Neural stem cell composition and method for treating neurodegenerative disorders

Abstract

Providing neural stem cell compositions and methods for treating neurodegenerative disorders. [Solution] Stem cell-based therapies for the treatment of neurodegenerative diseases such as Huntington's disease and CNS disorders are provided herein. The therapies improved motor deficits and rescued synaptic changes. The cells were shown to be electrophysiologically active and to improve motor dysfunction and late cognitive impairment. In some embodiments, the method further comprises genetically modifying the cells. In some embodiments, the cells are genetically modified by transgene insertion or CRISPR modification. In some embodiments, the transgene is ApiCCT1, a fragment thereof, or their respective equivalents, and is overexpressed in the cells as necessary.

Description

[Background technology]

[0001] Currently, there are no disease-modifying therapies available for many neurodegenerative disorders affecting the central or peripheral nervous system. Some studies suggest that human stem cells may offer a promising therapeutic strategy for certain neurodegenerative disorders (see Drouin-Ouellet, 2014; Golas and Sander, 2016; Kirkeby et al., 2017 for an overview).

[0002] For example, Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an elongated CAG repeat sequence that codes for a polyglutamine repeat sequence within the huntingtin protein (HTT) (The Huntington's Disease Collaborative Research Group, (1993). Involuntary movements, progressive intellectual decline, and mental disorders occur (Rossand Tabrizi, 2011), and neuropathology mainly involves degeneration of medium spiny neurons (MSNs) in the striatum and cortical atrophy (Vonsatteland DiFiglia, 1998). Neurodegenerative diseases and disorders such as HD There is a need in the art to find a solution. This disclosure satisfies this need and also provides related advantages. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Ross CA et al. Lancet Neurol. (2011)10:83~98 [Non-Patent Document 2] Vonsattel JP et al., J. Neuropathol. Exp. Neurol. (1998) 57:369~384 [Overview of the project] [Means for solving the problem]

[0004] A method for preparing human neuronal stem cells (hNSCs) from human embryonic stem cells (hESCs), a) A step of isolating at least one stem cell rosette from a population of embryoid bodies (EBs) cultured in differentiation medium, b) A step of culturing at least one individual cell isolated from the rosette in step a) for a certain period of time under conditions that provide for the formation of at least one rosette, c) The step of isolating individual cells from the rosette in step b) and making them individual cells, d) A step of culturing at least one individual cell isolated from step c) for a certain period of time under conditions that provide for the generation of a confluent population of hNSCs. Methods comprising, or alternatively essentially consisting of, or even comprising, are provided herein.

[0005] In some embodiments, the isolation of at least one individual cell from the rosette is performed manually. In other embodiments, the isolation of at least one individual cell from the rosette is performed enzymatically. In further embodiments, the isolation of at least one individual cell from the rosette in step a) is performed digitally, using digital two- or three-dimensional image recognition techniques as necessary. In further embodiments, the isolation of at least one individual cell in step c) is performed enzymatically.

[0006] In some embodiments, one or more of steps a) to c) which can be performed manually or, if necessary, mechanically at high throughput using digital two- or three-dimensional image recognition technology, are performed two or more times.

[0007] In some embodiments, the method further comprises generating embryoid bodies from ESI-017. In some embodiments, the method further comprises culturing embryoid bodies (EBs) in EB medium on an ultra-low adhesion surface. In some embodiments, the method further comprises performing step a) on an ornithine / laminin coated surface by replacing the EB medium with N2 medium after the EBs have been cultured for a sufficient time to produce at least one EB of step a).

[0008] In some embodiments, at least one individual cell isolated in step c) is cultured for an effective time in an ornithine / laminin-coated plate in N2 medium to generate a confluent cell population of hNSCs. In some embodiments, the method further comprises culturing the confluent population of hNSCs with an effective amount of N2 medium. In some embodiments, the method further comprises expanding and growing the cell population.

[0009] In some embodiments, the method further includes genetically modifying cells. In some embodiments, cells are genetically modified by insertion of a transgene or by CRISPR modification. In some embodiments, the transgene is ApiCCT1, a fragment thereof, or their respective equivalents, and is overexpressed in the cells as necessary.

[0010] In some cases, a) A step of isolating at least one stem cell rosette from a population of embryoid bodies (EBs) cultured in differentiation medium, b) A step of culturing at least one individual cell isolated from the rosette in step a) for a certain period of time under conditions that provide for the formation of at least one rosette, c) The step of isolating individual cells from the rosette in step b) and making them individual cells, d) A step of culturing at least one individual cell isolated from step c) for a certain period of time under conditions that provide for the generation of a confluent population of hNSCs. hNSCs prepared by including, or alternatively essentially consisting of, or even comprising, these are provided herein.

[0011] In some embodiments, hNSCs express BNDF. In some embodiments, hNSCs express BNDF upon differentiation. In some embodiments, cells are genetically modified by transgene insertion or CRISPR.

[0012] In some embodiments, a population of cells prepared according to the method described herein is provided herein. Also provided are compositions comprising isolated cells prepared according to the method described herein. In some embodiments, the composition further comprises a carrier. In some embodiments, the carrier is a preservative and / or cryoprotectant.

[0013] In some embodiments, a method is provided herein for delivering an introduced gene to a subject or for gene editing cells in a subject requiring such delivery, the method comprising administering an effective amount of isolated cells prepared according to the method described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0014] In one aspect, there is provided a method of treating neurodegenerative disorders or enhancing synaptic connections in a subject that requires it, the method comprising administering an effective amount of isolated cells prepared according to the methods described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the neurodegenerative disorder is Huntington's disease, stroke, Alzheimer's disease, Parkinson's disease, traumatic brain injury, brain inflammation, stroke, autoimmune disorders such as multiple sclerosis, primary or secondary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, chronic spinal cord injury, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumors, peripheral neuropathy, Guillain-Barré syndrome, spinal muscular atrophy, Friedreich's ataxia, amyotrophic lateral sclerosis, and selected from the group of Huntington's chorea.

[0015] In one aspect, there is provided a kit comprising hESCs and instructions regarding performing the methods described herein.

[0016] In one aspect, non-human animals are provided in which hNSCs are prepared according to the methods described herein and transplanted. In some embodiments, the animal is a murine or a sheep. The present invention provides, for example, the following items. (Item 1) A method for preparing human neural stem cells (hNSCs) from human embryonic stem cells (hESCs), comprising: a) isolating at least one stem cell rosette from a population of embryoid bodies (EBs) cultured in a differentiation medium; b) culturing at least one individual cell isolated from the rosette of step a) for a period of time under conditions that provide for the generation of at least one rosette; c) isolating individual cells from the rosette of step b) to obtain individual cells; and d) culturing the at least one isolated individual cell from step c) for a time under conditions that provide for the generation of a confluent population of hNSCs A method comprising. (Item 2) The method according to item 1, wherein the isolation of the at least one individual cell from the rosette is performed manually. (Item 3) The method according to item 1, wherein the isolation of the at least one individual cell from the rosette is performed enzymatically. (Item 4) The method according to item 1, wherein the isolation of the at least one individual cell from the rosette in step a) is performed manually. (Item 5) The method according to item 1 or 4, wherein the isolation of the at least one individual cell in step c) is performed enzymatically. (Item 6) The method according to item 1, wherein one or more of steps a) to c) are performed two or more times. (Item 7) The method according to item 1, wherein at least one of steps a) to d) is performed manually. (Item 8) The method according to item 1, wherein at least one of steps a) to d) is performed mechanically. (Item 9) The method according to item 1, wherein the isolation of the rosette is performed digitally. (Item 10) The method according to item 1, further comprising generating the embryoid body from ESI-017. (Item 11) The method according to item 9 or 10, further comprising culturing the embryoid body (EB) on an ultra-low attachment surface in EB medium. (Item 12) The method according to item 11, further comprising, after culturing the EB for an effective time, replacing the EB medium with N2 medium and further performing step a) on an ornithine / laminin-coated surface. (Item 13) The method according to item 12, further comprising: the EB being cultured in the EB medium for a time sufficient to produce at least one EB of step a), and then replacing the EB medium with N2 medium. (Item 14) The method according to item 1, wherein at least one of the individual cells isolated in step c) is cultured for an effective time in N2 medium on an ornithine / laminin coated plate to generate a confluent cell population of hNSCs. (Item 15) The method according to item 14, further comprising culturing the confluent population of hNSCs using an effective amount of N2 medium. (Item 16) The method according to item 15, further comprising expanding and growing the aforementioned population of cells. (Item 17) The method according to item 1, further comprising genetically modifying the cells. (Item 18) The method according to item 17, wherein the cells are genetically modified by insertion of an introduced gene or modification by CRISPR. (Item 19) The method according to item 18, wherein the transgene is ApiCCT1, a fragment thereof, or an equivalent thereof, which is overexpressed in the cells as necessary. (Item 20) hNSCs prepared by the method described in any one of items 15 to 19, and expressing BNDF as appropriate. (Item 21) hNSCs prepared by the method described in item 10, which express BNDF upon differentiation. (Item 22) hNSCs as described in item 21, which have been genetically modified by transgene insertion or CRISPR. (Item 23) A group of cells as described in item 18. (Item 24) A composition comprising the isolated cells described in item 20. (Item 25) A composition comprising the group and carrier described in item 24. (Item 26) The composition according to item 24 or 25, further comprising a preservative and / or cryoprotectant. (Item 27) A method for delivering an introduced gene to a subject or for gene editing cells in a subject requiring such delivery, comprising administering an effective amount of cells as described in item 20 or 21. (Item 28) The method according to item 27, wherein the subject is a mammal. (Item 29) The method described in item 28, wherein the subject is a human. (Item 30) A method for treating a neurodegenerative disorder or enhancing synaptic connections in a subject requiring treatment of the disorder or enhancement of synaptic connections, comprising administering to the subject an effective amount of isolated cells as described in item 20 or 21. (Item 31) The method according to item 30, wherein the neurodegenerative disorder is selected from the group consisting of Huntington's disease, stroke, Alzheimer's disease, Parkinson's disease, traumatic brain injury, encephalitis, stroke, autoimmune disorders, such as multiple sclerosis, primary or secondary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, chronic spinal cord injury, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumors, peripheral neuropathy, Guillain-Barré syndrome, spinal muscular atrophy, Friedreich's ataxia, amyotrophic lateral sclerosis, and Huntington's chorea. (Item 32) The method according to item 30 or 31, wherein the subject is a mammal. (Item 33) The method according to item 32, wherein the subject is a human. (Item 34) A kit comprising hESC and instructions for carrying out the method described in any one of items 1 through 17. (Item 35) A kit comprising the hESC described in item 20 or 21 and instructions for carrying out the method described in any one of items 1 through 17. (Item 36) Non-human animals to which hNSCs described in item 20 or 21 have been implanted. (Item 37) A non-human animal, such as a rodent or sheep, as described in item 36. [Brief explanation of the drawing]

[0017] [Figure 1-1] Figures 1A–1D show that ESI-017 hNSCs implanted in R6 / 2 mice improve behavior and present evidence of differentiation into immature neurons and astrocytes. (A) The rotarod task demonstrates a deficit in R6 / 2 mice compared to non-transgenic littermates (NTs), with hNSC-treated R6 / 2 mice showing increased mean latency to fall at 1 week (black bars) and 3 weeks (gray bars) after implantation compared to vehicle-treated (Veh) mice. (B) The pole test demonstrates a deficit in R6 / 2 mice compared to NTs. hNSC-treated R6 / 2 mice descended faster than Veh mice at 4 weeks after implantation (gray bars), but not at 2 weeks after implantation (black bars). (C) Grip strength demonstrates a deficit in R6 / 2 mice compared to NTs. hNSC-treated R6 / 2 mice had a greater intensity of Gram at 4 weeks compared to Veh mice (black bars), but not at 2 weeks (gray bars). (D) Immunohistochemistry (IHC). hNSCs embedded in the striatum of R6 / 2 mice with neuronal-limited precursor cell markers (doublecortin [DCX]) and astrocytes (SC121 and GFAP) co-localized (human marker SC121). One-way ANOVA, followed by Tukey's HSD test including Scheffé, Bonferroni, and Holm multiple comparison calculations performed post-hoc. *p<0.05, **p<0.01 (n=15). Graphs show mean ± SEM. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4]Same as above.

[0018] [Figure 2] Figures 2A-2F show that IHC demonstrates that ESI-017 hNSCs implanted in R6 / 2 mice differentiate. (A) hNSCs (SC121) implanted in R6 / 2 mice differentiate into neuronal-limited precursor cells (doublecortin [DCX]) and astrocytes (SC121 and GFAP). (B) High magnification showing differentiation (633): hNSCs (human nuclear marker Ku80) implanted in R6 / 2 mice differentiate into neuronal-limited precursor cells (DCX) and some astrocytes (Ku80 and GFAP). (C) hNSCs (Ku80) and neuronal-limited precursor cells (DCX). (D) hNSCs (Ku80) and neuronal-limited precursor cells (βIII-tubulin); mouse cell nuclei shown with DAPI. (E) hNSCs (Ku80) and neuronal-limited precursor cells (MAP-2); mouse cell nuclei shown with DAPI. (F)hNSC(Ku80) does not co-localize with the differentiated post-mitotic neuronal marker (NeuN).

