB disruption.

[0213] In some embodiments, the agent comprises hypertonic saline (e.g., sodium chloride with or without sodium acetate) or mannitol. In preferred embodiments, the agent comprises hypertonic saline with or without sodium acetate. In some embodiments, the hypertonic saline is 2% NaCl, 3% NaCl, 5% NaCl, 7% NaCl, or 23% NaCl, preferably 3% NaCl. In some embodiments, 3% NaCl is administered at about 2-3.5 ml / kg.

[0214] In some embodiments, the agent is administered intravenously or by infusion injection about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 minutes, preferably about 5 minutes, before or after administration of the pharmaceutical composition, which administration may optionally be repeated.

[0215] sleep Studies have shown that glymphatic function is improved during sleep and impaired by sleep disorders. Xie et al., Science 342:373-377(2013). Sleep deprivation leads to changes in AQP4 expression. Kylkilahti et al., Journal of Cerebral Blood Flow & Metabolism 0():1-13(2021).

[0216] In one embodiment, the agent is a non-anesthetic agent that increases slow wave sleep. In one embodiment, the agent is selected from the group consisting of GAT-1 inhibitors, selective extrasynaptic GABAA agonists, α2-δ sites in voltage-gated calcium ion channels, GABAB / GHB agonists, partially selective 5HT2A receptor antagonists and serotonin 2A receptor antagonists (ASTAR). In one embodiment, the agent is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine and trazodone. Additional agents that increase slow wave sleep are described in Walsh, JK Enhancement of Slow Wave Sleep: Implications for Insomnia. Journal of Clinical Sleep Medicine. 2009, 5(2):S27-S32, which is incorporated by reference in its entirety.

[0217] Enhancement of VEGF-C Studies show that blood endothelial growth factor C (VEGF-C) binds to receptors in lymphatic endothelial cells and also affects the endothelium. Joukov et al. EMBO J. 15(2):290-298(1996). Expression of VEGF-C in mice leads to lymphatic vessel growth. Da Mesquita et al., Nature 560(7717):185-191(2018).

[0218] In some embodiments, the agent that enhances glymphatic influx comprises VEGF-C. In some embodiments, VEGF-C comprises (i) an amino acid sequence of any one of the sequences provided in Table 1, or a sequence with at least 95% sequence identity thereto (optionally, the sequence includes or does not include a linker (e.g., a glycine-serine linker) and / or a his-tag); and / or (ii) an amino acid substitution of C137A, numbered according to SEQ ID NO:1.

[0219] Table 1 VEGF-C and its variants The following sequence corresponds to the monomer. When a dimer is formed, two identical sequences are organized together via a cysteine ​​bridge. Note that the his tag is used for experimental purposes, but may not be required in all embodiments.

[0220] [Table 1]

[0221] [Table 2]

[0222] Studies have shown that placing adult macaques in Trendelenburg position for 5 or 10 minutes on a reclining table with the body in supine position and the head approximately 30° below the feet increases cervical delivery compared to lumbar delivery after intrathecal infusion of AAV9. This suggests that gravity influences vector distribution and that placing the head below the feet may improve delivery to the brain. Castle et al.,Sci Adv 4(11):eaau9859(2018).

[0223] In some embodiments, the subject is maintained in a hind limbs elevated position, e.g., Trendelenburg position, for about 5-10 minutes, about 10-30 minutes, about 30 minutes to 1 hour, about 1-2 hours, about 2-3 hours, or about 3-4 hours after administration of the pharmaceutical composition. In a preferred embodiment, the subject is maintained in a hind limbs elevated position, e.g., Trendelenburg position, for about 1-2 hours after administration of the pharmaceutical composition.

[0224] In one aspect, the disclosure provides a method for improving delivery of a pharmaceutical composition to the central nervous system. In some embodiments, the pharmaceutical composition comprises a viral vector, an antibody, an antisense oligonucleotide, or a nanoparticle.

[0225] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a viral vector used to deliver transgene sequences to neural cells or tissues. Examples of viruses used in vectors include, but are not limited to, retroviruses, adenoviruses, lentiviruses, adeno-associated viruses, and other hybrid viruses. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, a chimeric AAV vector, an adenovirus vector, a retrovirus vector, a lentivirus vector, a DNA virus vector, a herpes simplex virus vector, a baculovirus vector, or any mutant or derivative thereof.

[0226] Without being bound by theory, the viral vectors disclosed herein can insert their genome into the host cells they infect to deliver their nucleic acid sequences to the host. The inserted viral genome can be episomal or integrated into the host cell chromosome at a site that can be random or targeted. In one embodiment, the vector is a viral vector used to deliver transgene sequences to cells. Examples of viruses used in vectors include, but are not limited to, retroviruses, adenoviruses, lentiviruses, adeno-associated viruses, and other hybrid viruses. Warnock et al., (2011) Methods Mol. Biol., 737:1-25. Lentiviruses are a genus of retroviruses that can integrate significant amounts of viral DNA into host cells, making them an efficient method of gene delivery. Adenoviruses, on the other hand, introduce genetic material that is not integrated into the host cell chromosome, thus reducing the risk of destroying the host cell. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, a chimeric AAV vector, an adenoviral vector, a retroviral vector, a lentiviral vector, a DNA viral vector, a herpes simplex viral vector, a baculoviral vector, or any mutant or derivative thereof.

[0227] In some embodiments, the vector containing the transgene is or is derived from an adeno-associated virus (AAV). In some embodiments, the vector is a recombinant adeno-associated virus vector (rAAV). The rAAV genome may contain one or more AAV ITRs flanking the transgene sequence encoding a polypeptide (including but not limited to hPGRN polypeptide) or encoding siRNA, shRNA, antisense and / or miRNA directed to mutant proteins or regulatory sequences of their genes. The transgene sequence may be operably linked and linked by a sequence encoding one or more protease cleavage sites or a sequence encoding one or more self-cleaving peptides or combinations thereof. In embodiments, the vector further comprises other transcriptional control elements such as those disclosed herein, for example, a promoter, enhancer, PRE and / or polyA sequence that are functional in target cells to drive expression of the transgene sequence. The transgene sequence may also include an intron sequence that facilitates processing of the RNA transcript when expressed in a mammalian cell.

[0228] In various embodiments, the AAV vector, e.g., rAAV vector, is a self-complementary AAV vector (scAAV). As used herein, "self-complementary" means that the coding region is designed to form an intramolecular double-stranded template, e.g., at one or more inverted terminal repeats (ITRs). Without being bound by theory, the rate-limiting step of the AAV genome often involves second-strand synthesis, since a typical AAV genome is a single-stranded DNA template. Ferrari et al, (1996) J. Virology, 70(5): 3227-34; Fisher et al, (1996) J. Virology, 70(1): 520-32. However, in the case of the scAAV genome, upon infection, the two complementary halves of the scAAV can assemble to form one double-stranded DNA (dsDNA) unit that is ready for replication and transcription, rather than waiting for cell-mediated synthesis of the second strand. In some embodiments, the rAAV vectors disclosed herein are scAAV vectors, providing faster and / or increased expression.

[0229] In some embodiments, the rAAV vectors disclosed herein lack one or more (e.g., all) AAV rep and / or cap genes. The AAV vector can include nucleic acid sequences (e.g., DNA) from any suitable AAV serotype (e.g., in its ITRs). Suitable AAV serotypes include, but are not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAVrh8, AAVrh10, AAV.Anc80, AAV.Anc80L65, AAV-DJ and AAV-DJ / 8, AAVrh37, AAV-DJ, AAV-DJ / 8, AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB and AAV-PHP.S. For example, an AAV vector, such as an scAAV vector, can include a nucleic acid sequence from AAV2, such as an ITR sequence from AAV2. An AAV vector, such as an scAAV vector, can also include nucleic acids from multiple serotypes. The nucleotide sequences of the genomes of AAV serotypes are known in the art.For example, the entire genome of AAV1 is provided under GenBank Accession No. NC_002077; the entire genome of AAV2 is provided under GenBank Accession No. NC 001401 and Srivastava et al., Virol., 45:555-564 {1983); the entire genome of AAV3 is provided under GenBank Accession No. NC_1829; the entire genome of AAV4 is provided under GenBank Accession No. NC_001829; the AAV5 genome is provided under GenBank Accession No. AF085716; the entire genome of AAV-6 is provided under GenBank Accession No. NC_001862; at least portions of the AAV7 and AAV8 genomes are provided under GenBank Accession Nos. AX753246 and AX753249, respectively; and the AAV9 genome is provided by Gao et al. al., J. Virol., 78:6381-6388 (2004); the AAV10 genome is provided in Williams, (2006) Mol. Ther., 13(1):67-76; the AAV11 genome is provided in Mori et al., (2004) Virology, 330(2):375-383.

[0230] In some embodiments, functional inverted terminal repeat (ITR) sequences may be used, for example, to support rescue, replication, and packaging of AAV virions. Thus, the AAV vectors disclosed herein may include sequences that provide viral replication and packaging in cis (e.g., functional ITRs). The ITRs may be, but need not be, wild-type nucleotide sequences and may be altered, for example, by insertion, deletion, or substitution of nucleotides, so long as the sequences provide functional rescue, replication, and packaging. The ITRs may be from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11. The nucleotide sequences of the genomes of AAV serotypes are known in the art. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided under GenBank Accession No. NC 001401 and Srivastava et al., Virol., 45:555-564 {1983); the complete genome of AAV-3 is provided under GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829; the AAV-5 genome is provided under GenBank Accession No. AF085716; the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank Accession Nos. AX753246 and AX753249, respectively; and the AAV-9 genome is provided by Gao et al. al., (2004) J. Virol., 78:6381-6388; the AAV-10 genome is provided in Williams, (2006) Mol. Ther., 13(1):67-76; the AAV-11 genome is provided in Mori et al., (2004) Virology, 330(2):375-383. In one embodiment, the vector is an AAV-9 vector having ITRs from AAV-2.

