Enhanced brain transduction by gene therapeutics

Abstract

The present disclosure relates to chimeric adeno-associated virus capsids and related adeno-associated viruses targeting glial progenitor cells.

Description

[0001] Enhanced Brain Transduction by Gene Therapeutics

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No. 63 / 652,307, filed on May 28, 2024, which is hereby incorporated by reference in its entirety.

[0004] GOVERNMENT INTERESTS

[0005] This invention was made with government support under AG072298 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (161118.0695 ISeqlist.xml; Size: 99,210 bytes; and Date of Creation: May 12, 2025) is herein incorporated by reference in its entirety.

[0008] FIELD OF THE INVENTION

[0009] This disclosure relates to chimeric adeno-associated virus (AAV) capsids and related adeno-associated viruses targeting glial progenitor cells.

[0010] BACKGROUND

[0011] Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene delivery in therapeutic applications because of their ability to transduce both dividing and nondividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. However, gene delivery to the central nervous system (CNS) remains a significant challenge in gene therapy. Engineered AAV capsids with improved brain tropism represent an attractive solution to the limitations of CNS delivery. While AAVs have been evolved to target a number of cell types of the adult CNS, most such efforts have focused on mouse cells, and among those targeting human cells, most have been developed in vitro, as few systems appropriate for targeting human brain cells in vivo have been developed.

[0012] Glial progenitor cells (GPCs) comprise an especially important phenotype of the adult human CNS, as these multi-lineage competent cells can give rise to both oligodendrocytes and astrocytes in the human brain; under select circumstances they may give rise to some neuronal phenotypes as well. In addition, when anaplastic, they are a cell type of origin of malignant glial tumors. Yet to date, no AAVs able to selectively target human GPCs have been identified, much less human GPCs in vivo. There is a need for AAVs to selectively and efficiently deliver a transgene to human GPCs. SUMMARY OF THE INVENTION

[0013] This disclosure addresses the need mentioned above in a number of aspects.

[0014] In one aspect, the disclosure provides a modified adeno-associated virus (AAV) VP1 capsid protein. As disclosed herein, such modified capsid protein specifically targets human glial progenitor cells, astrocytes, and / or oligodendrocytes. This modified capsid protein comprises a chimeric sequence of an AAV serotype 5 (AAV5) capsid amino acid sequence into which a polypeptide sequence is inserted after the amino acid residue at position corresponding to 574 of SEQ ID NO: 53. The polypeptide sequence comprises a peptide sequence selected from the group consisting of SEQ ID NOs: 1-52. In some embodiments, the polypeptide sequence inserted may contain one adapter / linker sequence at its N-terminus or C- terminus. In some embodiments, the polypeptide sequence inserted may contain two adapter / linker sequences at its N-terminus and C-terminus respectively. Each adapter / linker sequence can be 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) amino acids in length. In some embodiments, the adapter / linker sequence comprises or consists of A or AS.

[0015] In one embodiment, the polypeptide sequence is inserted between the two amino acid residues at positions corresponding to 574 and 575 of SEQ ID NO: 53. In one embodiment, the peptide sequence is selected from the group consisting of SEQ ID NOs: 1-20. In one embodiment, the peptide sequence is selected from the group consisting of SEQ ID NOs: 21- 40. In one embodiment, the peptide sequence is selected from the group consisting of SEQ ID NOs: 41-52. In some embodiments, the modified AAV capsid protein is at least 70% (e.g., 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 53 or 55.

[0016] In a second aspect, the disclosure provides an AAV particle or virion comprising (i) an AAV capsid comprising a first AAV capsid protein as described above and (ii) an AAV vector genome packaged in the capsid. In some embodiments, the AAV vector genome comprises a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence encodes a polypeptide or an RNA. In some embodiments, the AAV vector genome further comprises a promoter that is operatively linked to the heterologous nucleic acid sequence. In some embodiments, the AAV capsid further comprises a second AAV capsid protein that is different from the first AAV capsid protein. In some embodiments, the second AAV capsid protein and the first AAV capsid protein may have different cell specificities and tropisms. In some embodiments, the promoter is tissue-specific, differentiation stage-specific, cell type-specific, or ligand inducible. In some embodiments, the promoter is selected from the group consisting of glial progenitor cell -expressed genes including GPR17, PCDH15, PDGFRA, CPSG4, NKX2.2, 0LIG1, 0LIG2, GPR56. In some embodiments, the promoter is selected from the group consisting of astrocyte-expressed genes including GFAP, HOPX, AQP4, SOX9, and GLUL. In some embodiments, the promoter is selected from the group consisting of oligodendrocyte-expressed genes including SOXIO, CNP, MYRF, MAG, MOG, and MBP.

[0017] In a further aspect, the disclosure provides a nucleic acid encoding the modified AAV capsid protein described above. Also within the scope of this disclosure are a vector comprising the nucleic acid and a host cell comprising the nucleic acid or the vector. In some embodiments, the host cell further comprises (i) an AAV vector genome or an AAV vector genome-encoding sequence and (ii) an AAV Rep protein or an AAV Rep-encoding sequence. In some embodiments, the AAV vector genome comprises a heterologous nucleic acid sequence as described above. In some embodiments, the heterologous nucleic acid sequence encodes a polypeptide or a RNA. In some embodiments, the host cell further comprises a helper plasmid.

[0018] In yet a further aspect, the disclosure provides a method of producing a recombinant AAV particle or virion. The method comprises providing in vitro a host cell described above and culturing the host cell under conditions permitting encapsidating the vector genome and assembly of the recombinant AAV particle or virion.

[0019] In a further aspect, the disclosure provides a method of introducing a heterologous nucleic acid sequence into a cell, comprising contacting the cell with the AAV particle or virion described above. In some embodiments, the cell is a glial cell or a glial progenitor cell. In some embodiments, the glial cell is an oligodendrocyte or an astrocyte.

[0020] In yet a further aspect, the disclosure provides a method of introducing a heterologous nucleic acid sequence into a subject, comprising administering to the subject the AAV particle or virion described above. In some embodiments, the subject has or is at risk of having a disorder associated with glial cell dysfunction or a neurodegenerative disease.

[0021] In another aspect, the disclosure provides a method of treating a disorder associated with glial cell dysfunction or a neurodegenerative disease in a subject, comprising administering to the subject the AAV particle or virion described above. In some embodiments, the disorder associated with glial cell dysfunction is selected from the group of hereditary disorders consisting of Pelizaeus-Merzbacher disease, vanishing white matter disease, Krabbe's disease, metachromatic leukodystrophy, gangliosidoses, mucopolysaccharidoses, adrenoleukodystrophy, Canavan disease, Alexander disease, pigmentary orthochromatic leukodystrophy, Zellweger disease, Angelman syndrome, 18q- syndrome, phenylketonuria, and aminoacidurias. In some embodiments, the disorder associated with glial cell dysfunction is selected from the group of acquired disorders consisting of cerebral palsy, multiple sclerosis, spinal cord injury, traumatic brain injury, and white matter stroke. In some embodiments, the disorder associated with glial cell dysfunction is selected from the group of neurodegenerative disorders consisting of Huntington disease, Alzheimer disease, Parkinson disease, Lewy body disease, multisystem atrophy, and schizophrenia. In some embodiments, the AAV particle or virion is administered by intracistemal magna administration. In some embodiments, the AAV particle or virion is administered by intra-striatal administration.

[0022] In another aspect, the disclosure provides a pharmaceutical composition comprising the above-described AAV particle or virion and a pharmaceutically acceptable carrier.

[0023] In one aspect, provided is a method for delivering a viral vector or a non-viral delivery vector to a central nervous system (CNS), one or more cellular targets therein, or a tissue therein of a subject, or (II) for treating a neurological disorder in the subject, comprising:

[0024] (1) enhancing glymphatic system influx of the subject by a process comprising administering an agent into the blood of the subject; and

[0025] (2) delivering a composition comprising the viral vector to the subject’s cerebrospinal fluid (CSF), wherein the agent comprises or is a hypertonic solution.

[0026] In one aspect, provided is a method (I) for delivering a viral vector or a non-viral delivery vector to a central nervous system (CNS), a one or more cellular targets therein, or a tissue therein of a subject, or (II) for treating a neurological disorder in the subject, comprising:

[0027] (1) enhancing glymphatic system influx of the subject; and

[0028] (2) delivering a composition comprising the viral vector or non-viral delivery vector to the subject’s cerebrospinal fluid (CSF) intraci sternally or intrathecally.

[0029] In one embodiment, the step of enhancing glymphatic system influx comprises administering an agent to the subject. In one embodiment, the agent comprises or is a hypertonic solution, and is administered into blood in the subject. In one embodiment, the solution comprises a hypertonic solution of NaCl or Mannitol. In one embodiment, the agent is administered intravenously to the subject. In one embodiment, the composition is a hypertonic composition.

[0030] In one embodiment, the viral vector comprises an adeno-associated virus (AAV). In one embodiment, the AAV is AAV5. In some embodiments, the cellular targets are selected from the group consisting of a neuron, a neuronal progenitor cell, a neural progenitor cell, a glial cell, a glial progenitor cell, a microglial cell, an astrocyte, an oligodendrocyte, a pericyte, or an endothelial cell. In some embodiments, the neurological disorder is selected from the group consisting of a myelin disorder, a viral or microbial infection, an inflammatory disorder, an ischemic lesion or post- ischemic state, a neurodegenerative disease, a behavioral disorder, a lysosomal or peroxisomal storage disease, or a brain cancer.

[0031] In some embodiments, the subject is placed into the Trendelenburg position before, during and / or after the step of enhancing, the step of delivering, or both. In some embodiments, the subject is anesthetized before the step of enhancing, the step of delivering, or both. In some embodiments, the composition is delivered at about the same time or within about 4 hours after the glymphatic system influx is enhanced.

[0032] In one embodiment, the non-viral delivery vector comprises a lipid nanoparticle (LNP).

[0033] In one aspect, provided is a hypertonic pharmaceutical composition comprising (i) a viral vector or a non-viral delivery vector and (ii) a pharmaceutically acceptable carrier or excipient.

[0034] In one aspect, provided is a kit for delivering a viral vector or a non-viral delivery vector to the CNS, a one or more cellular targets therein, or a tissue therein of a subject, comprising two or more of the following:

[0035] (i) the viral vector or the non-viral delivery vector;

[0036] (ii) a hypertonic pharmaceutical composition, and

[0037] (iii) a hypertonic solution.

[0038] In one embodiment, the viral vector or the non-viral delivery vector is in the hypertonic pharmaceutical composition. In one embodiment, the viral vector comprises an AAV. In one embodiment, the AAV is AAV5. In one embodiment, the non-viral delivery vector comprises an LNP.

[0039] The details of one or more embodiments of the disclosure are set forth in the description below. Other features, objectives, and advantages of the disclosure will be apparent from the description and from the claims.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and IB show the strategy for generating chimeric capsids. FIG. 1A is a schematic of the in vivo Strategy: modified M-CREATE strategy. FIG. IB shows the amplification of evolved AAV5 particles that specifically transduce hGPCs in vivo following CM or striatal injection. PCR amplicons are sequenced using Next Generation Sequencing (NGS) and data emanating from mouse or human cells are compared. Capsid sequence present in human GPC but not mouse cells were selected and ranked. Top hit capsids found only in hGPCs (also filtered against viral capsids found in liver) were then selected for a second round of screening and validation.

[0041] FIG. 2 shows the strategy based on PCR amplification of the genome of evolved AAV5 that specifically transduced hGPCs in vivo following Cisterna magna injections. Recipient mice: Pl-hGPCs chimerized mice (12wks); Cisterna magna injection / 10pl viral suspension; Mice were euthanized 1 week post injection. Ctr: untreated mice.

[0042] FIGS. 3A and 3B shows validation of selected modified capsid in vivo (FIG. 3A) and the liver showed only weak expression of EGFP in the viral capsids evolved following Cistema magna injection (FIG. 3B).

[0043] FIGS. 4A-4K illustrate that different capsid variants exhibit distinct glial tropisms, exhibit distinct glial tropisms. Based on the sequence data of Table 4, AAV5s were engineered whose capsids presented two of the candidate peptides selected on PDGFRA-Cre expressing hGPCs in vivo. This figure shows sagittal images of brains infected by one of two different capsids, AAV CM1 (FIGS. 4A-4E) and CM6 (FIGS. 4F-4H). In each case, virus was injected (10 pl; 1x1012 vg / ml) into the cisterna magna of 12 wk-old mice, that were rendered hypertonic (3% NaCl IV infusion) 15 min prior to intraci sternal injection, and killed 3 wks later. EGFP+ infected glia were recognized throughout the brain. FIG. 4A shows low power sagittal view of AAV CMl-infected, EGFP-reported infected cells, scattered throughout the brain. FIGS. 4B-4E show white matter fields in the corpus callosum (FIG. 4B), hypothalamus (FIG. 4C), thalamus (FIG. 4D) and cortex (FIG. 4E). In each example, most infected cells co-labeled for Olig2, which is expressed by GPCs as well as oligodendrocytes; few infected non-glia were noted in this animal. FIG. 4F shows low power view of AAV CM6-infected cells. FIG. 4G is a view from cortex through callosum to striatum, showing infected cells; most are GPCs, with a few astrocytes. FIG. 4H shows that most AAV CM6-infected cells expressed Sox9, indicating astrocytic phenotype. FIGS. 41- 4K illustrate that in all 3 regions assessed, including cortex (FIG. 41), corpus callosum (FIG. 4J) and striatum (FIG. 4K), AAV-CM1 preferentially infected Olig2-defined hGPCs and oligodendroglial lineage cells, while AAV-CM6 preferentially targeted Sox9-defined astrocytes. Two-way ANOVA with Sidak’s post-hoc tests. Means ± SEM. *p<0.05, **p<0.01, ***p<0.001. Scale, FIG. 4A, FIG. 4F: 1 mm. B-E, H: 50 pm. G: 200 pm.

[0044] FIGS. 5A-5H demonstrate that capsid-evolved AAV5s CM1 and CM6 transduce distinct human glial phenotypes in vivo. Human glial chimeric mice, given neonatal intrastriatal injections of hESC-derived hGPCs, were injected intraci sternally with AAV-CM1-CAG- EGFP or AAV-CM6-CAG-EGFP at 16 weeks of age, then examined 3 weeks later. EGFP expression in AAV-CM1 -injected mice preferentially colocalized with Olig2 (FIGS. 5A-5C), whereas AAV-CM6 primarily infected Sox9-defined astrocytes (FIGS. 5E-5G). FIGS. 5D- 5H show that AAV-CM1 and AAV-CM6 were significantly more efficient at targeting human glial progenitors and oligodendroglia (FIG. 5D) and astrocytes (FIG. 5H) than the unmodified AAV5 control vector. Two-way ANOVA with Sidak post-hoc tests. Means ± SEM. *p<0.05, **p<0.01, ***p<0.001. Scale: A-B, E-F: 100 pm, C, G: 25 pm.

[0045] FIGS. 6A-6D show that viral selection on human PDGFRA-Cre GPCs in vivo yields vectors selective for glia. FIG. 6A is a schematic of the viral constructs used to generate AAV- CM1 and AAV2 / 5 viruses. FIGS. 6B and 6B depict images of PDGFRa-Cre chimeric RagF" mice (transplanted with PDGFRA-Cre hGPCs on postnatal day 1) were injected at 12 months of age with a 10 pl viral cocktail containing AAV5 (AAV2 / 5 WT)-Flex-dtTomato and AAV- CMl-Flex-EGFP (1 : 1 ratio; 1 x 1012vg / ml). The mice were sacrificed three weeks after injection. Brain sections were immunostained with anti -EGFP, anti-mCherry (recognizing dtTomato), and anti-human nuclear antigen (hNA) antibodies. FIG. 6D is a graph that quantifies cells expressing EGFP and dtTomato in graft-derived hGPCs from the striatum, corpus callosum, and cortex. Means ± SEM. Two-way ANOVA with Sidak's post-hoc test (****p <0.0001). Scale, 50 pm.

[0046] FIGS. 7A-7E illustrate that AAV-CM1 selectively transduces human glia. Neonatally chimerized shiverer mice, with hESC-derived glia engrafted into the striatum, were injected with AAV-CM1-EGFP virus into the cistema magna at 17 weeks. Upon sacrifice and histological analysis three weeks later, we demonstrated that the virus preferentially transduces graft-derived human cells in the corpus callosum. FIG. 7A depicts immunostaining for human nuclear antigen (hNA, white), Olig2 (red), and EGFP (green), as well as a DAPI (blue) counterstain. FIG. 7B and its color-split insets show a high-power field revealing colocalization of EGFP and Olig2 in human (hNA+) cells. FIG. 7C is a dot-mapped distribution in the corpus callosum of infected (EGFP+) human (hNA) and mouse Olig2+cells. FIG. 7D is a graph of the preferential infection of human relative to mouse glia. FIG. 7E is a graph of the respective infection of human and mouse glia after normalization for the relative proportions of all human and mouse cells in the chimeric corpus callosa. Means ± SEM. **p <0.01 ****p <0.0001, two-tailed t-test. Scale bar: FIG. 7A: 10 pm; FIG. 7B: 100 pm.

[0047] FIGS. 8A-8D demonstrate that AAV-CM1 infects myelinated oligodendrocytes as well as their progenitors. AAV-CM1 exhibited efficient infection of mature myelinating oligodendrocytes, as well as of hGPCs. In this example, AAV5-CM1 was delivered intraci stemally, after induction of systemic hypertonicity, in 16-week-old human glial chimeric shiverer mice, in which most forebrain myelinating oligodendrocytes and glial progenitors are of human origin. When sacrificed 3 weeks later, robust infection of CNPase (cyclic nucleotide diphosphatase)-defined and myelin basic protein (MBP)-defined myelinated oligodendrocytes - as well as parenchymal astrocytes and persistent hGPCs - was noted in the callosal (FIGS. 8A and 8B) and corticostriatal (FIGS. 8C and 8D) white matter tracts.

[0048] FIGS. 9A-9H illustrate that colonization of mouse forebrain with human PDGFRA:Cre glia allows in vivo capsid selection. FIG. 9A shows mice that neonatally engrafted with hESC- derived PDGFRA:Cre-expressing glial progenitor cells (hGPCs) displayed robust colonization of the striatum by 14 weeks post-transplant. Human donor-derived cells either remained as hGPCs (FIG. 9B), or differentiated into Sox9-expressing astrocytes (FIG. 9C), but did not differentiate into neurons (FIG. 9D). FIG. 9E depicts RNAscope multiplex in situ hybridization on striata of engrafted mice showed that the CRE-specific probe (FIG. 9F) colocalized with PDGFRA (FIG. 9G) but not with that of NeuN (FIG. 9H). Scale: 100 pm (FIG. 9A); 25 pm (FIGS. 9D, 9E-9H)

[0049] FIGS. 10A-10C illustrate that hypertonic intracistemal delivery of tracer achieves widespread brain access. Cistema magna CSF tracer infusions (FITC-dextran, 2,000kDa, 2.5% w / v in saline; 10 pl delivered at 2 pl / min), were performed under ketamine / xylazine anesthesia with postural modification, osmotic manipulation via hypertonic saline (IM, 20 pl / g), or both. FIG. 10A is a graph of the quantification of coronal brain sections showing CSF tracer distribution in mice being positioned prone on either a level surface or a declined plane for 30 minutes post-infusion. There was no significant difference in tracer distribution with postural modification alone (p=.1775), unpaired Student’s t-test. FIG. 10B is a graph quantifying CSF tracer distribution after injection of either physiologic saline (PS) or hypertonic saline (HTS) from coronal sections. There was significant greater tracer influx following HTS (p=.0005; PS, n=5 mice, HTS, n=6). FIG. 10C is a graph quantifying tracer distribution under three conditions: Trendelenburg position alone, vs. HTS alone, and the combination. While both HTS and positioning increased influx, the combined intervention yields the greatest enhancement (One-way ANOVA with post hoc comparisons; p values as noted, individual mean and standard deviation were plotted, and each data point is an individual mouse (n= 5 level, 5 ramp, 5 PS, 6 HTS, 6 Ramp + HTS). Ramp = postural modification, HTS = hypertonic saline, PS = physiologic saline).

[0050] FIGS. 11A-11E demonstrate that intracisternally-delivered AAV pervades the forebrain parenchyma via glymphatic spread. To establish the efficacy of the intracistemal hypertonic delivery protocol using a well-described control vector before testing our modified capsid variants, we injected a set of adult mice with either AAV5-retro (FIGS. 11A and 11B) or WT AAV5 (FIGS. 11C-11E). For the AAV5-retro injections, of A-B, we inserted the neurotropic retro cassette (encoding the peptide LADQDYTKTA; SEQ ID NO: 111) in between Q574 and S575 of the AAV5 VP1 capsid protein; this locus presents an exposed sialic acid-binding loop domain by which target specificity may be modified. A single intracistemal injection under systemic hypertonicity yielded high-efficiency infection of forebrain hippocampal pyramidal and selected corticofugal neurons, as has been described for parenchymally-administered AAV2-based AAV retro (Tervo, D.G. et al. (2016). A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. Neuron 92, 372- 382). FIGS. 11C-11E, Similarly, intracistemal WT AAV5 control vims yielded predominantly neuronal labeling throughout the forebrain, most prominently so in the hippocampus (FIG. 11D) and cortex (FIG. HE). These results confirm that glymphatic-mediated intracistemal delivery supports efficient, brain-wide parenchymal access and neuronal gene transfer. Scale: FIG. HA, FIG. HC: 1 mm; FIG. HB, FIG. HD: 250 pm; E: 100 pm

[0051] FIGS. 12A and 12B illustrate that adventitious neuronal infection persists in gliotropic variants. Despite their strong relative gliotropism, some neuronal transduction persisted to a variable extent in each of these engineered viral variants. FIG. 12A is a graph of the percent of neurons among all EGFP+ cells in the striatum or cortex treated as indicated. Both AAV- CM1 and AAV-CM6 exhibited significantly less neuronal infection than the unmodified AAV5s from which they were derived, in both the neostriatum and the overlying neocortex. In contrast, in controls injected with unmodified AAV5 almost 50% of infected cells were neurons. FIG. 12B is a graph of the percent transduced neurons among total neurons in the striatum or cortex treated as indicated. In the same brains and regions, less than 1% of all neurons were infected, and as noted in FIG. 12A, these comprised <20% of all cells infected by either CM1 or CM6. 2-way ANOVA with Tukey’s post-hoc tests. Mean ± SEM. ns p > 0.05, ***p < 0.001, ****p < 0.0001.