[0019] [Figure 3]Figures 3A–3F show that embedding ESI-017 hNSCs reduces corticostriate excitability in R6 / 2 mice. (A) Biocitin-filled (arrow) hNSCs recorded in IHC in the striatum and SC121. Scale bar, 20 mm. (B) Upper trace: Recording of cellular attachment of spontaneously firing hNSCs. Lower trace: sEPSCs and sIPSCs from hNSCs. Recordings illustrate spontaneous internoclavicular and extracanal synaptic currents in hNSCs. (C) sEPSCs and sIPSCs recorded in MSNs. (D) Biocitin-filled MSNs near a cluster of hNSCs (SC121). Scale bar, 20 mm. (E) Recordings of sEPSCs in a subpopulation of R6 / 2 MSNs show “epileptic” activity after addition of bicuculine (10 mM), a GABAA receptor antagonist (first trace). Typically, these large-amplitude excitatory events are followed by high-frequency, small-amplitude sEPSCs. In mice with hNSC implantation, these events were significantly reduced in frequency (second trace). (F) In cells exhibiting “epileptic” activity (6-8 minutes after BIC), the R6 / 2 group with hNSC implantation showed a rightward shift in the probability distribution of the interval between events, corresponding to a significant reduction in high-frequency spontaneous events compared to the vehicle (p<0.001, two-way repeated measures ANOVA followed by Bonferroni post-hoc analysis; *p<0.05).

[0020] [Figure 4] Figures 4A-4B show that host-derived nerve terminals form synaptic connections with embedded hNSCs. (A) Unlabeled nerve terminals (U-NTs) contain synaptic vesicles and form synaptic-like connections with underlying labeled (SC121)hNSC dendrites (L-DENDs) (arrows). The connection may be symmetrical. (B) Unlabeled nerve terminals (U-NTs) contain synaptic vesicles and form asymmetrical synaptic connections with underlying labeled (SC121)hNSC dendrites (L-DENDs) (arrows). This asymmetric connection suggests an excitatory synaptic connection.

[0021] [Figure 5]Figures 5A–5G show that ESI-017 hNSCs implanted in Q140 mice improve behavior and present evidence of differentiation into immature neurons and astrocytes. (A) Transient improvement in motor coordination (pole task) 3 months after cell injection. WT Veh (n=20), Q140 Veh (n=18), Q140 hNSC (n=18). One-way ANOVA including Bonferroni post-hoc test: *p<0.05, **p<0.01. (B–D) Sustained improvement in running wheel deficit 5.5 months after treatment (n=5 / group). (B) Graph showing mean running wheel rotations / 3 minutes / night over 2 weeks in 7.5-month-old male WT or Q140 mice 5.5 months after treatment. Comparison by two-way ANOVA: group effect F=52.93, p<0.0001; nocturnal wheel effect F=17, p<0.0001. Bonferroni post-hoc test: *p<0.01, **p<0.001, and ***p<0.0001 compared to Q140 Veh. (C) Total mean nocturnal wheel rotations over two weeks. Two-way ANOVA including Bonferroni post-hoc test: *p<0.01, **p<0.001. (D) Slope of motor learning that is not significant among the three groups. (E and F) Novel object recognition. hNSC prevented deficits in Q140 mice at 5 months after treatment in the discriminative index of odor-smelling behavior time (E) or number of bouts (F), but not at 3 months. WT Veh n=18, Q140 Veh n=18, and Q140 hNSC n=19. One-way ANOVA with Bonferroni post-hoc test: *p<0.05, **p<0.01. (G) Survival and differentiation of hNSCs in Q140 mice by staining with human-specific antibodies (HNA; a and d) co-expressing with astrocytes (GFAP; b and c) or neuronal-limited precursor cells (DCX; e and f). Scale bar, 20 mm. All graphs show mean ± SEM.

[0022] [Figure 6]Figures 6A–6D show that ESI-017 hNSCs implanted in HD mice increase BDNF expression. (A) ESI-017 hNSCs (Ku80) show co-localization with BDNF; astrocytes are shown as GFAP-positive. (B) Veh-treated mice do not show BDNF or hNSCs but have GFAP. (C) BDNF levels by ELISA in the striatum of Q140 or WT mice 6 months after implantation. (D) hNSC treatment in Q140 mice reduced microglial activation. Data are presented as mean + 95% confidence interval (n=5 / group). Bars represent the percentage of cells for each diameter, and the gray area represents the confidence interval. Significant striatal microglial activation was observed in Q140 Veh compared to WT Veh. Q140 hNSC mice showed a significant reduction in microglial activation in the striatum compared to Q140 Veh mice. One-way ANOVA, including Bonferroni post-hoc tests, showed *p<0.05 and **p<0.01. The graph shows the mean ± SEM.

[0023] [Figure 7]Figures 7A–7F show that ESI-017 hNSCs implanted in R6 / 2 mice induce a reduction in scattered aggregates and inclusions and a reduction in huntingtin aggregates in Q140 mice. (A and B) ESI-017 hNSCs induce a reduction in scattered aggregates and inclusions in R6 / 2 mice (arrows in A). (A) Images of Ku80 using nickel, HTT marker EM48, and cresyl violet for non-hNSC nuclear staining. Stereoanalytic evaluation performed using StereoInvestigator. Contour drawing under a 53 objective lens (dotted line, example in the left panel) and counting at 1003. For six sections across the entire striatum, every three sections were counted (40 mm coronal section), in which case Ku80 can be seen between bregma 0.5 mm and bregma 0.34 mm. (B) Graph depicting the percentage of cells with aggregates or inclusions (n=4 / group), one-way ANOVA with Bonferroni post-hoc test, **p<0.01. (C and D) ESI-017 hNSCs reduce huntingtin aggregates in Q140 mice. (C) Image of HTT marker EM48 (arrows indicate inclusions). (D) HTT-stained nuclei and aggregates were analyzed with StereoInvestigator for aggregate type / section quantification. Data are shown as mean ± SEM (n=5 / group). One-way ANOVA with Bonferroni post-hoc test, *p<0.05. (E and F) hNSC transplantation modulates insoluble protein accumulation in R6 / 2 mice. Western blots of striatal lysate products were separated into surfactant-soluble and surfactant-insoluble fractions. (E) R6 / 2 mice enriched with insoluble accumulated mHTT compared to NT mice. hNSC transplantation in R6 / 2 mice results in a significant reduction in HTT with accumulated insoluble HMW compared to veh-treated animals. R6 / 2 striatum are also enriched with insoluble ubiquitin conjugate protein compared to NT mice. hNSC transplantation in R6 / 2 mice results in a significant reduction in ubiquitin-modified insoluble conjugate protein compared to veh treatment, and there is no significant effect in NT compared to veh controls. (F) Quantification of relative protein expression of mHTT and ubiquitin.Values ​​represent mean ± SEM. The statistical significance of relative insoluble accumulated mHTT and ubiquitin-conjugated protein expression in R6 / 2 was determined using one-way ANOVA followed by Bonferroni post-hoc tests (n=3 / treatment). *p<0.05, **p<0.01, ***p<0.001. Graphs show mean ± SEM.

[0024] [Figure 8-1] Figures 8A–8D show the characterization of ESI-017 hNSCs by monochromatic flow cytometry. (A) Staining of ESI-017 hNSCs positive for CD24, SOX1, SOX2, Nestin, and Pax6 NSC markers. Staining of ESI-017 hNSCs negative for the pluripotency marker SSEA4. Karyotyping analysis was performed on ESI-017 hNSCs, and metaphase was visualized by Giemsa staining of enriched chromosomes. The final karyotype was shown to have a high mitotic index along with a normal profile of 46 XX. (B) Flowchart of the NSC production process: hNSCs are produced by embryoid body (EB) formation, followed by plating of the generated EBs onto poly-ornithine-laminin (Poly-O) coated plates, and then neural rosette formation. The rosettes are manually cut and transferred to fresh Poly-O plates where they are attached. Next, the expanded neural rosettes are enzymatically cleaved and then plated onto fresh poly-O plates. The cells are then grown to confluence and enzymatically subcultured onto more poly-O plates. After expanding to a sufficient number, the resulting hNSCs are finally harvested and cryopreserved. (C) Immunocytochemistry of cultured ESI-017 hNSCs shows positive NSC staining and DAPI nuclear staining for Nestin, a stem cell marker for neuroectoderm. Scale bar equals 30 μm. (D) is a photograph of the rosette. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0025] [Figure 9]Figure 9 shows clasping behavior, demonstrating that R6 / 2 mice (n=15) treated with ESI-017 hNSCs exhibit delayed clasping behavior after implantation. Non-transgenic (NT) mice do not demonstrate this phenotype. Mice were tested daily for phenotype, and the graph depicts the percentage of each group exhibiting clasping over the course of the study. The significance of the clasping assay was determined by Fisher's exact test.

[0026] [Figure 10] Figures 10A–10E show low-magnification immunohistochemistry of ESI-017. hNSC-embedded R6 / 2 mice: hNSCs (human marker SC121) embedded in R6 / 2 mice co-localize with the marker for neuronal-limited precursor cells (double cortin DCX). To screen for hNSCs, IHC is performed on section numbers 34, 37, 40, 43, 46, and 49 (equal to bregma 0.38 mm, 0.26 mm, 0.14 mm, 0.02 mm, -0.10 mm, and -0.22 mm, respectively). S2 is a reuse of the image shown in Figure 1D for comparison with other coronal sections. ESI-017 hNSC embedding in R6 / 2 mouse immunohistochemistry: (A) hNSCs (human marker Ku80) embedded in R6 / 2 mice do not co-localize with the oligodendrocyte marker (Olig2) mouse cell nucleus, shown together with DAPI. High magnification (63×) showing differentiation: (B) hNSCs (human nuclear marker Ku80 and cytoplasmic marker SC121 blue) show co-localization (lit. blue) with neuronal-limited precursor cells (BIII-tubulin). (C) hNSCs (human nuclear marker Ku80 and cytoplasmic marker SC121) show co-localization with neuronal-limited precursor cells (MAP-2). (D) hNSCs (human nuclear marker Ku80) do not co-localize with huntingtin marker (EM48). (E) S1-6 shows collected and immunostained coronal sections, starting at bregma 1.70 mm and 40 μm per section.

[0027] [Figure 11]Figures 11A–11B show that ESI-017 hNSCs implanted in the striatum did not improve deficits in Q140 mice in open-field or cage climbing tests. Mice were tested for 15 minutes in open field (A) and 5 minutes in cage climbing (B) 0.5 months prior to implantation, or 3 and 5 months after implantation. Data are expressed as mean ± SEM; Wt Veh (n=18), Q140 Veh (n=18), and Q140 hNSC (n=17). In two-way ANOVA including Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 compared to vehicle-treated Wt mice at the same time point.

[0028] [Figure 12] Figures 12A-12C show in vitro BDNF expression in ESI-017 hNSCs. ESI-017 hNSCs were cultured in neural stem cell medium (A) or differentiated (B), and then stained for the BDNF human nuclear marker Ku80 and doublecortin DCX (C). qPCR comparing RNA levels from cultured ESI-017 hNSCs showed increased BDNF expression with differentiation. In comparison, the stem cell marker Nestin decreased with differentiation, while DCX increased.

[0029] [Figure 13]Figures 13A–13E show that (A and B) synaptophysin levels increase in the striatum of Q140 mice with ESI-017 hNSCs. (A) Images were taken with a microarray scanner and quantified for fluorescence intensity. White scale bars equal 10 μm. (B) Data are shown as mean ± SEM, and the statistical test used was one-way ANOVA with Bonferroni post-hoc test, *p<0.05, n=5 mice / group. hNSC treatment in R6 / 2 mice does not alter microglial activation. Data are expressed as mean + 95% confidence interval (n=5 / group). Bars represent cell percentages for each diameter, and colored portions represent confidence intervals. (C) Significant striatal microglial activation observed in vehicle-treated R6 / 2 mice (R6 / 2 Veh) compared to non-transgenic controls (NT Veh). (D) Comparison of NT+vehicle and NT+hNSC. (E) R6 / 2 mice treated with hNSC (R6 / 2 NSC) did not show a significant reduction in microglial activation in the striatum compared to R6 / 2 Veh mice.

[0030] [Figure 14] Figure 14 shows real-time PCR of human HTT transgene expression in R6 / 2 mice. Differences in gene expression in cDNA samples were normalized using RPLPO (large ribosomal protein) endogenous control. No significance was observed when determined by one-way ANOVA including Bonferroni post-hoc test.