[0231] In some embodiments, the rAAV vectors disclosed herein comprise one or more ITRs, e.g., two ITRs, one upstream and one downstream of the transgene and / or other nucleic acid elements discussed above. In some embodiments, e.g., in scAAV vectors, the nucleic acids disclosed herein comprise a first ITR located 5' and a second ITR located 3' to the promoter, transgene, post-transcriptional regulatory element, and / or polyA, e.g., the ITRs are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 150, 200, 250 nucleotides 5' and / or 3' of the other element. The ITR sequence can be wild-type or can comprise one or more mutations, e.g., so long as it retains one or more functions of the wild-type ITR. In some embodiments, the wild-type ITR can be modified to include a deletion of a terminal resolution site. In some embodiments, the scAAV disclosed herein may comprise two ITR sequences, where both are wild-type, mutant or modified AAV ITR sequences. In some embodiments, at least one ITR sequence is a wild-type, mutant or modified AAV ITR sequence. In some embodiments, the two ITR sequences are both wild-type, mutant or modified AAV ITR sequences. In some embodiments, the "left" or 5'-ITR is a modified AAV ITR sequence that allows for the production of a self-complementary genome, and the "right" or 3'-ITR is a wild-type AAV ITR sequence. In some embodiments, the "right" or 3'-ITR is a modified AAV ITR sequence that allows for the production of a self-complementary genome, and the "left" or 5'-ITR is a wild-type AAV ITR sequence. In some embodiments, the ITR sequences are wild-type, mutant or modified AAV2 ITR sequences. In some embodiments, at least one ITR sequence is a wild-type, mutant or modified AAV2 ITR sequence. In some embodiments, the two ITR sequences are both wild-type, mutant or modified AAV2 ITR sequences.In some embodiments, the "left" or 5'-ITR is a modified AAV2 ITR sequence that allows for the production of a self-complementary genome, and the "right" or 3'-ITR is a wild-type AAV2 ITR sequence. In some embodiments, the "right" or 3'-ITR is a modified AAV2 ITR sequence that allows for the production of a self-complementary genome, and the "left" or 5'-ITR is a wild-type AAV2 ITR sequence. Exemplary sequences that may be used for one or more ITRs are described herein. In some embodiments, the AAV vector comprises SEQ ID NO: 12 and SEQ ID NO: 23. In some embodiments, the AAV vector comprises SEQ ID NO: 85 and SEQ ID NO: 90. The embodiments of AAV ITRs provided in PCT / US2018 / 058744 are incorporated herein by reference in their entirety and may be used for any AAV ITR disclosed herein.

[0232] In some embodiments, the rAAV vector lacks one or more (e.g., all) AAV rep and / or cap genes. The AAV vector may comprise (e.g., in its ITRs) a nucleic acid sequence (e.g., DNA) from any suitable AAV serotype. Suitable AAV serotypes include, but are not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11. For example, the AAV vector, e.g., a scAAV vector, may comprise a nucleic acid sequence from AAV-2, e.g., an ITR sequence from AAV-2. The AAV vector, e.g., a scAAV vector, may also comprise nucleic acids from multiple serotypes. GenBank Accession No. NC 001401 and Srivastava et al., Virol., 45:555-564 {1983); GenBank Accession No. NC_1829; GenBank Accession No. NC_001829; GenBank Accession No. AF085716; GenBank Accession No. NC_001862; GenBank Accession Nos. AX753246 and AX753249; Gao et al., J. Virol., 78:6381-6388 (2004); Williams, (2006) Mol. Ther., 13(1):67-76; and Mori et al., (2004) Virology, 330(2):375-383.

[0233] In some embodiments, functional inverted terminal repeat (ITR) sequences in a viral vector containing a transgene can be used to support, for example, rescue, replication, and packaging of AAV virions. Thus, the AAV vectors disclosed herein can include sequences that provide viral replication and packaging in cis (e.g., functional ITRs). The ITRs do not have to be wild-type nucleotide sequences, but can be altered, for example, by nucleotide insertion, deletion, or substitution, so long as the sequences provide functional rescue, replication, and packaging. The ITRs can be from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11. GenBank Accession No. NC_002077; GenBank Accession No. NC 001401 and Srivastava et al., Virol., 45:555-564 {1983); GenBank Accession No. NC_1829; GenBank Accession No. NC_001829; GenBank Accession No. AF085716; GenBank Accession No. NC_001862; GenBank Accession Nos. AX753246 and AX753249, respectively; Gao et al., (2004) J. Virol., 78:6381-6388; Williams, (2006) Mol. Ther., 13(1):67-76; and Mori et al., (2004) Virology, 330(2):375-383. In one embodiment, the vector is an AAV-9 vector with ITRs from AAV-2.

[0234] In some embodiments, AAV virus vector is AAV1、AAV2、AAV2G9、AAV3、AAV3a、AAV3b、AAV3-3、AAV4、AAV4-4、AAV5、AAV AV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV 9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27. 3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV 42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20 、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223 .4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-3 / rh.61 AV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r 11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu .10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127. 2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV A3.3、AAV A3.4、AAV A3.5、AAV A3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2 、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAV hu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.2 5、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu. u.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu. AVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20 、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R 2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、 AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R A586R、AAVrh8R R533A、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAV hEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, A AV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-L K16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 Capsid proteins from AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPEN AAV10 and / or Japanese AAV10 serotypes and variants thereof.

[0235] In some embodiments, the AAV viral vector comprises a capsid protein from AAV9.

[0236] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein.

[0237] In some embodiments, the AAV viral vector comprises a polynucleotide encoding the methyl-CpG binding protein 2 (MECP2) protein.

[0238] In some embodiments, the AAV viral vector comprises a polynucleotide encoding a small hairpin RNA (shRNA) that targets superoxide dismutase 1 (SOD1).

[0239] In some embodiments, the AAV viral vector comprises two ITRs (e.g., a modified AAV2 ITR and an unmodified AAV2 ITR), a promoter (e.g., the chicken beta actin (CB) promoter), an enhancer (e.g., the cytomegalovirus (CMV) immediate early / early enhancer), an intro (e.g., a modified SV40 late 16s intron), and a polyadenylation signal (e.g., the bovine growth hormone (BGH) polyadenylation signal).

[0240] In some embodiments, the pharmaceutical composition comprises 1×10 10 ~1×10 15 viral vector genomes, e.g. 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 The vector comprises a viral vector genome.

[0241] In some embodiments, the pharmaceutical composition comprises 1×10 10 ~1×10 15 viral vector genomes / milliliter (vg / ml), e.g., 1 x 10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 Contains viral vector genomes / milliliter (vg / ml).

[0242] In various embodiments, the nucleic acids and vectors discussed herein may be present in one or more viral particles, such as recombinant viral particles. Recombinant viruses are viruses that have been produced by recombinant means. A variety of different virus types may be used, such as retroviruses, adenoviruses, lentiviruses, AAV, murine leukemia viruses, and the like. Without being bound by theory, vectors delivered from retroviruses, such as lentiviruses, may provide long-term gene transfer and low immunogenicity, as they allow long-term stable integration and transmission of the transgene to daughter cells. Other suitable retroviruses include gamma retroviruses. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen-limited focus-forming virus (SFFV) and myeloproliferative sarcoma virus (MPSV) and vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., "Gamma retroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun;3(6):677-713. In some embodiments, the virus is a recombinant adenovirus comprising a nucleic acid or vector disclosed herein. In some embodiments, the virus is a recombinant AAV comprising a nucleic acid or vector disclosed herein.

[0243] In some embodiments, the nucleic acid or vector disclosed herein is for use in the manufacture of a recombinant virus. In some embodiments, the nucleic acid or vector disclosed herein is for use in the manufacture of a rAAV. Thus, in various embodiments, a viral composition (also called a virion), such as a rAAV viral composition comprising a viral vector or a nucleic acid disclosed above, is also disclosed herein. In some embodiments, the recombinant virus is an adeno-associated virus (AAV) or any mutant or derivative thereof. In some embodiments, the recombinant virus is a chimeric AAV or any mutant or derivative thereof. In some embodiments, the recombinant virus is an adenovirus or any mutant or derivative thereof. In some embodiments, the recombinant virus is a retrovirus or any mutant or derivative thereof. In some embodiments, the recombinant virus is a lentivirus or any mutant or derivative thereof. In some embodiments, the recombinant virus is a DNA virus or any mutant or derivative thereof. In some embodiments, the recombinant virus is a herpes simplex virus or any mutant or derivative thereof. In some embodiments, the recombinant virus is a baculovirus or any mutant or derivative thereof.