[0052] FIGS. 13A-13C demonstrates that selected capsid-evolved AAVs manifest little systemic infection. Using PCRto assess the hepatic viral genome concentrations (vg / g) allowed estimation of the degree of off-target infection by each capsid variant. Only those candidates lacking appreciable hepatic infection were developed further. When assessed a week after intracistemal delivery, two capsid candidates that survived this initial filtration, AAV-CM1 and AAV-CM6, exhibited little or no infection of the liver (FIG. 13A), or of the kidney (FIG. 13B) or spleen (FIG. 13C), indicating the strong phenotypic selectivity afforded by capsid evolution on hGPCs in vivo. One-way ANOVA with Tukey post hoc tests. Means ± SEM. **p<0.01, ***p<0.001; ****p<0.0001.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] This disclosure relates to modified AAV capsid proteins, modified AAV capsids, and uses thereof for delivering a transgene into a target cell or environment (e.g., a cell-type or tissue) in a subject when they are administered to a subject. At least some aspects of this disclosure are based on unexpected identifications of modified AAV capsid proteins that specifically target human glial progenitor cells, astrocytes, and / or oligodendrocytes. Provided are evolved AAV capsids against human glial progenitor cells, as well as their derived astrocytes and oligodendrocytes, by serial selection of successfully-infecting viral capsid variants in glial chimeric mice that had been neonatally transplanted with human glial progenitor cells engineered to express Cre recombinase under the regulatory control of the PDGFRA promoter, which is selectively expressed by glial progenitor cells. Accordingly, in some embodiments, this disclosure provides a new set of AAVs, evolved from AAV serotype AAV5, that selectively and efficiently transfect or infect GPCs or GPCs in a subject such a human, with little off-target CNS or systemic infection.

[0055] The modified AAV capsid proteins of the present disclosure comprise at least one insertion or substitution of an amino acid in a corresponding parental AAV capsid protein that confers a desired tropism such as an increased infection specificity (e.g., to human GPCs) or decreased infection specificity (e.g., to human liver cells) as compared to a reference AAV capsid protein, or increased or decreased transgene transduction efficiency as compared to a reference AAV capsid protein. This disclosure is based, at least in part, on an unexpected discovery of AAV VP 1 capsid proteins comprising a chimeric sequence of an AAV serotype 5 (AAV5) capsid amino acid sequence, which specifically targets prospectively-defined cell types of interest, such as GPCs or progenies thereof, e.g., oligodendrocytes or astrocytes.

[0056] AAVs enable stable expression of therapeutic transgenes in cell types of interest, but natural AAV serotypes can infect many different cell types, leading to nonspecific infection of, as well as transgene expression by, undesired cellular targets. Transgene expression may be restricted by cell type-specific regulatory elements. However, the range of host cell infection - while serotype-specific - is nonetheless typically far broader than specific phenotypes of interest, leading to undesired effects and toxicities due to off-target infection.

[0057] As disclosed herein, to address this deficiency in current technology, a number of strategies have been developed for evolving serotype binding domains to specifically target prospectively-defined cell types of interest. While AAVs have been evolved to target a number of cell types of the adult central nervous system, most such efforts have focused on mouse cells, and among those targeting human cell, most have been developed in vitro, as few systems appropriate for targeting human brain cells in vivo have been developed.

[0058] Multi-lineage competent glial progenitor cells comprise an especially important phenotype of the adult human CNS, as these cells can give rise to both oligodendrocytes and astrocytes in the human brain; under select circumstances they may give rise to some neuronal phenotypes a swell. In addition, when anaplastic, they are a cell type of origin of malignant glial tumors as well. Yet to date, no AAVs able to selectively target GPCs have been identified, much less human GPCs in vivo. This disclosure addresses the need mentioned above in a number of aspects.

[0059] In some examples, to establish efficient in vivo transduction of human glial progenitor cells (hGPCs) with therapeutic transgenes, PDGFRA-driven Cre-recombinase expressing hGPCs was targeted in human glial chimeric mice using a library of capsid-modified, recombination-reported adeno-associated viruses (AAVs). PCR-based screening for gliotrophy and filtering against off-target expression identified a subset of AAV5-based vectors that preferentially infect human GPCs and their astrocytic or oligodendrocytic progeny in vivo, with minimal systemic spread. To enhance intracerebral distribution while minimizing viral dose and extracerebral exposure, intracistemal AAVs delivery was paired with systemic hypertonicity. This method exploits glymphatic flow to bypass the blood-brain barrier and distribute AAVs directly into the brain parenchyma. Glymphatic delivery of capsid-modified AAV5s evolved on human GPCs thus enables efficient, brain-wide transgene delivery to human glia and their progenitors in the adult brain, with minimal off-target infection.

[0060] A broad variety of neurological disorders are characterized by atrophy and involution of the cerebral white matter, with oligodendrocytic loss and demyelination unaccompanied by adaptive or compensatory remyelination. In a number of these conditions - including disorders as varied as progressive multiple sclerosis (SPMS), Huntington disease, juvenile onset schizophrenia and the periventricular leukomalacia of cerebral palsy - dysmyelination evolves despite the persistence of a pool of parenchymal glial progenitor cells (GPCs). These glial progenitors, which are typically referred to as oligodendrocyte progenitor cells, but which in humans may give rise to astrocytes as well as oligodendrocytes, appear to lose mitotic and differentiation competence in the setting of these disorders, whether by virtue of mitotic exhaustion, as in PMS and late aging, or due to cell-intrinsic defects in maturational pathways, as in HD and schizophrenia (Huynh, N.P.T. et al. (2024). Shared patterns of glial transcriptional dysregulation link Huntington's disease and schizophrenia. Brain. 10.1093 / brain / awael66; Osipovitch, M. et al. (2019). Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation. Cell Stem Cell 24, 107-122 el07; Windrem, M.S. et al. (2017). Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia. Cell Stem Cell 21, 195- 208 el96). In order to target these cells for therapeutic manipulation, vectors able to deliver genetic payloads to these cells, whether specifically so or to neurons as well, and to target the CNS only, with little or no systemic transduction, are needed.

[0061] Adeno-associated viruses (AAVs) enable stable expression of therapeutic transgenes in cell types of interest, but natural AAV serotypes can infect many different cell types, leading to both nonspecific infection and transgene expression by undesired cellular targets. While transgene expression may be restricted by cell type-specific regulatory elements, the range of host cell infection by AAVs, while serotype-specific, is nonetheless typically far broader than specific phenotypes of interest. The resultant promiscuity in infection leads to undesired off- target effects and toxicities, which have impeded the clinical development of therapeutic AAV vectors.

[0062] To address this issue, a number of strategies have been developed for evolving serotype binding domains to target prospectively-defined cell types of interest. In particular, previous studies have developed approaches towards capsid evolution based on Cre recombinasedependent identification of viruses evolved to target cells of interest (Deverman, B.E et al. (2016). Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204-209). These studies have included multiplexed assessment of recombination-dependent viral variants with randomly mutagenized VP1 capsid sequences, allowing high throughput screening of variants for their relative infectivity of given cell types of interest (Kumar, S.R. et al. (2020). Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat Methods 17, 541-550). However, most of these studies have evolved viral variants either in murine transgenic reporters with phenotype- selective Cre expression in vivo, or on human cells in vitro,' none have been specifically evolved against human cells in vivo. Yet the surface epitopes presented by human cells are quite distinct from those presented by mice, and epitopes expressed in vivo may differ substantially from those targeted in vitro. Moreover, while the brain has been an especially attractive target for such studies, most investigators have focused on developing AAVs able to target neuronal populations; few studies have focused on targeting glial cells, much less human glia in vivo. No vectors have yet been reported that efficiently target glial progenitor cells (GPCs), the precursor to both oligodendrocytes and astrocytes, that comprises 3-4% of all brain cells in adult humans and mice alike. To thus develop vectors targeting human GPCs and their progeny in vivo, this disclosure adapted the previously described M-CREATE selection strategy of Deverman, Gradinaru and colleagues (Deverman, B.E. et al. (2016). Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204- 209; Kumar, S.R. et al. (2020). Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat Methods 17, 541-550), by which Cre recombinaseexpressing cells are used to screen recombination-reported viral variants for cell tropism. However, in this disclosure capsid evolution on Cre-expressing human GPCs in vivo was achieved. In particular, viral candidates were screened in human glial chimeric mice, that had been neonatally engrafted with human embryonic stem cell-derived GPCs expressing Cre under the control of PDGFRA, which in the brain is selectively expressed by hGPCs. The viral candidates were engineered to include Iox66 / lox71 -flanked sequences, whose Cre-dependent recombination in targeted Cre-expressing host cells allowed the PCR-based identification of those viral variants that had most efficiently infected hGPCs.

[0063] Yet the engineering of capsid-evolved, vectors address only one aspect of the larger challenge of vector delivery to the brain; viral vectors, however efficient and cell type- selective, are only as good as their tissue access. AAV delivery to the brain has typically been limited by poor blood-brain barrier permeability and broad systemic leakage, requiring high - and hence toxic - doses. The blood-brain barrier (BBB) in particular poses a formidable obstacle to the efficient transduction of neurons and glia by systemically-administered viral vectors, as it hinders viral entry from the systemic circulation. While serotypes such as AAV9- PHP.B and capsid-modified variants have been engineered to traverse the barrier, their clinical use has nonetheless been limited by off-target transduction and the attendant toxicity of systemic infection, as well as by the immunotoxicity associated with high dose intravenous administration. Thus, despite the development of a number of creative strategies towards circumventing the BBB, the targeting efficiencies for desired phenotypes remain relatively low, and no vector has yet been reported to accomplish both BBB permeance and cell type-specific glial infection. To establish a practicable strategy for circumventing the BBB using cell type-selective vectors, a method was developed to co-opt the brain’s glymphatic system (Plog, B.A. et al. (2018). Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain. JCI Insight 3. 10.1172 / jci. insight.126138) for viral delivery. By combining intraci sternal injection with systemic hyperosmolarity (Plog, B.A. et al. (2018). Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain. JCI Insight 3. 10.1172 / jci. insight.126138; Xavier, A.L.R. et al. (2018). Cannula Implantation into the Cisterna Magna of Rodents. J Vis Exp. 10.3791 / 57378), broad AAV distribution throughout the brain was achieved, while avoiding BBB impedance. This delivery platform enabled in vivo screening of a capsid library targeting hGPC in glia chimeric mice. Candidate vectors were filtered by excluding those with significant off-target infection in liver or spleen, identified variants that selectively and efficiently transduced hGPCs and their astrocytic and oligodendrocytic progeny. This approach yielded a panel of gliotropic viral vectors, each optimized to target a specific state along the human macroglial lineage. Combined with glymphatic delivery, these vectors demonstrate high on-target efficiency with minimal systemic spread, offering a powerful platform for gene delivery to human glial cells in vivo.

[0064] 1. AAV Capsids and Capsid Proteins

[0065] In certain aspects, this application discloses recombinant AAV (rAAV) capsid proteins and rAAV capsids comprising such recombinant rAAV capsid proteins, wherein the recombinant rAAV capsid proteins are modified capsid proteins (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are generated using the methods disclosed herein. In some embodiments, the rAAV capsid proteins can be used in methods of delivering a therapeutic nucleic acid (e.g., a transgene) to a subject. In some instances, the rAAV capsid proteins have desired AAV expression rendering them particularly suitable for certain therapeutic applications, e.g., the treatment of a disease or disorder in a subject such as those disclosed herein.

[0066] The rAAV capsid proteins are engineered for optimized transfection or infection in the CNS cells or tissues, for example the brain, of a subject upon administration of the rAAV to the subject. The rAAV capsid proteins described herein have, in some cases, an insertion of an amino acid that is heterologous to the parental AAV capsid protein at amino acid positions in the 574 loop of AAV5 VP1. In some embodiments, the amino acid is not endogenous to the parental AAV capsid protein at the amino acid position of the insertion. The amino acid may be a naturally occurring amino acid in the same or equivalent amino acid position as the insertion of the substitution in a different AAV capsid protein.

[0067] In one embodiment, this disclosure reports the development of a new set of AAVs, evolved from AAV serotype AAV5, that selectively and efficiently transfect or infect GPCs or GPCs in a subject such as a human, with little off-target CNS or systemic infection. Accordingly, the rAAV capsid proteins are engineered proteins that include the insertions provided in Tables 1-3. Such rAAV capsid proteins achieve efficient transduction of an encapsidated transgene. In particular, the rAAV capsid proteins have increased transduction activity in the glial cells of a subject, including GPCs or progenies thereof, e.g., oligodendrocytes or astrocytes.

[0068] As disclosed herein, in some embodiments, these rAAVs evolved by repetitive rounds of selection upon introducing them intraci sternally into mice that had been neonatally chimerized with human glial progenitor cells, which were themselves derived from human pluripotent stem cells (PSCs) - whether embryonic stem cells or induced pluripotent stem cells. These cells were edited to express Cre recombinase under the regulatory control of the PDGFRA promoter, so that only human GPCs - which specifically express the PDGF-alpha receptor encoded by PDGFRA - express Cre. These human glial chimeric mice were injected - both intraci sternally and intrastriatally, in separate groups of chimeric mice - with a library of genetically barcoded AAVs (e.g., the rAAV vector transgene comprises nucleic acid sequences encoding modified or rAAV capsid proteins that are contained within the rAAV capsid infecting the GPCs). Each of these viral preparations expressed a random heptapeptide insertion into an AAV binding domain that regulates target cell adhesion, and each harbored an inverted sequence, the BGH Poly-adenylation sequence, flanked by two different Lox sequences (Lox66 and Lox71), that upon recombination in PDGFRa-Cre expressing cells, allows the specific amplification and expression of those nucleic acid molecules encoding the rAAV capsid proteins contained in the rAAVs that successfully transduced human GPCs.

[0069] In some embodiments, this application discloses one or more the heptapeptide sequences that are inserted into the amino acid 574-575 position - a loop domain - of the AAV5 capsid protein sequence. The resulting modified AAV5 capsid proteins permit human- selective infection of glial progenitor cells in vivo, with minimal non-glial infection. Listed in Tables 1-3 are exemplary heptapeptide sequences. Table 1. AAV5 evolution for targeting hGPCs- Selected targets following Cisterna magna injection (hGPC-PDGFRa-Cre chimerized mice).

[0070] Table 2. AAV5 evolution for targeting hGPCs- Selected targets following striatal injection (hGPC-PDGFRa-Cre chimerized mice).

[0071] Table 3. AAV5 evolution for targeting hGPCs- Selected targets following serial transduction of HEK293expressing PCDH15, PDGFR, GPR17 or AQP4 in vitro. The rAAV capsid proteins of the present disclosure comprise an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids within an AAV capsid protein. The AAV capsid, from which an rAAV capsid protein of the present disclosure is produced, is referred to as a “parental” AAV capsid.

[0072] In some cases, the parental AAV is derived from an AAV with a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. The AAV capsid protein that is “derived” from another may be a variant AAV capsid protein. A variant may include, for example, a heterologous amino acid in an amino acid sequence of the AAV capsid protein. The heterologous amino acid may be non-naturally occurring in the AAV capsid protein. The heterologous amino acid may be naturally occurring in a different AAV capsid protein. In some instances, examples of the parental AAV capsids include those described in US Pat Publication 2020 / 0165576 and PCT / US20 / 20778, the content of each of which is incorporated herein.

[0073] Preferably, the parental AAV capsid is a AAV-5 capsid, such as SEQ ID NO: 53 below. An exemplary AAV-5 genome is provided in GenBank Accession No. AF085716. In some embodiments, the parental AAV capsid protein sequence is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 53.

[0074] AA V5 VP1 Capsid, wild-type (SEQ ID NO: 53; underlined and bold Q574 and S575)

[0075] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRG EPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKK RVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQ PASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRWTKSTRTWVLPSYNN HQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVK I FNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYWGNGTEGCLPAFPPQVFTLPQY GYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLA NPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSA FATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMI FNSQPANPGTTATYLEGNMLI TSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAK IPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEM EWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL*

[0076] In some embodiments, the parental AAV capsid protein comprises the entire VP1 region provided in SEQ ID NO: 53 (e.g., amino acids 1-724). In some instances, the parental AAV capsid protein comprises a region in SEQ ID NO: 53, which is the common region found in VP1, VP2 and VP3 AAV5 capsid proteins. In some instances, the AAV capsid protein comprises the region in SEQ ID NO: 1, which is the common region found in VP1 and VP2.

[0077] Disclosed herein are insertions of an amino acid sequence in an AAV capsid protein sequence. Where the sequence numbering designation “AA574-575” is noted for AAV5, for example AAV5 VP1, the invention also includes insertions in similar locations in the other AAV serotypes. As used herein, “AA574-575” indicates that the insertion of the amino acid (or amino acid sequence) is immediately after an amino acid (AA) at position 574 and immediately before an AA at position 575 within an amino acid sequence of a parental AAV VP capsid protein (VP1 numbering).

[0078] Amino acids 570-579 include a motif comprising “ATNNQSSTTA” (SEQ ID NO: 54) as set forth in SEQ ID NO: 53. Exemplary insertion sequences are provided in Table 1, 2, or 3. Shown below is an exemplary recombinant or chimeric AAV capsid protein of this disclosure, where the CM1 sequence (TLRDSTT, SEQ ID NO: 1) is inserted at AA570-579 in an AAV5 capsid amino acid sequence along with two adapter sequences (or linker sequences), AS and A (bold and italic) at the N- and C-termini of SEQ ID NO: 1 (bold) respectively.

[0079] AAV5 VP1 with CM1 and Adapter (SEQ ID NO: 55):

[0080] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGE PVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRV LEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPAS SLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRWTKSTRTWVLPSYNNHQYR EIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKI FNIQ VKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYWGNGTEGCLPAFPPQVFTLPQYGYATLN RDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQY LYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRME LEGASYQVPPQPNGMTNNLQGSNTYALENTMI FNSQPANPGTTATYLEGNMLITSESETQPV NRVAYNVGGQMATNNQASTLRDSTTASSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAK IPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEME WELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL*

[0081] The insertions described herein may, in some cases, comprise a 7-mer insertion (e.g., one of those shown in Tables 1-3) at AA574-575. With the addition of the adaptors / linker, it is envisioned that any insertion disclosed herein may comprise at least an 8, 9, 10, 11, 12, 13, 14, or 15 mer. The insertion sequences may include, but are not limited to, sequences that are not exactly the same as the sequences disclosed herein, but which have, in addition to the substitutions explicitly described for various sequences listed herein, additional substitutions of amino acid residues which substantially do not impair the activity or properties of the sequences described herein, such as those predicted by homology software e.g. BLOSUM62 matrices.

[0082] Disclosed herein are AAV capsid proteins with an insertion described above in a parental AAV capsid protein that confers an increased transduction in GPCs or CNS tissues such as the brain in a subject, even when delivered systemically. One of the many advantages of the AAV capsid proteins described herein is their ability to target tissue and cells within the brain. The tissue can be the brain. Non-limiting examples of brain cells include a neuron and a glial cell. Glial cells can be selected from a GPC, an oligodendrocyte, an ependymal cell, an astrocyte and a microglia.

[0083] Another advantage among certain rAAV capsid proteins described herein is a detargeting effect with respect to liver tissue relative to the parental AAV capsid protein. AAV capsid proteins from native AAV serotypes with tropisms includes the liver activating the innate immune response, which in some cases causes a severe inflammatory response in a subject, which can lead to multi-organ failure. By improving transduction of a native AAV serotype for a target in vivo tissue (e.g., brain), and potentially decreased transduction in off target tissue (e.g., liver), the rAAV particles of the present disclosure reduce the immunogenic properties of AAV-mediated transgene delivery and prevent activation of the innate immune response.

[0084] In some embodiments, the rAAV capsid protein comprises an insertion / substitution of at least or about seven, eight, nine, ten or eleven amino acids of a parental amino acid sequence. In some cases, the rAAV capsid protein has an increased viral transduction enrichment in brain. In some cases, the rAAV capsid protein has a decreased viral transduction enrichment in liver.

[0085] The rAAV capsid proteins described herein may be isolated and purified. The rAAV may be isolated and purified by methods standard in the art such as by column chromatography, iodixanol gradients, or cesium chloride gradients. Methods for purifying AAV from helper virus are known in the art and may include methods disclosed in, for example, Clark et al., Hum. Gene Then, 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427- 443 (2002); U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0086] In one embodiment, this disclosure provides a set of rAAV viruses that selectively infect and transduce human glial progenitor cells, including bipotential oligodendrocyte and astrocyte progenitors.

[0087] In one embodiment, this disclosure provides a set of rAAV viruses that selectively infect and transduce human oligodendrocytic progenitor cells. In one embodiment, this disclosure provides a set of rAAV viruses that selectively infect and transduce human glioma or glioblastoma tumor progenitor cells, which derive from glial progenitor cells and express PDGF alpha receptor.

[0088] In one embodiment, this disclosure provides the use of these rAAVs with included cellspecific regulatory sequences (e.g., promoters and enhancers) that selectively drive gene expression in glial progenitor cells and / or oligodendrocyte progenitor cells. Thus allowing cell-type specific infection as well as transgene expression, thus further ensuring specificity and hence safety).

[0089] 2. Nucleic Acids encoding the rAAV capsid proteins

[0090] In certain aspects, this application discloses the nucleic acid comprises a nucleotide sequence that encodes for a modified capsid protein described herein. In certain embodiments, the nucleic acid comprises a nucleotide sequence that encodes for a modified capsid protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino add sequence set forth in SEQ ID NO: 53 or 55.

[0091] In certain embodiments, the nucleic acid may be optimized, e.g., using codon optimization, replacement and / or removal of certain elements, to improve, e.g. expression of the capsid protein. Various methods to optimize nucleic acid sequences are known in the art by those of ordinary skill in the art. For example, certain nucleotides in the nucleic acid can be mutated without altering the amino acid sequence encoded by the nucleic add sequence, utilizing the degeneracy of the genetic code. For example, a nucleic acid can be optimized by using an alternative codon for an identical amino acid. In certain embodiments, optimization methods can increase expression of the encoded capsid protein relative to the expression of the capsid encoded by nucleic acid sequences that have not been optimized.

[0092] 3. AAV Particles and AAV Vectors

[0093] AAV is a widely used gene therapy vector due to its clinical safety record, non- pathogenic nature, ability to infect non-dividing cells, and ability to provide long-term gene expression after a single administration. Because of their safety, nonpathogenic nature, and ability to infect neurons, AAVs such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9 are commonly used gene therapy vectors for CNS applications. However, after direct CNS infusion, these serotypes exhibit a dominant neuronal tropism and expression in glial cells such as oligodendrocytes is low, especially when gene expression is driven by a constitutive promoter, which restricts their potential for use in treating disorders related to glial cells, such as white matter diseases. The approach described herein to alleviate these issues includes using AAV serotypes with high tropism for glial cells, including GPC, oligodendrocytes, and astrocytes.

[0094] As discussed above, the terms “adeno-associated virus” and / or “AAV” refer to parvoviruses with a linear single-stranded DNA genome and variants thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. Parvoviruses, including AAV, are useful as gene therapy vectors as they can penetrate a cell and introduce a nucleic acid (e.g., transgene) into the nucleus. In some embodiments, the introduced nucleic acid (e.g., rAAV vector genome) forms circular concatemers that persist as episomes in the nucleus of transduced cells. In some embodiments, a transgene is inserted in specific sites in the host cell genome, for example at a site on human chromosome 19. Site-specific integration, as opposed to random integration, is believed to likely result in a predictable long-term expression profile. The insertion site of AAV into the human genome is referred to as AAVS1. Once introduced into a cell, RNAs or polypeptides encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, a nucleic acid delivered by AAV can be used to express a therapeutic RNA or polypeptide for the treatment of a disease, disorder and / or condition in a human subject.

[0095] Multiple serotypes of AAV exist in nature with at least fifteen wild type serotypes having been identified from humans thus far (z.e., AAV1-AAV15). Naturally occurring and variant serotypes are distinguished by having a protein capsid that is serologically distinct from other AAV serotypes. Examples include AAV1, AAV2, AAV, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrhlO, AAVrh74 (see WO 2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and recombinantly produced variants (e.g., capsid variants with insertions, deletions and substitutions, etc.), such as variants referred to as AAV2i8, NP4, NP22, NP66, DJ, DJ / 8, DJ / 9, LK3, RHM4-1, among many others. “Primate AAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non- primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, and so on.