[0031] [Figure 15]Figures 15A–15F show that R6 / 1 mice at 5 weeks of age were injected with AAV expressing sApiCCT1 or mCherry control into the bilateral striatum. In two separate experiments, mice were injected with 12 × 10⁹ genomic copies of AAV2 / 1 and harvested at 17 weeks of age. (A) Schematic diagram. (B, C) Agarose gel electrophoresis and subsequent Western blot quantification show a significant reduction in oligomeric mHTT in the animals. (D) Immunohistochemistry shows sApiCCT1 (anti-HA) expression. (E) Mice injected with sApiCCT1 show approximately 40% reduction in visible mHTT inclusions (anti-EM48) by stereochemistry. (F) Mice injected with sApiCCT1-AAV2 / 1 show improvement in the rotarod movement task, *p<0.05, **p>0.01.

[0032] [Figure 16]Figures 16A–16D show that ESI-017 hNSCs produce ApiCCT. (A) ESI-017 hNSCs transduced with sApiCCT lentivirus at MOI of 0, 5, 10, or 15 were cultured for 48 hours post-transduction, lysed, and Western blotted with HA antibody, then stripped and re-probed with alpha-tubulin antibody for loading controls. (B) ApiCCT secreted from hNSCs enters PC12 Htt14A2.6 cells. Conditional media from ESI-017 hNSCs transduced with sApiCCT lentivirus were applied to 14A2.6 cells induced by ponasterone in EtOH to express HTT-GFP or controls treated with EtOH alone. ApiCCT1 was detected in cell lysates, supporting the feasibility of manipulating hNSCs to express a secreted form of ApiCCT1 that can be taken up by adjacent cells after transplantation. Western blots using HA antibody are shown. In treated PC12 cell lysates, higher MOI and greater amounts of ApiCCT1 were detected. (C) ApiCCT1 secreted from hNSCs did not alter monomeric HTT in PC12 Htt14A2.6 cells. Conditional medium from ESI-017 hNSCs transduced with sApiCCT lentivirus was applied to ponasterone-induced 14A2.6 cells or controls treated with EtOH alone. Treatment with secreted ApiCCT1 did not cause alteration in the monomeric mHTT-GFP transgene. Western blots using GFP antibody are shown, followed by stripping and re-probing for alpha-tubulin as a loading control. (D) ApiCCT1 secreted from hNSCs alters oligomeric HTT species in PC12 Htt14A2.6 cells. Conditional media from ESI-017 hNSCs transduced with sApiCCT1 lentivirus and applied to ponasterone-induced 14A2.6 cells or controls treated with EtOH alone resulted in a reduction of oligomeric HTT at the maximum MOI (red box). Western blots of representative samples using GFP antibody are shown.

[0033] [Figure 17] Figures 17A and 17B show that IHC demonstrates that the virus for ApiCCT is transduced and that ESI-017 hNSCs embedded in the striatum of R6 / 2 mice express ApiCCT. (A) hNSCs (human nuclear antigen [HNA]) embedded in R6 / 2 mice differentiate into neuronal-limited precursor cells (doublecortin [DCX]) and express HA-tagged ApiCCT (HA). (B) High magnification (95×) taken from the area in the white box indicated by A, showing differentiation and ApiCCT expression: hNSCs (HNA) embedded in R6 / 2 mice differentiate into neuronal-limited precursor cells (DCX) and express HA-tagged ApiCCT (HA). [Modes for carrying out the invention]

[0034] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods, devices, and materials are described herein. All technical and patent publications referenced herein are incorporated herein by whole by reference. Nothing herein should be construed as an acknowledgment that the present invention does not have prior rights to such disclosures for the sake of prior art.

[0035] Throughout this application and within this application, technical and patent documents are referenced by means of reference. For some of these references, the identification citation is found at the end of this application, immediately preceding the claims. All publications are incorporated into this disclosure by means of reference to more fully illustrate the level of the art to which this disclosure pertains.

[0036] Unless otherwise indicated, the implementation of this disclosure will utilize conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the scope of the art. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition;the series Ausubel et al. eds. (2007) CurrentProtocols in MolecularBiology;the series Methods in Enzymology (AcademicPress, Inc., NY);MacPhersonet al. (1991) PCR 1: A Practical Approach (IRLPress at Oxford University Press);MacPhersonet al. (1995) PCR 2: A PracticalApproach;Harlow and Lane eds. (1999)Antibodies, A Laboratory Manual;Freshney(2005) Culture of Animal Cells: AManual of Basic Technique, 5 th edition;Gait ed. (1984)Oligonucleotide Synthesis;US Patent No. 4,683,195;Hames and Higgins eds. (1986));Perbal (1984) A Practical Guide toMolecular Cloning;Miller andCalos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makridesed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);Herzenberget al. eds (1996) Weir's Handbook of Experimental Immunology;Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd See edition (Cold Spring HarborLaboratory Press (2002)); Sohail (ed.) (2004) GeneSilencing by RNAInterference: Technology and Application (CRC Press).

[0037] All numerical specifications, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary by (+) or (-) in increments of 0.1 or 1.0, as appropriate. It should be understood that, although not always explicitly stated, all numerical specifications are preceded by the term "approximately." It should also be understood that, although not always explicitly stated, the reagents described herein are merely illustrative, and equivalents of such reagents are known in the art.

[0038] As used herein and in the claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise. For example, the term “a cell” refers to multiple cells, including mixtures thereof.

[0039] Where used herein, the terms “comprising” or “comprises” are intended to mean that a composition and method includes the elements described but does not exclude other elements. “Essentially consisting of” when used to define a composition and method shall mean excluding other elements that have any essential significance to the combination for the stated purpose. Thus, a composition essentially consisting of the elements defined herein does not exclude trace impurities from isolation and purification methods, as well as pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, etc. “Consists of” shall mean excluding elements of other components in amounts greater than trace amounts, and substantial method steps for administering the composition of the present invention or process steps for producing the composition or achieving the desired result. Embodiments defined by each of these transitional terms are within the scope of the present invention.

[0040] When used herein in relation to nucleic acids such as DNA or RNA, the term “isolated” refers to a molecule separated from other DNA or RNA present in the natural source of the respective macromolecular weight. The term “isolated nucleic acid” includes nucleic acid fragments that do not exist naturally as fragments and cannot be found in their natural state. The term “isolated” is also used herein to refer to polypeptides, proteins, and / or host cells isolated from other cellular proteins, and includes both purified polypeptides and recombinant polypeptides. In other embodiments, the term “isolated” means that cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof have been separated from cells and other components that normally associate in nature. For example, isolated cells are cells separated from tissues or cells of a different phenotype or genotype. As will be apparent to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that do not exist naturally do not require “isolation” to distinguish them from their naturally occurring counterparts.

[0041] The term "isolation" refers to the process of separating a composition or component from other things that are very close to it or associated with it. Cells can be isolated manually (e.g., by hand using a pipette or other tool), enzymatically by the use of chemical agents, or digitally by the use of digital techniques based on cellular or rosette morphology. See, for example, cellavision.com / en / introducing-digital-cell-morphology-by-cellavision (accessed May 22, 2018).

[0042] A "differentiation medium" refers to a cell culture medium containing certain growth factors or other factors that promote the differentiation of immature cells into more mature phenotypes, such as the differentiation of embryonic stem cells into neural cells.

[0043] As used herein, the term “confluent population” refers to a population of cells that are in continuous contact with neighboring cells.

[0044] In some embodiments, "ultra-low adhesion surfaces" refer to cell or tissue culture surfaces containing a hydrophilic and charge-neutral covalently bonded hydrogel layer. Because proteins and other biomolecules passively adsorb to the polystyrene surface via either hydrophobic or ionic interactions, this hydrogel surface naturally inhibits nonspecific fixation via these forces and therefore inhibits subsequent cell adhesion. These surfaces are commercially available from various vendors, such as Millipore-Sigma, Fisher-Scientific, and S-bio. Methods relating to the manufacture of cell culture plates and surfaces are known in the art.

[0045] A "transgene" refers to a polynucleotide that has been added to a cell, tissue, or organism. An example of a transgene is ApiCCT1.

[0046] "ApiCCT1" refers to the apical domain of CCT1 and / or the polynucleotide encoding the said apical domain of CCT1 (incorporated herein by reference, Sontag, E. Proc Natl Acad Sci US A. 2013 Feb 19;110(8):3077-82). ApiCCT1 is a molecular chaperone that is a member of the TCP1-containing chaperonin complex (CCT), also known as the TCP1 ring complex (TRiC). This complex consists of two identical stacked rings, each containing eight different proteins. Unfolded polypeptides enter the cavity at the center of the complex and fold in an ATP-dependent manner. The complex folds various proteins, including actin and tubulin. In some embodiments, ApiCCT1 is 20 kDa in size. In humans, the TCP1 ring complex is encoded by the TCP1 gene (Entrez gene 6950). Non-limiting examples of TCP1 mRNA and protein sequences are provided herein as Sequence IDs 1-4. The apical domain is involved in substrate binding. (Incorporated herein by reference, Pappenberger, G. et al. J Mol Biol. 2002 May 17;318(5):1367-79). Non-limiting examples of ApiCCT1 sequences A typical example is provided below (SEQ ID NO: 7): [ka]

[0047] "sApiCCT1" refers to the secreted form of ApiCCT1. Non-limiting examples of nucleic acid and amino acid sequences of sApiCCT are provided below. Underlined sequences correspond to the HA tag. In some embodiments, sApiCCT1 does not include a tag. sApiCCT1 mRNA (SEQ ID NO: 8) [ka] sApiCCT1 peptide (SEQ ID NO: 9) [ka]

[0048] As used herein, “BDNF” refers to brain-derived neurotrophic factor (BDNF) and its equivalents, and / or polynucleotides encoding BDNF or its equivalents. BDNF acts on neurons in the central and peripheral nervous systems to support the survival of existing neurons and to promote the growth and differentiation of new neurons and synapses. BDNF is also active in the hippocampus, cortex, and basal forebrain—regions essential for learning, memory, and higher-order thinking. It is also expressed in the retina, motor neurons, kidneys, saliva, and prostate. The BDNF protein is encoded by the BDNF gene (Entrez gene: 627, mRNA: NM_001143805, NM_001143806, NM_001143807, NM_001143808, NM_001143809, NM_001143810, NM_001143811, NM_001143812, NM_001143813, NM_001143814, NM_001143815, NM_001143816, NM_001709, NM_170731, NM_170732, NM_170733, NM_170734, NM_170735). Non-limiting examples of BDNF mRNA and protein sequences are provided herein as SEQ ID NOs. 5-6.

[0049] As used herein, the term “CRISPR” refers to a sequence-specific gene engineering technique that relies on clustered, regularly spaced, short, palindromic repeat sequence pathways. CRISPR can be used to perform gene editing and / or gene regulation, as well as simply to target proteins to specific genomic locations. Gene editing refers to a type of genetic engineering in which the nucleotide sequence of a target polynucleotide is altered by introducing deletions, insertions, or base substitutions into the polynucleotide sequence. In some embodiments, CRISPR-mediated gene editing utilizes non-homologous end joining (NHEJ) or homologous recombination pathways to perform the editing. Gene regulation refers to increasing or decreasing the production of a specific gene product, such as a protein or RNA.

[0050] The terms “gRNA” or “guide RNA,” as used herein, refer to guide RNA sequences used to target specific genes for modification using CRISPR techniques. Techniques for designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J., et al. Nature biotechnology 2014; 32(12):1262-7, Mohr, S. et al. (2016) FEBSJournal 283: 3232-38, and Graham, D., et al. Genome Biol. 2015; 16: 260. gRNAs include, or alternatively essentially, a fusion polynucleotide containing CRISPR RNA (crRNA) and transactivated CRISPR RNA (tracrRNA), or a polynucleotide containing CRISPR RNA (crRNA) and transactivated CRISPR RNA (tracrRNA). In some embodiments, gRNA is synthetic (Kelley, M. et al. (2016) J of Biotechnology 233 (2016) 74-83). As used herein, bioequivalents of gRNA are not limited to these. Examples include polynucleotides or targeting molecules that can direct Cas9 or its equivalent to specific nucleotide sequences, such as specific regions of the cell genome.

[0051] CRISPR expression in cells can be achieved using conventional CRISPR / Cas systems and guide RNAs specific to target genes in cells. Suitable expression systems, such as lentiviral or adenoviral expression systems, are known in the art. It is further understood that CRISPR editing constructs can be useful for both knocking out endogenous genes and knocking in genes. Therefore, it is understood that CRISPR systems can be designed to achieve one or both of these objectives.