[0244] In some embodiments, the AAV disclosed herein may comprise one or more AAV capsid proteins. The AAV capsid proteins may be from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAVrh8, AAVfh10, AAV-DJ, AAV-DJ / 8, AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB, and AAV-PHP.S. In some embodiments, the one or more capsid proteins in the AAV are from AAV-9. Without being bound by theory, typically in AAV, three capsid proteins, VP1, VP2 and VP3, multimerize to form a capsid. The polypeptide sequences of capsid proteins are known in the art and can be derived from the genome of AAV. These can be used as exemplary capsids in the AAV virus compositions disclosed herein.For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided under GenBank Accession No. NC 001401 and Srivastava et al., Virol., 45:555-564 {1983); the complete genome of AAV-3 is provided under GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829; the AAV-5 genome is provided under GenBank Accession No. AF085716; the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank Accession Nos. AX753246 and AX753249, respectively; and the AAV-9 genome is provided by Gao et al. al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Williams, (2006) Mol. Ther., 13(1):67-76; the AAV-11 genome is provided in Mori et al., (2004) Virology, 330(2):375-383. The capsid proteins AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB or AAV-PHP.S are provided in Deverman et al., (2016) Nat. Biotech., 34:204-209 and Chan et al., (2017) Nat. Neurosci., 20:1172-1179. In some embodiments, the recombinant virus is an AAV that comprises one or more of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, AAV-DJ / 8, AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB, or AAV-PHP.S capsid serotypes or functional variants thereof. In some embodiments, the recombinant virus is an AAV that comprises a combination of capsids from multiple AAV serotypes.

[0245] In some embodiments, the AAV compositions disclosed herein contain one or more cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration, and are contained within the ITRs. In some embodiments, one or more of these sequences may also be present in trans, rather than in cis, e.g., on a separate plasmid during the virus production process in the host cell. Typically, three AAV promoters (named p5, p19, and p40 by their relative map positions) drive the expression of two AAV internal open reading frames that encode the rep and cap genes of wild-type virus. In some embodiments, one or more of these promoters and / or open reading frames are present in cis in the AAV vectors and / or AAV virions disclosed herein, or are present on a separate plasmid during the AAV virus production process, e.g., in the host cell that produces the virus. Two rep promoters (p5 and p19), coupled with specific splicing of a single AAV intron (nucleotides 2107 and 2227), can result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is typically expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is present at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, (1992) Curr. Topics Microbiol. Imm., 158:97-129.

[0246] In some embodiments, the AAV compositions disclosed herein comprise modified capsids with enhanced tropism to the human CNS or PNS. A variety of methods for modifying capsid proteins can be used, including, but not limited to, mutational methods, DNA barcoding, directed evolution, random peptide insertion, and capsid shuffling and / or chimeras.

[0247] Rational engineering and mutation methods have been used to direct AAV to target tissues. In rational design, structure-function relationships are used to determine the regions in which changes can be made to the capsid sequence. As non-limiting examples, surface loop structures, receptor binding sites and / or heparin binding sites can be mutated or otherwise altered to rationally design recombinant AAV capsids to facilitate targeting to target tissues. In one example of rational design, AAV capsids were modified by mutating surface-exposed tyrosines to phenylalanines to escape ubiquitination, reduce proteasomal degradation, and allow for increased AAV particle and viral genome expression (Lochrie MA, et al, J Virol. 2006 January; 80(2): 821-34; Santiago-Ortiz JL and Schaffer DV, J Control Release, 2016 October. 28; 240: 287-301, the contents of each of which are incorporated by reference in their entirety). Rational design also encompasses the addition of a targeting peptide to the parent AAV capsid sequence, where the targeting peptide may have affinity for a receptor of interest within the target tissue.

[0248] In certain embodiments, rational engineering and / or mutation methods were used to identify AAV capsids and / or targeting peptides that promoted transduction of target tissues (e.g., the CNS or PNS).

[0249] Capsid shuffling and / or chimerism describes a method in which fragments of at least two parental AAV capsids are combined to generate a new recombinant capsid protein; the number of parental AAV capsids used can be from 2 to 20 or more than 20.

[0250] In certain embodiments, capsid shuffling is used to identify AAV capsids and / or targeting peptides that promoted transduction of a target tissue (eg, the CNS or PNS).

[0251] Directed evolution involves the generation of an AAV capsid library (~10 4 -10 8 ) and selection of lead candidates based on their response to selection pressure with a property of interest (e.g., tropism). Directed evolution of AAV capsids allows for positive selection from a diverse pool of mutants without the need for extensive pre-characterization of the mutant library. Directed evolution libraries can be generated by any molecular biology technique known in the art and can include DNA shuffling, random point mutagenesis, insertional mutagenesis (e.g., targeting peptides), random peptide insertion, or ancestral sequence rearrangement. AAV capsid libraries can be subjected to multiple rounds of selection using directed evolution for further optimization. Directed evolution methods are most commonly used to identify AAV capsid proteins with enhanced transduction of target tissues. Capsids with enhanced transduction of target tissues have been identified for targeting human airway epithelium, neural stem cells, human pluripotent stem cells, retinal cells, and other in vitro and in vivo cells.

[0252] In certain embodiments, directed evolution methods were used to identify AAV capsids and / or targeting peptides that promoted transduction of target tissues (e.g., the CNS or PNS).

[0253] One method described for the high-throughput characterization of the phenotype of multiple AAV serotypes is known as AAV Barcode-Seq (Adachi K et al, Nature Communications 5:3075 (2014), the contents of which are incorporated herein by reference in their entirety). In this next-generation sequencing (NGS)-based method, AAV libraries are generated containing DNA barcode tags, which can be assessed by multiplexed Illumina barcode sequencing. This method can be used to identify AAV mutants with altered receptor binding, tropism, neutralization, and / or blood clearance when compared to wild-type or non-mutant sequences. Amino acids in the AAV capsid that are important for these functions can also be identified in this way.

[0254] AAV capsid libraries were generated as described by Adachi et al. 2014, where each mutant carried a pair of left and right 12-nucleotide long DNA barcodes downstream of the AAV cap gene and AAV2 polyadenylation signal (pA) derived from the wild-type AAV2 rep gene and a series of mutants or mutants. Thus, seven different DNA barcode AAV capsid libraries were generated. The capsid libraries were then provided to mice. At pre-defined time points, samples were collected, DNA extraction was performed, and PCR amplification was performed with AAV-clone specific viral barcodes and sample specific barcodes linked to PCR primers. All viral barcode PCR amplicons were sequenced on Illumina and converted to raw sequence read count data by computational algorithms. The core of the Barcode-Seq approach is a 96-nucleotide cassette containing the above DNA barcodes (left and right), three PCR primer binding sites, and two restriction enzyme sites. As an example, we used the AAV rep-cap genome, but this system can be applied to any AAV viral genome, including those lacking the rep and cap genes. The advantage of the Barcode-Seq method is the large set of data and the correlation to the desired phenotype with only a few copies and in a short time.

[0255] DNA Barcode-Seq methods can be similarly applied to RNA.

[0256] In certain embodiments, Barcode-Seq methods are used to identify AAV capsids and / or targeting peptides that promoted transduction of target tissues (e.g., the CNS or PNS).

[0257] In some embodiments, insertion of a targeting peptide into the parent AAV capsid sequence can be used to facilitate targeting to CNS or PNS tissue. Disclosed herein are targeting peptides and related AAV particles that include capsid proteins with one or more targeting peptide inserts for enhanced or improved transduction of target tissues (e.g., cells of the CNS or PNS).

[0258] In certain embodiments, the targeting peptide can direct the AAV particle to a cell or tissue of the CNS. The cell of the CNS can be, but is not limited to, a neuron (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), a glial cell (e.g., microglia, astrocytes, oligodendrocytes), and / or a supporting cell of the brain, such as an immune cell (e.g., T cell). The tissue of the CNS can be, but is not limited to, the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, putamen, caudate, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei.

[0259] The targeting peptides of the present disclosure may be identified and / or designed by any method known in the art. As a non-limiting example, the CREATE system as described in Deverman et al. (Nature Biotechnology 34(2):204-209(2016)), and in International Publication Nos. WO 2015038958 and WO 2017100671, the contents of each of which are incorporated by reference in their entirety, may be used as a means to identify targeting peptides in mice or other research animals, such as, but not limited to, any non-human primate.

[0260] Non-limiting examples of modified AAVs with enhanced targeting to CNS or PNS tissues can be found in U.S. Patent Application Publication Nos. 20180021364, 20210207167, 20210214749, 20210230632, and 20210277418, which are incorporated by reference in their entireties.

[0261] Treatment of Disease In various embodiments, the term "treating" includes administering to an animal (including a human) in need thereof an effective dose or effective multiple doses of a composition comprising a nucleic acid, vector, recombinant virus, or pharmaceutical composition as disclosed herein. If the dose is administered prior to the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments, an effective dose is a dose that detectably alleviates (either eliminates or attenuates) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to the disorder / disease state, slows or prevents progression of the disorder / disease state, reduces the extent of the disease, thereby remitting (partially or totally) the disease and / or prolonging survival. The term encompasses but does not require complete treatment (i.e., cure) and / or prevention. In some embodiments, an effective dose is a dose that detectably alleviates (either eliminates or attenuates) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression of the disorder / disease state, reduces the extent of the disease, thereby remitting (partially or totally) the disease and / or prolonging survival. 10 ~1×10 15In some embodiments, an effective dose comprises 1×10 vector genomes / milliliter (vg / ml) of virus as disclosed herein. 6 ~1×10 10 In some embodiments, an effective dose is 1×10 plaque forming units / milliliter (pfu / ml) of virus. 6 ~1×10 9 Transducing units / milliliter (TU / ml) of virus. Examples of disease conditions contemplated for treatment are described herein.