[0096] Serotype distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences and antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). However, some naturally occurring AAV or man-made AAV mutants (e.g., recombinant AAV) may not exhibit serological difference with any of the currently known serotypes. These viruses may then be considered a subgroup of the corresponding type, or more simply a variant AAV. Thus, as used herein, the term “serotype” refers to both serologically distinct viruses, as well as viruses that are not serologically distinct but that may be within a subgroup or a variant of a given serotype.

[0097] A comprehensive list and alignment of amino acid sequences of capsids of known AAV serotypes is provided by Marsic et al. (2014) Molecular Therapy 22(11): 1900-1909. Genomic sequences of various serotypes of AAV, as well as sequences of the native ITRs, rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001863 (AAV3B), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), NC_001862 (AAV6), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of which are incorporated by reference herein. See also, e.g., Srivistava etal. (1983) J. Virology 45:555; Chiorini etal. (1998) J. Virology 71 :6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221 :208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; WO 2013 / 063379; WO 2014 / 194132; WO 2015 / 121501, and U.S. Patent No. 6,156,303 and U.S. Patent No. 7,906,111.

[0098] As discussed herein, a “recombinant adeno-associated virus” or “rAAV” is distinguished from a wild-type AAV by replacement of all or part of the endogenous viral genome with a non-native sequence. The term a "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (z.e., nucleic acid sequence not of AAV origin) that are flanked by at least one AAV inverted terminal repeat sequence (ITR). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (z.e., AAV Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector may be referred to as a "pro-vector" which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. A rAAV vector can be in any of number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, e.g., an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)".

[0099] For the production of an rAAV capsid , the desired ratio of VP1 :VP2:VP3 can be in the range of about 1 : 1 : 1 to about 1 : 1 : 100, preferably in the range of about 1 : 1 :2 to about 1 : 1 :50, more preferably in the range of about 1 : 1 :5 to about 1 : 1 :20. Although the desired ratio of VP1 :VP2 can be 1 : 1, the ratio range of VP1 :VP2 could vary from 1 :50 to 50: 1.

[0100] The present disclosure provides for an rAAV vector comprising a polynucleotide sequence not of AAV origin (e.g., a transgene or a polynucleotide heterologous to AAV). The heterologous polynucleotide may be flanked by at least one, and sometimes by two, AAV terminal repeat sequences (e.g., inverted terminal repeats). The heterologous polynucleotide flanked by ITRs, also referred to herein as a “vector genome,” typically encodes an RNA or a polypeptide of interest, or a gene of interest, such as a target for therapeutic treatment. Delivery or administration of an rAAV vector to a subject (e.g. a patient) provides encoded RNAs / proteins / peptides to the subject. Thus, an rAAV vector can be used to transfer / deliver a heterologous polynucleotide for expression for, e.g., treating a variety of diseases, disorders and conditions. rAAV vector genomes generally retain 145 base ITRs in cis to the heterologous nucleic acid sequence that replaced the viral rep and cap genes. Such ITRs are useful to produce a recombinant AAV vector; however, modified AAV ITRs and non-AAV terminal repeats including partially or completely synthetic sequences can also serve this purpose. ITRs form hairpin structures and function to, for example, serve as primers for host-cell-mediated synthesis of the complementary DNA strand after infection. ITRs also play a role in viral packaging, integration, etc. ITRs are the only AAV viral elements which are required in cis for AAV genome replication and packaging into rAAV vectors. An rAAV vector genome optionally comprises two ITRs which are generally at the 5’ and 3’ ends of the vector genome comprising a heterologous sequence (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest including, but not limited to, an antisense, and siRNA, a CRISPR molecule, among many others). A 5’ and a 3’ ITR may both comprise the same sequence, or each may comprise a different sequence. An AAV ITR may be from any AAV including by not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV.

[0101] An rAAV vector of the disclosure may comprise an ITR from an AAV serotype that differs from the serotype of the capsid. Such an rAAV vector comprising at least one ITR from one serotype, but comprising a capsid from a different serotype, may be referred to as a hybrid viral vector (see U.S. Patent No. 7,172,893). An AAV ITR may include the entire wild type ITR sequence, or be a variant, fragment, or modification thereof, but will retain functionality.

[0102] In some embodiments, an rAAV vector genome is linear, single-stranded and flanked by AAV ITRs. Prior to transcription and translation of the heterologous gene, a single stranded DNA genome of approximately 4700 nucleotides must be converted to a double-stranded form by DNA polymerases (e.g., DNA polymerases within the transduced cell) using the free 3 ’-OH of one of the self-priming ITRs to initiate second-strand synthesis. In some embodiments, full length-single stranded vector genomes (z.e., sense and anti-sense) anneal to generate a full length-double stranded vector genome. This may occur when multiple rAAV vectors carrying genomes of opposite polarity (z.e., sense or anti-sense) simultaneously transduce the same cell. Regardless of how they are produced, once double- stranded vector genomes are formed, the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.

[0103] The efficiency of transgene expression from an rAAV vector can be hindered by the need to convert a single stranded rAAV genome (ssAAV) into double-stranded DNA prior to expression. This step can be circumvented by using a self-complementary AAV genome (scAAV) that can package an inverted repeat genome that can fold into double-stranded DNA without the need for DNA synthesis or base-pairing between multiple vector genomes. See, e.g., U.S. Patent No. 8,784,799; McCarty, (2008) Molec. Therapy 16(10): 1648-1656; and McCarty et al., (2001) Gene Therapy 8: 1248-1254; McCarty et al., (2003) Gene Therapy 10:2112-2118.

[0104] As used herein, the term an “rAAV virus" or "rAAV particle or virion" refers to a viral particle comprising at least one AAV or rAAV capsid protein and an encapsi dated rAAV vector genome. In certain embodiments, an rAAV particle or virion of the present disclosure comprises one or more rAAV capsid proteins described herein and an encapsidated rAAV vector genome containing a heterologous nucleic acid encoding a polypeptide or nucleic acid.

[0105] A viral capsid may be from a wild type AAV or a variant AAV such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAVrh74 (see W02016 / 210170), AAV12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO:5 of WO 2015 / 013313), RHM15-1, RHM15-2, RHM15- 3 / RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, snake AAV, goat AAV, shrimp AAV, ovine AAV and variants thereof (see, e.g., Fields et al., VIROLOGY, volume 2, chapter 69 (4thed., Lippincott-Raven Publishers). Capsids may be derived from a number of AAV serotypes disclosed in U.S. Patent No. 7,906,111; Gao et al. (2004) J. Virol. 78:6381; Morris et al. (2004) Virol. 33:375; WO 2013 / 063379; WO 2014 / 194132; and include true type AAV (AAV-TT) variants disclosed in WO 2015 / 121501, and RHM4-1, RHM 15-1 through RHM 15-6, and variants thereof, disclosed in WO 2015 / 013313. A full complement of AAV cap proteins includes VP1, VP2, and VP3. The ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV cap proteins may be provided.

[0106] In some embodiments, an rAAV capsid comprising a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype (e.g., wild type AAV serotypes, variant AAV serotypes) is referred to as a “chimeric vector” or “chimeric capsid” (See U.S. Patent No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, a chimeric capsid protein is encoded by a nucleic acid sequence derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes. In some embodiments, a recombinant AAV vector includes a capsid sequence derived from e.g., AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrhlO, AAV2i8, or variant thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the foregoing AAV serotypes (see, Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, a chimeric capsid can comprise a mixture of a VP1 from one serotype, a VP2 from a different serotype, a VP3 from yet a different serotype, and a combination thereof. For example, a chimeric virus capsid may include an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit.

[0107] In some embodiments, a chimeric capsid is an AAV5 based chimeric capsid as described herein. In some embodiments, chimeric vectors have been engineered to exhibit altered tropism or tropism for a particular tissue or cell type. The term “tropism” refers to preferential entry of the virus into certain cell (e.g., GPC, oligodendrocytes, or astrocytes) or tissue types and / or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types. AAV tropism is generally determined by the specific interaction between distinct viral capsid proteins and their cognate cellular receptors (Lykken et al. (2018) J. Neurodev. Disord. 10: 16). Preferably, once a virus or viral particle has entered a cell, sequences (e.g. , heterologous sequences such as a transgene) carried by the vector genome (e.g , an rAAV vector genome) are expressed.

[0108] A “tropism profile” refers to a pattern of transduction of one or more target cells in various tissues and / or organs. For example, a chimeric AAV capsid may have a tropism profile characterized by efficient transduction of oligodendrocytes with only low transduction of neurons, astrocytes and other CNS cells. See WO2014 / 052789, incorporated herein by reference. Such a chimeric capsid may be considered “specific for glial cells” exhibiting tropism for oligodendrocytes, and referred to herein as “glialtropism,” if when administered directly into the CNS, preferentially transduces glial cells over neurons and other CNS cell types. In some embodiments, at least about 80% of cells that are transduced by a capsid specific rAAV particle for glial cells are GPCs, e.g, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the transduced cells are GPCs. In some embodiments, at least about 80% of cells that are transduced by a capsid specific rAAV particle for glial cells are oligodendrocytes, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the transduced cells are oligodendrocytes.

[0109] In some embodiments, at least about 80% of cells that are transduced by a capsid specific rAAV particle for glial cells are astrocytes, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the transduced cells are astrocytes.

[0110] In some embodiments, an rAAV particle is useful for treating or preventing a “disorder associated with oligodendrocyte dysfunction.” As used herein, the term “associated with oligodendrocyte dysfunction” refers to a disease, disorder or condition in which oligodendrocytes are damaged, lost or function improperly compared to otherwise identical normal oligodendrocytes. The term includes diseases, disorders and conditions in which oligodendrocytes are directly affected as well as diseases, disorders or conditions in which oligodendrocytes become dysfunctional secondary to damage to other cells. In some embodiments, a disorder associated with oligodendrocyte dysfunction is demyelination.

[0111] In some embodiments, an rAAV particle comprising a chimeric AAV capsid described herein and a therapeutic transgene may be used to treat a disease, disorder or condition associated with glial cell dysfunction, such as oligodendrocyte dysfunction. In such a disease, disorder or condition, glial cells, such as oligodendrocytes, are damaged, lost or function improperly. This may be the result of a direct effect on the cells or result when the cells become dysfunctional secondary to damage to other cells. 4. Viral Vector Genome / Heterologous Nucleic Acids

[0112] In some embodiments, an AAV particle / virion described herein comprising an rAAV capsid described herein, may be used for the delivery of an rAAV vector genome to a tissue (e.g., CNS). In some embodiments, an rAAV particle comprising an rAAV capsid described herein can be used for delivery of an rAAV vector genome to a tissue or cell, e.g., CNS or glial / glial progenitor cells. In some embodiments, an rAAV particle of the present disclosure is an isolated rAAV particle.

[0113] The rAAV vector genome may encode any payload, such as but not limited to a polypeptide (e.g., a therapeutic polypeptide), an antibody, an enzyme, an inhibitory nucleic acid (e.g., an RNAi agent) and / or components of a gene editing system. In one embodiment, the rAAV particles described herein are used to deliver a payload to cells of the CNS. In another embodiment, the rAAV particles described herein are used to deliver a payload to glial progenitor or glial cells.

[0114] In one embodiment, the rAAV vector genome encodes an anti-Ap antibody. "Anti-Ap antibody" refers to an antibody that specifically binds to human Ap. A nonlimiting example of an anti-Ap antibody is crenezumab. Other non-limiting examples of anti- Ap antibodies are solanezumab, bapineuzumab, gantenerumab, aducanumab, ponezumab and any anti-Ap antibodies disclosed in the following publications: W02000162801, W02002046237, W02002003911, W02003016466, W02003016467, W02003077858, W02004029629, W02004032868, W02004032868, W02004108895, W02005028511, W02006039470, W02006036291, W02006066089, W02006066171, W02006066049, W02006095041, and W02009027105.

[0115] In some embodiments, an rAAV vector genome of an rAAV particle comprising an rAAV capsid, as described herein, comprises a nucleotide sequence comprising a transgene encoding a payload. In some embodiments, the rAAV vector genome comprises an inverted terminal repeat sequence (ITR). In some embodiments, the rAAV vector genome comprises two ITR sequences, one at the 5’ end of the viral genome (e.g., 5’ relative to the encoded payload) and one at the 3’ end of the viral genome (e.g., 3’ relative to the encoded payload). In some embodiments, the rAAV vector genome of an rAAV particle, described herein, may comprise a regulatory element (e.g., promoter), untranslated regions (UTR), a miR binding site, a polyadenylation sequence (poly A), a filler or stuffer sequence, an intron, and / or a linker sequence, e.g., for enhancing transgene expression. In some embodiments, the rAAV vector genome components are selected and / or engineered for expression of the payload in a target tissue such as the CNS (e.g., brain, spinal cord, or both).

[0116] Transgenes / Payloads

[0117] An rAAV particle / virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products (one or more heterologous gene products). In some cases, the gene product is a polypeptide. In some cases, the gene product is an RNA. In some cases, an rAAV particle or virion of the present disclosure comprises a heterologous nucleotide sequence encoding both a heterologous nucleic acid gene product and a heterologous polypeptide gene product. Where the gene product is an RNA, in some cases, the RNA gene product encodes a polypeptide. Where the gene product is an RNA, in some cases, the RNA gene product does not encode a polypeptide. In some cases, an rAAV particle / virion of the present disclosure comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding a single heterologous gene product. In some cases, an rAAV particle / vector / virion of the present disclosure comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding two heterologous gene products. Where the single heterologous nucleic acid encodes two heterologous gene products, in some cases, nucleotide sequences encoding the two heterologous gene products are operably linked to the same promoter. Where the single heterologous nucleic acid encodes two heterologous gene products, in some cases, nucleotide sequences encoding the two heterologous gene products are operably linked to two different promoters. In some cases, an rAAV particle / virion of the present disclosure comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding three heterologous gene products. Where the single heterologous nucleic acid encodes three heterologous gene products, in some cases, nucleotide sequences encoding the three heterologous gene products are operably linked to the same promoter. Where the single heterologous nucleic acid encodes three heterologous gene products, in some cases, nucleotide sequences encoding the three heterologous gene products are operably linked to two or three different promoters. In some cases, an rAAV particle / virion of the present disclosure comprises two heterologous nucleic acids, each comprising a nucleotide sequence encoding a heterologous gene product.

[0118] In some cases, the gene product is a polynucleotide-encoding RNA. In some cases, the gene product is an interfering RNA. In some cases, the gene product is a microRNA (miRNA). In some cases, the gene product is an aptamer. In some cases, the gene product is a polypeptide. In some cases, the gene product is a therapeutic polypeptide, e.g., a polypeptide that provides clinical benefit. In some cases, the gene product is a site-specific nuclease that provide for sitespecific knock-down of gene function. In some cases, the gene product is an RNA-guided endonuclease that provides for modification of a target nucleic acid. In some cases, the gene products are: (i) an RNA-guided endonuclease that provides for modification of a target nucleic acid; and (ii) a guide RNA that comprises a first segment that binds to a target sequence in a target nucleic acid and a second segment that binds to the RNA-guided endonuclease. In some cases, the gene products are: (i) an RNA-guided endonuclease that provides for modification of a target nucleic acid; (ii) a first guide RNA that comprises a first segment that binds to a first target sequence in a target nucleic acid and a second segment that binds to the RNA-guided endonuclease; and iii) a first guide RNA that comprises a first segment that binds to a second target sequence in the target nucleic acid and a second segment that binds to the RNA-guided endonuclease.

[0119] Polypeptide Gene Products

[0120] Where the gene product is an mRNA encoding a polypeptide, in some cases, the polypeptide enhances function of a glial cell (e.g., a glial progenitor cell) or a neuronal cell. Non-limiting examples of such polypeptide-encoding genes include: TCF7L2, OLIG2, SOXIO, MYRF, MBP, MAG, AQP4, UBE3A. BCL11A, TEAD2, TEAD3, TEAD4, MYC, MYCN, PIEZO 1, GALC, ARSA, ASP A, NPC1, NPC2, HEXB, SGSH, NAGLU, and HGSNAT, among others.

[0121] In some cases, the gene product is a polypeptide that induces differentiation of a stem cell or progenitor cells, e.g., induces the glial progenitor cell to differentiate into a further differentiated glial cell, an astrocyte, or an oligodendrocyte.

[0122] Inhibitory Nucleic Acids

[0123] In certain aspects of the disclosure, the viral genome comprises one or more inhibitory nucleic acids (e.g., inhibitory RNA molecules), polynucleotides encoding such inhibitory nucleic acids, and transgenes engineered to express such inhibitory nucleic acids. The one or more inhibitory nucleic acids may target the same gene (e.g., hybridize or specifically bind to a same mRNA sequence or different mRNA sequences of the same gene) or different genes (e.g., hybridize or specifically bind to mRNAs of different genes).

[0124] Typically, the inhibitory nucleic acid may target a human gene transcript whose expression is associated with a disease or disorder, or with premature glial senescence or differentiation arrest. Non -limiting examples include mutant HTT, FXYD1, E2F6, ZNF274, CDKN1A, and CDKN2A, among others. An inhibitory nucleic acid refers to a nucleic acid that can bind to a target nucleic acid (e.g., a target RNA) in a cell and reduce or inhibit the level or function of the target nucleic acid in the cell. Example of the inhibitory nucleic acid include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, small interfering (si)RNA compounds, single- or double-stranded RNA interference (RNAi) compounds, modified bases / locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that specifically hybridize to at least a portion of a target nucleic acid and modulate its level or function.

[0125] In some embodiments, the inhibitory nucleic acid can be an antisense RNA, an antisense DNA, a chimeric antisense oligonucleotide, an antisense oligonucleotide comprising modified linkages, an interference RNA (iRNA), a short or small interfering RNA (siRNA), a micro RNA or micro interfering RNA (miRNA), a small temporal RNA (stRNA), a short hairpin RNA (shRNA), a small RNA-induced gene activation agent (RNAa), a small activating RNA (saRNA), or combinations thereof. In some examples, the inhibitory nucleic acid is an inhibitory RNA molecule that mediates RNA interference.

[0126] RNA interference (RNAi) is a process discovered in 1998 (Fire et aL, 1998) by which cells regulate gene expression. A double-stranded RNA (dsRNA) in the cell cytoplasm triggers the RNAi pathway in which the double-stranded RNA is processed into small double-stranded fragments of approximately 21-23 nucleotides in length by the RNAse Ill-like enzyme DICER. These double-stranded fragments are integrated into a multi-subunit protein called the RNA- induced silencing complex (RISC). The RISC contains Argonaute proteins that unwind the double-stranded fragment into a passenger strand that is removed from the complex and a guide strand that is complementary to a target sequence in a specific mRNA and which directs the RISC complex to cleave or suppress the translation of the specific target mRNA molecule (Kotowska-Zimmer et al., 2021). In this way the gene that encoded the mRNA molecule is rendered essentially inactive or “silenced.”

[0127] RNAi technology may employ three kinds of tools: synthetic siRNAs, vector-based shRNAs, and artificial miRNAs (amiRNAs). Synthetic siRNAs are exogenous double stranded RNAs that must be delivered into cells and must overcome stability and pharmacokinetic challenges. shRNAs are artificial RNA molecules with a tight hairpin loop structure that are delivered to cells using plasmids or viral expression vectors. shRNAs are typically transcribed from strong pol III promoters (e.g., U6 or Hl) and enter the RNAi pathway as hairpins. However, transcription driven by strong pol III promoters can produce supraphysiologic levels of shRNA that saturate the endogenous miRNA biogenesis machinery, resulting in toxicity. AmiRNAs embed a target-specific shRNA insert in a scaffold based on a natural primary miRNA (pri-miRNA). This ensures proper processing and transport similar to endogenous miRNAs, resulting in lower toxicity (Kotowska-Zimmer et aL, 2021).

[0128] In some embodiments of this disclosure, the inhibitory RNA molecule can be an siRNA, a miRNA (including an amiRNA), or an shRNA.

[0129] An siRNA is known in the art as a double-stranded RNA molecule of approximately 19-25 (e.g., 19-23) base pairs in length that induces RNAi in a cell. In some embodiments, the siRNA sequence can also be inserted into an artificial miRNA scaffold ("shmiRNA").

[0130] An shRNA is known in the art as an RNA molecule comprising approximately 19-25 (e.g., 19-23) base pairs of double stranded RNA linked by a short loop (e.g., about 4-11 nucleotides) that induces RNAi in a cell.

[0131] An miRNA is known in the art as an RNA molecule that induces RNAi in a cell comprising a short (e.g., 19-25 base pairs) sequence of double-stranded RNA linked by a loop and containing one or more additional sequences of double-stranded RNA comprising one or more bulges (e.g., mis-paired or unpaired base pairs). As used herein, the term "miRNA" encompasses endogenous miRNAs as well as exogenous or heterologous miRNAs. In some embodiments, "miRNA" may refer to a pri-miRNA or a pre-miRNA. During miRNA processing, a pri-miRNA transcript is produced. The pri-miRNA is processed by Drosha- DGCR8 to produce a pre-miRNA by excising one or more sequences to leave a pre-miRNA with a 5' flanking region, a guide strand, a loop region, a non-guide strand, and a 3' flanking region; or a 5' flanking region, a non-guide strand, a loop region, a guide strand, and a 3' flanking region. The pre-miRNA is then exported to the cytoplasm and processed by Dicer to yield a siRNA with a guide strand and a non-guide (or passenger) strand. The guide strand is then used by the RISC complex to catalyze gene silencing, e.g., by recognizing a target RNA sequence complementary to the guide strand. Further description of miRNAs may be found, e.g., in WO 2008 / 150897. The recognition of a target sequence by a miRNA is primarily determined by pairing between the target and the miRNA seed sequence, e.g., nucleotides 1-8 (5' to 3') of the guide strand (see, e.g., Boudreau, R. L. etal. (2013) Nucleic Acids Res. 41 :e9).

[0132] In some embodiments of this disclosure, an inhibitory RNA molecule forms a hairpin structure. Generally, hairpin-forming RNAs are arranged into a self-complementary "stemloop" structure that includes a single nucleic acid encoding a stem portion having a duplex comprising a sense strand (e.g., passenger strand) connected to an antisense strand (e.g., guide strand) by a loop sequence. The passenger strand and the guide strand share complementarity. In some embodiments, the passenger strand and guide strand share 100% complementarity. In some embodiments, the passenger strand and guide strand share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% complementarity. A passenger strand and a guide strand may lack complementarity due to a base-pair mismatch. In some embodiments, the passenger strand and guide strand of a hairpin-forming RNA may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 at least 8, at least 9, or at least 10 mismatches. Generally, the first 2-8 nucleotides of the stem (relative to the loop) are referred to as "seed" residues and play an important role in target recognition and binding. The first residue of the stem (relative to the loop) is referred to as the "anchor" residue. In some embodiments, hairpin-forming RNA have a mismatch at the anchor residue.