[0052] As is well known to those skilled in the art, there are six classes of viruses. DNA viruses constitute classes I and II. RNA viruses and retroviruses make up the remaining classes. Class III viruses have a double-stranded RNA genome. Class IV viruses have a single-stranded positive-sense RNA genome, which itself functions as mRNA. Class V viruses have a single-stranded negative-sense RNA genome that is used as a template for mRNA synthesis. Class VI viruses have a single-stranded positive-sense RNA genome, but they contain DNA intermediates not only in replication but also in mRNA synthesis. Retroviruses carry genetic information in the form of RNA, but when a retrovirus infects a cell, its RNA is reverse-transcribed into a DNA form that is integrated into the genomic DNA of the infected cell. This integrated DNA form is called a provirus.

[0053] The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably and refer to polymeric forms of nucleotides of any length, which are either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs. Modifications of nucleotide structure, if present, can be conjugated before or after the assembly of polynucleotides. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeling components. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present invention that is a polynucleotide encompasses both a double-stranded form and two complementary single-stranded forms that are known or predicted to form a double-stranded form.

[0054] A polynucleotide consists of distinct sequences of four types of nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) instead of thymine. Therefore, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database on a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searches.

[0055] "Homologie," or "identity," or "similarity," refers to the sequence similarity between two peptides or two nucleic acid molecules. Homologie can be determined by comparing the positions of each sequence that can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "Irrelevant" or "non-homologous" sequences share less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.

[0056] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence means that, when aligned, that percentage of bases (or amino acids) are identical when comparing the two sequences. This alignment, and percentage homology or sequence identity, is known in software programs known in the art, for example, the software programs described in Ausubelet al. eds. (2007) Current Protocols in Molecular Biology. This can be determined using a program. Preferably, initial parameters are used for alignment. One alignment program is BLAST, which uses initial parameters. In particular, the programs BLASTN and BLASTP use the following initial parameters: genetic code = standard, filter = none, strand = 2, cutoff = 60, expected value = 10, matrix = BLOSUM62, display = 50 sequences, sorting = high score, database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0057] An equivalent or bioequivalent nucleic acid, polynucleotide, oligonucleotide, or peptide is one that has at least 80% sequence identity with a reference nucleic acid, polynucleotide, oligonucleotide, or peptide, or alternatively at least 85% sequence identity, or alternatively at least 90% sequence identity, or alternatively at least 92% sequence identity, or alternatively at least 95% sequence identity, or alternatively at least 97% sequence identity, or alternatively at least 98% sequence identity.

[0058] The term "polynucleotide amplification" includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR), and amplification methods. These methods are publicly known and widely practiced in the art. See, for example, U.S. Patent Nos. 4,683,195 and 4,683,202, as well as Inniset al., 1990 (regarding PCR), and Wu et al. (1989) Genomics 4:560-569 (regarding LCR). In general, PC The R procedure describes a method of gene amplification comprising (i) sequence-specific hybridization of primers to individual genes in a DNA sample (or library), (ii) subsequent amplification with multiple rounds of annealing, extension, and denaturation using DNA polymerase, and (iii) screening the PCR product for bands of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to initiate polymerization; that is, each primer is specifically designed to be complementary to each strand of the genomic locus being amplified.

[0059] Reagents and hardware for performing PCR are commercially available. Primers useful for amplifying sequences from specific gene regions are preferably complementary to the target region or adjacent regions and specifically hybridize. The nucleic acid sequences produced by amplification may be directly sequenced, or the amplified sequences may be sequence-analyzed after cloning. Methods for direct cloning and sequence-analysis of enzyme-amplified genomic segments are known in the art.

[0060] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that can encode a specific polypeptide or protein after transcription and translation.

[0061] The term "express" refers to the production of a gene product.

[0062] As used herein, “expression” refers to the process by which a polynucleotide is transcribed into mRNA, and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide originates from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0063] A "gene product," or alternatively, a "gene expression product," refers to an amino acid (e.g., a peptide or polypeptide) produced when a gene is transcribed and translated.

[0064] "Transcriptionally regulated" is a term well understood in the art, and it indicates that the transcription of a polynucleotide sequence, typically a DNA sequence, depends on the operative ligation of the sequence to an element that contributes to the initiation of transcription or promotes transcription. "Operationally ligated" means that the polynucleotide is positioned in a way that allows it to function in a cell. In one embodiment, the present invention provides a promoter that operatively ligates to a downstream sequence, such as a suicide gene, a polynucleotide encoding ApiCCT1, a fragment thereof such as sApiCCT1, or their respective equivalents.

[0065] The term “coding,” when applied to polynucleotides, refers to a polynucleotide that, in its original state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA of a polypeptide and / or its fragments. The antisense strand is the complementary strand of such nucleic acid, from which the coding sequence can be derived.

[0066] When used in the context of polynucleotide manipulation, "probe" refers to an oligonucleotide provided as a reagent for detecting a potentially present target in a sample of interest by hybridizing with the target. Typically, a probe may include a detectable label or means that allow the label to adhere either before or after the hybridization reaction. Alternatively, a "probe" may be a biocompound capable of binding to a potentially present target in the sample of interest, such as a polypeptide, antibody, or a fragment thereof.

[0067] Examples of "detectable labels" or "markers" include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins containing enzymes. Detectable labels can also be attached to polynucleotides, polypeptides, antibodies, or compositions as described herein.

[0068] A “primer” is generally a short polynucleotide having a free 3'-OH group that binds to a potentially present target or “template” in a sample of interest by hybridizing with the target, and subsequently promotes the polymerization of a polynucleotide complementary to the target. A “polymerase chain reaction” (“PCR”) is a reaction in which a replicate copy is made from a target polynucleotide using a “pair of primers” or “a set of primers” consisting of “upstream” and “downstream” primers, and a polymerization catalyst, such as DNA polymerase, and typically a thermostable polymerase enzyme. Methods for PCR are well known in the art and are taught, for example, in MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press). All processes for producing replicate copies of polynucleotides, such as PCR or gene cloning, are collectively referred to as “replication” herein. Primers can also be used as probes in hybridization reactions such as Southern or Northern blot analysis. See Sambrook and Russell (2001) below.

[0069] Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can arise by Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific method. The complex may consist of two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridized strand, or any combination thereof. Hybridization reactions may constitute a step in a broader process, such as the initiation of a PCR reaction or the enzymatic cleavage of polynucleotides by a ribozyme.

[0070] Hybridization reactions can be carried out under different "stringency" conditions. Generally, low-stringency hybridization reactions are carried out at approximately 40°C in 10×SSC or in a solution of equivalent ionic strength / temperature. Moderate-stringency hybridization is typically carried out at approximately 50°C in 6×SSC, and high-stringency hybridization reactions are generally carried out at approximately 60°C in 1×SSC. Additional examples of stringent hybridization conditions include low-stringency incubation temperatures of approximately 25°C to 37°C, hybridization buffer concentrations of approximately 6×SSC to 10×SSC, formamide concentrations of approximately 0% to 25%, and washing solutions of approximately 4×SSC to 8×SSC. Examples of moderate hybridization conditions include incubation temperatures of approximately 40°C to 50°C, buffer concentrations of approximately 9×SSC to 2×SSC, formamide concentrations of approximately 30% to 50%, and washing solutions of approximately 5×SSC to 2×SSC. Examples of high stringency conditions include incubation temperatures of approximately 55°C to 68°C, buffer concentrations of approximately 1×SSC to 0.1×SSC, formamide concentrations of approximately 55% to 75%, and washing solutions of approximately 1×SSC, 0.1×SSC, or deionized water. In general, the hybridization incubation time, including one, two, or more washing steps, is 5 minutes to 24 hours, and the washing incubation time is approximately 1, two, or 15 minutes. SSC is 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems may be used. Hybridization reactions can also be carried out under “physiological conditions” that are well known to those skilled in the art. Non-limiting examples of physiological conditions include temperature, ionic strength, pH, and Mg, which are typically found in cells. 2+ This is the concentration.

[0071] When hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration, the reaction is called "annealing," and the polynucleotides are described as "complementary." Double-stranded polynucleotides can be "complementary" or "homologous" to another polynucleotide if hybridization can occur between one strand of the first polynucleotide and the second polynucleotide. "Complementarity" or "homonymy" (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion of bases on opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base-pairing rules.

[0072] The terms “increase” or “grow” mean to make cells or a group of cells grow. The term “grow” also refers to the proliferation of cells in the presence of maintenance medium, nutrients, growth factors, supporting cells, or any chemical or biocompound necessary to obtain a desired number of cells or cell type.

[0073] The term "culturing" refers to the in vitro growth of cells or organisms on or in various types of culture media. It is understood that offspring of cells grown in culture may not be entirely identical to the parent cells (i.e., morphologically, genetically, or phenotypically).

[0074] As used herein, the term “vector” refers to a non-chromosomal nucleic acid containing a complete replicon so that the vector can be replicated, for example, when placed in a cell by a transformation process. Vectors may be viral or nonviral. Viral vectors include retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papoviruses, and Examples of nonviral vectors for nucleic acid delivery include: naked DNA; DNA complexed with cationic lipids, either alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles containing heterogeneous polylysine, oligopeptides of a defined length, and cationic polymers such as polyethyleneimine, sometimes contained in liposomes; and the use of ternary complexes containing viruses and polylysine-DNA.

[0075] A "viral vector" is defined as a recombinantly produced virus or viral particle containing polynucleotides that are delivered to host cells in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and alphaviral vectors. Alphaviral vectors, such as those based on Semryki Forest virus and Sindbis virus, are also being developed for use in gene therapy and immunotherapy. (Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439, and Ying, et al. (1999) Nat.) See Med. 5(7):823-827.

[0076] In embodiments where gene transfer is mediated by a lentiviral vector, the vector construct refers to a polynucleotide containing the lentiviral genome or a portion thereof and the therapeutic gene. As used herein, “lentiviral-mediated gene transfer” or “lentiviral transduction” are synonymous and refer to the process by which a gene or nucleic acid sequence is stably transferred into a host cell in order for the virus to enter the cell and integrate its genome into the host cell genome. Viruses can enter host cells via normal infection mechanisms or can be modified to enter cells by binding to different host cell surface receptors or ligands. Retroviruses carry genetic information in the form of RNA, but when a retrovirus infects a cell, its RNA is reverse-transcribed into a DNA form that is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, a lentiviral vector refers to a viral particle that can introduce exogenous nucleic acids into cells via a virus or virus-like entry mechanism. A “lentiviral vector” is a type of retroviral vector well known in the art that has certain advantages in transduction into non-dividing cells compared to other retroviral vectors. See Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag BerlinHeidelberg.

[0077] The lentiviral vectors of the present invention are based on or derived from onchoretroviruses (a subgroup of retroviruses containing MLV) and lentiviruses (a subgroup of retroviruses containing HIV). Examples include ASLV, SNV, and RSV, all of which are divided into packaging and vector components for lentiviral vector particle production systems. The lentiviral vector particles of the present invention may also be based on specific retroviruses in genetically or otherwise modified forms (e.g., by specific selection of packaging cell systems).

[0078] The statement that the vector particles of this invention are "based on" a specific retrovirus means that the vector originates from that specific retrovirus. The genome of the vector particle contains components from that retrovirus as its backbone. The vector particle contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Typically, essential vector components include gag and pol proteins derived from a specific retrovirus. Therefore, while most of the structural components of the vector particle are usually considered to originate from that retrovirus, they may be modified genetically or otherwise to achieve desired useful properties. However, certain structural components, particularly the env protein, may originate from a different virus. The range of vector host and cell type to be infected or transduced can be modified by using different env genes in the vector particle production system to give the vector particles different specificities.

[0079] The term "promoter" refers to a region of DNA that initiates the transcription of a particular gene. Promoters include the core promoter, which is the smallest part of the promoter necessary for proper transcription initiation, and may also include regulatory elements such as transcription factor binding sites. Regulatory elements may either promote or inhibit transcription. Regulatory elements in a promoter may be binding sites for transcription activators or repressors. Promoters can be constitutive or inductive. A constitutive promoter is one that is always active and / or constantly directs the transcription of a gene beyond basal level transcription. Non-exclusive examples of such promoters include the phosphoglycerin kinase 1 (PGK) promoter; SSFV, CMV, MNDU3, SV40, Ef1a, UBC, and CAGG. An inductive promoter is one that can be induced by a molecule or factor attached to or expressed in a cell. Inductive promoters can still produce basal level transcription in the absence of induction, but induction typically results in significantly greater protein production than in its absence. Promoters can also be tissue-specific. Tissue-specific promoters enable protein production in a specific population of cells that possess the appropriate transcription factors to activate the promoter.

[0080] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing a sequence that can perform both promoter and enhancer functions. For example, the long terminal repeat sequences of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous," "exogenous," or "heterogeneous." An "endogenous" enhancer / promoter is an enhancer / promoter that naturally aligns with a given gene in the genome. An "exogenous" or "heterogeneous" enhancer / promoter is an enhancer / promoter that is placed proximal to a gene by genetic engineering (i.e., molecular biological techniques) so that the transcription of the gene is directed by the aligning enhancer / promoter.