[0262] In some embodiments, the method of treating comprises delivering a therapeutically effective amount of a nucleic acid disclosed herein to a subject in need thereof. In some embodiments, the method of treating comprises delivering a therapeutically effective amount of a vector disclosed herein to a subject in need thereof. In some embodiments, the method of treating comprises delivering a therapeutically effective amount of a recombinant virus disclosed herein to a subject in need thereof. In some embodiments, the method of treating comprises delivering a therapeutically effective amount of a pharmaceutical composition disclosed herein to a subject in need thereof. In some embodiments, the nucleic acid, vector, recombinant virus or pharmaceutical composition disclosed herein is used in the manufacture of a medicament for treating a subject in need thereof.

[0263] In various embodiments, the nucleic acid, vector, recombinant virus or pharmaceutical composition disclosed herein may be delivered to a subject in need thereof by intravenous administration, direct brain administration (e.g., intrathecal, intracerebral and / or intraventricular administration), intranasal administration, intraaural administration or intraocular route of administration or any combination thereof. In some embodiments, the nucleic acid, vector, recombinant virus or pharmaceutical composition is delivered by intrathecal administration. In some embodiments, the nucleic acid, vector, recombinant virus or pharmaceutical composition is delivered by intracerebral or intracerebroventricular administration route. In some embodiments, the administered nucleic acid, vector, recombinant virus or pharmaceutical composition is ultimately delivered to the brain, spinal cord, peripheral nervous system and / or CNS, either directly or by translocation, after administration into a separate tissue or bodily fluid, such as blood.

[0264] In one aspect, the methods and materials are indicated for the treatment of nervous system or neurodegenerative diseases, such as Rett Syndrome, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, or for the treatment of nervous system injuries, including traumatic injuries of the spinal cord and brain, stroke, and brain cancer. In one embodiment, the use of the methods and materials is indicated for the treatment of spinal muscular atrophy (SMA).

[0265] There are four types of SMA, which are traditionally classified by the age of onset and the peak motor function achieved. All forms of SMA are autosomal recessive and caused by mutations in the survival motor neuron 1 (SMN1) gene. Humans also carry a second, nearly identical copy of the SMN gene, called SMN2. Lefebvre et al.“Identification and characterization of a spinal muscular atrophy-determining gene.”Cell,80(1):155-65.Monani et al.'Spinal muscular atrophy: a deficiency in a ubiquitous protein; a motor-neuron specific disease”Neuron,48(6):885-896.Both SMN1 and SMN2 genes express SMN protein, but SMN2 contains a translationally silent mutation in exon 7 that results in ineffective inclusion of exon 7 in the SMN2 transcript. Thus, SMN2 produces both full-length SMN protein and a truncated form of SMN lacking exon 7, with the truncated form being the predominant form produced. As a result, the amount of functional full-length protein produced by SMN2 is significantly (70-90%) less than that produced by SMN.Lorson et al.“A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy.” PNAS,96(11)6307-63 1.Monani et al,“A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2.”Hum Mol Genet 8(7):177-83. Although SMN2 cannot fully compensate for the loss of the SMN1 gene, patients with milder SMA generally have higher SMN2 copy numbers.Lefebvre et al., "Correlation between severity and SMN protein level in spinal muscular atrophy" Nat Genet 6(3):265-269. Park et al, "Spinal muscular atrophy: new and emerging insights from model mice" Curr Neurol Neurosci Rep 10(2):108-117. Of note, SMN2 copy number is not the only phenotype modifier. In particular, the c859GC variant in exon 7 of the SMN2 gene has been reported as a positive disease modifier. Patients with this particular mutation have a less severe disease phenotype. Prior et al., "A positive modification of spinal muscular atrophy in the SMN2 gene." Am J Hum Genet 85(3):408-413.

[0266] Type I SMA (also called infantile onset or Werdnig-Hoffmann disease) is when SMA symptoms are present at birth or by 6 months of age. In this type, infants typically have low muscle tone (hypotonia), a weak cry, and respiratory distress. These infants often have difficulty swallowing and sucking, and do not reach the developmental milestone of being able to sit unassisted. These infants often exhibit one or more of the SMA symptoms selected from hypotonic delayed motor skills, poor head control, hunched shoulders, and hypermobility of the joints. Typically, these infants have two copies of the SMN2 gene, one on each chromosome 5. More than half of all new SMA cases are of this type.

[0267] Type II or intermediate SMA is when SMA has its onset between 7 months and before the child can stand or walk independently. Children with type 2 SMA generally have at least three SMN2 genes. In late-onset SMA (also known as types III and IV SMA, mild SMA, adult-onset SMA, and Kugelberg-Welander disease), there are variable levels of weakness. Type III SMA develops after 18 months, and these children can stand and walk independently, but may require assistance. Type IV SMA onset is in adulthood, and children can walk throughout adulthood. People with type III or IV SMA generally have four to eight SMN2 genes, and from these genes, significant amounts of full-length SMN protein can be produced.

[0268] In one embodiment, the term "treatment" includes administering an effective dose or effective multiple doses of a composition comprising a rAAV as disclosed herein to an animal (including a human) in need thereof, either intravenously or via an intrathecal route. If the dose is administered prior to the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In an embodiment, an effective dose is a dose that alleviates (either eliminates or attenuates) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to the disorder / disease state, slows or prevents progression of the disorder / disease state, reduces the extent of the disease, thereby remitting (partially or totally) the disease and / or prolonging survival. Examples of disease states contemplated for treatment are provided herein.

[0269] In one embodiment, a composition comprising a rAAV of the disclosure is administered intravenously to a patient in need thereof having SMA, hi another embodiment, a composition comprising a rAAV of the disclosure is administered intrathecally to a patient in need thereof having SMA II, III, or IV.

[0270] Disclosed herein are methods of treating SMA type 1 in a patient in need thereof by administering an AAV9 viral vector via an intrathecal or intravenous route. In some embodiments, the patient is 0-9 months old. In some other embodiments, the patient is 0-6 months old. In some embodiments, when a viral vector is used to treat an SMA type in a patient, the patient's weight is determined. In some embodiments, the patient's weight is less than 8.5 kg. In some embodiments, the patient's weight is greater than 2.6 kg. In some embodiments, the patient's weight is between 2.6 and 8.5 kg.

[0271] In some embodiments, the patient has a mutation, e.g., a null mutation, in one copy of the SMN1 gene (including any mutation that confers encoded SM1 non-functional). In some embodiments, the patient has a mutation, e.g., a null mutation, in two copies of the SMN1 gene. In some embodiments, the patient has a mutation, e.g., a null mutation, in all copies of the SMN1 gene. In some embodiments, the patient has a deletion in one copy of the SMN1 gene. In some embodiments, the patient has a deletion in two copies of the SMN1 gene. In some embodiments, the patient has a biallelic SMN1 mutation, i.e., either a deletion or a replacement of SMN1 on both alleles of the chromosome. In some embodiments, the patient has at least one functional copy of the SMN2 gene. In some embodiments, the patient has at least two functional copies of the SMN2 gene. In some embodiments, the patient has at least two functional copies of the SMN2 gene. In some embodiments, the patient has at least three functional copies of the SMN2 gene. In some embodiments, the patient has at least four functional copies of the SMN2 gene. In some embodiments, the patient has at least five functional copies of the SMN2 gene. In some embodiments, the patient does not have a c.859G>C substitution in exon 7 of at least one copy of the SMN2 gene. In some embodiments, the gene sequence of the SMN1 or SMN2 gene may be determined by full genome sequencing. In other embodiments, the gene sequence and copy number of the SMN1 or SMN2 gene may be determined by high throughput sequencing. In some embodiments, the gene sequence and copy number of the SMN1 or SMN2 gene may be determined by microarray analysis. In some embodiments, the gene sequence and copy number of the SMN1 or SMN2 gene may be determined by Sanger sequencing. In some embodiments, the copy number of the SMN1 or SMN2 gene may be determined by fluorescent in situ hybridization (FISH).

[0272] In some embodiments, the patient exhibits one or more SMA symptoms. SMA symptoms may include hypotonia, delayed motor skills, poor head control, hunched shoulders and hypermobility of joints. In some embodiments, poor head control is determined by placing the patient in a ring in a seated position with support for the shoulders (front and back). Head control is assessed by the patient's ability to hold the head upright. In some embodiments, locomotor activity is observed while the patient is supine and motor skills are assessed by the patient's ability to lift elbows, knees, hands and feet off a surface. In some embodiments, the patient's grip strength is measured by placing fingers in the patient's palm and lifting the patient until the patient's shoulders are off the surface. Hypotonia and grip strength are measured by how quickly / long the patient holds on to the grip. In some embodiments, head control is assessed by placing the patient's head in the maximum available rotation and measuring the patient's ability to turn the head back to midline and back. In some embodiments, shoulder posture may be assessed by having the patient sit with head and torso supported and observing whether the patient bends the elbow or shoulder to reach for a stimulus placed at arm's length at shoulder level. In some embodiments, shoulder posture may also be assessed by having the patient lie on his / her side and observing whether the patient bends the elbow or shoulder to reach for a stimulus placed at arm's length at shoulder level. In some embodiments, motor skills are assessed by observing whether the patient bends their hip or knee when their foot is stroked, tickled or pinched. In some embodiments, shoulder flexion, elbow flexion, lumbar adduction, cervical flexion, head extension, cervical extension and / or spinal flexion may be assessed by known clinical measures, such as the CHOP INTEND. Other SMA symptoms may be assessed according to known clinical measures, such as the CHOP INTEND.