[0133] In some embodiments, an inhibitory RNA molecule is processed in a cell (or subject) to form a "mature miRNA". Mature miRNA is the result of a multistep pathway which is initiated through the transcription of primary miRNA from its miRNA gene or intron, by RNA polymerase II or III generating the initial precursor molecule in the biological pathway resulting in miRNA. Once transcribed, pri-miRNA (often over a thousand nucleotides long with a hairpin structure) is processed by the Drosha enzyme which cleaves pri-miRNA near the junction between the hairpin structure and the ssRNA, resulting in precursor miRNA (pre- miRNA). The pre-miRNA is exported to the cytoplasm where is further reduced by Dicer enzyme at the pre-miRNA loop, resulting in duplexed miRNA strands.

[0134] Of the two strands of a miRNA duplex, one arm, the guide strand (miR), is typically found in higher concentrations and binds and associates with the Argonaute protein which is eventually loaded into the RNA-inducing silencing complex. The guide strand miRNA-RISC complex helps regulates gene expression by binding to its complementary sequence of mRNA, often in the 3' UTR of the mRNA. The non-guide strand of the miRNA duplex is known as the passenger strand and is often degraded, but may persist and also act either intact or after partial degradation to have a functional role in gene expression.

[0135] In some embodiments, a transgene is engineered to express an inhibitory nucleic acid (e.g., an miRNA) having a guide strand that targets a human gene. "Targeting" refers to hybridization or specific binding of an inhibitory nucleic acid to its cognate (e.g., complementary) sequence on a target gene (e.g., mRNA transcript of a target gene). In some embodiments, an inhibitory nucleic acid that targets a gene transcript shares a region of complementarity with the target gene that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, a region of complementarity is more than 30 nucleotides in length. Typically, the guide strand may target a human gene transcript associated with a disease or disorder. Examples include that for FXYD1.

[0136] In some embodiments, the inhibitory nucleic acid is 5 to 300 bases in length (e.g., 10- 30, 15-25, 19-22, 25-50, 40-90, 60-90, 75-100, 90-150, 110-200, 150-250, 200-300, etc. nucleotides in length). The inhibitory nucleic acid sequence encoding a pre-miRNA or mature miRNA may be 10-50, or 5-50 bases length.

[0137] CRISPR / Cas System

[0138] In one aspect, suppressing or knocking down of one or more of the genes described herein can also be achieved via a CRISPR-Cas guided nuclease using a CRISPR / Cas system and related methods known in the art. See, e.g., US11225659B2, WO2021168799A1, WO2022188039A1, WO2022188797A1, WO2022068912A1, and WO2022047624A1. See also Gimenez et al., “CRISPR-on System for the Activation of the Endogenous human INS gene,” Gene Therapy 23: 543-547 (2016); Wiedenheft etal., “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nature 482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering Using CRISPR / Cas Systems,” Science 339(6121):819-23 (2013); and Gaj et al., “ZFN, TALEN, and CRISPR / Cas-based Methods for Genome Engineering,” Cell 31(7):397-405 (2013), which are hereby incorporated by reference in their entirety.

[0139] CRISPR-Cas system is a genetic technique which allows for sequence-specific control of gene expression in prokaryotic and eukaryotic cells by guided nuclease double-stranded DNA cleavage. It is based on the bacterial immune system -derived CRISPR (clustered regularly interspaced palindromic repeats) pathway.

[0140] In another aspect, this application provides a complex comprising: (i) a protein composition that comprise a Cas protein, or orthologs, homologs, derivatives, conjugates, functional fragments thereof, conjugates thereof, or fusions thereof; and (ii) a polynucleotide composition, comprising a CRISPR RNA and a programmable spacer sequence or guide sequence complementary to at least a portion of a target RNA or DNA. The programmable guide RNA, CRISPR RNA and the Cas protein together form a CRISPR / Cas-based module for sequence targeting and recognition.

[0141] The target RNA can be any RNA molecule of interest, including naturally-occurring and engineered RNA molecules. The target RNA can be an mRNA, a tRNA, a ribosomal RNA (rRNA), a microRNA (miRNA), an interfering RNA (siRNA), a ribozyme, a riboswitch, a satellite RNA, a microswitch, a microzyme, or a viral RNA.

[0142] In some embodiments, the target nucleic acid is associated with a condition or disease, such as a condition or disorder as described herein. Thus, in some embodiments, the systems described herein can be used to treat such a condition or disease by targeting these nucleic acids.

[0143] For instance, the target nucleic acid associated with a condition or disease may be an RNA molecule that is overexpressed in a diseased cell, an old or older cell, or a senescent cell. The target nucleic acid may also be a toxic RNA and / or a mutated RNA (e.g., an mRNA molecule having a splicing defect or a mutation). The target nucleic acid may also be an miRNA. For example, the target nucleic acid may be that of a gene whose increased activity has been linked to loss of [K+]e homeostasis and pathological increases in extracellular K+which contribute to hyperexcitability and neuronal loss in neurodegenerative diseases.

[0144] Various Cas proteins can be used in this invention. A Cas protein, CRISPR-associated protein, or CRISPR protein, used interchangeably, refers to a protein of or derived from a CRISPR-Cas Class 1 or Class 2, including type I, type II, type III, type IV, type V, or type VI system, which has an RNA-guided DNA-binding. Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CaslOd, Casl3, Casl3e, Casl3f, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966. See e.g., US11225659B2, WO2021168799A1, WO2022188039A1, WO2022188797A1, WO2022068912A1, WO2022047624A1, WO2014144761,

[0145] WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entireties.

[0146] Site-Specific Endonucleases

[0147] In some cases, a gene product of interest is a site-specific endonuclease that provides for site-specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a glial disease. For example, where a dominant allele encodes a defective copy of a gene that, when wild-type, is a glial structural protein and / or provides for normal glial function, a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele. In some cases, a site-specific endonuclease is an RNA-guided endonuclease.

[0148] A site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, e.g., a subject rAAV virion can be used to deliver a site-specific endonuclease that knocks out a defective allele, and can be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional neural protein. In some cases, a subject rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes a site-specific endonuclease; and a heterologous nucleotide sequence that encodes a functional copy of a defective allele, where the functional copy encodes a functional protein.

[0149] Site-specific endonucleases that are suitable for use include, e.g., zinc finger nucleases (ZFNs); meganucleases; and transcription activator-like effector nucleases (TALENs), where such site-specific endonucleases are non-naturally occurring and are modified to target a specific gene. Such site-specific nucleases can be engineered to cut specific locations within a genome, and non-homologous end joining can then repair the break while inserting or deleting several nucleotides. Such site-specific endonucleases (also referred to as “INDELs”) then throw the protein out of frame and effectively knock out the gene. See, e.g., U.S. Patent Publication No. 2011 / 0301073. Suitable site-specific endonucleases include engineered meganuclease re-engineered homing endonucleases. Suitable endonucleases include an I-Tevl nuclease. Suitable meganucleases include I-Scel (see, e.g., Bellaiche et al. (1999) Genetics 152: 1037); and I-Crel (see, e.g., Heath el al. (1997) Nature Structural Biology 4:468).

[0150] Inverted Terminal Repeats (ITRs)

[0151] In some embodiments, the rAAV particle described herein comprises an rAAV vector genome comprising an ITR and a transgene encoding a payload. In some embodiments, the rAAV vector genome comprises two ITRs. In some embodiments, the two ITRs flank the nucleotide sequence encoding the pay load at the 5’ and 3’ ends. In some embodiments, the ITRs function as origins of replication comprising recognition sites for replication. In some embodiments, the ITRs comprise sequence regions which can be complementary and symmetrically arranged. In some embodiments, the ITRs incorporated into the rAAV vector genomes as described herein may be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0152] In some embodiments, the ITR may be from the same serotype as the capsid polypeptide, e.g. , capsid variant, selected from any of the known serotypes, or a variant thereof. In some embodiments, the ITR may be of a different serotype than the capsid. In some embodiments, the viral genome comprises two ITR sequence regions, wherein the ITRs are of the same serotype as one another. In some embodiments, the viral genome comprises two ITR sequence regions, wherein the ITRs are of different serotypes. Non-limiting examples include zero, one, or both of the ITRs having the same serotype as the capsid. In some embodiments, both ITRs of the viral genome of the AAV particle are AAV5 ITRs.

[0153] Each ITR may be about 100 to about 150 nucleotides in length. An ITR may be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131-135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146- 150 nucleotides in length. In some embodiments, the ITRs are 140-142 nucleotides in length. Nonlimiting examples of ITR length are 102, 105, 130, 140, 141, 142, 145 nucleotides in length.

[0154] Regulatory elements

[0155] In some embodiments, the rAAV vector genome of an AAV particle described herein comprises at least one element to regulate or enhance the payload target specificity and / or expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in their entirety). Nonlimiting examples of elements to enhance payload target specificity and expression include promoters, endogenous miRNAs, post- transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.

[0156] Promoters

[0157] In some embodiments, an rAAV particle described herein comprises an rAAV vector genome comprising a nucleic acid that comprises a transgene encoding a payload, wherein the transgene is operably linked to a promoter. In some embodiments, the promoter is a speciesspecific promoter, an inducible promoter, a tissue-specific promoter, a cell differentiation-stage specific promoter, or a cell specific promoter (e.g., a promoter as described in Parr et al., Nat. Med.3: 1145-9 (1997); the contents of which are herein incorporated by reference in their entirety).

[0158] As used herein, the term “promoter,” such as a “eukaryotic promoter,” refers to a nucleotide sequence that initiates transcription of a particular gene, or one or more coding sequences in eukaryotic cells (e.g., an oligodendrocyte). A promoter can work with other regulatory elements or regions to direct the level of transcription of the gene or coding sequence(s). These regulatory elements include, for example, transcription binding sites, repressor and activator protein binding sites, and other nucleotide sequences known to act directly or indirectly to regulate the amount of transcription from the promoter, including, for example, attenuators, enhancers and silencers. The promoter is most often located on the same strand and near the transcription start site, 5’ of the gene or coding sequence to which it is operably linked. A promoter is generally 100 - 1000 nucleotides in length. A promoter typically increases gene expression relative to expression of the same gene in the absence of a promoter.

[0159] As used herein, a “core promoter” or “minimal promoter” refers to the minimal portion of a promoter sequence required to properly initiate transcription. It may include any of the following: a transcription start site, a binding site for RNA polymerase and a general transcription factor binding site. A promoter may also comprise a proximal promoter sequence (5 ’ of a core promoter) that contains other primary regulatory elements (e.g. , enhancer, silencer, boundary element, insulator) as well as a distal promoter sequence (3’ of a core promoter).

[0160] Examples of suitable a promoter include adenoviral promoters, such as the adenoviral major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus promoter; the Rous Sarcoma Virus (RSV) promoter; the albumin promoter; inducible promoters, such as the Mouse Mammary Tumor Virus (MMTV) promoter; the metallothionein promoter; heat shock promoters; the a- 1 -antitrypsin promoter; the hepatitis B surface antigen promoter; the transferrin promoter; the apolipoprotein A-l promoter; chicken P-actin (CBA) promoter, the elongation factor la promoter (EFla), the hybrid form of the CBA promoter (CBh promoter), and the CAG promoter (cytomegalovirus early enhancer element and the promoter, the first exon, and the first intron of chicken beta-actin gene and the splice acceptor of the rabbit beta-globin gene) (Alexopoulou et al. (2008) BioMed. Central Cell Biol. 9:2).

[0161] A promoter may be constitutive, tissue-specific or regulated. Constitutive promoters are those which cause an operably linked gene to be expressed at all times. In some embodiments, a constitutive promoter is active in most eukaryotic tissues under most physiological and developmental conditions.

[0162] Regulated promoters are those which can be activated or deactivated. Regulated promoters include inducible promoters, which are usually “off’ but which may be induced to turn “on,” and “repressible” promoters, which are usually “on” but may be turned “off.” Many different regulators are known, including temperature, hormones, cytokines, heavy metals and regulatory proteins. The distinctions are not absolute; a constitutive promoter may often be regulated to some degree. In some cases, an endogenous pathway may be utilized to provide regulation of the transgene expression, e.g., using a promoter that is naturally downregulated when the pathological condition improves. A tissue-specific promoter is a promoter that is active in only specific types of tissues, cells or organs. Typically, a tissue-specific promoter is recognized by transcriptional activator elements that are specific to a particular tissue, cell and / or organ. For example, a tissue-specific promoter may be more active in one or several particular tissues (e.g., two, three or four) than in other tissues. In some embodiments, expression of a gene modulated by a tissue-specific promoter is much higher in the tissue for which the promoter is specific than in other tissues. In some embodiments, there may be little, or substantially no activity, of the promoter in any tissue other than the one for which it is specific.

[0163] In some embodiments, the promoter may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include those derived from viruses, plants, mammals, or humans. In some embodiments, the promoters may be those derived from human cells or systems. In some embodiments, the promoter may be truncated or mutated, e.g., a promoter variant.

[0164] In some embodiments, the promoter is a ubiquitous promoter, e.g., capable of expression in multiple tissues. In some embodiments the promoter is a human elongation factor la-subunit (EFla) promoter, the cytomegalovirus (CMV) immediate -early enhancer and / or promoter, the chicken P-actin (CBA) promoter and its derivative CAG, glucuronidase (GUSB) promoter, or ubiquitin C (UBC) promoter. In some embodiments, the promoter is a cell or tissue specific promoter, e.g., capable of expression in tissues or cells of the central or peripheral nervous systems, targeted regions within (e.g., frontal cortex), and / or sub-sets of cells therein (e.g., glial cells). In some embodiments, the promoter is a cell-type specific promoters capable of expression of a payload in glial cells such as microglia, GPCs, astrocytes, oligodendrocytes, and / or Schwann cells.

[0165] In some embodiments, the promoter is tissue-specific, differentiation stage-specific, cell type-specific, or ligand inducible. In some embodiments, the promoter is selected from the group consisting of glial progenitor cell -expressed genes including GPR17, PCDH15, PDGFRA, CPSG4, NKX2.2, 0LIG1, 0LIG2, GPR56. In some embodiments, the promoter is selected from the group consisting of astrocyte-expressed genes including GFAP, HOPX, AQP4, SOX9, and GLUL. In some embodiments, the promoter is selected from the group consisting of oligodendrocyte-expressed genes including SOXIO, CNP, MYRF, MAG, MOG, and MBP.

[0166] In some embodiments, the promoter is a tissue-specific promoter for payload expression in a tissue or cell of the central nervous system. In some embodiments, the promoter is a synapsin (Syn) promoter, glutamate vesicular transporter (VGLUT) promoter, vesicular GABA transporter (VGAT) promoter, parvalbumin (PV) promoter, sodium channel Nav1.8 promoter, tyrosine hydroxylase (TH) promoter, choline acetyltransferase (ChaT) promoter, methyl -CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) or heavy chain (NFH) promoter, neuron-specific enolase (NSE) promoter, P-globin minigene np2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, and excitatory amino acid transporter 2 (EAAT2) promoter, or a fragment thereof. In some embodiments, the promoter is a cell-type specific promoter capable of expression in an astrocyte, e.g., a glial fibrillary acidic protein (GFAP) promoter and a EAAT2 promoter, or a fragment thereof. In some embodiments, the promoter is a cell-type specific promoter capable of expression in an oligodendrocyte, e.g., a myelin basic protein (MBP) promoter or a fragment thereof.

[0167] Untranslated Regions (UTRs)

[0168] In some embodiments, wild type untranslated regions (UTRs) of a gene are transcribed but not translated. Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.

[0169] Features typically found in abundantly expressed genes of specific target organs (e.g., CNS tissue or glial cells) may be engineered into UTRs to enhance stability and protein production. As a non-limiting example, a 5’ UTR from mRNA normally expressed in the brain may be used in the viral genomes of the AAV particles described herein to enhance expression in glial cells. Examples of the 5’ UTRs include sequences which play roles in translation initiation, such as Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another ‘G’.

[0170] In another aspect, an rAAV vector genome described herein can further comprise an enhancer to increase expression of the transgene. Typically, an enhancer element is located upstream of a promoter element but may also be located downstream or within another sequence (e.g., a transgene). An enhancer may be located 100 nucleotides, 200 nucleotides, 300 nucleotides or more upstream or downstream of the promoter. An enhancer typically increases expression of a transgene beyond the increased expression provided by a promoter element alone. Many enhancers are known in the art, including, but not limited to, the cytomegalovirus major immediate-early enhancer. More specifically, the CMV MIE promoter comprises three regions: the modulator, the unique region and the enhancer (Isomura and Stinski (2003) J. Virol. 77(6):3602-3614). The CMV enhancer region can be combined with another promoter, or a portion thereof, to form a hybrid promoter to further increase expression of a nucleic acid operably linked thereto. For example, a CBA promoter, or a portion thereof, can be combined with a CMV promoter / enhancer, or a portion thereof, to make a version of CBA termed the “CBh” promoter, which stands for chicken beta-actin hybrid promoter, as described in Gray et al. (2011, Human Gene Therapy 22: 1143-1153). Like promoters, enhancers may be constitutive, tissue-specific or regulated.

[0171] Fillers, Spacers and Staffers

[0172] As disclosed herein, a vector genome may include an additional nucleic acid element to adjust the length of the nucleic acid to near, or at the normal size (e.g., approximately 4.7 to 4.9 kilobases), of the viral genomic sequence acceptable for AAV packaging into an rAAV vector (Grieger and Samulski (2005) J. Virol. 79(15):9933-9944). Such a sequence may be referred to interchangeably as filler, spacer or stuffer. In some embodiments, filler DNA is an untranslated (non-protein coding) segment of nucleic acid. In some embodiments, a filler or stuffer polynucleotide sequence is a sequence between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90-90-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1000, 1000-1500, 1500-2000, 2000-3000 or more in length.

[0173] AAV vectors typically accept inserts of DNA having a size ranging from about 4 kb to about 5.2 kb or about 4.1 to 4.9 kb for optimal packaging of the nucleic acid into the AAV capsid. In some embodiments, an rAAV vector comprises a vector genome having a total length between about 3.0 kb to about 3.5 kb, about 3.5 kb to about 4.0 kb, about 4.0 kb to about 4.5kb, about 4.5 kb to about 5.0 kb or about 5.0 kb to about 5.2 kb. In some embodiments, an rAAV vector comprises a vector genome having a total length of about 4.7 kb. In some embodiments, an rAAV vector comprises a vector genome that is self-complementary. While the total length of a self-complementary (sc) vector genome in an rAAV vector is equivalent to a singlestranded (ss) vector genome (i.e., from about 4 kb to about 5.2 kb), the nucleic acid sequence (z.e., comprising the transgene, regulatory elements and ITRs) encoding the sc vector genome must be only half as long as a nucleic acid sequence encoding a ss vector genome in order for the sc vector genome to be packaged in the capsid.

[0174] Introns and Exons In some embodiments, an rAAV vector genome disclosed herein includes, for example, an intron, exon and / or a portion thereof. An intron may function as a filler or stuffer polynucleotide sequence to achieve an appropriate length for vector genome packaging into an rAAV vector. An intron and / or an exon sequence can also enhance expression of a transgene as compared to expression in the absence of the intron and / or exon element (Kurachi et al. (1995) J. Biol. Chem. 270 (10):576-5281; WO 2017 / 074526). Furthermore, filler / stuffer polynucleotide sequences (also referred to as “insulators”) are well known in the art and include, but are not limited to, those described in WO 2014 / 144486 and WO 2017 / 074526.

[0175] Polyadenylation Signal Sequence (poly A)

[0176] Further regulatory elements may include a stop codon, a termination sequence, and a polyadenylation (poly A) signal sequence, such as, but not limited to a bovine growth hormone poly A signal sequence (BHG poly A). A polyA signal sequence drives efficient addition of a poly-adenosine “tail” at the 3’ end of a eukaryotic mRNA which guides termination of gene transcription (see, e.g., Goodwin and Rottman J. Biol. Chem. (1992) 267(23): 16330-16334). A polyA signal acts as a signal for the endonucleolytic cleavage of the newly formed precursor mRNA at its 3 ’ end and for addition to this 3 ’ end of an RNA stretch consisting only of adenine bases. A polyA tail is important for the nuclear export, translation and stability of mRNA. In some embodiments, a poly A can be a SV40 early polyadenylation signal, a SV40 late polyadenylation signal, an HSV thymidine kinase polyadenylation signal, a protamine gene polyadenylation signal, an adenovirus 5 Elb polyadenylation signal, a growth hormone polyadenylation signal, a PBGD polyadenylation signal or an in silico designed polyadenylation signal.

[0177] 5. Production of AAV

[0178] A viral vector (e.g., rAAV vector) carrying a transgene (e.g., one encoding an RNA or protein disclosed herein) can be assembled from a polynucleotide encoding a transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction. Examples of a viral vector include but are not limited to adenoviral, retroviral, lentiviral, herpesvirus and AAV vectors, and in particular rAAV vector.

[0179] A vector genome component of an rAAV vector according to the disclosure include at least one transgene (e.g., a polynucleotide encoding the RNA or protein molecule) and associated expression control sequences for controlling expression of the RNA or protein. In a preferred embodiment, the vector genome includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and / or replaced by a transgene and its associated expression control sequences. The transgene is typically inserted adjacent to one or two (z.e., is flanked by) AAV ITRs or ITR elements adequate for viral replication, in place of the nucleic acid encoding viral rep and cap proteins. Other regulatory sequences suitable for use in facilitating tissue-specific expression of the transgene in the target cell (e.g., oligodendrocyte) may also be included.

[0180] A. Packaging cell

[0181] One skilled in the art would appreciate that an rAAV vector comprising a transgene, and lacking virus proteins needed for viral replication (e.g., cap and rep), cannot replicate since such proteins are necessary for virus replication and packaging. Cap and rep genes may be supplied to a cell (e.g., a host cell, e.g., a packaging cell) as part of a plasmid that is separate from a plasmid supplying the vector genome with the transgene.

[0182] Packaging cell or producer cell means a cell or cell line which may be transfected with a vector, plasmid or DNA construct, and provides in trans all the missing functions which are required for the complete replication and packaging of a viral vector. The required genes for rAAV vectors assembly into rAAV particles include the vector genome (e.g., a transgene encoding an RNA or polypeptide, regulatory elements, and ITRs, etc.), AAV rep gene, AAV cap gene, and certain helper genes from other viruses such as, e.g., adenovirus. One of ordinary skill would understand that the requisite genes for AAV production can be introduced into a packaging cell in various ways including, for example, transfection of one or more plasmids. However, in some embodiments, some genes (e.g., rep, cap, helper) may already be present in a packaging cell, either integrated into the genome or carried on an episome. In some embodiments, a packaging cell expresses, in a constitutive or inducible manner, one or more missing viral functions.

[0183] Any suitable packaging cell known in the art may be employed in the production of a packaged viral vector. Mammalian cells or insect cells are preferred. Examples of cells useful for the production of a packaging cell in the practice of the disclosure include, for example, human cell lines, such as PER.C6, WI38, MRC5, A549, HEK293 cells (which express functional adenoviral El under the control of a constitutive promoter), B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080 cell lines. Suitable non-human mammalian cell lines include, for example, VERO, COS-1, COS-7, MDCK, BHK21-F, HKCC or CHO cells. In some embodiments, a packaging cell is capable of growing in suspension culture. In some embodiments, a packaging cell is capable of growing in serum-free media. For example, HEK293 cells are grown in suspension in serum free medium. In another embodiment, a packaging cell is a HEK293 cell as described in U.S. Patent No. 9,441,206 and deposited as American Type Culture Collection (ATCC) No. PTA 13274. Numerous rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO 2002 / 46359.