[0081] As used herein, “stem cells” define cells that have the ability to divide indefinitely in culture to produce specialized cells. For convenience of explanation, stem cells are classified as somatic (adult) or embryonic. Somatic stem cells are undifferentiated cells found in differentiated tissues that can self-replicate (clonal) and differentiate (with certain limitations) to produce all the specialized cell types of the tissue from which they originate. Embryonic stem cells are primitive (undifferentiated) cells of embryonic origin that have the potential to become a wide variety of specialized cell types. Embryonic stem cells are cells cultured in vitro under conditions that allow for undifferentiated proliferation for several months to several years. A clone is a line of cells that is genetically identical to the originating cell, in this case the stem cell.

[0082] A "stem cell rosette" refers to a cluster of stem cells that resembles a group of flower petals under proliferative conditions. See Figure 8D, for example.

[0083] A population of cells represents a set of two or more cells that are phenotypic and / or genotypic identical (clonal) or non-identical. A substantially homogeneous population of cells is one that, as measured by a pre-selected marker, has phenotypic identity of at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98%.

[0084] As used herein, “embryonic stem cells” refers to stem cells derived from tissue formed after fertilization and before late pregnancy, including pre-embryonic tissue (e.g., blastocyst), embryonic tissue, or fetal tissue obtained at any time during pregnancy, but not necessarily, typically before approximately 10-12 weeks of gestation. In most cases, embryonic stem cells are pluripotent cells derived from an early embryo or blastocyst. Embryonic stem cells can be obtained directly from preferred tissues, including but not limited to human tissue, or from established embryonic cell lines. “Embryonic stem cells” refers to cells that share one, but not all, of the characteristics of embryonic stem cells.

[0085] Neural stem cells are cells that can be isolated from the adult central nervous system of mammals, including humans. These cells have been shown to generate neurons, migrate, extend axons and dendrites, integrate into existing neural circuits, and contribute to normal brain function. An overview of research in this field can be found in Miller (2006) *The Promise of Stem Cells for Neural Repair*, BrainRes. Vol. 1091(1):258-264;Pluchino et al. (2005) Neural Stem Cells and TheirUse asTherapeutic Tool in Neurological Disorders, Brain Res. Brain Res. Rev.,Vol.48(2):211-219; and Goh, et al. (2003) Adult Neural Stem Cells and Repair of theAdult Central Nervous System, J. See Hematother. Stem Cell Res., Vol.12(6):671-679. It will be released.

[0086] "Differentiation" refers to the process by which unspecialized cells acquire the characteristics of specialized cells, such as heart, liver, or muscle cells. "Directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a specific cell type. "Dedifferentiated" defines a cell that returns to a less relevant position in the cell lineage. As used herein, the terms "differentiating" or "differentiated" define a cell that takes on a more relevant ("differentiated") position in the cell lineage. As used herein, "cells that differentiate into a mesoderm (or ectoderm or endoderm) lineage" define cells that become involved in the respective mesoderm, ectoderm, or endoderm lineages. Examples of cells that differentiate into a mesoderm lineage or give rise to individual mesoderm cells include, but are not limited to, cells that are adipogenic, smooth myogenic, chondrogenic, cardiogenic, dermatogenic, hematopoietic, angiogenic, myogenic, nephrogenic, urogenital, osteogenic, pericardiogenic, or stromal.

[0087] As used herein, the terms “differentiated” or “differentiated” define a cell that takes on a more relevant (“differentiated”) position in the cell lineage. “Dedifferentiated” defines a cell that returns to a less relevant position in the cell lineage. Induced pluripotent stem cells are an example of dedifferentiated cells.

[0088] As used herein, the “lineage” of a cell defines the heredity of a cell, i.e., the ancestors and descendants of that cell. The lineage of a cell places the cell within the genetic system of development and differentiation.

[0089] "Multilineage stem cells" or "pluripotent stem cells" refer to stem cells that regenerate and produce at least two types of progeny cells further differentiated from distinct developmental lineages. These lineages may originate from the same germ layer (i.e., mesoderm, ectoderm, or endoderm) or from different germ layers. An example of two progeny cells with distinct developmental lineages from the differentiation of multilineage stem cells is myogenic cells and adipogenic cells (both of mesoderm origin but giving rise to different tissues). Another example is neurogenic cells (of ectoderm origin) and adipogenic cells (of mesoderm origin).

[0090] The term "precursor" or "progenitor cell" is intended to mean a cell that has the potential to differentiate into a specific type of cell. Progenitor cells may be stem cells. Progenitor cells may also be more specific than stem cells. Progenitor cells may be unipotent or multipotent. Compared to adult stem cells, progenitor cells may be in a later stage of cell differentiation. Examples of progenitor cells include, but are not limited to, progenitor neurons.

[0091] "Parthenogenetic stem cells" refer to stem cells arising from the parthenogenetic activation of an egg cell. Methods for creating parthenogenetic stem cells are well known in this field. See, for example, Cibelli et al. (2002) Science 295(5556):819 and Vrana et al. (2003) Proc. Natl. Acad. Sci.USA 100(Suppl. 1)11911-6.

[0092] As used herein, “pluripotent cells” define less differentiated cells that can produce at least two distinct (genotypic and / or phenotypic) further differentiated progeny cells. In another embodiment, “pluripotent cells” include non-pluripotent cells, typically stem cells artificially induced from adult somatic cells, and induced pluripotent stem cells (iPSCs) that have been produced by inducing the expression of one or more stem cell-specific genes. Such stem cell-specific genes include, but are not limited to, octamer transcription factors, i.e., the Oct-3 / 4 family; Sox genes, i.e., the Sox1, Sox2, Sox3, Sox15, and Sox18 family; Klf genes, i.e., the Klf1, Klf2, Klf4, and Klf5 family; Myc genes, i.e., the c-myc and L-myc family; Nanog genes, i.e., the OCT4, NANOG, and REX1 family; or LIN28. Examples of iPSCs include Takahashi et al. (2007) Cell advance online publication 20 November 2007; Takahashi & Yamanaka (2006) Cell 126:663-76; Okita et al. (2007) Nature 448:260-262; Yu et al. (2007) Science advance online publication 20 November 2007; and Nakagawa et al. (2007) Nat. Biotechnol. Advance online publication 30 November 2007.

[0093] An embryoid body (EB) is a three-dimensional (3D) aggregate of embryonic stem cells that forms during culture, facilitating subsequent differentiation. When grown in suspension culture, EB cells form small aggregates of cells surrounded by an outer layer of visceral endoderm. As they grow and differentiate, EBs develop into cystic embryoid bodies with a fluid-filled cavity and an inner layer of ectoderm-like cells.

[0094] "Composition" is intended to mean a combination of an active polypeptide, polynucleotide, or antibody with another inactive compound or composition (e.g., a detectable label), or another active compound or composition (e.g., a gene delivery vehicle).

[0095] A "pharmaceutical composition" is intended to include a combination of an active polypeptide, polynucleotide, or antibody and an inactive or active carrier, such as a solid support, which makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

[0096] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers, such as phosphate-buffered salt solutions, water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0097] "Subject," "individual," or "patient" are interchangeable terms used herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, rodents, rats, rabbits, monkeys, cattle, sheep, pigs, canids, felines, domestic animals, sports animals, companion animals, equids, and primates, particularly humans. In addition to its usefulness in human treatment, the present invention is also useful in the veterinary treatment of companion mammals, exotic animals, and domesticated animals, including mammals and rodents, that are susceptible to neurodegenerative diseases. In one embodiment, mammals include horses, dogs, and cats. In another embodiment of the present invention, a human is an adolescent or infant under the age of 18.

[0098] "Host cell" refers not only to a specific target cell but also to the offspring or potential offspring of such a cell. While certain modifications may occur during generational continuation due to mutation or environmental influences, and such offspring may not be identical to the parent cell in practice, they are still included within the scope of the terminology used herein.

[0099] "Treating" a disease or "treating" a disease includes (1) preventing the disease, i.e., preventing the development of clinical symptoms of the disease in patients who may be susceptible to the disease but have not yet experienced or shown any symptoms of the disease; (2) suppressing the disease, i.e., preventing or reducing the development of the disease or its clinical symptoms; or (3) alleviating the disease, i.e., causing a regression of the disease or its clinical symptoms.

[0100] The term "to contract" refers, when used in conjunction with the term "treatment," to a patient or individual diagnosed with or susceptible to an infectious disease or an infectious disease. A patient may also be said to be "at risk of contracting" the disease due to an active or latent infection. This patient has not yet developed the characteristic disease pathology.

[0101] An "effective dose" is an amount sufficient to produce a beneficial or desired result. An effective dose may be administered in one or more doses, applications, or dosages. Such delivery depends on several variables, including the time period over which individual dosage units are to be used, the bioavailability of the therapeutic agent, and the route of administration. However, it is understood that the specific dose level of the therapeutic agent of the present invention for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, overall health, sex, and diet, the time of administration, the rate of excretion, drug combinations, and the severity of the particular disorder being treated, as well as the form of administration. Treatment dosages can generally be dose-set to optimize safety and efficacy. Typically, dose-response relationships from in vitro and / or in vivo studies can provide early useful guidance regarding appropriate doses for patient administration. Generally, it is considered desirable to administer an amount of the compound effective in achieving a serum level corresponding to the concentration found to be effective in vitro. Determining these parameters is well within the scope of the art of the present invention. These considerations, as well as effective formulations and administration procedures, are well known in the art and described in standard textbooks. Where used herein without contradiction to this definition, the term “therapeutic dose” is a quantity sufficient to inhibit RNA viral replication ex vivo, in vitro, or in vivo.

[0102] The term "administration" includes, but is not limited to, oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisional injection or infusion, subcutaneous injection, or implantation), nasal spray inhalation, vaginal, rectal, sublingual, urethral (e.g., urethral suppositories), intracranial, or topical administration routes (e.g., gels, ointments, creams, aerosols, etc.), which can be formulated alone or together in suitable dosing unit formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each administration route. The present invention is not limited by the route of administration, formulation, or dosing schedule.

[0103] Huntington's disease (HD) is a genetic disorder that causes progressive destruction (degeneration) of nerve cells in the brain. Huntington's disease has a wide range of effects on a person's functional abilities, including loss of motor and cognitive function, as well as mental disorders. Treating or improving the symptoms of HD is intended to improve the patient's mental, cognitive, or motor function, or to mitigate the adverse effects of this genetic disorder. The symptoms and course of the disease are known to those skilled in the art; see mayoclinic.org / diseases-conditions / huntingtons-disease / symptoms-causes / syc-20356117 (accessed May 21, 2018).

[0104] Central nervous system (CNS) disorders or conditions refer to a group of neurological disorders that affect the structure of function of the brain or spinal cord and may result in degeneration of one or more parts of the brain or spinal cord. Non-exclusive examples include HD, Alzheimer's disease, Parkinson's disease, traumatic brain injury, stroke, autoimmune disorders such as multiple sclerosis, primary or secondary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, encephalitis, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumors, peripheral neuropathy, Guillain-Barré syndrome, spinal muscular atrophy, Friedreich's ataxia, amyotrophic lateral sclerosis, and Huntington's disease. Treating or improving the symptoms of CNS injury is intended to improve the patient's neurological function or mitigate the adverse effects of the inherited or acquired disease, injury, or disorder. The symptoms and course of the disease are publicly known to those skilled in the art; please refer to hopkinsmedicine.org / healthlibrary / conditions / nervous_system_disorders / overview_of_nervous_system_disorders_85,P00799 (accessed May 21, 2018).

[0105] "Neurodegenerative disease or disorder" is a disease or phenotype characterized by the degeneration of neurons in the nervous system, particularly in the central nervous system (CNS).

[0106] "Enhancing synaptic connections" means promoting connections between neurons or between receptors on neurons.

[0107] A synapse is a junction between two nerve cells, consisting of a tiny gap through which an impulse passes via the diffusion of neurotransmitters.

[0108] Forms for implementing this disclosure A method for preparing human neuronal stem cells (hNSCs) from human embryonic stem cells (hESCs), a) A step of isolating at least one stem cell rosette from a population of embryoid bodies (EBs) cultured in differentiation medium, b) A step of culturing at least one individual cell isolated from the rosette in step a) for a certain period of time under conditions that provide for the formation of at least one rosette, c) The step of isolating individual cells from the rosette in step b) and making them individual cells, d) A step of culturing at least one individual cell isolated from step c) for a certain period of time under conditions that provide for the generation of a confluent population of hNSCs. Methods comprising, or alternatively essentially consisting of, or even comprising, are provided herein.