[0273] In some embodiments, the patient is treated after exhibiting symptoms (e.g., one or more symptoms) of SMA type I as determined using one of the tests described herein. In some embodiments, the patient is treated before exhibiting symptoms of SMA type I. In some embodiments, the patient is diagnosed with SMA type I based on genetic testing before exhibiting symptoms, and combination therapy is also contemplated herein. Combination therapy, as used herein, includes either simultaneous or sequential treatment. Combination methods may include the addition of certain standard medical treatments (e.g., riluzole in ALS) when combined with a novel treatment. For example, other treatments for SMA include antisense oligonucleotides (ASOs) that alter binding to pre-mRNAs and alter their splicing patterns. Singh et a!, “A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes.” P os One, 7(11):e49595. In one embodiment, nusinersen (U.S. Patent Nos. 8,361,977 and 8,980,853, incorporated herein by reference) may be used. Nusinersen, an approved ASO, targets intron 6, exon 7, or intron 7 of SM2 pre-mRNA, modulating the splicing of SMN2 to produce full-length SMN protein with higher efficiency. In some embodiments, the method of treatment comprising AAV9 viral vector is administered in combination with a muscle enhancer. In some embodiments, the method of treatment comprising AAV9 viral vector is administered in combination with a neuroprotective agent. In some embodiments, the method of treatment comprising AAV9 viral vector is administered in combination with an antisense oligonucleotide-based drug targeting SMN. In some embodiments, the method of treatment comprising AAV9 viral vector is administered in combination with nusinersen. In some embodiments, the method of treatment comprising AAV9 viral vector is administered in combination with a myostatin inhibitor.In some embodiments, methods of treatment comprising an AAV9 viral vector are administered in combination with stamlumab.

[0274] While postnatal delivery to an individual in need thereof is contemplated, in utero delivery to the fetus is also contemplated.

[0275] Methods of treating SMA type I patients using pharmaceutical compositions comprising the viral vectors are contemplated. In some embodiments, the viral vectors are administered in amounts of about 1-8×10 13 In some embodiments, the viral vector is formulated at a concentration of about 1.7-2.3 x 10 AAV9 viral vector genomes / mL (vg / mL). 13 In some embodiments, the viral vector is formulated at a concentration of about 1.9-2.1 x 10 vg / mL. 13 In some embodiments, the viral vector is formulated at a concentration of about 2.0×10 13 It is formulated at a concentration of v mL.

[0276] In some embodiments where a viral vector is used to treat SMA type I in a patient, the AAV viral vector (e.g., AAV SMN) is administered in an amount of about 1.0-2.5×10 14vg / kg. In some embodiments where a viral vector is used to treat SMA type I in a patient, the AAV viral vector is administered to the patient at a dose of about 1.014 vg / kg. In some embodiments where a viral vector is used to treat SMA type I in a patient, the AAV viral vector is infused into the patient over about 45-70 minutes. In some embodiments where a viral vector is used to treat SMA type I in a patient, the AAV viral vector is infused into the patient over about 60 minutes. In some embodiments where a viral vector is used to treat SMA type I in a patient, the AAV viral vector is infused into the patient using an infusion pump, a peristaltic pump, or any other device known in the art. In some embodiments where a viral vector is used to treat SMA type I in a patient, the AAV viral vector is infused into the patient using a syringe pump.

[0277] In one embodiment, the methods and materials described herein may be used for the treatment of neurodevelopmental disorders such as Rett syndrome. Rett syndrome is a rare neurological disorder that is first recognized in infancy, resulting in 90-95% of cases from mutations in the MECP2 gene on the X chromosome. Ruthie et al., “Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-bindin protein 2.” Nature Genetics, 23:185-188. Boys with only one copy of the X chromosome generally die shortly after birth, while girls with two copies of the X chromosome usually have one functional copy of the gene. Patients begin to show symptoms at 6-18 months of age, with characteristic symptoms such as hand rubbing or squeezing movements, clapping, rubbing, washing, or putting hands in the mouth. The disease is progressive, with significant impairments that may include autistic-like behavior, irregular breathing, feeding and swallowing difficulties, developmental delay, and epileptic seizures. There are 200 known mutations in the MECP2 gene, and disease severity varies widely from patient to patient, depending on the level of X-inactivation and dosage correction. Mouse studies have shown that MECP2 mutations do not cause neuronal death, suggesting that it is not a neurodegenerative disorder. Guy et al. “Reversal of Neurological Defects in a Mouse Model of Rett Syndrome.” Science, 315(5815)” 1:143-1147.

[0278] For embodiments relating to Rett syndrome, the rAAV (e.g., rAAV9) genome can, for example, encode methylcytosine-binding protein 2 (MeCP2). Representative AAV, e.g., scAAV9, constructs comprising polynucleotides encoding MeCP2 are provided in U.S. Pat. No. 9,415,121, the contents of which are hereby incorporated in their entirety. In some embodiments, AAV constructs comprising polynucleotides encoding MeCP2 can be prepared using methods disclosed herein. In some embodiments, these AAV constructs can be used to treat Rett syndrome. In some embodiments, MeCP2 AAV exhibits less than 10%, e.g., less than 7%, 5%, 4%, 3%, 2%, or 1% empty capsids. In some embodiments, MeCP2 AAV exhibits low amounts of residual host cell proteins, host cell DNA, plasmid DNA, and / or endotoxins, e.g., at levels discussed herein for preparation and purification of AAV vectors.

[0279] In one embodiment, the methods and materials described herein may be used for the treatment of ALS. ALS is a neurodegenerative disease that results in progressive loss of motor neurons in the brain and spinal cord, with symptoms including loss of ability to speak, eat, move, and eventually breathe. The disease typically results in death within 3-5 years of diagnosis. While 90-95% of ALS cases are unknown, some cases of ALS are caused by genetic mutations in the superoxide dismutase 1 (SOD1) gene, which results in a toxic overt gain of function. Mouse studies have shown that SOD knockouts do not result in disease, and thus treatments that knock down levels of mutant SOD1 are believed to alleviate disease symptoms.

[0280] In some embodiments, the AAV vector encodes an shRNA targeting SOD1 for ALS. Representative AAV, e.g., scAAV9, constructs encoding shRNAs against SOD1 are provided in WO 201 503 1392 and US 2016272976, the contents of which are incorporated herein in their entirety. In some embodiments, AAV constructs encoding shRNAs against SOD can be prepared using the methods disclosed herein. In some embodiments, these AAV constructs can be used to treat ALS. In some embodiments, the SOD1 AAV exhibits less than 10%, e.g., less than 7%, 5%, 4%, 3%, 2%, or 1% empty capsid. In some embodiments, the SOD1 AAV exhibits low amounts of residual host cell proteins, host cell DNA, plasmid DNA, and / or endotoxins, e.g., at the levels discussed herein for AAV vector preparation and purification.

[0281] In some embodiments, the methods and materials described herein may be used for the treatment of neurodegenerative and / or neurodevelopmental disorders and to enhance clinical trials such as those shown in Table 2.

[0282] [Table 3]

[0283] [Table 4]

[0284] [Table 5]

[0285] [Table 6]

[0286] AAV toxicity While AAV9 vectors have shown remarkable potential for delivery to the CNS following systemic delivery, resulting in clinical success in pediatric patients with spinal muscular atrophy type 1, systemic injection of high doses of AAV vectors can lead to the induction of T cell responses that can eliminate transduced cells2. There is one report in monkeys where high systemic doses of AAV9-like vectors caused toxicity and animals died from systemic inflammation. Hinderer et al., Hum. Gene. Ther. 29(3):285-298 (2018). The reason for the need for high doses is the relatively low efficiency of AAV to provide adequate transgene expression in a significant number of target cells per vector genome copy. As disclosed herein, the low efficiency of AAV can be corrected by enhancing glymphatic influx. Thus, the methods disclosed herein allow for more efficient transduction at lower doses, resulting in better therapeutic efficacy while reducing safety issues such as immunotoxicity. In one aspect, the disclosure provides a method of reducing systemic exposure of a CNS-targeted pharmaceutical composition in a subject in need thereof to reduce liver and / or dorsal root ganglion (DRG) toxicity in the subject, the method comprising administering to the subject an agent that enhances glymphatic influx in combination with the pharmaceutical composition.

[0287] Reducing variability in brain distribution As described herein, immunohistochemistry for GFP expression in animals administered AAV9 encoding GFP shows variable levels of expression in brain sections. By enhancing glymphatic influx upon intrathecal administration, higher vector levels can be achieved in the interstitial fluid, resulting in improved or more uniform transduction of targeted cell types.

[0288] In one aspect, the disclosure provides a method of reducing variability in brain distribution of a viral vector among a population of patients treated with a pharmaceutical composition comprising the viral vector, the method comprising administering to a subject an agent that enhances glymphatic influx in combination with the pharmaceutical composition.

[0289] Pharmaceutical Compositions In various embodiments, pharmaceutical compositions are disclosed. In some embodiments, the pharmaceutical compositions comprise one or more of the nucleic acids, vectors and / or viruses disclosed herein. In some embodiments, the pharmaceutical compositions comprise a pharma- ceutically acceptable carrier.