[0184] A cell line for use as a packaging cell includes insect cell lines. Any insect cell which allows for replication of AAV and which can be maintained in culture can be used in accordance with the present disclosure. Examples include Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings concerning use of insect cells for expression of heterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O’Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al. (1989) J. Virol. 63:3822-3828; Kajigaya et al. (1991) Proc. Nat’l. Acad. Sci. USA 88: 4646-4650; Ruffing et al. (1992) J. Virol. 66:6922-6930; Kimbauer et al. (1996) Virol. 219:37-44; Zhao et al. (2000) Virol. 272:382- 393; and U.S. Pat. No. 6,204,059.

[0185] As a further alternative, viral particles or virion of the disclosure may be produced in insect cells using baculovirus vectors to deliver the rep / cap genes and rAAV template as described, for example, by Urabe et al. (2002) Human Gene Therapy 13: 1935-1943. When using baculovirus production for AAV, in some embodiments, a vector genome is self- complementary. In some embodiments, a host cell is a baculovirus-infected cell (e.g., an insect cell) comprising, optionally, additional nucleic acids encoding baculovirus helper functions, thereby facilitating production of a viral capsid.

[0186] A packaging cell generally includes one or more viral vector functions along with helper functions and packaging functions sufficient to result in replication and packaging of the viral vector. These various functions may be supplied together, or separately, to the packaging cell using a genetic construct such as a plasmid or an amplicon, and they may exist extrachromosomally within the cell line, or integrated into the host cell’s chromosomes. B. Helper function

[0187] AAV cannot replicate in a cell without co-infection of the cell by a helper virus. Helper functions include helper virus elements needed for establishing active infection of a packaging cell, which is required to initiate packaging of the viral vector. Helper viruses include, typically, adenovirus or herpes simplex virus. Adenovirus helper functions typically include adenovirus components adenovirus early region 1A (Ela), Elb, E2a, E4, and viral associated (VA) RNA. Helper functions (e.g, Ela, Elb, E2a, E4, and VA RNA) can be provided to a packaging cell by transfecting the cell with one or more nucleic acids encoding various helper elements. Alternatively, a host cell (e.g., a packaging cell) can comprise a nucleic acid encoding the helper protein. For instance, HEK293 cells were generated by transforming human cells with adenovirus 5 DNA and now express a number of adenoviral genes, including, but not limited to El and E3 (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59-72). Thus, those helper functions can be provided by the HEK 293 packaging cell without the need of supplying them to the cell by, e.g., a plasmid encoding them.

[0188] In some embodiments, a packaging cell is transfected with at least (i) a plasmid comprising a vector genome comprising a transgene and AAV ITRs and further comprising at least one of the following regulatory elements: an enhancer, a promoter, an exon, an intron, and a poly A, (ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene and (iii) a plasmid comprising a helper function.

[0189] Any method of introducing a nucleotide sequence carrying a helper function into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In some embodiments, helper functions are provided by transfection using a virus vector, or by infection using a helper virus, standard methods for producing viral infection may be used.

[0190] The vector genome may be any suitable recombinant nucleic acid, such as a DNA or RNA construct and may be single stranded, double stranded, or duplexed (i.e., self- complementary as described in WO 2001 / 92551).

[0191] C. Production of Packaged Viral Vector

[0192] Packaged viral vectors can be made by several methods known to skilled artisans (see, e.g., WO 2013 / 063379). A preferred method is described in Grieger, et al. (2015) Molecular Therapy 24(2):287-297, the contents of which are incorporated by reference herein for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, wherein an adherent HEK293 cell line from a qualified clinical master cell bank is used to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allow for rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96 / 40240), a HEK293 cell line suspension can generate greater than IxlO5vector genome containing particles (vg) / cell, or greater than IxlO14vg / L of cell culture, when harvested 48 hours posttransfection. More specifically, triple transfection refers a method whereby a packaging cell is transfected with three plasmids: one plasmid encodes the AAV rep and cap genes, another plasmid encodes various helper functions (e.g., adenovirus or HSV proteins such as Ela, Elb, E2a, E4, and VA RNA, and another plasmid encodes a transgene (e.g., an RNA described herein) and various elements to control expression of the transgene.

[0193] Single-stranded vector genomes are packaged into capsids as the plus strand or minus strand in about equal proportions. In some embodiments of an rAAV vector, a vector genome is in the plus strand polarity (z.e., the sense or coding sequence of the DNA strand). In some embodiments an rAAV vector, a vector is in the minus strand polarity (z.e., the antisense or template DNA strand). Given the nucleotide sequence of a plus strand in its 5’ to 3’ orientation, the nucleotide sequence of a minus strand in its 5’ to 3’ orientation can be determined as the reverse-complement of the nucleotide sequence of the plus strand.

[0194] To achieve the desired yields, a number of variables are optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density.

[0195] An rAAV particle may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV particles are known in the art and include methods described in Clark et al. (1999) Human Gene Therapy 10(6): 1031-1039; Schenpp and Clark (2002) Methods Mol. Med. 69:427-443; U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0196] A universal purification strategy, based on ion exchange chromatography methods, may be used to generate high purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids. In some embodiments, this process can be completed within one week, results in high full to empty capsid ratios (>90% full capsids), provides post-purification yields (>lxl013vg / L) and purity suitable for clinical applications. In some embodiments, such a method is universal with respect to all serotypes and chimeric capsids. Scalable manufacturing technology may be utilized to manufacture GMP clinical and commercial grade rAAV vectors (e.g., for the treatment of an inherited or acquired disorder of myelin). After rAAV particles of the present disclosure have been produced and purified, they can be titered (e.g., the amount of rAAV particle in a sample can be quantified) to prepare compositions for administration to subjects, such as human subjects with an inherited or acquired disorder of myelin. rAAV particle titering can be accomplished using methods known in the art.

[0197] In some embodiments, the number of viral particles, including particles containing a vector genome and “empty” capsids that do not contain a vector genome, can be determined by electron microscopy, e.g., transmission electron microscopy (TEM). Such a TEM-based method can provide the number of vector particles (or virus particles in the case of wild type AAV) in a sample.

[0198] In some embodiments, rAAV vector genomes can be titered using quantitative PCR (qPCR) using primers against sequences in the vector genome, for example ITR sequences, and / or sequences in the transgene or regulatory elements. By performing qPCR in parallel on dilutions of a standard of known concentration, such as a plasmid containing the sequence of the vector genome, a standard curve can be generated permitting the concentration of the rAAV vector to be calculated as the number of vector genomes (vg) per unit volume such as microliters or milliliters. By comparing the number of vector particles as measured by, e.g., electron microscopy, to the number of vector genomes in a sample, the number of empty capsids can be determined. Because the vector genome contains the therapeutic transgene, vg / kg or vg / ml of a vector sample may be more indicative of the therapeutic amount of the vector that a subject will receive than the number of vector particles, some of which may be empty and not contain a vector genome. Once the concentration of rAAV vector genomes in the stock solution is determined, it can be diluted into or dialyzed against suitable buffers for use in preparing a composition for administration to subjects (e.g., subjects with an inherited or acquired disorder of myelin).

[0199] 6. Uses and Treatment Methods

[0200] An AAV particle or vector as disclosed herein may be used for gene therapy treatment and / or prevention of a disease, disorder or condition.

[0201] In particular, it can be used for treating or preventing a disease, disorder or condition associated with deficiency or dysfunction of glial cell (e.g., oligodendrocyte) or a disease / disorder / condition of myelin by targeting a particular target gene, and of any other condition and or illness in which reducing the expression of the related target gene may produce a therapeutic benefit or improvement, e.g., a disease, disorder or condition mediated by, or associated with, an increase in the level or function of the related protein compared with the level or function of the protein in an otherwise healthy individual. The vector genome and / or an rAAV particle described herein can be used for gene therapy treatment and / or prevention of the same disease, disorder or condition.

[0202] In some embodiments, methods of the disclosure include use of an rAAV particle, or a pharmaceutical composition thereof, in the treatment of the disease, disorder or condition in a subject. In some embodiments, methods of the disclosure include use of an rAAV particle, or pharmaceutical composition thereof, to decrease the level of a gene of interest in a subject in need thereof.

[0203] In one aspect, provided is a method (I) for delivering a viral vector or a non-viral delivery vector to a central nervous system (CNS), one or more cellular targets therein, or a tissue therein of a subject, or (II) for treating a neurological disorder in the subject, comprising:

[0204] (1) enhancing glymphatic system influx of the subject by a process comprising administering an agent into the blood of the subject; and

[0205] (2) delivering a composition comprising the viral vector to the subject’s cerebrospinal fluid (CSF), wherein the agent comprises or is a hypertonic solution.

[0206] In one aspect, provided is a method (I) for delivering a viral vector or a non-viral delivery vector to a central nervous system (CNS), a one or more cellular targets therein, or a tissue therein of a subject, or (II) for treating a neurological disorder in the subject, comprising:

[0207] (1) enhancing glymphatic system influx of the subject; and

[0208] (2) delivering a composition comprising the viral vector or non-viral delivery vector to the subject’s cerebrospinal fluid (CSF) intraci sternally or intrathecally.

[0209] In some embodiments, the neurological disorder is selected from the group consisting of a myelin disorder, a viral or microbial infection, an inflammatory disorder, an ischemic lesion or post-ischemic state, a neurodegenerative disease, a behavioral disorder, a lysosomal or peroxisomal storage disease, or a brain cancer.

[0210] The nucleic acid, a vector genome, and / or an rAAV particle described above can be used in the preparation of a medicament for use in the treatment and / or prevention of a disease, disorder or condition associated with or caused by deficiency or dysfunction of glial cells, oligodendrocytes or myelin and of any other condition or illness in which up-regulation or down-regulation of the related protein(s) may produce a therapeutic benefit or improvement. In some embodiments, the disorder associated with glial cell dysfunction is selected from the group of hereditary disorders consisting of Pelizaeus-Merzbacher disease, vanishing white matter disease, Krabbe's disease, metachromatic leukodystrophy, gangliosidoses, mucopolysaccharidoses, adrenoleukodystrophy, Canavan disease, Alexander disease, pigmentary orthochromatic leukodystrophy, Zellweger disease, Angelman syndrome, 18q- syndrome, phenylketonuria, and aminoacidurias.

[0211] In some embodiments, the disorder associated with glial cell dysfunction is selected from the group of acquired disorders consisting of cerebral palsy, multiple sclerosis, spinal cord injury, traumatic brain injury, and white matter stroke.

[0212] In some embodiments, the disorder associated with glial cell dysfunction is selected from the group of neurodegenerative disorders consisting of Huntington disease, Alzheimer disease, Parkinson disease, Lewy body disease, multisystem atrophy, and schizophrenia.

[0213] In some embodiments, the AAV particle or virion is administered by intraci sternal magna administration. In some embodiments, the AAV particle or virion is administered by intra-striatal administration.

[0214] As used herein a disorder of myelin, a disease of myelin, a myelin-related disorder, a myelin-related disease, a myelin disorder, and a myelin disease are used interchangeably. They include any disease, condition (e.g., those occurring from traumatic spinal cord injury and cerebral infarction), or disorder related to demyelination, insufficient myelination and remyelination, or dysmyelination in a subject. Such a disorder can be inherited or acquired or both. It can arise from a myelination related disorder or demyelination resulting from a variety of neurotoxic insults. "Demyelination" as used herein, refers to the act of demyelinating, or the loss of the myelin sheath insulating the nerves, and is the hallmark of some neurodegenerative autoimmune diseases, including multiple sclerosis, transverse myelitis, chronic inflammatory demyelinating polyneuropathy, and Guillain-Barre Syndrome. Leukodystrophies are caused by inherited enzyme deficiencies, which cause abnormal formation, destruction, and / or abnormal turnover of myelin sheaths within the CNS white matter. Both acquired and inherited myelin disorders share a poor prognosis leading to major disability. Thus, some embodiments of the present disclosure can include methods for the treatment of neurodegenerative autoimmune diseases in a subject. Remyelination of neurons requires oligodendrocytes. The term "remyelination", as used herein, refers to the re-generation of the nerve's myelin sheath by replacing myelin producing cells or restoring their function.

[0215] Myelin related diseases or disorders which may be treated or ameliorated by the methods of the present invention include diseases, disorders or injuries which relate to dysmyelination or demyelination in a subject's brain cells, e.g., CNS neurons. Such diseases include, but are not limited to, diseases and disorders in which the myelin which surrounds the neuron is either absent, incomplete, not formed properly, or is deteriorating. Such disease include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Wallerian Degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillian-Barre syndrome, Marie-Charcot-Tooth disease and Bell's palsy.

[0216] Myelin related diseases or disorders which may be treated or ameliorated by the methods of the present invention include a disease or disorder characterized by a myelin deficiency. Insufficient myelination in the central nervous system has been implicated in a wide array of neurological disorders. Among these are forms of cerebral palsy in which a congenital deficit in forebrain myelination in children with periventricular leukomalacia, contributes to neurological morbidity (Goldman et cd.. 2008) Goldman, S. A., Schanz, S., and Windrem, M. S. (2008). Stem cell-based strategies for treating pediatric disorders of myelin. Hum Mol Genet. 17, R76-83. At the other end of the age spectrum, myelin loss and ineffective repair may contribute to the decline in cognitive function associated with senescence (Kohama et al. , 2011) Kohama, S. G., Rosene, D. L., and Sherman, L. S. (2011) Age (Dordr). Age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline. Therefore, it is contemplated that effective compositions and methods of enhancing myelination and / or remyelination may have substantial therapeutic benefits in halting disease progression and restoring function in a wide array of myelin-related disorders.

[0217] In some embodiments, the compositions of the present invention can be administered to a subject that does not have, and / or is not suspected of having, a myelin related disorder in order to enhance or promote a myelin dependent process. In some embodiments, compositions described herein can be administered to a subject to promote myelination of CNS neurons in order to enhance cognition, which is known to be a myelin dependent process, in cognitive healthy subjects. In certain embodiments, compositions described herein can be administered in combination with cognitive enhancing (nootropic) agents. Exemplary agents include any drugs, supplements, or other substances that improve cognitive function, particularly executive functions, memory, creativity, or motivation, in healthy individuals. Non limiting examples include racetams (e.g., piracetam, oxiracetam, and aniracetam), nutraceuticals (e.g., bacopa monnieri, panax ginseng, ginko biloba, and GABA), stimulants (e.g., amphetamine pharmaceuticals, methylphenidate, eugeroics, xanthines, and nicotine), L-Theanine, Tolcapone, Levodopa, Atomoxetine, and Desipramine.

[0218] The overall dosage of a therapeutic agent (e.g., an rAAV particle, or a cell) will be a therapeutically effective amount depending on several factors including the overall health of a subject, the subject's disease state, severity of the condition, the observation of improvements and the formulation and route of administration of the selected agent(s). Determination of a therapeutically effective amount is within the capability of those skilled in the art. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition.

[0219] In certain embodiments, the cell or nucleotide compositions described herein may be administered in an amount effective to enhance myelin production in the CNS of a subject by an increase in the amount of myelin proteins (e.g., MBP) of at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% as compared to the level of myelin proteins of an untreated subject.

[0220] In other embodiments, the cell or nucleotide compositions may be administered in an amount effective to promote survival of CNS neurons in a subject by an increase in the number of surviving neurons of at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% as compared to the number of surviving neurons in an untreated CNS neurons or subject.

[0221] Another strategy for treating a subject suffering from myelin-related disorder is to administer a therapeutically effective amount of a cell or a vector or a particle composition described herein along with a therapeutically effective amount of an oligodendrocyte differentiation and / or proliferation inducing agent(s) and / or anti-neurodegenerative disease agent. Examples of anti-neurodegenerative disease agents include L-dopa, cholinesterase inhibitors, anticholinergics, dopamine agonists, steroids, and immunomodulators including interferons, monoclonal antibodies, and glatiramer acetate. Therefore, in a further aspect of the disclosure, the compositions described herein can be administered as part of a combination therapy with adjunctive therapies for treating neurodegenerative and myelin related disorders.

[0222] The phrase "combination therapy" embraces the administration of GPCs or oligodendrocyte precursor differentiation inducing compositions described herein and a therapeutic agent as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents. When administered as a combination, the precursor cells differentiation inducing compound and a therapeutic agent can be formulated as separate compositions. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).

[0223] 7. Pharmaceutical Compositions

[0224] The present disclosure provides a pharmaceutical composition, or medicament, for preventing or treating an inherited or acquired disorder of myelin. In some embodiments, a pharmaceutical composition comprises one or more of the above-described rAAV particles or virions described herein, and host cells. In one aspect, provided is a hypertonic pharmaceutical composition comprising (i) a viral vector or a non-viral delivery vector as described herein and (ii) a pharmaceutically acceptable carrier or excipient. In one embodiment, the viral vector comprises an AAV. In one embodiment, the AAV is AAV5. In one embodiment, the non-viral delivery vector comprises an LNP.

[0225] The pharmaceutical composition further comprises a pharmaceutically-acceptable carrier, adjuvant, diluent, excipient and / or other medicinal agents. A pharmaceutically acceptable carrier, adjuvant, diluent, excipient or other medicinal agent is one that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing undesirable biological effects which outweigh the advantageous biological effects of the material. Any suitable pharmaceutically acceptable carrier or excipient can be used in the preparation of a pharmaceutical composition according to the invention (See e.g., Remington The Science and Practice of Pharmacy, Adeboye Adejare (Editor) Academic Press, November 2020).

[0226] A pharmaceutical composition is typically sterile, pyrogen-free and stable under the conditions of manufacture and storage. A pharmaceutical composition may be formulated as a solution (e.g., water, saline, dextrose solution, buffered solution, or other pharmaceutically sterile fluid), microemulsion, liposome, or other ordered structure suitable to accommodate a high product (e.g., viral vector particles, microparticles or nanoparticles) concentration. In some embodiments, a pharmaceutical composition comprising the above-described rAAV particles or virions or host cells of the disclosure is formulated in water or a buffered saline solution. A carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of a coating such as lecithin, by maintenance of a required particle size, in the case of dispersion, and by the use of surfactants. In some embodiments, it may be preferable to include isotonic agents, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged adsorption of an injectable composition can be brought about by including, in the composition, an agent which delays absorption, e.g., a monostearate salt and gelatin. In some embodiments, a nucleic acid, vector and / or host cell of the disclosure may be administered in a controlled release formulation, for example, in a composition which includes a slow-release polymer or other carrier that protects the product against rapid release, including an implant and microencapsulated delivery system.

[0227] In some embodiments, a pharmaceutical composition of the disclosure is a parenteral pharmaceutical composition, including a composition suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and / or intracistemal magna (ICM) administration. In some embodiments, a pharmaceutical composition of this disclosure is formulated for administration by ICV or ICM injection. In some embodiments, an rAAV vector may be formulated in 350 mM NaCl and 5% D-sorbitol in PBS.

[0228] 8. Methods of Administration

[0229] The above-described rAAV particle may be administered to a cell or a subject (e.g., a patient) in order to treat the subject. Administration of the viral particle to a human subject, or an animal in need thereof, can be by any means known in the art for administering a vector. A target cell of the rAAV particle of the present disclosure includes cells of the CNS, preferably glial cells, such as GPCs, oligodendrocytes, or astrocytes.

[0230] In one embodiment, the step of enhancing glymphatic system influx for a method described herein comprises administering an agent to the subject. In one embodiment, the agent comprises or is a hypertonic solution, and is administered into blood in the subject. In one embodiment, the agent is administered intravenously to the subject. In one embodiment, the composition is a hypertonic composition. In some embodiments, the subject is placed into the Trendelenburg position before, during and / or after the step of enhancing, the step of delivering, or both. In some embodiments, the subject is anesthetized before the step of enhancing, the step of delivering, or both. In some embodiments, the composition is delivered at about the same time or within about 4 hours after the glymphatic system influx is enhanced.

[0231] The rAAV particle of the present disclosure can be administered in addition to, and as an adjunct to, the standard of care treatment. That is, the vector can be co-administered with another agent, compound, drug, treatment or therapeutic regimen, either simultaneously, contemporaneously, or at a determined dosing interval as would be determined by one skilled in the art using routine methods. Uses disclosed herein include administration of an rAAV vector of the disclosure at the same time, in addition to and / or on a dosing schedule concurrent with, the standard of care for the disease as known in the art.

[0232] In some embodiments, a combination composition includes one or more immunosuppressive agents. In some embodiments, a combination composition includes an rAAV particle comprising a transgene (e.g., a polynucleotide encoding a nucleic acid molecule or polypeptide disclosed herein) and one or more immunosuppressive agents. In some embodiments, a method includes administering or delivering an rAAV particle comprising the transgene to a subject and administering an immunosuppressive agent to the subject either prophylactically prior to administration of the vector, or after administration of the vector (z.e., either before or after symptoms of a response against the vector and / or the protein provided thereby are evident).

[0233] In one embodiment, the particle of the disclosure (e.g., an rAAV particle) is administered systemically. Exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic and intramuscular administration, and the like, as well as direct tissue or organ injection. One skilled in the art would appreciate that systemic administration can deliver a nucleic acid to all tissues. In some embodiments, direct tissue or organ administration includes administration to areas directly affected by oligodendrocyte deficiency (e.g., brain and / or central nervous system). In some embodiments, vectors of the disclosure, and pharmaceutical compositions thereof, are administered to the brain parenchyma (z.e., by intraparenchymal administration), to the spinal canal or the subarachnoid space so that it reaches the cerebrospinal fluid (CSF) (z.e., by intrathecal administration), to a ventricle of the brain (z.e., by intracerebroventricular administration) and / or to the cistema magna of the brain (z.e., by intracistemal magna administration).

[0234] Accordingly, in some embodiments, the viral particle of the present disclosure is administered by direct injection into the brain (e.g., into the parenchyma, ventricle, cisterna magna, etc.) and / or into the CSF (e.g., into the spinal canal or subarachnoid space) to treat a disorder of myelin. A target cell of a vector of the present disclosure includes a cell located in the cortex, subcortical white matter of the corpus callosum, striatum and / or cerebellum. In some embodiments, a target cell of a vector of the present disclosure is a glial cell, such as a GPC, an oligodendrocyte, or an astrocyte. Additional routes of administration may also comprise local application of a vector under direct visualization, e.g., superficial cortical application, or other stereotaxic application.

[0235] In some embodiments, the viral particle of the disclosure is administered by at least two routes. For example, the viral particle is administered systemically and also directly into the brain. If administered via at least two routes, the administration of a vector can be, but need not be, simultaneous or contemporaneous. Instead, administration via different routes can be performed separately with an interval of time between each administration.

[0236] The above-described rAAV particle or virion comprising the polynucleotide may be used for transduction of a cell ex vivo or for administration directly to a subject (e.g., directly to the CNS of a patient with a disease). In some embodiments, a transduced cell (e.g., a host cell) is cultured and then administered to a subject to treat or prevent a disease, disorder or condition (e.g., cell therapy for the disease). An rAAV particle comprising a therapeutic nucleic acid (e.g., encoding the nucleic acid molecule or polypeptide) is preferably administered to a cell in a biologically-effective amount. In some embodiments, a biologically-effective amount of a particle is an amount that is sufficient to result in reducing the expression of a related gene in a target cell.

[0237] In some embodiments, the disclosure includes a method of decreasing the level and / or activity of a gene in a cell by administering to a cell (in vivo, in vitro or ex vivo) a polynucleotide encoding an RNA molecule described herein, either alone or in a vector (including a plasmid, a virus vector, a nanoparticle, a liposome, or any known method for providing a nucleic acid to a cell).