[0109] In one embodiment, the isolation of at least one individual cell from the rosette is performed manually. In another embodiment, the isolation of at least one individual cell from the rosette is performed enzymatically. In a further embodiment, the isolation of at least one individual cell from the rosette in step a) is performed manually, enzymatically, and / or digitally, one or more of these methods. In a further embodiment, the isolation of at least one individual cell in step c) is performed enzymatically. Methods and techniques for digitally identifying three-dimensional or two-dimensional images are known in the art; see, for example, U.S. Patents 7,689,043, 6,907,140, ​​and 5,020,112.

[0110] In one embodiment, one or more of steps a) to c), which can be carried out using either a manual method or a high-throughput mechanical method, are performed two or more times. In further embodiments, the isolation of the rosettes is carried out digitally. Methods and techniques for digitally identifying three-dimensional or two-dimensional images are known in the art; see, for example, U.S. Patents 7,689,043, 6,907,140, ​​and 5,020,112.

[0111] In one embodiment, embryoid bodies are generated from the cell line ESI-017, which is available from BioTime (see esibio.com / esi-017-human-embryonic-stem-cell-line-46-xx / [last accessed June 6, 2018]).

[0112] In one aspect of the present disclosure, the method further comprises culturing embryoid bodies (EBs) in EB medium on an ultra-low adhesion surface. In another aspect, the method further comprises performing step a) on an ornithine / laminin coated surface by replacing the EB medium with N2 medium after the EBs have been cultured for a sufficient time to produce at least one EB of step a).

[0113] The method can be further modified by culturing at least one individual cell isolated in step c) for an effective time in an ornithine / laminin-coated plate in N2 medium to generate a confluent cell population of hNSCs. As is known to those skilled in the art, a confluent cell population is a cell population in which a considerable number of cells are in contact with other cells in the population. This method can be further modified by culturing the confluent population of hNSCs with an effective amount of N2 medium.

[0114] Cells or populations of cells prepared by the methods described herein are also provided herein. Neuronal cells and differentiated cells produce or overexpress BDNF.

[0115] The cells of the population can be expanded and / or genetically modified, for example, by transgene insertion or CRISPR. In one embodiment, the transgene is a fragment thereof, such as ApiCCT1, sApiCCT, or their respective equivalents. The cells and / or transgene can be detected and labeled as needed. The transgene can be inserted by inserting it into a vector using well-known and conventional recombination techniques, and the transgene is under the control of regulatory elements, such as a promoter, and optionally an enhancer element. The cells and / or vector containing the transgene can be detected and labeled. As detailed below, the transgene sApiCCT is inserted into a specific cell population of hNSCs to provide further protection to the hNSCs or tissues when implanted as a therapeutic agent. The sApiCCT transgene can also be inserted into hESCs, or other embryonic cell lines, fetal cell lines, mesenchymal cell lines, neuron cell lines, and other stem cell derivatives and differentiated cell types, including but not limited to these.

[0116] Populations of these cells, and non-human animals containing these cells, are further provided. The populations may be substantially homogeneous, substantially heterogeneous, or clonal. The populations can be detectably labeled. The populations can be combined with a carrier, such as a pharmaceutically acceptable carrier.

[0117] For example, compositions comprising isolated cells having a carrier are further provided. In a further embodiment, the composition further comprises a preservative and / or cryoprotective agent. Non-limiting examples of cryoprotective agents include DMSO and glycerol, which are commercially available, e.g., streck.com / collection / streck-cell-preservative / (last accessed May 22, 2018) Please refer to ).

[0118] Cells are useful in therapeutic methods. In one embodiment, a method is provided for delivering a transgene to a subject or for gene editing cells in a subject requiring such delivery by administering one or more of the cells, populations, or compositions described herein. In another embodiment, a method is provided for treating neurodegenerative disorders or enhancing synaptic connections in a subject requiring such treatment by administering one or more of the cells, populations, or compositions to the subject. In yet another embodiment, a method is provided for treating neurodegenerative disorders or enhancing synaptic connections or treating CNS damage in a subject requiring such treatment, comprising administering one or more of the cells, populations, or compositions to the subject. Any suitable method of administration may be used, and non-limiting examples of such methods are provided herein.

[0119] Non-limiting examples of neurodegenerative disorders are selected from the group of conditions including Huntington's disease, stroke, CNS injury, chronic spinal cord injury, spinal cord injury, aneurysm, surgery, arteriovenous malformation (AVM), radiation, spinal muscular atrophy, Friedreich's ataxia, amyotrophic lateral sclerosis (ALS), muscle sclerosis, primary or secondary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, vascular dementia, epileptic seizures, cerebral vasospasm, Alzheimer's disease, acute or traumatic brain injury, encephalitis, and cerebral hypoxia as a result of, for example, cardiopulmonary arrest or drowning, or any other CNS injury resulting in acute physical injury to CNS tissue, as well as combinations thereof.

[0120] In certain embodiments, CNS injury is injury caused by a stroke. “Stroke” means any condition that results in physical injury to the central nervous system due to impaired blood supply or oxygen to the brain. This may be caused by thrombosis or embolism or by ischemia (lack of blood supply or oxygen) resulting from bleeding.

[0121] kit Kits are also provided. In one embodiment, the kit includes an hESC and instructions for carrying out the method described herein. In a further embodiment, the kit includes neuronal cells prepared using the method described herein and instructions for use. The kit may further include compositions and reagents for carrying out the instructions provided with the kit.

[0122] The agents described herein may, in some embodiments, be assembled into pharmaceutical, diagnostic, or research kits to facilitate the use of the agents in therapeutic, diagnostic, or research applications. In one embodiment, the kit includes an hESC and instructions for carrying out the method described herein. In further embodiments, the kit includes neuronal cells prepared using the method described herein and instructions for use. The kit may further include compositions and reagents for carrying out the instructions provided with the kit.

[0123] In some embodiments, the kit further includes instructions for use. Specifically, such a kit may include one or more of the agents described herein, along with instructions describing the intended application and appropriate use of these agents. For example, in one embodiment, the kit may include instructions for mixing one or more of the components of the kit, and / or isolating and mixing samples for application to a subject. In certain embodiments, the agents in the kit are pharmaceutical formulations and dosages suitable for a particular application and method of administering the agent. A research kit may contain components in concentrations or quantities appropriate for performing a variety of experiments.

[0124] The kit may be designed to facilitate the use of the methods described herein and can take many forms. Each of the kit's compositions may be provided in liquid form (e.g., solution) or solid form (e.g., dry powder), where applicable. In certain cases, some compositions may be constitutable by the addition of a suitable solvent or other chemical species (e.g., water or cell culture medium), which may or may not be provided with the kit, or otherwise processable (e.g., into an active form). In some embodiments, the compositions may be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor® (Stem Cell Technologies, Vancouver, Canada). In some embodiments, the preservation solution contains a certain amount of a metalloproteinase inhibitor.

[0125] As used herein, “Instructions” can define components of a description and / or progress, and typically include written instructions on or accompanying the packaging of the claimed method or composition. Instructions may also include any oral or electronic instructions provided in any form that the user can clearly recognize as relating to the kit, e.g., audiovisual devices (e.g., videotape, DVD, etc.), the internet, and / or online communications. In some embodiments, written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, and may also reflect an agency approval for manufacture, use, or sale for animal administration.

[0126] In some embodiments, the kit contains one or more of the components described herein in one or more containers. Thus, in some embodiments, the kit may include a container for containing the drug described herein. The drug may be in liquid, gel, or solid (powder) form. The drug may be sterile-prepared, packaged in syringes, and transported under refrigeration. Alternatively, the drug may be contained in vials or other containers for storage. A second container may contain other sterile-prepared drugs. Alternatively, the kit may contain activators that are pre-mixed and transported in syringes, vials, tubes, or other containers. The kit may have one or more or all of the components necessary for administering the drug to a subject, such as syringes, topical application devices, or IV needles and bags.

[0127] The therapies described herein can be combined with appropriate diagnostic techniques for identifying and selecting patients for treatment. For example, genetic testing may be provided to identify mutations in muscular dystrophy genes. Thus, patients with mutations can be identified as suitable for treatment.

[0128] The following embodiments are intended to be illustrative and not to limit the scope of this disclosure. [Examples]

[0129] Experimental Procedure [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] Procedure for generating NSC from ESC Passaging of ESCs for embryoid body (EB) formation. On day 1, differentiated colonies were manually removed from the ESC culture using a P1000 tip. The ESC medium was then replaced with 2 mL / well of EB medium. Using a P1000 tip, the ESC colonies were scraped off the wells using a back-and-forth motion, first horizontally and then vertically. The scraped colonies from each well were transferred to one well of an ultra-low adhesion 6-well plate using a 10 mL serological pipette. The wells of the ESC plate were washed with EB medium using a P1000 micropipette, and the washing solution was added to each well of the ultra-low adhesion 6-well plate to obtain a final volume of 3 mL / well. The EB plate was moved to an incubator at 37°C and 5% CO2. The EB plate was incubated at 37°C and 5% CO2 for the duration of day 2. On day 3, a half volume change of EB medium was performed. Floating colonies were gently swirled to the center of the wells. Using a P1000 micropipette, 1 ml of medium was removed from each well and discarded. 1.5 mL of fresh EB medium was gently added to each well. The plate was then returned to the incubator. Nothing was done on day 4. Stirring was continued while incubating at 37°C and 5% CO2. On day 5, EB derived from ESCs was plated onto laminin-coated plates. Poly-L-ornithine diluted 1:3 in PBS was coated into an appropriate number of wells in a 6-well plate at room temperature for 1 hour. N2 medium was prepared according to Table 2 (N2 medium is good for 1 week after preparation). After 1 hour, the poly-L-ornithine solution was removed and discarded. The wells were washed twice with PBS. Laminin diluted 1:100 in PBS was coated into the wells at room temperature for 1 hour. The EB suspension was transferred from each well to its respective 15 mL conical tube using a 10 mL serological pipette. EB was allowed to settle from the suspension at room temperature for 15 minutes. Laminin was aspirated and discarded from the wells of the coated 6-well plate. 1 mL of N2 medium was added to each well. EB medium was aspirated from each 15 mL conical tube. EB was gently resuspended in 2 mL of N2 medium using a 10 mL serological pipette.EB was added to coated wells containing 1 mL of N2 medium to a total volume of 3 mL. The plate was gently shaken back and forth to distribute the EB, and then placed in an incubator. Rosette formation and isolation of plated EB (Ros1, Round 1) were performed from day 6 to day 14. Cells were examined over the following 4 to 6 days to check for rosette formation. Rosettes were harvested at any time depending on the quality of formation. If rosette formation was not yet present, the N2 medium was changed every other day until rosettes appeared. To harvest rosettes, a 12-well plate was coated with poly-L-ornithine diluted 1:3 in PBS for 1 hour at room temperature. After 1 hour, the poly-L-ornithine solution was removed and discarded. The wells were washed three times with PBS. Then, the wells were coated with laminin diluted 1:100 in PBS for 1 hour at room temperature. If necessary, one vial of N2 medium was prepared according to Table 2. After 1 hour, the laminin was removed, 1 mL of N2 medium was added to each well to maintain moisture, and the wells were stored in an incubator for rosette dissection. Around days 10–12, rosettes formed from previously plated EB cells were observed. Under a dissecting microscope, the rosettes were dissected by tracing the rosette outline using an 18-gauge needle attached to a 1 mL syringe. The mixture was then transferred to a laminin-coated plate in N2 medium using a p200 micropipette. After transfer, the mixture was stored in an incubator overnight and labeled as Ros1. The medium was changed every other day during storage in the incubator. On days 15–18, the rosettes were dissociated into single cells. Two to three days after isolating Ros1 (Round 1), the rosette with the fewest impurities was dissociated into single cells. N2 medium was aspirated from each desired well, and 0.05% trypsin in 0.5 mL of EDTA was added to each well. The mixture was incubated at 37°C and 5% CO2 for 90 seconds. After 90 seconds, 0.5 mL of DTI was added. Using a 1000 μL micropipette, the rosettes were dissociated in the wells, and the volume of each well was added to the respective 15 mL conical tubes. Each well, or "clone," remained isolated throughout all passages.The conical tubes were centrifuged at 1000 RPM for 5 minutes. The supernatant was added and discarded. Each pellet was resuspended in 1 mL of fresh N2 medium. Each 1 mL suspension was plated into each well of a poly-L / laminin-coated 12-well plate, and the plate was labeled as (NSC Passage 0). The plate was returned to the incubator and the culture was monitored, with the medium replaced with fresh N2 medium every other day. When the cells reached approximately 85% confluence, each "clone" was transferred to each well of a 6-well plate using the same procedure as above, except that the medium was replaced with 3 mL of N2 medium every other day, with 1 mL of 0.05% trypsin and 1 mL of DTI. The cells were maintained and passaged 10 times. 6 The process was either continued at the same cell / well ratio, or cell cryopreservation (discussed below) was performed.