[0290] The nucleic acid, vector and / or recombinant virus (e.g., viral particle) according to the present disclosure can be formulated to prepare a pharma- ceutically useful composition. Exemplary formulations include those disclosed, for example, in U.S. Pat. Nos. 9,051,542 and 6,703,237, which are incorporated by reference in their entirety. The compositions of the present disclosure can be formulated for administration to a mammalian subject, for example, a human. In some embodiments, the delivery system can be formulated for intramuscular, intradermal, mucosal, subcutaneous, intravenous, intrathecal, injectable depot-type device, or topical administration.

[0291] In some embodiments, when the delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, for example, an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized and / or sterile filtered. The resulting aqueous solution can be packaged for immediate use or lyophilized. In some embodiments, lyophilized preparations are combined with a sterile solution before administration.

[0292] In some embodiments, the compositions, e.g., pharmaceutical compositions, may contain pharma- ceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc., e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the pharmaceutical composition contains a preservative. In other embodiments, the pharmaceutical composition does not contain a preservative.

[0293] The details of one or more embodiments of the present disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. Other features, objectives, and advantages of the present disclosure will become apparent from this description and the claims. In this specification and the appended claims, the singular forms include plural references unless otherwise clearly indicated by the context. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All patents and publications cited herein are incorporated by reference where applicable, unless otherwise indicated. The following examples are given to more specifically illustrate preferred embodiments of the present disclosure. These examples should not be construed as limiting the scope of the disclosed subject matter in any way, which scope is defined by the appended claims. EXAMPLES

[0294] Example 1: AAV biodistribution in non-human primates method Samples to be evaluated 3.0 × 10 by intrathecal (IT) route through lumbar puncture (LP) or intracranial magnum (ICM) administration 13Tissues were collected from cynomolgus macaques administered scAAV9-CB-GFP in 1000 mg / kg and compared to vehicle control animals. For dosing, animals were placed under anesthesia with ketamine / dexmedetomidine and maintained in Trendelenburg position for 10 min, followed by administration of atipamezole (an alpha-2 adrenergic antagonist) as an antagonist. Tissues were collected at necropsy, fixed in formalin, and then routinely processed into paraffin for histological evaluation and molecular localization studies.

[0295] immunohistochemistry Immunohistochemistry staining for GFP was performed using standard Ventana Discovery XT reagents (Ventana, Indianapolis, IN) on a Ventana Discovery XT autostainer, including deparaffinization and antigen retrieval steps. Slides were deparaffinized and then subjected to heat-induced antigen retrieval by covering them with Cell Conditioning 1 (CC1 / pH8) solution according to the standard Ventana retrieval protocol. Slides were incubated with primary antibodies (rabbit monoclonal anti-GFP antibody clone EPR14104-89 at 0.372ug / mL) or non-immune isotype-matched control (rabbit monoclonal IgG clone DA1E at 0.372ug / ml) as indicated in Table 3 for 1 hour. Visualization was performed by incubation with the appropriate Ventana Discovery OmniMap HRP reagent as indicated below, followed by Ventana Discovery ChromoMap 3,3'-diaminobenzidine (DAB). Counterstaining was performed using Ventana Hematoxylin and Ventana Bluing reagents for 4 minutes each. Slides were dehydrated, cleared, and coverslipped with synthetic mounting medium. Immunohistochemistry slides were examined and assigned a negative, minimal, moderate, or strong reaction score based on no staining, <1% stained area, 1-10% stained tissue, 11-50% stained tissue, or >50% stained tissue, respectively.

[0296] [Table 7]

[0297] Image analysis of GFP immunohistochemical staining Image analysis was performed on 20x images scanned on an Aperio AT2 scanner (Leica Biosystems) using the HALO platform by Indica Labs (v3.0.311.149). Tissue was manually annotated to remove nonspecific background staining. The Area Quantification algorithm v2.1.3 using pixel-based deconvolution was optimized for positive GFP immunohistochemistry signal and run on the annotated images. Results were based on positive signal normalized to total tissue area, resulting in % positive signal / total area. Analysis and graphing were performed in GraphPad Prism version 8.1.2. Differences in percent pixel positive area between IT and ICM groups were compared using a multiple Mann-Whitney test of group analysis.

[0298] In situ hybridization to vector sequences In situ hybridization to detect GFP antisense (AS) and sense (S) sequences encoded in AAV vectors and Macaca fascicularis (Mf)-PPIB (AS) (positive control and tissue quality control) and DapB (AS) (negative control) genes was performed in selected blocks using reagents and equipment supplied by Advanced Cell Supply Diagnostics (ACD) (Hayward, CA) and Ventana Medical Systems (Roche, Tuscon AZ). In situ hybridization RNAScope® probes were designed by ACD. A list of probes is given in Table 4. Positive PPIB and negative DAPB control probe sets were included to ensure mRNA quality and specificity, respectively. Hybridization methods followed protocols established by ACD and Ventana systems using Ventana mRNA Red chromogens. Briefly, 5 μm sections were baked at 60°C for 60 min and used for hybridization. Deparaffinization and rehydration protocols were performed using a Sakura Tissue-Tek DR5 stainer with the following steps: xylene, 3 times for 5 min each; 100% alcohol, 2 times for 2 min; air-drying for 5 min; offline manual pretreatment in 1x recovery buffer at 98-104°C for 15 min. The results were first evaluated by PPIB and DAPB hybridization signals, followed by optimization by using the same conditions for all slides. After pretreatment, slides were transferred to a Ventana Ultra automated stainer to complete the ISH procedure, including protease pretreatment; hybridization at 43°C for 2 h, followed by amplification; and detection by HRP and hematoxylin counterstaining.

[0299] [Table 8]

[0300] result Immunohistochemistry for GFP protein expression was performed on selected blocks of brain, spinal cord, lumbar dorsal root ganglion (DRG), and systemic tissues, and scored for the degree of GFP immunohistochemical staining (Table 5). Staining of control tissues yielded no signal, and no signal or nonspecific signal was observed with rabbit IgG control antibody DA1E. Compared to DRG and spinal cord, overall lower and more variable expression levels were detected in brain sections from all evaluated animals administered scAAV9-CB-GFP (Figure 1A-G). GFP protein detection was multifocal in distribution, with significant areas of the brain parenchyma showing no expression. In some regions, strong expression was observed in the pia mater, but not in the underlying neuropil. Robust GFP expression was observed in systemic tissues such as liver and skeletal muscle. Despite administration of vectors directly into the CSF via the intrathecal route, these findings are consistent with a significant barrier to distribution of vectors into the interstitial fluid of the brain parenchyma, a step required before the vector can interact with glycan and protein receptors on the surface of the targeted cell types.

[0301] Morphologically, the majority of cells expressing GFP protein appeared consistent with astrocytes. To validate this interpretation, double-labeling immunohistochemistry experiments were performed for the astrocyte marker GFAP and the GFP reporter (Figure 2). These results supported the morphological interpretation and confirmed transduction and protein expression primarily in astrocytes. Quantitative image analysis was performed on brain, spinal cord, and DRG sections stained for GFP by immunohistochemistry and reported as percent GFP-positive pixels (Figure 3). Maximum expression levels were detected in the spinal cord and DRG, with lower levels detected in the brain. There were no statistically significant differences between LP IT and ICM animals administered at 3.0 × 1013 vg / animal across the regions evaluated. Consistent with these findings, limited GFP protein expression was observed in Purkinje neurons and neurons of the deep cerebellar nuclei (Figure 4).

[0302] To confirm the protein expression pattern, in situ hybridization was performed using GFP sense and antisense probes to detect vector sequences in selected regions of the brain. In situ hybridization detected similar patterns of vector localization compared to immunohistochemistry for GFP, often revealing signals in vascular and perivascular patterns. No differences were observed between LP IT and ICM-treated animals.

[0303] In addition to the multifocal perivascular distribution, limited periventricular GFP protein expression was evident in some animals. In this pattern, protein expression was detected primarily in astrocytes and was generally restricted to 500-1000 um of the adjacent neuropil (Figure 5). These findings are consistent with limited spread of the vector from the ventricular CSF.

[0304] Further evaluation of the multifocal GFP expression pattern revealed that positive astrocytes often displayed a perivascular distribution along perforating arterial vessels (Figures 6 and 7). Detection of GFP immunohistochemistry positive cells through image analysis highlighted the distribution along these vessels and the linear nature of expression (Figure 8). This perivascular transduction of astrocytes is consistent with intrathecally administered vector reaching the interstitial fluid of the brain parenchyma via glymphatic influx.

[0305] [Table 9]

[0306] The glymphatic system is a recently recognized system in which CSF is drawn into deeper regions of the brain along periarterial spaces formed by astrocytes adjacent to blood vessels, where it may interact with interstitial fluid before exiting the brain in the corresponding perivenular spaces. This system is thought to play a major role in fluid movement and removal of macromolecules from the brain parenchyma. Larger particles such as lipoproteins, which are comparable in size to AAV vectors, move through the glymphatic system. The GFP distribution pattern observed in this study is consistent with limited vector diffusion across membranes lining the brain surface and vector entry occurring primarily through glymphatic influx.