[0238] The dosage amount of an rAAV particle depends upon, e.g., the mode of administration, disease or condition to be treated, the stage and / or aggressiveness of the disease, individual subject's condition (age, sex, weight, etc.), particular viral vector, stability of protein to be expressed, host immune response to the vector, and / or gene to be delivered. Generally, doses range from at least 1 x 108, or more, e.g., 1 x 109, 1 x IO10, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014, 1 x IO15or more vector genomes (vg) per kilogram (kg) of body weight of the subject to achieve a therapeutic effect.

[0239] In some embodiments, a polynucleotide encoding an RNA or polypeptide / protein molecule described herein may be administered as a component of a DNA molecule (e.g, a recombinant nucleic acid) having a regulatory element (e.g, a promoter) appropriate for expression in a target cell (e.g., oligodendrocytes). The polynucleotide may be administered as a component of a plasmid or a viral vector, such as an rAAV particle or virion. An rAAV particle or virion may be administered in vivo by direct delivery of the particle or virion (e.g., directly to the CNS) to a patient in need of treatment. An rAAV particle or virion may be administered to a patient ex vivo by administration of the vector in vitro to a cell from a donor patient in need of treatment, followed by introduction of the transduced cell back into the donor (e.g., cell therapy).

[0240] In some embodiments, an agent described herein (such as a hypertonic solution) is administered into blood in a subject. In one embodiment, the agent is administered intravenously to the subject.

[0241] The methods described herein include administering to a subject a pharmaceutical composition that is hypertonic with respect to plasma or blood. As hypertonic solutions, once injected into blood, may cause fluid shifts out of cells and a variety of negative effects, care should be taken to select a proper osmolality that are not so hypertonic as to cause significant thrombosis and / or vessel irritation. In one embodiment, the solution / preparation is considered to have suitable osmolality if 30 minute after injection into a subject in the manner described in the working example below, the resulting plasma osmolality is greater than about 320 mOsml.kg'1and less than about 600 mOsml.kg'1, e.g., greater than about 340 or 350 and less than about 375, 400, 425, 450, 475, 500, or about 575 mOsml.kg'1. In general, hypertonic solutions useful in this invention exhibit a tonicity that is greater than about 320 mOsml.kg'1, e.g., 340 to 3,000 (e.g., 500 to 2,000, 1,000 to 2,000, 1,500 to 1,800) mOsml.kg'1. Solutions with an osmolality that is greater than about 600 mOsml.kg'1should be used with care in injections.

[0242] Various primary bulking agents can be used for preparing a hypertonic solution / preparation for intravenous injection. Examples include ionizing agents, e.g., NaCl, and nonionizing. Examples of non-ionizing bulking agents include, but are not limited to, mannitol, glycine, sucrose, lactose, other disaccharides, therapeutic proteins or the active ingredient of a formulation itself, or other bulking agents known to one skilled in the art. The concentrations of non-ionizing bulking agents do not significantly affect whether a solution has a sufficient ionic strength. However, their concentrations do have an effect on osmolarity, and therefore, their concentrations can have an effect on tonicity. In certain examples, NaCl or mannitol is used. The osmotic diuretic mannitol or hypertonic saline can establish an osmotic gradient between plasma and brain cells and draws water across the BBB into the vascular compartment. Exemplary dosages for mice were described in the working examples below. The human equivalent doses (HED) can be obtained using methods known in the art. See e.g., Nair AB, Jacob S. J Basic Clin Pharm. 2016 Mar;7(2):27-31. doi: 10.4103 / 0976-0105.177703 and the FDA’s Guidance for Industry. Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy. For example, to a human subject, NaCl may be administered at 30 mg / kg or more (e.g., 30 to 300 mg / kg) and mannitol may be administered at 130 mg / kg or more (e.g., 130 to 1300 mg / kg).

[0243] 9. Kit

[0244] The present disclosure provides a kit with packaging material and one or more components therein. In one aspect, provided is a kit for delivering a viral vector or a non-viral delivery vector to the CNS, a one or more cellular targets therein, or a tissue therein of a subject, comprising two or more of the following:

[0245] (i) the viral vector or the non-viral delivery vector;

[0246] (ii) a hypertonic pharmaceutical composition, and

[0247] (iii) a hypertonic solution.

[0248] A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., the above-described polynucleotide, nucleic acid, expression cassette, expression vector (e.g, viral vector genome, expression vector, rAAV vector), viral particle and host cell, etc., and optionally a second active agent such as a compound, therapeutic agent, drug or composition.

[0249] A kit refers to a physical structure that contains one or more components of the kit. Packaging material can maintain the components in a sterile manner and can be made of material commonly used for such purposes (e.g, paper, glass, plastic, foil, ampules, vials, tubes, etc.).

[0250] A label or insert can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredients(s) including mechanism of action, pharmacokinetics and pharmacodynamics. A label or insert can include information identifying manufacture, lot numbers, manufacture location and date, expiration dates. A label or insert can include information on a disease (e.g., an inherited or acquired disorder of myelin) for which a kit component may be used. A label or insert can include instructions for a clinician or subject for using one or more of the kit components in a method, use or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency of duration and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimens described herein.

[0251] A label or insert can include information on potential adverse side effects, complications or reaction, such as a warning to a subject or clinician regarding situations where it would not be appropriate to use a particular composition.

[0252] Glial progenitor cells comprise an especially important phenotype of the adult human CNS, as these cells can give rise to both oligodendrocytes and astrocytes; under select circumstances they may also give rise to neurons as well. As such, this is an important phenotype to be able to target with gene therapeutics, both efficiently and specifically so. While AAVs have been evolved to target a number of cell types of the adult central nervous system, most such efforts have focused on mouse cells; among those studies focused on human cells, most were necessarily conducted in vitro, as few systems appropriate for targeting human brain cells in vivo have been developed. In this disclosure, provided is the development of a new set of AAVs, evolved from AAV serotype AAV5, that selectively and efficiently infect human glial progenitor cells (hGPCs) and their derivatives in vivo, with little off-target CNS or systemic infection. To do so, candidate viruses were selected in vivo, introducing libraries of randomly mutagenized, Cre recombinase-reported AAV5s intraci sternally into mice that had been neonatally chimerized with Cre recombinase-expressing hGPCs. By this means, the methodology of the CREATE strategy, as first described by Deneen and colleagues, was combined with human glial chimeric brains as a platform for in vivo viral selection. Critically, robust glial infection was achieved in these mice using delivery via the paravascular glymphatic system, so as to avoid the blood-brain barrier and hence enable efficient parenchymal entry of the engineered viruses, using a clinically feasible strategy.

[0253] The hGPCs in these chimerized mice were derived from hESCs that had been edited to express Cre recombinase under the regulatory control of the PDGFRA promoter, so that only human GPCs - which specifically express PDGFaR - expressed Cre. The resultant human glial chimeras were injected intracistemally with a library of AAVs, each of which expressed a random heptapeptide insertion into an AAV binding domain that regulates target cell adhesion. Each also harbored an inverted BGH Poly-adenylation sequence, flanked by two different Lox sequences (Lox66 and Lox71), which upon recombination in PDGFRA-Cre expressing cells, allowed the specific amplification of those viruses that had successfully transduced human GPCs. By this means, variants that selectively infected human GPCs and their derivatives in vivo were identified, and which exhibited minimal infection of non-neural or other systemic phenotypes.

[0254] The persistent neuronal infection by these glial-evolved capsid variants was an expected result of this approach, since the capsids modifications included the addition of random sequences and selection for those that bound glia, but the remaining VP1 sequence was not deleted (or mutagenized) and its other exposed loops were not modified, which are together responsible for its neuronal avidity. As such, significant glial tropism was added to these vectors, without necessarily removing neuronal infectivity; the subsequent filtration against other tissues (liver, spleen, kidney) was intended to exclude variants cross-reactive with non- CNS tissue, while allowing inclusion of variants that retained neuronal infectivity. Since many of the potential disease targets of interest involve both neuronal and glial pathology, this is a desirable feature in most instances. In some disorders though, glial specificity might be preferred. In such cases where glial-specific gene expression to the exclusion of neurons is a priority, one may add cell type-selective regulatory elements - enhancers and / or promoters - to further specify gene expression to glial phenotypes of interest. In this regard, a number of enhancers have been identified that can direct gene expression to astrocytes and / or oligodendrocytes, whose pairing with capsids permitting cell type-selective infection may provide especially robust vectors for safe clinical use.

[0255] These vectors can be used in a broad set of clinical indications, as an increasing numbers of neurological disorders become recognized as either glial in etiology, or with attendant and contributory glial pathology. These include the adult myelin disorders, such as progressive multiple sclerosis and age-related white matter loss, for which hGPCs and their oligodendrocytic progeny are clear targets. In addition, the hGPC-targeted viruses may selectively infect neoplastic stem and progenitor cells within malignant glial tumors, since human glioma cells may arise from resident hGPCs, and express high levels of the PDGFA receptor (Persson, A.I. et al. (2010). Non-stem cell origin for oligodendroglioma. Cancer Cell 18, 669-682). Most broadly though, these selectively gliotropic vectors may find value in treating the neurodegenerative disorders, given the importance of astrocytic and oligodendroglial pathology in diseases as diverse as Huntington’s, schizophrenia, Parkinson’s, and Alzheimer’s. For each of these, potential transcriptional targets have been identified, but the means of selectively and safely transducing a high proportion of the relevant glia in any given brain region in vivo, much less throughout the entire brain and CNS, have not previously been available. Described herein is a vector selection platform that enables the production of AAV5s targeting human phenotypes of interest in vivo, while validating a glymphatic delivery pathway that both minimizes extra-cerebral viral spread and efficiently circumvents the bloodbrain barrier, potentially enabling the delivery of therapeutic transgenes to brain glia in a clinically safe and effective manner.

[0256] 10. Definitions

[0257] As used herein, the terms “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” refer interchangeably to any molecule composed of or comprising monomeric nucleotides connected by phosphodi ester linkages. A nucleic acid may be an oligonucleotide or a polynucleotide. Nucleic acid sequences are presented herein in the direction from the 5’ to the 3’ direction. A nucleic acid sequence (i.e., a polynucleotide) of the present disclosure can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule and refers to all forms of a nucleic acid such as, double stranded molecules, single stranded molecules, small or short hairpin RNA (shRNA), micro interfering RNA or micro RNA (miRNA), small or short interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, ribosomal RNA. Where a polynucleotide is a DNA molecule, that molecule can be a gene, a cDNA, an antisense molecule or a fragment of any of the foregoing molecules. Nucleotides are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U). A nucleotide sequence may be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Each of these sequences is distinguished from naturally- occurring DNA or RNA by changes to the backbone of the molecule. Also, phosphorothioate nucleotides may be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3’-P5’-phosphoramidates, and oligoribonucleotide phosphorothioates and their 2’-0-allyl analogs and 2’-0-methylribonucleotide methylphosphonates which may be used in a nucleotide sequence of the disclosure.

[0258] "Anti-sense" refers to a nucleic acid sequence, regardless of length, that is complementary to the coding strand or mRNA of a nucleic acid sequence. Antisense RNA can be introduced to an individual cell, tissue or organanoid. An anti-sense nucleic acid can contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural intemucleoside linkages. In some embodiments, a protein or a nucleic acid is isolated. As used herein, the term "isolated" means artificially produced. As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. As used herein with respect to proteins or peptides, the term "isolated" refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.). In some embodiments, any one or more thymidine (T) nucleotides or uridine (U) nucleotides in a sequence provided herein may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide. For example, T may be replaced with U, and U may be replaced with T.

[0259] “Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0260] The term "transgene" refers to a heterologous polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and / or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA.

[0261] As used herein, the term “recombinant,” refers to a vector, polynucleotide (e.g., a recombinant nucleic acid), polypeptide or cell that is the product of various combinations of cloning, restriction or ligation steps (e.g. relating to a polynucleotide or polypeptide comprised therein), and / or other procedure that results in a construct that is distinct from a product found in nature. A recombinant virus or vector (e.g., rAAV vector) comprises a vector genome comprising a recombinant nucleic acid (e.g., a nucleic acid comprising a transgene and one or more regulatory elements). The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0262] As used herein, the term “operably linked” refers to a linkage of nucleic acid sequence (or polypeptide) elements in a functional relationship. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or other transcription regulatory sequence (e.g., an enhancer) is operably linked to a coding sequence if it affects the transcription of the coding sequence. In some embodiments, operably linked means that nucleic acid sequences being linked are contiguous. In some embodiments, operably linked does not mean that nucleic acid sequences are contiguously linked, rather intervening sequences are between those nucleic acid sequences that are linked.

[0263] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one, and in embodiments two, AAV inverted terminal repeat sequences. Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e. N Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector may be referred to as a "pro-vector" which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. An rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, particularly an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)".

[0264] As used herein, the term “vector” refers to a plasmid, virus (e.g., an rAAV), cosmid, or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid (e.g., a recombinant nucleic acid). A vector can be used for various purposes including, e.g., genetic manipulation (e.g., cloning vector), to introduce / transfer a nucleic acid into a cell, to transcribe or translate an inserted nucleic acid in a cell. In some embodiments a vector nucleic acid sequence contains at least an origin of replication for propagation in a cell. In some embodiments, a vector nucleic acid includes a heterologous nucleic acid sequence, an expression control element(s) (e.g., promoter, enhancer), a selectable marker (e.g., antibiotic resistance), a poly-adenosine (poly A) sequence and / or an ITR. In some embodiments, when delivered to a host cell, the nucleic acid sequence is propagated. In some embodiments, when delivered to a host cell, either in vitro or in vivo, the cell expresses the polypeptide encoded by the heterologous nucleic acid sequence. In some embodiments, when delivered to a host cell, the nucleic acid sequence, or a portion of the nucleic acid sequence is packaged into a capsid. A host cell may be an isolated cell or a cell within a host organism. In addition to a nucleic acid sequence (e.g., transgene) which encodes an RNA, or a polypeptide or a protein, additional sequences (e.g., regulatory sequences) may be present within the same vector (z.e., in cis to the gene) and flank the gene. In some embodiments, regulatory sequences may be present on a separate (e.g., a second) vector which acts in trans to regulate the expression of the gene. Plasmid vectors may be referred to herein as “expression vectors.”

[0265] As used herein, the term “vector genome,” or “viral vector genome,” or “rAAV vector genome” refers to a recombinant nucleic acid sequence that is contained within an rAAV vector and packaged or encapsidated within a capsid. Typically, an rAAV vector genome includes a heterologous polynucleotide sequence, e.g., a transgene, regulatory elements, ITRs not originally present in the capsid. In cases where a recombinant plasmid is used to construct or manufacture a recombinant vector (e.g, rAAV vector), the vector genome does not include the entire plasmid but rather only the sequence intended for delivery by the viral vector. This nonvector genome portion of the recombinant plasmid is typically referred to as the “plasmid backbone,” which is important for cloning, selection and amplification of the plasmid, a process that is needed for propagation of recombinant viral vector production, but which is not itself packaged or encapsidated into an rAAV particle or virion.

[0266] As used herein, the term “viral vector” generally refers to a nucleic acid delivery vehicle and which comprises a vector genome (e.g, comprising a transgene instead of a nucleic acid encoding an AAV rep and cap) packaged within the viral particle (z.e., capsid) and includes, for example, lenti- and parvo- viruses, including AAV serotypes and variants (e.g., rAAV vectors). A recombinant viral vector does not comprise a vector genome comprising a rep and / or a cap gene.

[0267] As used herein, the term "hypertonic" is relative to physiological osmolality, but can diverge from this so long as the ultimate goal of an osmotic differential or gradient is achieved between two compartments (such as the blood plasma and the central nervous system) so as to promote the influx of glymphatic flow into central nervous system. Accordingly, a “hypertonic solution” refers any physiologically and / or pharmaceutically acceptable solution that is hypertonic with respect to physiological osmolality, including hypertonic saline or sugar solutions. As mentioned herein, hypertonic solutions preferred in this invention do not cause BBB disruption.

[0268] The methods of the invention provide an agent (e.g., a pharmaceutical preparation) for injection that is hypertonic with respect to blood. To determine whether a pharmaceutical preparation is hypertonic with respect to blood, one calculates the osmolarity for all chemical components of a solution including the diluent. Tonicity can be calculated for fluids and dissolved or diluted medications, which are expressed in a numerical value of milliosmoles per liter of fluid (mOsm / L) or per kilogram of solvent (mOsm / kg). These two values also known as osmolarity and osmolality, respectively. The osmolarity of blood ranges between 285 and 310 mOsm / L and the osmolality of blood ranges between 275 and 299 mOsm / kg.

[0269] Solution osmolarity is based in part on the concepts of osmosis and osmotic pressure. Osmosis is the diffusion of solutes (dissolved particles) or the transfer of fluid through semipermeable membranes such as blood vessels or cell membranes. Osmotic pressure, which facilitates the transport of molecules across membranes, is expressed in osmolar concentrations and is referred to as hypo-osmotic (hypotonic), iso-osmotic (isotonic), or hyper-osmotic (hypertonic) when compared with biologic fluids such as blood or plasma. The term "tonicity" and "osmotic pressure" are often considered synonymous.

[0270] The osmotic pressure is the hydrostatic (or hydraulic) pressure required to oppose the movement of water through a semipermeable membrane in response to an 'osmotic gradient' (z.e., differing particle concentrations on the two sides of the membrane). Serum osmolality can be measured by use of an osmometer or it can be calculated as the sum of the concentrations of the solutes present in the solution.

[0271] As used herein, tonicity and osmotic pressure are to be considered synonymously, and are to be understood broadly. Tonicity can mean the effective osmolality and is equal to the sum of the concentrations of the solutes in a solution that have the capacity to exert an osmotic force across a membrane, including a cell membrane. In the strict sense, osmolality is a property of a particular solution and is independent of any membrane. Tonicity is a property of a solution in reference to a particular membrane. However, the invention shall refer to solutions being isotonic, hypertonic, or hypotonic with respect to biological solutions such as blood or plasma, and this referencing shall include the meaning that the particular solution is isotonic hypertonic, or hypotonic with blood or plasma with respect to a cell membrane of a cell in the blood or plasma or other biological solution.

[0272] An operational definition of tonicity can be used to explain the term. This can be based on an experiment of adding a test solution to whole blood and observing the result. If the RBCs in whole blood swell and rupture, the test solution is said to be hypotonic compared to normal plasma. If the RBCs shrink and become crenate, the test solution is said to be hypertonic compared to normal plasma. If the RBCs stay the same, the test solution is said to be isotonic with plasma. The RBC cell membrane can be the reference membrane. For example, whole blood placed in normal saline (z.e., 0.9% sodium chloride) will not swell, and hence normal saline is said to be isotonic

[0273] As used herein, the "osmolality" of a solution is the number of osmoles of solute per kilogram of solvent. Osmolality is a measure of the number of particles present in solution and is independent of the size or weight of the particles. It can be measured only by use of a property of the solution that is dependent only on the particle concentration. These properties are vapour pressure depression, freezing point depression, boiling point elevation, and osmotic pressure, and are collectively referred to as colligative properties. The "osmolarity" of a solution is the number of osmoles of solute per liter of solution.

[0274] A functional variant or equivalent of a reference peptide, polypeptide, or protein refers to a polypeptide derivative of the reference peptide, polypeptide, or protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains substantially the activity to of the reference peptide, polypeptide, or protein. In general, the functional equivalent is at least 60% (e.g., any number between 60% and 100%, inclusive, e.g., 60%, 70 %, 80%, 85%, 90%, 95%, and 99%) identical to the reference peptide, polypeptide, or protein. In certain embodiments, a point mutation can be a conservative modification.

[0275] As used herein, the term "conservative modification" refers to amino acid modifications that do not significantly affect or alter the biological characteristics of a polypeptide or protein. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a polypeptide or protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0276] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the nonlimiting examples below.

[0277] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0278] The terms “substantial identity” and “substantially identical” indicate that a polypeptide or nucleic acid comprises a sequence with between 55-100% sequence identity to a reference sequence, with at least 55% sequence identity, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity or any percentage of value within the range of 55-100% sequence identity relative to the reference sequence. The percent sequence identity may occur over a specified comparison window. Optimal alignment may be ascertained or conducted using the homology alignment algorithm of Needleman and Wunsch, supra.

[0279] As used herein, a "subject" or "individual" means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, sheep, goats, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g, trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g, a human or a non -human mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disorders. The terms, "individual," "patient" and "subject" can be used interchangeably herein. A subject can be male or female. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model.

[0280] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition or disorder in need of treatment or one or more complications related to such a condition or disorder, and optionally, have already undergone treatment for such a condition or disorder or the one or more complications related to the condition or disorder. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition or disorder or one or more complications related to the condition or disorder. For example, a subj ect can be one who exhibits one or more risk factors for the condition or disorder or one or more complications related to the condition or disorder or a subject who does not exhibit risk factors.

[0281] A "subject in need" of treatment for a particular condition or disorder can be a subject having that condition or disorder, diagnosed as having that condition or disorder, or at risk of developing that condition or disorder.

[0282] As used herein, the term "administering," refers to the placement of an agent as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the agents disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. The terms "administering" and "administration" in general refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, ophthalmic administration, intraaural administration, intracerebral administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0283] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced. That is, "treatment" includes not just the improvement of symptoms or markers, but also a slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0284] The terms “prevent,” “preventing,” “prevention” and the like are used interchangeably herein to mean inhibit, hinder, retard, reduce or otherwise delay the development of and / or progression of a condition or disorder or a symptom thereof, in a subject. In the context of the present disclosure, the term “prevent” and variations thereof does not necessarily imply the complete prevention of the specified event. Rather, the prevention may be to an extent, and / or for a time, sufficient to produce the desired effect. Prevention may be inhibition, retardation, reduction or otherwise hindrance of the event, activity or function. Such preventative effects may be in magnitude and / or be temporal in nature.

[0285] An "effective amount" generally means an amount that provides the desired effect on treating a bone fracture or enhancing bone repair. For example, an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result. The effective amounts can be provided all at once in a single administration or in fractional amounts that provide the effective amount in several administrations. The precise determination of what would be considered an effective amount may be based on factors individual to each subject, including their size, age, bone fracture being treated, and the amount of time since the fracture occurred. One skilled in the art will be able to determine the effective amount for a given subject based on these considerations which are routine in the art. As used herein, "effective dose" means the same as "effective amount."

[0286] As used herein, the term “glial cells” refers to a population of non-neuronal cells that provide support and nutrition, maintain homeostasis, either form myelin or promote myelination, and participate in signal transmission in the nervous system. “Glial cells” as used herein encompasses fully differentiated cells of the glial lineage, such as oligodendrocytes or astrocytes, and as well as glial progenitor cells. Glial progenitor cells are cells having the potential to differentiate into cells of the glial lineage such as oligodendrocytes and astrocytes.

[0287] The glial progenitor cells described herein may be derived from any suitable source of pluripotent stem cells, such as, for example and without limitation, human induced pluripotent stem cells (iPSCs) and embryonic stem cells, as described in more detail herein. In one example, glial progenitor cells can be cells rejuvenated from glial progenitor cells or progenies thereof as described herein.

[0288] In some embodiments, to treat a subject in need thereof, glial progenitor cells or rejuvenated cells are young glial or glial progenitor cells, or are younger than the counterparts in the subject to be treated.