[0130] NSC cryopreservation. Cryopreservation medium or frozen medium was prepared according to Table 3, ensuring that the frozen medium was cooled to 4°C for the entire time before use. The NSC plate was removed from the incubator and placed in a biological safety cabinet. The old culture medium was completely aspirated and discarded in a waste container. 1 mL of 0.05% trypsin was added to each well and incubated at 37°C for 90 seconds. After 90 seconds, 1 mL of DTI was added to each well to inactivate the trypsin. Using a 1000 μL pipette tip, the mixture was pipetted to wash the cells from the surface of each well, and then the mixture was transferred to a 15 mL conical tube. The 15 mL conical tube was centrifuged at 1000 RPM for 3 minutes. The conical tube was returned to the biological safety cabinet and the supernatant was completely aspirated. The cells were resuspended in 5 mL of fresh N2 culture medium, and cell counting was performed using 0.4% trypan blue and a hemocytometer. The cells were centrifuged at 1000 rpm for 3 minutes in a benchtop centrifuge. An appropriate volume of 4°C frozen medium was added, with a cell concentration of 3.0 × 10⁶. 6Cells were added to the medium to a concentration of cells / mL. 1 mL of cell suspension in frozen medium was added to each frozen vial using a 10 mL serological pipette. One vial of frozen medium without cells was prepared for the frozen probe. The vials were tightly capped and immediately transferred to a pre-cooled, rate-controlled freezer-freezing rack. The probe was inserted into a vial containing only frozen medium and placed in the rack. The vials were transferred from the rate-controlled freezer to a pre-cooled -80°C, fully labeled freezing box and immediately to the LN2 storage unit.

[0131] mouse All procedures followed the NIH guidelines for the management and use of laboratory animals, as well as animal research protocols approved by the animal experimentation committees of UCI and UCLA, which are AAALAC-accredited facilities. R6 / 2 mice and their NT littermates (transgene-free C57Bl6 / CBA) were obtained from breeding colonies maintained at UCI (strain 6494, approximately 120±5 CAG repeat sequences) or at UCLA (strain 2810, approximately 150±5 CAG repeat sequences). Homozygous Q140 mice or WT (C57Bl6) littermates were from UCLA breeding colonies, and procedures were performed at UCLA. All mice were housed with ad libitum access to food and water under a 12 / 12 light / dark schedule. Mice were housed in mixed treatment groups, and individually only for running on the wheel. The length of CAG repeat sequences was examined for R6 / 2 mice (Laragen, Los Angeles, CA), and the frequency distribution was not significantly different for Q140 mice (Hickey et al., 2012b). The evaluation of differences in outcomes was based on previous experience and published results regarding HD models (Hickey et al., 2005; Hockly et al., 2003), and power analysis was performed. By applying G Power [psycho.uni-duesseldorf.de / abteilungen / aap / gpower3 / ], the applicants obtained the smallest n values ​​of n=10 for behavior and n=4 for biochemical analysis.

[0132] hNSC isolation The use of hNSCs was approved by the Human Stem Cell Research Monitoring Committees (hSCROs) of UCI, UCLA, and UC Davis, and the cells were derived from Biotime's ESI-017 hESCs. hESC colonies were transferred to EB medium containing nogging, then to NP medium, rosettes were cleaved, dissociated, and seeded with hNSC medium to generate hNSCs (Figure 8B). Rosettes were manually cleaved, plated in NSC medium on growth factor-reduced Matrigel coated plates, then dissociated using accutase, and plated on polyornithine / laminin coated plates containing NSC medium.

[0133] transplant surgery Bilateral intrastriatal injections of hNSC or veh were performed using a stereotactic device and coordinates relative to Bregma: anterior-posterior, 0.00; endo-exterior, ±2.00; dorsal-ventral, -3.25. Mice were anesthetized, placed in a stereotactic frame, and injected with either 100,000 hNSC / side (2 μL / injection) or veh (2 μL of Hanks equilibrium salt solution containing 20 ng / mL of human epidermal growth factor [STEMCELL Technologies, #78003] and human fibroblast growth factor [STEMCELL, #78006]) using a 5 μL Hamilton microsyringe (33 gauge) at an injection rate of 0.5 μL / min. The wounds were sealed, and the mice were allowed to recover in cages equipped with heated pads. Immunosuppressants were administered to all mice the day before surgery and continued throughout the surgery.

[0134] Behavioral evaluation R6 / 2 Mice were semi-randomly assigned, and behavioral tests were conducted over a period of 6–9 weeks. Researchers were blinded to genotype and treatment during the tests and data collection. To minimize experimenter variability, one investigator conducted each test. Behavioral tasks in R6 / 2 mice were performed as previously described by Ochaba et al. (2016).

[0135] Q140 Except for the running wheel, both males and females were used; for the running wheel, only males were used because the estrous cycle affects running activity. Genotype or treatment was unknown to the experimenters. All tests were conducted during the light phase, except for the running wheel which was conducted during the dark phase. Behavioral tasks in Q140 mice were performed as previously described by Hickey et al. (2008).

[0136] Electrophysiology in R6 / 2 brain slices R6 / 2 strains (strain 2810, 150±10 CAG repeat sequences) and NT littermates expressing phenotypes similar to those of the 6494 strains used for behavioral experiments were employed (Cummingset al., 2012). The procedure was the same as that described by Andre et al. (2011), with modifications detailed herein.

[0137] Immunohistochemistry and electron microscopy Three male R6 / 2 mice (n=3) implanted with hNSCs for five weeks were anesthetized and perfused with EM fixative (2.5% glutaraldehyde, 0.5% paraformaldehyde, and 0.1% picric acid in 0.1M phosphate buffer [pH 7.4]). The brains were then harvested and left overnight in EM fixative at 4°C, and washed in PBS until 60 mm sections of the striatum containing hNSCs (corresponding to +1.4 to +0.14 mm from the bregma) (Franklin and Paxinos, 2007) were sequentially prepared using a vibratome (Leica Microsystems). Pre-embedded IHC of striatal tissue treated for EM with diaminobenzidine (DAB) (Sigma, St. Louis, MO) and hNSC antibody (Stem121, 1:100, StemCells) is as previously described (Spinelli et al., 2014; Walker et al., 2012), and striatal slices are placed on two ACLAR (Electr The resin was polymerized by embedding the tissue flat overnight in a 60°C oven (on Microscopy Sciences, Hatfield, PA). The region containing hNSCs was microdissected from the embedded slice and bonded to the block with cyanoacrylate adhesive for sectioning.

[0138] Images of DAB-labeled structures (i.e., hNSC-labeled cells and dendrites) at a final magnification of 346,200 using a JEOL1400 transmission electron microscope (JEOL, Peabody, MA) were captured using a digital camera (AMT, Danvers, MA). Since DAB labeling is limited to the tips of the tissue sections, only the areas showing DAB labeling were photographed.

[0139] Biochemical, molecular, and immunohistochemical analysis in R6 / 2 mice Mice were euthanized by pentobarbital overdose and perfused with 0.01 M PBS. The striatum and cortex were cleaved from the left hemisphere, rapidly frozen for RNA, and proteins were isolated in TRIzol using the manufacturer's procedure (Life Technologies, Grand Island, NY) or homogenized as described below. The other half was post-fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and cut to 40 μm in a sliding vibratome for IHC. The sections were rinsed three times and placed in blocking buffer (PBS, 0.02% Triton X-100, 5% goat serum) for 1 hour, and the primary antibody was placed in the block solution overnight (ON) at 4°C. The sections were rinsed, incubated in Alexa Fluor secondary antibody for 1 hour, and mounted using Fluoromount G (Southern Biotechnology). The primary antibody is listed in the supplementary experimental procedure.

[0140] Soluble / Insoluble Fractionation Striatal tissue was processed as previously described (Ochaba et al., 2016). Antibody: Anti-H TT (Millipore, #MAB5492;RRID:AB_347723) and anti-ubiquitin (Santa Cruz Biotechnology, #sc-8017;RRID:AB_628423). Band quantification was performed using ImageJ software and densitometry applications from the NIH program.

[0141] Confocal microscopy and quantitative analysis Sections were imaged using a Bio-Rad Radiance2100 confocal system in lambda stroving mode. Images represent either single confocal z-slices or z-stacks. All unbiased stereochemical evaluations were performed using StereoInvestigator software (MicroBrightField, Williston, VT). Average cell number, number of scattered aggregates, and number of inclusions were estimated using optical fractionation probes.

[0142] RNA isolation and real-time qPCR Striatal cells were homogenized with TRIzol (Invitrogen) followed by the RNEasy Mini kit (Qiagen). RIN values ​​were greater than 9 for each sample (Agilent Bioanalyzer). RT was performed using oligo(dT) primers and 1 mg of total RNA with the SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed as described by Vashishtha et al. (2013).

[0143] Biochemical, molecular, and immunohistochemical analysis of Q140 mice Male Q140 was euthanized six months after the procedure by cervical dislocation (n=7, cryotherapy) or paraformaldehyde perfusion (n=5, IHC).

[0144] IHC Mice were perfused with 0.1 M PBS and 4% paraformaldehyde. Brains were removed, fixed overnight in 4% paraformaldehyde, cryoprotected in 30% sucrose, frozen, and coronal sections were cut to 40 μm in a cryostat (Leica CM, 1850). Sections were blocked at room temperature for 1 hour, then primary antibody was applied ON. After several washes, sections were incubated in Alexa Fluor secondary antibody and counterstained with DAPI. IHC for the quantification of HTT aggregates and microglia was performed as described by Menalled et al. (2003) and Watson et al. (2012), respectively.

[0145] HTT-stained nuclei and aggregates Sections were analyzed using StereoInvestigator 5.00 software (Microbrightfield, Colchester, VT) (Hickey et al., 2012a). To visualize the striatal contours, the software superimposed a 200 × 200 μm grid containing 20 × 20 μm counting frames used for the quantification of various aggregates in each section.

[0146] Quantification of IBA-1 positive cells in the striatum of Q140 mice The analysis was performed as described (Watsonet et al. 2012) using a Leica DM-LB microscope with StereoInvestigator software (MicroBrightField) to determine the diameter of microglia reflecting activation. 5 To capture the contours of the striatum at double the magnification, the software overlaid a 200x200 μm grid containing a 20x20 μm counting frame in the upper left corner, enabling unbiased sampling and quantification.

[0147] Biochemical analysis of Q140 mice The frozen striatal processing for ELISA was carried out using the Biosensis BDNF Rapid kit (Biosensis BEK-2211, SA, Australia) according to the manufacturer's instructions.

[0148] Statistical analysis Results for R6 / 2 mice were from a single cohort, using the same batch of cells, except for electrophysiology and EM data which were from different subsets. The number with sufficient power was determined using analysis prior to the study (described above). Statistical significance was achieved using rigorous analysis as described. All findings were reproducible. Multiple statistical methods are further detailed in the description of the figures above. Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, ****p<0.0001. In R6 / 2 mice, data were expressed as mean ± SEM, and statistical tests for behavioral tasks were performed using one-way ANOVA followed by Tukey's HSD test, including Scheffé, Bonferroni, and Holm's post-hoc multiple comparisons. The data satisfied the assumptions of the statistical tests used, and p-values ​​less than 0.05 were considered significant. All mice were randomly assigned, and the tasks were performed randomly using individuals blinded for genotype and treatment. Statistical comparisons of densitometry results were performed by one-way ANOVA followed by Bonferroni's multiple comparison test. Student's t-test was used for comparing aggregate numbers from EM48 stereoanalytic studies. Significance in clasping was determined by Fisher's exact probability. Statistical analysis of Q140 mice was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA) for significance (p<0.05) in behavioral and postmortem data, employing one-way ANOVA and Bonferroni post-hoc tests. Two-way ANOVA followed by Bonferroni post-hoc tests were used on the graph representing the average rotations per wheel / 3 minute test, and bootstrap statistics using custom MATLAB® functions were used for IBA-1 analysis. All error bars in the graph represent SEM.