[0307] Based on these findings, a model of CNS and systemic distribution of AAV vectors can be proposed after IT administration (Figure 9). CSF is constantly produced, with a half-life of approximately 5 h in cynomolgus macaques, and then drains from the intrathecal space through the arachnoid granulations and nerve roots, enters the meningeal lymphatics, and then enters the systemic circulation. Based on vector DNA copy numbers quantified in tissues, a total of 3.0 × 10 13 Only 0.01% of the vector dose was detectable in the brain one month after administration, compared with 1.3% in the liver after IT delivery. This is consistent with the majority of vector draining from the intrathecal space into the systemic circulation before interacting with the interstitial fluid in the brain parenchyma through glymphatic influx. By enhancing glymphatic influx upon intrathecal administration, higher levels of vector are achieved in the interstitial fluid, resulting in improved and more uniform transduction of targeted cell types. Furthermore, reduced vector distribution to systemic organs may reduce safety issues in these tissues.

[0308] Example 2: The effect of glymphatic flow modulation on AAV9 brain transduction after a single intrathecal injection in cynomolgus monkeys over a 4-week observation period Previous non-clinical NHP studies have shown low and variable transformation of the brain parenchyma following intrathecal dosing of AAV gene therapy vectors. Using complementary molecular localization approaches (immunohistochemistry and in situ hybridization) to assess brain transduction, the majority of transduced cells detected in the parenchyma appear to be astrocytes localized adjacent to the perivascular space, indicating that the vector may enter the brain parenchyma through the glymphatic system. A number of facilitating factors, such as time of administration relative to the sleep cycle, anesthesia regimen, arterial pulse wave, and peripheral osmolality, may affect glymphatic influx and influence vector particle entry within the interstitial space of the brain parenchyma. Manipulation of these factors may improve transduction levels of targeted cell types in the brain and reduce overall variability. Further improvement of CNS tissue distribution and transduction may also reduce systemic distribution and associated safety signals such as liver and dorsal root ganglion toxicity.

[0309] Therefore, the aim of this study is to explore the timing of administration, anesthesia regime and plasma hyperosmolality to reduce variability and increase brain transduction levels after intrathecal injection of AAV vectors when administered to cynomolgus monkeys as a single dose. Electroencephalographic activity will be monitored by EEG to assess the depth of anesthesia compared to low frequency high amplitude delta wave patterns and to improve the timing of dose administration. After administration, animals will be observed for at least 4 weeks after administration and changes will be compared to a control group in which the vector was administered in a standard manner.

[0310] [Table 10]

[0311] Rationale for Dosing The intrathecal injection route of administration was chosen because it is the targeted human therapeutic route and is the preferred route of administration for achieving widespread transduction of the central nervous system while limiting systemic exposure.

[0312] Dosage Justification A dose of 3e13vg / animal has been used previously to characterize the transduction profile of AAV9-CB-GFP in the brain parenchyma, and therefore serves as a benchmark. This dose level has generally been well tolerated in previous studies using similar test articles, with no serious adverse events reported. Previously tolerated findings at this dose included liver enzyme elevations, and neuropathological changes in the dorsal root ganglia were observed (findings identified as related to the AAV platform).

[0313] Species Selection Cynomolgus monkeys have been used historically in AAV biodistribution and safety evaluation studies and are the preclinical model of choice from a scientific standpoint. Cynomolgus monkeys were selected as an appropriate species due to the similarity of CNS anatomy between monkeys and humans.

[0314] [Table 11]

[0315] Anesthesia method Methods for Cohort 1 Prior to dosing, anesthetize animals with ketamine (10 mg / kg) followed by dexmedetomidine (0.02 mg / kg) 10-15 days prior to dosing. After completion of dose administration, maintain animals in a dorsal rump position with hind limbs elevated (Trendelenburg-like position) for 10-15 minutes. Administer atipamezole (0.2 mg / kg IM). Dosing occurs between 8:00 and 10:00 AM standard times.

[0316] Methods for Cohorts 2 and 3 Prior to dosing, 10-15 min prior to dosing, anesthetize the animals with ketamine (10 mg / kg), followed by dexmedetomidine (0.02 mg / kg), followed by sevoflurane inhalation anesthesia.

[0317] Depth of anesthesia is monitored by EEG and medication is administered when a deep state of anesthesia (maximum delta power and minimal alpha power) is reached.

[0318] For cohort 3, administer an intravenous injection of hypertonic saline (HTS) (NaCl 3% AT 2-3.5 ml / kg) with medication administered 5 min after HTS administration. Fluids and electrolytes require monitoring with administration of all hyperosmolar fluids, with particular attention to serum sodium, potassium and fluid in / out.

[0319] After completion of dose administration, the animal is maintained in a dorsal breech position with the hind limbs elevated (Trendelenburg-like position) and maintained under anesthesia throughout the procedure, 1-2 hours after dosing.

[0320] Administration will take place between 2:00 and 4:00 PM.

[0321] Clinical Observations Animal Health Monitoring-At least twice a day (am and pm); at least once on transfer / exit days.

[0322] Cageside observations - once daily before and during the dosing period

[0323] Post-dose examination - day of dosing for each animal dosed. Time point is 1 hour post-dose. Observations are based on time of dosing completion for each animal.

[0324] Body weight-pre-dose period: at least once. Dosing period: once on days 1, 8, 15, 22 and 28.

[0325] Food Consumption - Pre-dose and daily during the dosing period, except on animal arrival / moving days or unless fasted for other study procedures as required.

[0326] Anti-AAV9 capsid immunogenicity analysis Serum samples are collected at least once during the pre-dose period and once pre-dose on day 1 and on days 8, 15, and 22 and on the day of scheduled euthanasia (only for animals scheduled for sacrifice on that day) during the dosing period.

[0327] If sufficient sample is available, serum samples will be analyzed for anti-AAV9 capsid immunogenicity. If planned testing cannot be completed, the reason will be recorded.

[0328] Biodistribution analysis Blood samples are collected prior to dosing on day 1, once on day 8 and on the day of scheduled euthanasia during the dosing phase (only for animals scheduled for sacrifice on that day).

[0329] If the sample is of sufficient volume, the blood cell pellet and plasma will be analyzed for DNA (vector genome) using a non-GLP method. If the sample volume is insufficient for analysis, it will be recorded.

[0330] NF1 and GFAP analysis Plasma samples are collected pre-dose on day 1 and once on days 8, 15, 22 and 28 during the dosing period.

[0331] If the sample is of sufficient volume, plasma will be analyzed by using non-GLP methods for NfL and GFAP analysis. If the sample volume is insufficient for analysis, it will be recorded.

[0332] Anti-AAV9 capsid immunogenicity, biodistribution and cerebrospinal fluid for NfL and GFAP analysis CSF samples are collected prior to dosing on Day 1 and on the scheduled euthanasia day during the dosing phase (only for animals scheduled for sacrifice on that day).

[0333] Tube 1: Analyze the CSF sample for DNA (vector genome) by using a non-GLP method if sample volume is sufficient. Record if sample volume is insufficient for analysis.

[0334] Tube 2: Analyze the CSF sample by using non-GLP methods for NfL and GFAP analysis if sample volume is sufficient. Record if sample volume is insufficient for analysis.

[0335] Tubes 3 and 4: Analyze CSF samples for anti-AAV9 capsid immunogenicity if sufficient sample is available (method information to be added by amendment). If planned testing cannot be completed, record the reason.

[0336] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of the specification and the appended claims.

[0337] Furthermore, when features or aspects of the disclosure are described in terms of a Markush group, those of skill in the art will recognize that the disclosure is also thereby described with respect to any individual members or subgroups of members of the Markush group.

[0338] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety (or where context indicates) to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

Claims

1. A method for improving delivery of a pharmaceutical composition comprising an adeno-associated virus (AAV) viral vector to the central nervous system of a subject in need of improved delivery of the pharmaceutical composition to the central nervous system, the method comprising administering to the subject, in combination with the pharmaceutical composition, an agent that enhances glymphatic influx.

2. A method for improving the transduction efficiency and / or distribution of an adeno-associated virus (AAV) viral vector in the brain, comprising administering to a subject in need thereof the adeno-associated virus (AAV) viral vector in combination with an agent that enhances glymphatic influx, thereby improving the transduction efficiency of the adeno-associated virus (AAV) viral vector in the subject.

3. A method for improving the efficacy of a pharmaceutical composition containing an adeno-associated virus (AAV) viral vector and delivered intrathecally, comprising administering to a subject in need thereof the pharmaceutical composition in combination with an agent that enhances glymphatic influx.

4. A method for reducing variability in brain distribution of an adeno-associated virus (AAV) viral vector among a patient population treated with a pharmaceutical composition comprising the AAV viral vector, the method comprising administering to the subject, in combination with the pharmaceutical composition, an agent that enhances glymphatic influx.

5. A method for reducing systemic exposure of a CNS-targeted pharmaceutical composition comprising an adeno-associated virus (AAV) viral vector in a subject in need of reduced systemic exposure of the CNS-targeted pharmaceutical composition in order to reduce liver and / or DRG toxicity in the subject, the method comprising administering to the subject an agent that enhances glymphatic influx in combination with the pharmaceutical composition.

6. 6. The method of any one of claims 1 to 5, wherein the agent is administered simultaneously with, sequentially with, before or after the adeno-associated virus (AAV) vector or the pharmaceutical composition comprising the adeno-associated virus (AAV) vector.