[0289] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall 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 than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

[0290] As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0291] EXAMPLES

[0292] Example 1

[0293] To specifically target glial cells, an in vivo Cre-dependent capsid evolution and selection strategy (FIGS. 1A and IB) was designed to drive positive selection of AAV variants that infect glial cells. Certain aspects of this strategy were based on and modified from the CREATE strategy (CRE-dependent AAV Targeted Evolution), as reported in Nature Biotechnology 34(2):204-209, 2016. doi: 10.1038 / nbt.3440 as well as US 20220220502, both of which are incorporated by reference in their entireties.

[0294] To obtain AAV variants that infect human glial cells, human glial chimeric mice established with Cre-expressing human GPCs were used as the host within which to define and serially evolve hGPC-selective AAVs.

[0295] More specifically, to develop AAVs evolved to selectively target human glial progenitor cells (hGPCs) and their derivatives in vivo, candidate viruses were evolved by repetitive rounds of selection in vivo, introducing them intraci sternally into mice that had been neonatally chimerized with hGPCs, which were themselves derived from human embryonic stem cells (hESCs). These cells were edited to express Cre recombinase under the regulatory control of the PDGFRA promoter, so that only human GPCs - which specifically express the PDGF-alpha receptor encoded by PDGFRA - express Cre. These human glial chimeric mice were injected - both intraci sternally and intrastriatally, in separate groups of chimeric mice - with a library of genetically barcoded AAVs. Each of these viral preparations expressed a random heptapeptide insertion into an AAV binding domain that regulates target cell adhesion. Each also harbored an inverted sequence, the BGH Poly-adenylation sequence, flanked by two different Lox sequences (Lox66 and Lox71), that upon recombination in PDGFRa-Cre expressing cells, allowed the specific amplification of those viruses that successfully transduced human GPCs. By screening a capsid library of AAV variants in vivo and on human cells, those variants able to selectively infect human glial progenitor cells in vivo were identified, and which exhibited the least nonspecific infection of non-glial cells, including systemic phenotypes such as liver.

[0296] AA V capsid library construction and amplification

[0297] Library capsid construction To generate a viral library, the previously reported M-CREATE strategy1>2. The plasmid to receive the 7-mer insert library was created using the backbone of the pAAV-MCS Expression Vector from Agilent (Cat. VPK-410). This backbone, including the ITRs (AAV2), was fused to an expression cassette. In this expression cassette, a portion of the AAV2-P40- Rep2 was included for maintaining the expression of the capsid proteins and their proper splicing. The capsid sequence was modified by inserting a GCT codon (Alanine) between Q574 and S575 of the AAV5 VP1 capsid to introduce an Nhel cut site for insertion of a random Heptamer as well as a silent mutation at L543 to destroy one of the Xhol sites and make it unique. The library is then created by amplifying the region between Nhel and Xhol with AAV5-Cap-XhoI-Forward primer (5’ -ccacgaccaataggatggagc-3 ’ ; SEQ ID NO: 108) and AAV5-Cap-NNN-NheI-Reverse primer (5’- gggcagtggtggagctagcNNNNNNNNNNNNNNNNNNNNNactagcctggttgttggtggc-3 ’ ; SEQ ID NO: 109). The PCR amplicon is then inserted into the capsid opened Nhel and Xhol. Downstream of the Capsid gene, the construct also included two polyadenylation sequences: SV40 (sense) and bGH (anti-sense), both flanked by Lox66 and Lox71 to allow for a Cre- dependent single flip. The resulting library preps contained 4xl05variants by limiting dilution of the bacterial clones.

[0298] AA V2-Rep construct

[0299] In order to abolish the expression in cis of the native capsid and generate viral particle exclusively from the engineered capsid library. To this end, pAAV2 / 5 (Addgene #104964) was subjected to PCR-directed mutagenesis followed by recombination-mediated ligation ( InFusion, Takara). In this construct (pAAV2 / 5-Rep2-ACap5-3STOP), the expression of Cap proteins was abolished by introduction of nonsense mutations at the start of each of VP1 (Glul2Ter), VP2 (Lys6Ter), and VP3 (Glnl4Ter). All the clonings were performed using TakaraBio’s In-Fusion Snap Assembly Cloning Kit (Catalog #638952).

[0300] Generating capsids with evolved peptide inserts

[0301] After enriched capsid variants were recovered, their 21 bp sequences were ordered as primers and ligated into AAV5 Rep-Cap containing the Nhel and Xhol cut sites (swapped into pAAV2 / 5 (Addgene #104964)) to create pAAV2 / 5-574-Empty -Recipient plasmid. Individual 21 bp, heptamer coding sequences for each viral variant (i.e., CM1 or CM6) were then inserted by PCR at the Nhel end and ligated to the vector open Nhel / Xhol, to create a separate rep-cap plasmid for each variant.

[0302] Viral Amplification For amplification of the final AAV library preps, HEK293FT cells were cultured in a 5% CO2 incubator at 37°C using 10% fetal bovine serum (FBS) formulated in high glucose DMEM media (Thermo Fisher). Previously published protocols3were followed to produce the AAVs. Briefly, per viral prep, ten 150-mm plates of approximately 80% confluency HEK293FT cells were transfected with 0.4 mg of DNA-mix containing the Capsid, Helper (Addgene #112867), and AAV shuttle plasmids (pAAV-CAG-EGFP , pAAV-CAG-FLEX- EGFP and pAAV-CAG-FLEX-tdTomato; Addgene #37825, #28306 and #28304, respectively), at a respective ratio of 0.6:0.3:0.1 along with PEI transfection reagent at a ratio of 1 :4. The media was replaced at 24 and 72 hrs post-transfection and the cells and media were harvested at 120 hrs. At both the 72 and 120 hrs timepoints, the media was treated with polyethylene glycol (PEG) for precipitating the virus. Cell pellets were lysed using Salt Active Nuclease, and the viral particles were separated by iodixanol density gradient solution spun using Type 60Ti rotor, at 59,000 rpm for 3h. The resulting viral band was further washed on an Amicon filter (MW cut-off 30 kDa; Millipore, UFC910024). The virus was then aliquoted and stored at -80°C. Viral titers were established by way of a qPCR AAV titer kit (ABM, G931), as per the manufacturer’s instructions.

[0303] Generation of WA09 PDGFRa-Cre hGPCs

[0304] A Cre recombinase was inserted immediately downstream of the PDGFRa coding sequence and Intra-ribosomal entry site (IRES) for bicistronic expression (SEQ ID NO: 110). The knockin was performed by HDR-mediated insertion of the following donor DNA. The PDGFRa gene locus was targeted using the following target 5’ -ctgtaactggcggattcgag-3 ’ (SEQ ID NO: 110) inserted upstream of a U6 promoter in the pSpCas9(BB)-2A-Puro (PX459) plasmid (Addgene #62988), which contains spCas9 expressed under a CBH promoter. Both donor and the guide / Cas9 plasmid were electroporated into WA09 undifferentiated hESCs following as previously described4. The cells were selected with Puromycin (0.5ug / ml; Thermo Fisher Scientific, Al 113803) until colonies are large enough to be picked up for expansion and genotyping. The genomic integrity of selected homozygous clones was assessed via karyotype and array chromosomal genome hybridization (aCGH) before hGPC production, as previously described5.

[0305] Production of hGPCs and establishment of human glial chimeric mice

[0306] Glial progenitor cells were produced from the PDGFRA-Cre transduced human ESCs using the previously described protocol (Wang, S et al. (2013). Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252-264). At 130-140 DIV, hGPCs were collected in Ca2+ / Mg2+-free Hanks’ balanced salt solution (HBSS ; Thermo Fisher Scientific, 14170112), then mechanically dissociated to small clusters by gentle pipetting, spun, and resuspended in cold HBSS at 1 x 105cells / pl. Newborn mice (postnatal day 1) received bilateral intrastriatal injection of 1 pl cell suspension under cryoanesthesia, z.e., IxlO5hGPCs / hemisphere, under cryoanesthesia, as previously described (Wang, S et al. (2013). Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252-264; Windrem, M.S. et al. (2008). Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell 2, 553-565; Windrem, M.S. et al. (2014). A Competitive Advantage by Neonatally Engrafted Human Glial Progenitors Yields Mice Whose Brains Are Chimeric for Human Glia. J. Neurosci. 34, 16153-16161). The injection tracks permitted donor cell infiltration and chimerization of the overlying corpus callosum and cortex as well.

[0307] Cisterna magna viral delivery

[0308] At 12 weeks of age, the neonatally-chimerized mice were anesthetized with ketamine (lOOmg / kg) and xylazine (lOmg / kg) and monitored for anesthetic depth via cessation of toe pinch reflex. Mice were affixed in a stereotaxic frame and had their cistema magna exposed. Surgical dissection procedures were conducted as previously described (Xavier, A.L.R. et al. (2018). Cannula Implantation into the Cisterna Magna of Rodents. J Vis Exp. 10.3791 / 57378). A 30-gauge cannula, connected by PE10 tubing to a 100 pl Hamilton syringe (gas-tight 1700 series), was implanted into the cistema magna and secured with cyanoacrylate glue and dental cement. 10 pl of high titer (>1012gc / ml) AAV injectate was then infused into the cisterna magna at 2 pl / min using a syringe micropump (Harvard Apparatus). Concurrent with infusion start, the mice were administered 20ul / g body weight IP injection of IM hypertonic saline as previously described (Plog, B.A. et al. (2018). Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain. JCI Insight 3. 10.1172 / jci. insight.120922). Once the infusion was complete, mice were removed from the stereotaxic frame and placed in a prone Trendelenburg position on a -45-degree slope for 30 min, then returned to their home cage. Anesthetic depth was maintained for 1 hour following the time at which the infusion began. Animals were given analgesic and allowed to recover for either 7 or 21 days, depending on the experiment, at which time the mice were anesthetized and sacrificed via transcardiac perfusion with 20 ml of cold phosphate buffered saline (PBS) followed by 20 ml 4% paraformaldehyde (PF A). Tissues were then harvested, and immersion fixed in 4% PFA for an additional two hours.

[0309] AA V5 library generation and analysis

[0310] One week post injection, mice were sacrificed and livers, corpora callosa, and striata dissected. Genomic DNA was then extracted from either liver or pooled callosal and striatal cells using the Wizard SV Genomic DNA Purification System (A2361). Next Generation Sequencing Libraries were generated by using a nested PCR and PrimeSTAR GXL DNA Polymerase (Takarabio, R050A) to recover either recombined or non-recombined viral genomes from the samples. This PCR consisted of 10 cycles of outer PCR after which primers with added Illumina adapters and sample indices were spiked into the reactions and run for 25 more cycles to give each sample a unique barcode. These were run on a 1% agarose gel and the expected 352 bp band was purified. Final samples were indexed using NexteraXT Indexing and sequencing on an Illumina MiSeq V2 Nano (150bp paired end).

[0311] Following sequencing and demultiplexing all subsequent analysis was carried out in R; the 21bp DNA sequences, corresponding to the random heptamer coding sequences, were extracted based on their flanking consistent sequences. Each sample was then normalized by sequencing depth and converted into 7aa peptide sequences. Within animal matched libraries, recombined brain libraries were then evaluated for enrichment vs non-recombined brain or liver libraries. From each animal, the top 5 most enriched and specific heptamers were used to generate viral particles for downstream testing and validation. All code for this analysis may be found at: github.com / CTNGoldmanLab / hGPC_AAV_Capsid_Evolution.

[0312] Brain tissue processing and whole brain imaging

[0313] Mice were transcardially perfused with HBSS (Thermo Fisher Scientific) followed by 4% paraformaldehyde (PFA) in 0. IM PBS at 3 weeks following AAV delivery. The brains and livers were dissected, post-fixed in 4% PFA for 2h, and stored in lx PBS. The dorsal and ventral surface of whole brains were imaged using FV3000 confocal microscope (Evident Scientific). eGFP expression was imaged in an 8x8 grid of z-stack images across brain surfaces using a resonant scanning mirror with 5x averaging (Objective: UPlanXApo lOx). Individual z-stacks were stitched together in FluoView imaging software using the Multi-area time-lapse setting. Stitched images were z-projected for presentation. Brains were cryopreserved in 0.1M PBS + 30% sucrose before embedding in OCT. Cryopreserved brains were sagittally sectioned into 20 |im -thick serial sections using a cryostat. Immunohistochemistry

[0314] Brain sections were blocked with 5% normal goat serum in 0.1 M PBS + 0.3% Triton- X for 1 hr at RT and incubated overnight at 4°C with primary antibodies against EGFP (chicken, 1 : 1000, Thermo Fisher Scientific), human nuclear antigen (mouse, 1 :400, Abeam ab254080), PDGFRa (rabbit, 1 :600, Cell Signaling Technology), Olig2 (rabbit, 1 :500, Millipore), NeuN (rabbit, 1 :800, Millipore), Sox9 (rabbit, 1 :500, Abeam), MBP (1 :400, Abeam ab40390), anti-dtTomato (1 :400, Invitrogen, Ml 1217), CNPase (1 :800, Abeam abl8527), NeuN (1 :400, Millipore ABN78MI), or HA tag (mouse, 1 :400, Invitrogen). Immunostaining was visualized with secondary species-specific fluorescent antibodies conjugated with AlexaFluor-488, AlexaFluor-568 (both diluted 1 :400, Invitrogen) or AlexaFluor-647 (diluted 1 :200, Invitrogen). Lastly, sections were coverslipped with mounting media containing DAPI (Vector Laboratories). Slides were sealed and imaged with Olympus Slideview vs200 or a Leica DM6000B confocal microscope.

[0315] Generation of PDGFRA-Cre hESCs

[0316] The human embryonic stem cells (hESC) line GENEA019 (GENEA Biocells, Sydney, Australia; a female line), was used to generate hGPCs in this study. First at the hESC stage, Cre recombinase was inserted immediately downstream of the PDGFRA coding sequence and an intra-ribosomal entry site (IRES), the latter to allow bicistronic expression. The knock-in was performed by HDR-mediated insertion of the donor DNA, with the sgRNA targeting the PDGFRA gene using the following sequence, 5’ -ctgtaactggcggattcgag-3 ’ (SEQ ID NO: 110). This was targeted immediately after the stop of the coding sequence, and the construct was inserted upstream of a U6 promoter in the pSpCas9(BB)-2A-Puro (PX459) plasmid (Addgene 62988) (Ran, F.A. et al. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc S, 2281-2308). This plasmid contains spCas9 expressed under a CBH promoter. Both the donor and the guide / Cas9 plasmid were electroporated into undifferentiated GENEA19 hESCs, as was previously described (Vieira, R. et al. (2024). Young glial progenitor cells competitively replace aged and diseased human glia in the adult chimeric mouse brain. Nature biotechnology 42, 719-730). The cells were selected with puromycin (0.5pg / ml; Thermo Fisher, Al 113803) until colonies were large enough to be picked up for expansion and genotyping. The genomic integrity of selected homozygous clones was assessed via karyotype and array chromosomal genome hybridization (aCGH), and a lack of major structural variants was confirmed before hGPC production, all as described (Wang, S. etal. (2013). Human iPSC- derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252-264), and a lack of major structural variants was confirmed before hGPC production, all as described (Wang, S. et al. (2013). Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252-264).

[0317] Mouse Models

[0318] For screening viruses in wild-type, non-chimeric mice (C57BL / 6J) were purchased from Jax Laboratories and maintained in temperature and humidity-controlled housing (64- 79°F; 30-70% humidity), in a pathogen-free colony room on a 12: 12 hr light cycle. For screening viruses in human glial chimeras, with an emphasis on targeting hGPCs and astrocytes, Ragl" ' immunodeficient mice (Taconic) chimerized neonatally with hESC GPCs were used; the brains of these mice typically exhibited widespread colonization hGPCs and their derived astrocytes.

[0319] For screening potential oligodendrocyte-tropic viruses, Rag2-null immunodeficient and myelin-deficient mice chimerized neonatally with hESC hGPCs were used. These mice reliably produced a largely humanized white matter. For this purpose, homozygous MBPshl / shlshiverer mice (Jackson Laboratory) were bred with homozygous Rag2' / _immunodeficient mice on the C3h background (Taconic) to produce myelin-deficient and immunodeficient mice, as previously described (Windrem, M.S. etal. (2008). Neonatal Chimerization with Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse. Cell Stem Cell 2, 553-565).

[0320] All mice were housed in a temperature and humidity-controlled environment (64-73 °F, 30-70% humidity) within a pathogen-free colony room on a 12: 12-hour light cycle. They were given ad libitum access to Modified ProLab RMH 3000, 5P00 containing 0.025% trimethoprim / 0.124% sulfamethoxazole (Mod LabDiet 5P00), and autoclaved acid water (pH 2.5-3.0).

[0321] Quantitative PCR

[0322] Sample tissue from spleen, kidney or liver were homogenized and DNA extracted using the Wizard® Genomic DNA Purification Kit following the manufacturer protocol (Promega). qPCR was run using TaqMan probes for the 18s subunit ribosomal gene and EGFP (ThermoFisher, IDs Mr04329676 and Mm03928990 respectively). Data were analyzed in R as One-way ANOVAs with Tukey post hoc tests and visualized using ggplot2.

[0323] Fluorescence in situ hybridization (FISH)

[0324] One-year-old mice engrafted at PND1 with G19-Cre GPCs were anesthetized and rapidly decapitated. Brains were then harvested, frozen in chilled isopentane, embedded in OCT (TissueTek), sectioned sagittally at 10 pm on a cryostat and stored at -80°C. Slides with tissue sections were immersed in 4% PFA for 15 min, rinsed twice with IX PBS, and dehydrated at room temperature in increasing ethanol solutions: 50%, 70%, 100%, 100% for 5mins each. Using the RNAscope® Multiplex Fluorescent Reagent Kit (323100, ACDBio), tissue sections were incubated in hydrogen peroxide for lOmins in a humidified box, rinsed with distilled water, and incubated with protease III for 30mins at room temperature. Hybridization probes against Cre recombinase (312281-C1, ACDBio), human PDGFRa (604481-C2, ACDBio), and human Olig2 (424191-C3, ACDBio) or human NeuN (415591- C3, ACDBio) were then incubated for 2hrs at 40°C. Control probes were also incubated in separate tissue sections. Probe signals were then serially amplified for 30mins at 40°C each and individually developed starting with Opal 520 (1 :750; FP1487001KT, PerkinElmer), followed by Opal 570 (1 :750; FP1488001KT, PerkinElmer), then Opal 690 (1 : 1500; FP1497001KT, PerkinElmer). Slides were coverslipped with Vectashield with DAPI for imaging.

[0325] Citations

[0326] 1. Ravindra Kumar, S., Miles, T.F., Chen, X., Brown, D., Dobreva, T., Huang, Q., Ding, X., Luo, Y., Einarsson, P.H., Greenbaum, A., et al. (2020). Multiplexed Cre- dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat Methods 77, 541-550. 10.1038 / s41592-020-0799-7.

[0327] 2. Deverman, B.E., Pravdo, P.L., Simpson, B.P., Kumar, S.R., Chan, K.Y., Banerjee, A., Wu, W.L., Yang, B., Huber, N., Pasca, S.P., and Gradinaru, V. (2016). Cre- dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204-209. 10.1038 / nbt.3440.

[0328] 3. Challis, R.C., Ravindra Kumar, S., Chan, K.Y., Challis, C., Beadle, K., Jang, M.J., Kim, H.M., Rajendran, P.S., Tompkins, J.D., Shivkumar, K., et al. (2019). Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat Protoc 14, 379-414. 10.1038 / s41596-018-0097-3.

[0329] 4. Vieira, R., Mariani, J.N., Huynh, N.P.T., Stephensen, H.J.T., Solly, R., Tate, A., Schanz, S., Cotrupi, N., Mousaei, M., Sporring, J., et al. (2024). Young glial progenitor cells competitively replace aged and diseased human glia in the adult chimeric mouse brain. Nat Biotechnol 42, 719-730. 10.1038 / s41587-023-01798-5.

[0330] 5. Wang, S., Bates, J., Li, X., Schanz, S., Chandler-Militello, D., Levine, C., Maherali, N., Studer, L., Hochedlinger, K., Windrem, M., and Goldman, S.A. (2013). Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 72, 252-264.

[0331] 10.1016 / j. stem.2012.12.002.

[0332] 6. Xavier, A.L.R., Hauglund, N.L., von Holstein-Rathlou, S., Li, Q., Sanggaard, S., Lou, N., Lundgaard, I., and Nedergaard, M. (2018). Cannula Implantation into the Cistema Magna of Rodents. J Vis Exp. 10.3791 / 57378.

[0333] 7. Plog, B.A., Mestre, H., Olveda, G.E., Sweeney, A.M., Kenney, H.M., Cove, A., Dholakia, K.Y., Tithof, J., Nevins, T.D., Lundgaard, I., et al. (2018). Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain. JCI Insight 3. 10.1172 / jci. insight.120922. Example 2

[0334] Using the in vivo Cre-dependent modified M-CREATE evolution and selection strategy described above, a number of chimeric AAV capsid proteins were obtained. Each of these capsid proteins has a heptapeptide sequence (7-mer) inserted between the amino acid 574-575 position, - a loop domain - of the AAV capsid protein, which permit human-selective infection of glial progenitor cells in vivo, with minimal non-glial infection. The sequences of these capsid proteins are listed in Table 1. The results are shown in FIG. 2.

[0335] Example 3

[0336] Assays were carried out to validate the ability of chimeric AAV capsid proteins for selective infection of glial progenitor cells in vivo.

[0337] To that end, AAV capsid protein with the CM1, CM6, CM11, STR1, or STR14 heptapeptide sequence was tested. The results are shown in FIGS. 3A and 3B. As shown in FIG. 3A, AAVs having CM1, CM6, CM11, STR1, or STR14-containing capsid-infected glial cells in the brain. In contrast, the liver showed only weak expression of EGFP in the viral capsids evolved following cisterna magna injection of AAV5 having CM1, CM6, CM11, or STR1 -containing capsid. See FIG. 3B.

[0338] AAV5 viruses having a capsid containing CM1, CM6, or CM11 were further examined for glial transduction following intracistemal delivery.

[0339] It was found that the expression pattern of AAV5 / CM1-CAG-EGFP following Cisterna magna injection is consistent with glial transduction throughout the brain. Also, the CM1 virus predominantly transduced the mouse OPCs following CM injection. Most OPCs are doublelabeled and co-express AAV-EGFP and OPC / oligodendroglial Olig2. In addition, the CM1 virus efficiently transduced cell of the oligo lineage in the cortex and thalamus. The CM1 virus efficiently transduced cell of the oligo lineage in the motor cortex, visual cortex, thalamus, striatum, fimria, hindbrain, and Corpus Callosum.

[0340] Similarly, assays were carried out to examine transduction pattern of the CAG-EGFP expressing CM6 virus injected in the Cisterna Magna (CM) (lOpl viral suspension) as described herein. The mice were sacrificed 3 weeks post injection. The expression pattern of AAV5 / CM6-CAG-EGFP following Cisterna magna injection was consistent with glial transduction throughout the brain. More specifically, the CM6 virus efficiently transduced cortical glia. The AAV5-CM6 virus also efficiently transduced the choroid plexus. These CM6 capsid variant virus efficiently transduced the astrocytes in the cortex too. eAAV-CM6 virus transduced hGPCs in vivo as well. Additional assays were carried out to examine the expression pattern of AAV5 / CM11- CAG-EGFP following Cisterna magna injection. To that end, CAG-EGFP expressing virus (lOpl viral suspension) was injected into the Cisterna magna of ~12wks mice. The mice were sacrificed 3 weeks post injection and examined. The pattern is consistent with glial transduction throughout the brain. It was found that eAAV-CM6 transduced Olig2-expressing glial progenitor cells in vivo. It was also found that the CM11 virus does not efficiently transduce mouse cortical OPCs following CM injection. Only scattered Olig2-expressing cells also expressed EGFP, indicating that this virus selectively targets astrocytes.

[0341] Example 4. Human glial chimeric mice may be established with Cre-expressing human GPCs.

[0342] To establish human glial chimeric mice in which human GPCs could be identified as such following cell-specific CRE-dependent recombination, human embryonic stem cells (hESCs) were engineered to express Cre recombinase under the control of PDGFRA, which is selectively expressed by glial progenitor cells in the adult brain (Sim, F.J. etal. (2011). CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells. Nature biotechnology 29, 934-941). To this end, the CRE gene was knocked into the endogenous PDGFRA locus of hESCs (Geneal9 line) by homology directed repair (HDR)-mediated insertion of donor DNA (Ran, F.A. et al. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281-2308). After sequence confirmation of the insert, and confirmation by array CGH that no major structural variants had been introduced into the Cre-expressing hESC line during editing, the cells were then differentiated into hGPCs using previously described protocols (Wang, S. et al. (2013). Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252-264). At 140-160 DIV, the hGPCs were transplanted (2 x 105cells / brain, in 4 divided injections of 5 x 104cells each) into the forebrains of neonatal Rag l ' ' immunodeficient mice; this technique allows the large-scale replacement of mouse by human GPCs, yielding chimeras in which most GPCs, and large numbers of their derived astrocytes, are human (Wang, S. et al. (2013). Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252-264; Windrem, M.S. et al. (2008). Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell 2, 553-565; Windrem, M.S. et al. (2014). A competitive advantage by neonatally engrafted human glial progenitors yields mice whose brains are chimeric for human glia. J Neurosci 34, 16153-16161), while none differentiate as neurons (FIGS. 9A-9D). A total of 6 human glial chimeric mice were generated for this set of experiments, that were colonized with PDGFRA-expressing hGPCs expressing Cre recombinase. Histological assessment of the hGPC-engrafted mice at 14 weeks of age confirmed widespread engraftment by human donor cells, and fluorescence in situ hybridization using RNAScope confirmed the expression of Cre recombinase mRNA by these cells (FIGS. 9E-9H)

[0343] Example 5. Hypertonic intracisternal delivery of AAV5 achieves widespread brain access.

[0344] It was next asked whether the glymphatic could be harnessed to access perivascular routes on the brain side of the BBB, enabling efficiently delivering AAV5 directly to the brain parenchyma. The glymphatic system comprises the network of perivascular channels, mediates the influx of cerebrospinal fluid (CSF) into the brain and facilitates its exchange with interstitial fluid to clear metabolic and protein wastes (Iliff, J. J. et al. (2012). A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Science translational medicine 4, 147ral l l; Iliff, J. J. et al. (2015). Implications of the discovery of brain lymphatic pathways. Lancet Neurol 14, 977-979). Previous work had shown that the glymphatic influx pathways can serve as a conduit for viral delivery, analogous to the efflux pathways used for metabolite clearance (Plog, B.A. et al. (2018). Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain. JCI Insight 3.). It was therefore asked if the glymphatic network might also serve as a route for viral delivery, circumventing the blood-brain barrier to allow direct parenchymal entry. The utility and perivascular flow patterns of viral delivery via injection into the cistema magna was assessed, which permits direct access to the glymphatic system via anterograde CSF flow (Xavier, A.L.R. et al. (2018). Cannula Implantation into the Cistema Magna of Rodents. J Vis Exp. 10.3791 / 57378). Critically, intracisternal viral injection was paired with both the systemic delivery of hypertonic saline and head-down prone (Trendelenburg) positioning, which together greatly increase glymphatic influx (Plog, B.A. et al. (2018). Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain. JCI Insight 3. 10.1172 / jci. insight.126138). An FITC-coupled dextran was used as a tracer by which to validate the efficacy of this delivery approach, and observed widespread tracer entry throughout the forebrain (FIGS. 10A-10C). As a positive control, a neurotropic capsid variant of AAV5-retro vector (AAV5 incorporating the AAVrg capsid variant) (Tervo, D.G. et al. (2016). A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. Neuron 92, 372-382) was delivered, and robust transduction of hippocampal pyramidal neurons and corticofugal projection neurons AAV5 was observed, using wild-type AAV5 yielded only sparse infection.

[0345] The utility of this pathway for viral administration was next evaluated. Forebrain infection was assessed following the intraci sternal hypertonic delivery of both an unmodified AAV5-serotyped AAV2 ITR-based vector (AAV2 / 5 (Louboutin, J.P. et al. (2005). Gene transfer into skeletal muscle using novel AAV serotypes. J Gene Med 7, 442-451), hereafter referred to as AAV5), and a modified AAV5 into whose VP1 capsid protein was inserted the neurotropic capsid sequence of AAV-retrograde (AAVrg) (Tervo, D.G. et al. (2016). A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. Neuron 92, 372-382), yielding AAV5-retro. Using the injection protocol, widespread parenchymal infection was observed by each of these control viruses. AAV5-retro in particular infected high proportions of hippocampal pyramidal and corticofugal neurons following a single intracistemal injection (FIGS. 10A-10C), yet unmodified AAV5 similarly infected primarily neurons; both control vectors exhibited relatively sparse glial labeling (FIGS. 10D-10F). Together, these experiments indicated that intracistemal administration of AAV5 under hypertonic conditions enables its rapid, brain-wide perivascular spread, circumventing the blood-brain barrier to allow direct viral entry into the brain with widespread, efficient cellular transduction. They also confirm prior observations that both unmodified AAV5 and modified AAVs bearing the retro capsid insert are primarily neurotropic (Castle, M.J. et al. (2016). Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids. Methods Mol Biol 1382, 133-149).

[0346] Example 6. AAV5 capsids may be evolved to target human glial progenitor cells in vivo.

[0347] Once chimeric mice whose incorporated human glia expressed Cre recombinase were established, and a delivery strategy for delivering AAVs bearing recombination-sensitive sequences was optimized, human glial chimeric mice were intraci sternally injected with a viral library of candidate AAV5 capsids (1 pl at 1 x 1012vg / ml, hence 1 x 106vg / inj ection). The library was generated by the ligation of a 21 random (N) bp sequence inserted after Q574 of VP1, a recognition loop on the sialic acid receptor binding pocket, to randomly change the binding specificity of the capsid. A GCT codon (Alanine) was inserted between Q574 and S575 of the AAV5 VP1 capsid, to introduce an Nhel cut site for insertion of the heptamer-encoding sequences of the library; the engineered AAVs thus carried random cap sequences mediating their binding specificity. As such, each of these viral candidates expressed a random heptapeptide insert in the VP1 binding domain, thereby permuting its differential adhesion to target cells. The resultant library to encode 4 x 105of these capsid variants was estimated. Importantly, the resultant AAV Cap sequences were placed upstream to two polyA tails, BGH and SV40 poly-adenylation sequences inserted in opposite directions, both flanked by two different Lox sequences (Lox66 and Lox71); this allowed the viral DNA to be detected by PCR as either unperturbed, or as the product of Cre-dependent reorientation, using PCR primers within each polyA sequence. In virally-infected PDGFRA-Cre expressing human GPCs, the recombination of these sites allowed for the specific amplification of those viruses that had successfully transduced the human cells, while viruses that infected mouse cells remained unchanged.

[0348] As noted, one microliter of this viral capsid library (1 pl, 1 x 1012vg / ml) was then injected intra-cistemally into each of four 16-week-old mice, after inducing systemic hypertonicity with with an intraperitoneal infusion of NaCl (IM, or 5.8%; 20 pl / g b.w.), and with each mouse in a prone Trendelenburg position. The mice were euthanized a week after viral delivery, and their forebrain viral sequences were assessed by PCR using two sets of primers: one corresponding to the rearranged virion DNA resulting from recombination in hGPCs, where PDGFRA-driven Cre was active at the time of infection, and a second set corresponding to the native viral DNA sequence, which remained unchanged in the infected mouse cells. Importantly, to exclude viral capsids with significant off-target systemic affinities, the liver was also harvested from the infected mice, gDNA was extracted, and those capsids that had infected the liver were separately assessed by PCR for each mouse. This step enabled the exclusion of capsid variant sequences that targeted liver as well as brain, regardless of their relative affinity for hGPCs. Those capsids most selective for human brain cells, and for hGPCs and their derivatives in particular were identified, while manifesting the least off-target systemic hepatic infection. Choosing the top 5-most human glia-enriched capsids recovered from each mouse enabled the identification of a set of 20 different capsid variants, which were designated CM1-20, that efficiently infected GPCs, and exhibited differential infectivity of human GPCs relative to mouse (Table 4).

[0349] Table 4. Capsid insert sequences. Peptide insertions in the AAV2 / 5 VP1 capsid that were most recovered from infected PDGFRA-Cre hGPCs after Cre-mediated recombination, comprising the 20 most human glial-selective peptide sequences that were recovered. CM1, 6, 11 and 16 (in bold) were the most human and glial selective among the recovered capsid inserts; among these, CM1 and CM6 were those that survived filtration against systemic organs, that exhibited the greatest human selectivity, and which infected most efficiently.

[0350] Example 7. A family of capsid evolved AAV5s selectively infect GPCs and astrocytes.

[0351] A number of glial ectodomains are co-expressed by astrocytes as well as their progenitors, and as such may also be recognized by viruses that infect PDGFaR-expressing GPCs. As a result, the hGPC-selective clones manifested some degree of astrocytic targeting as well - some minimal, such as CM1, and some profoundly so, such as CM6 (FIG. 4A) such as CM6, which selectively targeted both GPCs and astrocytes efficiently (FIGS. 4F-4G). Focusing on these two vectors, after initial screening that identified them as transducing mouse as well as human glia, it was found that while some neuronal transduction was exhibited by each, both demonstrated marked gliotropism relative to the unmodified AAV5s from which they were derived (FIGS. 4I-4K). In contrast, in controls injected with unmodified WT AAV5, almost 50% of infected cells were neurons (FIGS. 12A and 12B); thus, both AAV-CM1 and AAV-CM6 exhibited significantly more avidity for glia than did WT AAV5 (n=3-4 mice / group; p<0.0001, 1-way ANOVA with post hoc Tukey’s t-tests). That said, neuronal infection by these AAV5-derived vectors was quantitatively weak; in the same brains and regions in which glial infection was scored, less than 1% of all neurons were infected (FIG. 12B). Together, these data serve to highlight both the relative gliotropism and greater net glial infectivity of our modified vectors, relative to unmodified AAV5.

[0352] Example 8. Capsid-evolved AAVs were filtered to exclude those manifesting systemic infection.

[0353] While there is scant literature concerning gliotropic AAVs, a number of neurotropic AAVs that have advanced to trails - including those modified from AAV9 that cross the bloodbrain barrier - have been associated with significant toxicity, largely due to adventitious infection of the liver and dorsal root ganglia, yielding hepatic dysfunction and pain syndromes respectively (Hinderer, C. et al. (2018). Severe Toxicity in Nonhuman Primates and Piglets Following High-Dose Intravenous Administration of an Adeno-Associated Virus Vector Expressing Human SMN. Hum Gene Ther 29, 285-298). This may be the product of the high systemic doses needed to achieve BBB passage and the large fraction of those doses that adventitiously infect off-target tissue, such as the liver and spleen, with consequent immune activation. Use of glymphatic delivery to circumvent the BBB allows significantly lower viral doses, thus mitigating toxicity. Nonetheless, viral escape from the CNS may permit viral access to these same off-target tissues.

[0354] To minimize the possibility of non-CNS infection, the vectors were screened to identify those with the least infection of systemic tissues. To this end, PCR screening was included for viral sequence of the livers of each animal in which viral candidates were assessed, in light of that organ’s frequent off-target infection by AAVs and associated toxicity. The hepatic viral genome concentrations (vg / g) were compared to that of the targeted brains of the same animals, so as to assess the degree of off-target infection by each of the candidate capsid variants. All candidates manifesting appreciable hepatic infection were excluded from further analysis; this step served as a gatekeeper to identify those capsid variants appropriate for further development and in vivo assessment. It was found that the capsid candidates that survived this filtration, delivered intraci sternally, exhibited little or no infection of the liver, or of the spleen or kidneys either (FIGS. 13A-13C). Two of these vectors in particular, CM1 and CM6, appeared to allow phenotype-restricted delivery of reporter transgenes to brain glia in vivo, with minimal visceral infection. Together, these data indicate that hGPC-targeted capsid-modified AAV5s, in particular the prototypic vectors CM1 and CM6, exhibit substantially greater glial infectivity than wild-type AAV5, with lesser though persistent infectivity of neuronal populations, and little if any off-target visceral infection. Example 9. Capsids evolved on human GPCs exhibited efficient human glial infectivity in vivo.

[0355] The data described thus far describe the relative infectivities of the hGPC capsid- evolved vectors on mouse glia in non-chimeric mice; these experiments demonstrated the marked gliotropism of these vectors relative to the wild-type, unmodified AAV5 from which the variants were derived. It was next asked if these vectors could efficiently target human GPCs and their derivatives in vivo, and if so, whether human cells were selectively infected relative to their murine counterparts. To that end, the glial targeting efficacy of the capsid- modified AAV5-CM1 was compared to that of wild-type (WT) unmodified AAV5 (AAV2 / 5), which was done in human glial cells in vivo. In particular, WT AAV5 and AAV5-CM1 vectors were both engineered to express distinct fluorescent reporters in a Cre-dependent manner, and then compared the relative infectivities of these viruses head-to-head in hGPCs in vivo, in PDGFRa-Cre human glial chimeric mice. For this purpose, the flex-EGFP reporter (Schnutgen, F etal. (2003). A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol 21, 562-565) was packaged into AAV-CM1, while the flex- dtTomato reporter was packaged into the WT AAV2 / 5 virus (FIG. 6A); the two reporter viruses were prepared in equal concentrations, and equal volumes then mixed together. A total of 10 pl (1 x 1012vg / ml) of this 1: 1 mixture was then injected into the cistema magna of 12- month-old mice (n=3), each of which had been neonatally chimerized with PDGFRa-Cre expressing hGPCs. Three weeks later the mice were sacrificed, and the relative expression of each vector was assessed in the engrafted human cells (FIGS. 6B-6C). This analysis revealed that AAV-CM1-EGFP transduced a significantly and substantially higher proportion of hGPCs than did AAV5-dtTomato (FIG. 6D), confirming the superior glial avidity of the AAV-CM1 vector for human glia in vivo.

[0356] Having established the enhanced tropism of AAV-CM1 for human glia in vivo, relative to wild-type AAV, the respective phenotypic biases of the most efficient vectors, CM1 and CM6, was next assessed. Adult human glial chimeric mice were used that had been perinatally engrafted with unmodified hESC GPCs to assess the avidity of these vectors. This was done by injecting them intraci stemally at 16 weeks of age with either AAV-CM1 or AAV-CM6, and sacrificing them 3 weeks later to assess both their transduction efficiencies and preferred phenotypic targets. It was found that CM1 exhibited significant bias towards hGPC and oligodendroglial lineage infection, while CM6 was especially gliotropic for astrocytes (FIGS. 5A-5C; FIGS. 5E-5G). In the corpus callosum, AAV-CM1 transduced 5.3-fold more human 01ig2+-defined glia than did the parental WT AAV5: 34.9% ± 5.7 of all human Olig2+glia in the scored field were infected by a single injection of AAV-CM1, whereas only 6.5% ± 1.1 of these cells were infected in WT AAV5-injected mice (n=4 / group; means ± SEM, p=0.0014, 2- way ANOVA with Sidak’s multiple comparison test) (FIG. 5D). In contrast, AAV-CM6 transduced 3.8-fold more Sox9+-defined astroglia than did WT AAV5 (AAV-CM6: 22.2% ± 3.9; AAV5: 5.9% ± 2.6; n=4 / group, means ± SEM, p=0.002, 2-way ANOVA) (FIG. 5H). Thus, while neither CM1 nor CM6 was exclusive for its preferred target, their respective very strong biases towards hGPCs and astrocytes were each highly significant.

[0357] The phenotypic selectivity of these vectors was all the more remarkable in that each exhibited greater tropism to human than mouse cells of each phenotype (FIGS. 7A-7E). To assess the human-selectivity of these vectors, over and above their glial tropism, the relative infection of mouse and human GPCs in the corpus callosa of chimeric shiverer mice (MBPshl / shlx rag2‘ ') was assessed that were neonatally engrafted, injected with AAV-CM1 at 16-17 weeks, and killed 3 weeks later. In these immunodeficient and myelin-deficient mice, hGPCs competitively dominate the host mouse GPCs (Windrem, M.S. et al. (2014). A Competitive Advantage by Neonatally Engrafted Human Glial Progenitors Yields Mice Whose Brains Are Chimeric for Human Glia. J. Neurosci. 34, 16153-16161), such thatjust over half(52.2 ± 6.0%, n=3) of all Olig2-defined hGPCs and oligodendroglia were of human origin at the time of sacrifice, consistent with our past studies of hGPC colonization of the neonatally-engrafted shiverer brain (Windrem, M.S. et al. (2020). Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain. Cell Rep 31, 107658) (FIGS. 7A-7D). It was found that of all Olig2-expressing cells in the scored corpus callosa, 87 ± 3.3% of the infected cells were human, and 13 ± 3% murine (n=3 mice / group; <0.0001 by t-test; FIG. 7D). After normalization for the incidence of each species’ GPCs in the chimeric white matter, it was found that of all human Olig2-expressing cells in the scored corpus callosa, 30.2 ± 1.1% were infected by CM1. In contrast, just 5.0 ± 0.2% of Olig2+ mouse cells were infected (n=3 mice / group; p<0.001 by t-test) (FIG. 7E). As such, when co-resident in the same regions of corpus callosum in roughly the same incidence, the human GPCs were roughly 6-fold more likely to be infected by AAV CM1 than were mouse cells. This hGPC-evolved vector thus manifested strong selective tropism for human GPCs relative to mouse GPCs. Example 10. CM1 exhibited marked infection of mature oligodendrocytes as well as of their progenitors.

[0358] In light of the efficient infection by AAV5-CM1 of hGPCs and Olig+oligodendroglial lineage cells broadly defined, it was next determined if this vector could efficiently infect mature oligodendrocytes, an especially important clinical target for which few competent delivery vectors have been described, and then only in rodent models. To this end, mice with largely humanized adult white matter were established by transplanting hGPCs into neonatal immunodeficient shiverer mice (MBPshl / shlx rag2"); this model permits the robust oligodendrocytic maturation and myelination by their engrafted hGPCs (Wang, S. etal. (2013). Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252-264; Windrem, M.S. et al. (2008). Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell 2, 553-565). When the mice reached 18-19 weeks of age, by which time they typically exhibit a mature, largely humanized forebrain white matter, AAV5-CM1 was delivered intraci stemally, after induction of systemic hypertonicity. When sacrificed 3 weeks later, robust infection of myelinated oligodendrocytes - as well as parenchymal astrocytes and persistent hGPCs - was noted in the corpus callosum and major forebrain white matter tracts (FIGS. 8A-8D). These data indicate the potential for therapeutic transgene delivery via AAV5-CM1 to mature myelinated oligodendroglia as well as to their progenitors, thus enabling a strategy for widespread transduction of human oligodendroglia in vivo, via a readily applicable route of intraci sternal delivery.

[0359] The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present disclosure as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present disclosure as set forth in the claims. Such variations are not regarded as a departure from the scope of the disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

Claims

CLAIMSWhat is claimed is:

1. A method (I) for delivering a viral vector or a non-viral delivery vector to a central nervous system (CNS), one or more cellular targets therein, or a tissue therein of a subject, or (II) for treating a neurological disorder in the subject, comprising:(1) enhancing glymphatic system influx of the subject by a process comprising administering an agent into the blood of the subject; and(2) delivering a composition comprising the viral vector to the subject’s cerebrospinal fluid (CSF), wherein the agent comprises or is a hypertonic solution.

2. A method (I) for delivering a viral vector or a non-viral delivery vector to a central nervous system (CNS), a one or more cellular targets therein, or a tissue therein of a subject, or (II) for treating a neurological disorder in the subject, comprising:(1) enhancing glymphatic system influx of the subject; and(2) delivering a composition comprising the viral vector or non-viral delivery vector to the subject’s cerebrospinal fluid (CSF) intraci sternally or intrathecally.

3. The method of claim 2, wherein the step of enhancing glymphatic system influx comprises administering an agent to the subject.

4. The method of claim 3, wherein the agent comprises or is a hypertonic solution, and is administered into blood in the subject.

5. The method of claim 1 or 4, wherein the solution comprises a hypertonic solution of NaCl or Mannitol.

6. The method of claim 5, wherein the agent is administered intravenously to the subject.

7. The method of any one of the preceding claims, wherein the composition is a hypertonic composition.

8. The method of any one of the preceding claims, wherein the viral vector comprises an adeno-associated virus (AAV).

9. The method of claim 8, wherein the AAV is AAV5.

10. The method of any of the preceding claims, wherein the cellular targets are selected from the group consisting of a neuron, a neuronal progenitor cell, a neural progenitor cell, a glial cell, a glial progenitor cell, a microglial cell, an astrocyte, an oligodendrocyte, a pericyte, or an endothelial cell.

11. The method of any one of the preceding claims, wherein the neurological disorder is selected from the group consisting of a myelin disorder, a viral or microbial infection, an inflammatory disorder, an ischemic lesion or post-ischemic state, a neurodegenerative disease, a behavioral disorder, a lysosomal or peroxisomal storage disease, or a brain cancer.

12. The method of any one of the preceding claims, wherein the subject is placed into the Trendelenburg position before, during and / or after the step of enhancing, the step of delivering, or both.

13. The method of any one of the preceding claims, wherein the subject is anesthetized before the step of enhancing, the step of delivering, or both.

14. The method of any one of the preceding claims, wherein the composition is delivered at about the same time or within about 4 hours after the glymphatic system influx is enhanced.

15. The method of any one of the preceding claims, wherein the non-viral delivery vector comprises a lipid nanoparticle (LNP).

16. A hypertonic pharmaceutical composition comprising (i) a viral vector or a non-viral delivery vector and (ii) a pharmaceutically acceptable carrier or excipient.

17. A kit for delivering a viral vector or a non-viral delivery vector to the CNS, a one or more cellular targets therein, or a tissue therein of a subject, comprising two or more of the following:(i) the viral vector or the non-viral delivery vector;(ii) a hypertonic pharmaceutical composition, and(iii) a hypertonic solution.

18. The kit of claim 17, wherien the viral vector or the non-viral delivery vector is in the hypertonic pharmaceutical composition.

19. The composition of claim 16 or the kit of any one of claims 17-18, wherein the viral vector comprises an AAV.

20. The composition or the kit of claim 19, wherein the AAV is AAV5.

21. The composition of claim 16 or the kit of any one of claims 17-18, wherein the non- viral delivery vector comprises an LNP.

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