7. The method of claim 1, wherein the adeno-associated virus (AAV) vector or the pharmaceutical composition comprising the adeno-associated virus (AAV) vector is administered by intrathecal (IT) administration, intracisternal (ICM) administration and / or intracerebroventricular (ICV) administration, intravenous infusion, intravenous injection, inhalation, intraperitoneal, oral, subcutaneous or intramuscular route.

8. The method of any one of claims 1 to 5, wherein the agent promotes interstitial fluid circulation within the blood-brain barrier, for example, the agent comprises an aquaporin 4 (AQP4) promoter, such as TGN-073.

9. A method according to any one of claims 1 to 5, wherein i) the agent comprises a compound that upregulates AQP4 expression (e.g., sevoflurane) or a compound that alters the intracellular localization of AQP4, or ii) the agent comprises an alpha-2 adrenergic agonist, such as clonidine, cizanidine, or dexmedetomidine (e.g., Precedex or Dexdomitol), or iii) the agent comprises one or more FDA-approved anesthetics that enhance glymphatic influx, in particular, the anesthetics are ketamine, dexmedetomidine, xylazine, or a combination thereof.

10. 10. The method of claim 9, wherein the medicament comprises a combination of ketamine and dexmedetomidine, and particularly wherein the subject is first administered ketamine, followed by the pharmaceutical composition, and then by dexmedetomidine.

11. 11. The method of claim 10, wherein ketamine is administered about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, preferably about 10-15 minutes, prior to said administration of said pharmaceutical composition.

12. 11. The method of claim 10, wherein ketamine is administered at about 100 mg / kg, about 90 mg / kg, about 80 mg / kg, about 70 mg / kg, about 60 mg / kg, about 50 mg / kg, about 40 mg / kg, about 30 mg / kg, about 20 mg / kg, about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, preferably about 10 mg / kg.

13. 11. The method of claim 10, wherein dexmedetomidine is administered at about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.09 mg / kg, about 0.08 mg / kg, about 0.07 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, about 0.009 mg / kg, about 0.008 mg / kg, about 0.007 mg / kg, about 0.006 mg / kg, about 0.005 mg / kg, preferably about 0.02 mg / kg.

14. 11. The method of claim 10, wherein the subject is further administered sevorlan followed by dexmedetomidine, particularly wherein sevorlan is administered as an inhalant.

15. 6. The method of any one of claims 1 to 5, wherein the agent induces plasma hyperosmolality, in particular the agent comprises hypertonic saline (e.g. sodium chloride with or without sodium acetate) or mannitol, more particularly the agent comprises hypertonic saline with or without sodium acetate.

16. 16. The method of claim 15, wherein the hypertonic saline is 2% NaCl, 3% NaCl, 5% NaCl, 7% NaCl or 23% NaCl, preferably 3% NaCl, particularly 3% NaCl administered at about 2-3.5 ml / kg.

17. 16. The method of claim 15, wherein the agent is administered by intravenous or infusion injection about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 minutes, preferably about 5 minutes, before or after administration of the adeno-associated virus (AAV) vector or the pharmaceutical composition comprising the adeno-associated virus (AAV) vector, and optionally, the administration may be repeated.

18. 6. The method of any one of claims 1 to 5, wherein i) the drug enhances glymphatic influx by increasing slow wave sleep, and in particular the drug is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine and trazodone or a combination thereof, or ii) the drug comprises VEGF-C.

19. 6. The method of any one of claims 1 to 5, wherein the subject is maintained in a hind limbs elevated position, such as Trendelenburg, for about 1 to 2 hours after the administration of the pharmaceutical composition.

20. The above-mentioned ААV virus vector .

1. V9.

2. A122-10、1122-11、1122-12、1122-13、1122-11、1122-aa V43-21、A3-23、A33-22、A33-5、A3..22.2.2.

2. 、AAV223.5、AAV223.6、AAV--7 / rh.48、A0-0 / rh.49、AA-2 / / rh.6222. 2-- / / rh.50、AAO2-5 / rh.51、A-2. / / u.6、A-- / / rh.9、A--9 / rh.522.11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AA V128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAV A3.3, AAV A3.4, AAV A3.5, AAVA3. rh.72、AAVhu.8、AAVrh68、AAVrh70、AAVp.1、AAVp.3、AAVp.2、AAVp. h.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh47、AAVrh.69、AAVrh.55 AVrh.59、AAVu.12、AAVH6、AAVLK03、AArH- / / u.1、AAVH / / / 5. -10 / rh.40、AAVLG- / / rh.38、AAVLG-9 / u.39、AAVN721-0 / rh.33- . . hu44、AAVhu.5、AAVhu.6、AAVh577、AAVh.9、AAVh.10、AAVh1111111 . AAVu.22、AAVu.2322、AAVu.24、AAVu.25、AAVu.27、AAVu.28、 . AAVu.39、AAVu.40、AAVu.41、AAVu.42、AAVu.43、AAVu.44、AAVu.42 4R1、AAVu.44R2、AAVu.44R3、AAVu.45、A5Vu.46、AAVu.47、A5V5. 48、AAVu.48R1、AAVu.48R2、AAVu.48R3、AAVu.49、AAVu.515111 52、AAVu.54、AAVu.55、AAVu.56、AAVu.57、AAVu.58、505505 hu.61、AAVhu.63、AAVhu.64、AAVu.66、AAVu.67、AAVh.1 / 99219、AAVrh.2、AAVrh.2R、1AVrh.8、1AVrh8R、1AVrh.10、1AVrh.12222 (.13、11Vr(13R、11Vr(111、11Vr(17、11Vr(111、11Vr(191111111 、AAVrh.21、AAVrh.22、AAVrh23、AVrh.24、AVrh.25、AAVrh.31、AVr h.32、AAVrh.33、AAVrh.34、A0Vrh.35、AVrh.36、AAVrh.37、AVrh.3R 2. AVrh.48.1.2、AAVrh.48.2、AVrh.49、AVrh.51、10Vrh.52、AVrh.53、 AAVrh.54、AAVrh.56、AAVrh.57、AVrh.58、AVrh.51、AAVrh.64、AVrh . A586R sudden change,AAVrh8R2533.26.26.3.26.6.2.

6. The 6hEr1.18、2262(5161616) 16181818 6hEr1.135、2266(51017、22612(51.136) 626 2.29、22.29、22.44、22.1616. 22.30、22.31222.31、2261222.

362. h221.23、2262(513.1、2262.544、226000 The 5004, 605, 606, 607,000,000,000 2008, 100,000,000,000,000 11、26-6-12、26-6-113、26-6-144. 6-6-15, 6-16, 6-17, 16-17 、¡¡V-L,19、AAV-!!!-32、AAV--44444AA 6-6, 6-6, 6-7, 6-8, 6-8. 3111、226-12、2262-20112 1011、26-6-81、26-6-81、26-6-1 37 100-26, 100-16, 100,000 The Theャッフル100-48、266シャッフル1000-22、26 37 100-1、26 37 100-48、226 337 100-3 1000-100、380061 236、380622263 266、26610000000000000000000000000000000000000000000000000000 Away (2000) 26000000 62, 48, 119, 111 The 9.39、2.26544.5444.23.23.26.24.2 / hu.222、22644477b 654444、2264441 / hu.211、22644444244447、26446.2 / hu.28、226466.64b22.29、226.281 / hu\.43、トゥルータイプ226(5000)The method according to any one of claims 1 to 5, comprising capsid proteins from AAV10 and / or Japanese AAV10 serotypes and variants thereof, in particular from AAV9.

21. The method of any one of claims 1 to 5, wherein the AAV viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein.

22. The method of any one of claims 1 to 5, wherein the AAV viral vector comprises two ITRs (e.g., a modified AAV2 ITR and an unmodified AAV2 ITR), a promoter (e.g., a chicken beta actin (CB) promoter), an enhancer (e.g., a cytomegalovirus (CMV) immediate-early / early enhancer), an intro (e.g., a modified SV40 late 16s intron), and a polyadenylation signal (e.g., a bovine growth hormone (BGH) polyadenylation signal).

23. i) The pharmaceutical composition comprises 1 x 10 １０ ~1 x 10 １５ viral vector genomes, e.g., 1 x 10 １２ , 5 x 10 １２ , 1×10 １３ , 5 x 10 １３ , 1×10 １４ , 5 x 10 １４ , 1×10 １５ 6. The method of any one of claims 1 to 5, wherein the pharmaceutical composition comprises 1x10 to 1x10 vector genomes per milliliter (vg / ml), for example 1x10, 5x10, 1x10, 5x10, 1x10, 5x10, 1x10, 5x10, 1x10 vector genomes per milliliter (vg / ml).

24. 1. A method for treating a neurological disease, comprising administering to a subject in need thereof a pharmaceutical composition, said pharmaceutical composition comprising an AAV encoding a gene associated with said neurological disease, wherein said administration of said pharmaceutical composition is coincident with CSF inflow during a sleep cycle.

25. 25. The method of claim 24, wherein the pharmaceutical composition is administered when the subject falls asleep, e.g., as indicated by electroencephalogram (EEG) monitoring.

26. 26. The method of claim 24 or 25, wherein the subject is administered a sleep-promoting drug in combination with the pharmaceutical combination, and in particular the sleep-promoting drug is selected from the group consisting of tiagabine, gaboxadol, gabapentin, pregabalin, GHB, ritanserin, eplivanserin, mirtazapine, olanzapine and trazodone or a combination thereof.

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