[0149] hNSC isolation. Daily (D) cultures were as follows: D1: ESC colonies were enzymatically "loosened" using collagenase IV until the colony edges began to bulge. Colonies were manually scraped from the wells and transferred to low-adhesion plates and cultured overnight in EB medium (ESC medium without bFGF). D2: EB culture medium was supplemented with 500 ng / ml noggin and 10 μM SB431542 and cultured for a further 2 days. D4: Medium change. D5: EB was plated in the same medium in a growth factor-reduced Matrigel-coated 6-well plate. D6: Medium was changed and NPC differentiation was driven using NP medium. Medium was changed every 2 days until day 12. D12-14: Rosettes were visually isolated under a dissection scope, manually detached using an 18-gauge needle, and plated in NSC medium in a growth factor-reduced Matrigel-coated 6-well plate. After 2-3 days, the rosettes were dissociated using acetase and plated onto polyornithine / laminin-coated plates containing NSC medium and the Y27632 compound. Cytogenetic analysis of ESI-017 hNSCs revealed that the cells were stable in terms of karyotype and no abnormalities were observed. Monochromatic flow cytometry was performed on CD271 (Brilliant Violet 510 -- BD Horizon catalog number 563451), CD24 (Brilliant Violet 711 -- BD Horizon catalog number 563401), Pax6 (PE -- BD Pharmingen catalog number 561552), Nestin (Alexa Fluor 647 -- BD Pharmingen catalog number 560341), SOX1 (PerCP CY5.5 -- BD Pharmingen catalog number 561549), SOX2 (V450 -- BD Horizon catalog number 561610), and CD44 (APC-H7 -- BD The tests were conducted on Pharmingen catalog number 560532, CD184 (PE-CY7 - Biolegend catalog number 306514), or SSEA4 (lexa Fluor700 - Invitrogen catalog number SSEA429).

[0150] Transplantation surgery. Bilateral intrastriatal injection of hNSC or vehicle was performed using a stereotactic device and the following coordinates relative to Bregma: AP: 0.00, ML: + / - 2.00, and DV - 3.25. Mice were placed in a stereotactic frame and injected with either 100,000 hNSCs (2 μl / injection) or a vehicle (2 μl of HBSS containing 20 ng / ml of hEGF and hFGF) per side as a control treatment, using a 5 μl Hamilton microsyringe (33 gauge) and an injection rate of 0.5 μl / min. R6 / 2 mice were anesthetized with isoflurane, and Q140 mice were anesthetized with pentobarbital sodium (60 mg / kg Nembutal, ip in sterile 0.9% saline). For all mice, to maintain the surgical surface under anesthesia, isoflurane (1-2% in 100% oxygen, 0.5 L / min) was administered via a nose cone, oxygen was administered throughout the surgery, the mice's body temperature was maintained on an electronically controlled heating pad, and monitored using a rectal probe thermometer (Physitemp). Precise placement of the injection to the target area was confirmed for all animals by visualization of the needle path in brain sections. The wound was sealed using bone wax on the skull and closed with Dermabond or sutures. After surgery, mice were placed on heating pads in individual cages until they recovered from anesthesia. A single daily dose of the immunosuppressant CSA was administered intravenously to hNSCs, vehicle-implanted R6 / 2, and non-transgenic mice at a concentration of 10 mg / kg, starting the day before surgery. To further immunosuppress the mice, an additional regimen of ip's weekly dose of CD4 antibody (BioXcell, Lebanon, NH) was administered at 10 mg / kg. Q140 mice or wt littermates received immunosuppression with CSA (2 mg / kg / day) administered via a subcutaneous osmotic minipump (Alzet#1004) which was replaced monthly to ensure continuous delivery of CSA throughout the study. The surgery to remove and replace the minipump was as follows. Mice were anesthetized with isoflurane (3% for induction and 1.75% for maintenance in 100% oxygen).After sterilizing the incision site, the mini-pump was removed through a small incision in the back, a new mini-pump was implanted, and the incision was sutured closed.

[0151] R6 / 2: Mice were semi-randomly assigned to multiple groups. The behavioral tests listed below were performed at 6, 7, 8, or 9 weeks of age, depending on the task. Mouse body weight was measured daily, and no significant differences were observed between the treatments. Researchers were blinded to which mice received hNSC transplants during the experiment and data collection. To minimize experimenter variability, one investigator performed each behavioral test. Mice were obtained from UCI breeding colonies using ovarian transplanted female mice (Jackson labs).

[0152] Using a rotarod apparatus, forelimb and hindlimb motor coordination and balance were measured, and mice were tested over three consecutive days using a 300-second accelerated assay. The rotarod test was performed twice, one week apart, at 6 and 8 weeks of age. For the pole test, mice were placed head-down on a pole and then descended upside down the length of the pole. The total time taken to descend from the starting point of the setup was measured. The pole test was performed twice, one week apart, at 7 and 9 weeks of age. Using an IITC Life Science instrument, forelimb gripping force was measured via a digital force transducer, which displays readings in 1-gram increments. Grip strength was measured twice, one week apart, at 7 and 9 weeks of age.

[0153] Q140: Climbing test and pole test. To evaluate motor coordination and spontaneous activity during climbing, mice were placed at the bottom of a cylindrical wire cage and their spontaneous activity was videotaped. For the pole test, each mouse was positioned face up at the top of the pole, and the time it took for them to invert their entire body into a downward position was measured. The time it took for them to descend the pole and enter their respective home cages was also measured.

[0154] Electrophysiology in R6 / 2 mice In short, mice were anesthetized, perfused transcardially with a high-sucrose-based slice solution, and then coronal slices (300 μm) were transferred to an incubation chamber containing ACSF. MSNs and NSCs were visualized using infrared illumination with differential interference contrast optics. All recordings were made in or around the injection site (recorded MSNs were adjacent to the graft between 150 and 200 μm). Biocitin was added to a patch pipette for cell visualization. Spontaneous postsynaptic currents were recorded in the total cellular composition in standard ACSF. Membrane currents were recorded in gap-free mode. Cells were potential-fixed at +10 mV, and spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in ACSF. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in ACSF at -70mV (baseline) in the presence of the GABAA receptor blocker bicucrimetobromide (Tocris, Minneapolis, MN) to isolate glutamatergic excitatory events. Spontaneous synaptic currents were analyzed using MiniAnalysis software (version 6.0, Synaptosoft, Fort Lee, NJ). After recording, slices were fixed and then transferred to 30% sucrose at 4°C until IHC treatment. To identify recording cells and hNSCs filled with biocitin, fixed slices were washed, permeabilized, blocked for 4 hours, and then incubated with SC121 (1:1000, StemCells, Inc.). After washing, slices were incubated in streptavidin conjugated with goat anti-mouse Alexa-488 (1:1000, Life Technologies, Carlsbad, CA, catalog number: A-11001) and Alexa-594 (1:1000, Life Technologies, catalog number: S11227). The slices were washed, mounted, and the cells were visualized using a Zeiss LSM510 confocal microscope.

[0155] Biochemical, molecular, and immunohistochemical analyses in R6 / 2 mice. Confocal microscopy and quantification. Sections were imaged using a Bio-Rad Radiance2100 confocal system in lambda stroving mode. Images represent either single confocal Z slices or Z stacks. All unbiased stereoanalytic evaluations were performed using StereoInvestigator software (MicroBrightField, Williston, VT). Average cell count, scattered aggregate count, and inclusion count were estimated using optical fractionation probes. A guard zone was set to 3% of the measured thickness, including a minimum optical dissector height of 14 μm. Contour plotting was performed with a 5x objective lens, and counting was performed with a 100x objective lens. For each section, plotting was performed approximately 70 μm away from the edge of the stem cell patch. Counting was performed on every three sections (40 μm coronal sections) across six sections spanning the entire striatum, where Ku80-labeled cells were found between bregma 0.5 mm and bregma -0.34 mm. All counting was performed in only one hemisphere, using a 50 × 50 μm counting frame and a 250 × 250 μm sampling grid. The CE values ​​for each individual mouse ranged from 0.03 to 0.06. Sections were stained first for Ku80 using the ABC kit and DAB substrate kit (Vector Laboratories) with nickel, and then for EM48 using only the ABC and DAB kits. Sections were stained with cresyl violet for non-stem cell nuclear staining. Aggregates and cells in vehicle-implanted mice were counted using the same stereochemical parameters. Using this stereochemical evaluation of Ku80-positive cells in implanted R6 / 2 brain sections, the number of ESI-017 NSC implanted viable cells averaged 63,975 cells in male mice (n=3) and 18,673 cells in female mice (n=3), representing 64% (males) and 18.6% (females) of the initially transplanted cells. Overall, there were an average of 41,323 cells in mice (n=6, 3 males and 3 females), representing approximately 41% of the initially transplanted cells.The difference in the number of implanted cells between males and females may be due to the technical difficulty of implanting cells in smaller females at 5 weeks.

[0156] Primary antibodies used for IHC: GFAP (Abcam ab4674), NeuN (Millipore MAB377), SC121 (STEM121, a human-specific cytoplasmic marker, Clontech AB-121-U-050), Ku80 (Abcam, Cambridge, United Kingdom ab80592), Doublecortin (Millipore AB2253), Olig2 (R&D Systems AF2418), BIII tubulin (Abcam ab107216), MAP-2 (Abcam ab5392), BDNF (Icosagen, 329-100), and EM48 (Millipore MAB5374).

[0157] RNA isolation and real-time quantitative PCR. Brain tissue was homogenized in TRIzol (Invitrogen), and total RNA was isolated using the RNEasy Mini kit (QIAGEN). DNase treatment was incorporated into the RNEasy procedure to remove residual DNA. RIN values ​​were greater than 9 for each sample (Agilent Bioanalyzer). Reverse transcription was performed using oligo(dT) primers and 1 μg of total RNA with the SuperScript III first-strand synthesis system (Invitrogen). Quantitative PCR (qPCR) was performed as previously described (Vashishtha, Ng et al. 2013), and ddCT values ​​were quantified and analyzed against RPLPO. The primers used to amplify the R6 / 2-Htt transgene were: [ka] The other primers used were: [ka] That was the case.

[0158] Immunohistological analysis in Q140 mice Primary antibodies used for IHC: HNA (Millipore MAB1281), DCX (abcam ab18723), GFAP (Dako Z033401), or synaptophysin (Millipore 04-1019). For IHC for the quantification of HTT aggregates, the described monoclonal antibody EM48 (Millipore MAB5374) was used (Menalled et al., 2003), and for IHC for the quantification of microglia, The rabbit anti-Iba-1 (Wako 019-19741) described in the text was used (Watsonet al., 2012). For cell counting, HNA+ cells were counted across the entire striatal region of six coronal sections. 2100 HNA-labeled cells were quantified, and the percentage of cells double-labeled with a neuronal marker (DCX, Abcam ab18723) or a glial marker (GFAP, Dako Z033401) was quantified. The final number was expressed as the mean ± SEM of 5 mice per group.

[0159] ESI-017 hNSCs, when implanted in R6 / 2 mice, modify behavior, enable survival, and differentiate. To evaluate the efficacy of hNSC transplantation in a transgenic model of HD, the applicants used exon 1 HTT R6 / 2 mice (CAG repeat sequence at rv120) (Cummingset al., 2012), which exhibit rapidly progressing motor and metabolic deficiencies and premature death (rv12-14 weeks) (Mangiarini et al., 1996), and prior This may provide an initial evaluation of treatment methodologies in clinical research (Hickey and Chesselet, 2003; Hockly et al., 2003). ESI-017 hNSC improves behavior.

[0160] Figures 8A and 8B show diagrams of the manufacturing process and quality control for GMP-grade hNSC strains. Flow cytometry showed appropriate staining for hNSC proliferation and pluripotency markers (Figure 8A). Immunocytochemistry confirmed staining for Nestin, a neuroectodermal stem cell marker (Figure 8C). ESI-017 hNSCs were obtained as frozen aliquots (UC Davis), thawed, cultured without subculturing using the same culture media and reagents as in the GMP facility, and then administered. Five-week-old mice were administered 100,000 hNSCs per hemisphere via stereotactic delivery within the striatum. The study included male (M) and female (F) R6 / 2 and non-transgenic (NT) littermate of the same age, as well as vehicle controls (veh) (n=8 R6 / 2 hNSC M, 6 R6 / 2 hNSC F, 7 NT hNSC M, 7 NT hNSC F, 7 R6 / 2 veh M, 6 R6 / 2 veh F, 8 NT veh M, and 6 NT veh F). Immunosuppression was administered to all mice. Behavioral analysis was performed, and the mice were euthanized at 9 weeks of age immediately after the behavioral tests.

[0161] veh-treated mice developed HD-related behaviors as previously described (Mangiariniet (al., 1996). Briefly, the behavior of R6 / 2 mice was indistinguishable from that of NT mice at 5 weeks of age. Up to 8 weeks, neurological abnormalities included progressive stereotypic hindlimb grooming, clasping, and irregular gait. When the tail is raised, normal mice spread both hind and forelimbs, but if a mouse grips its limbs and holds them against its abdomen, the mouse is considered to be "clasping." Delayed onset of R6 / 2 clasping was observed in all hNSC-treated mice, and veh-treated mice clasped by 3 weeks after implantation. hNSC-treated mice did not clasp at this point, and only 50% of hNSC-treated mice clasped at the time of euthanasia (4 weeks after implantation) (Figure 9). Two types of walking motion assays were performed. Rotarod tests the ability to walk on an accelerating rotating rod. hNSC-treated R6 / 2 mice showed a statistically significant improvement in rotarod performance compared to veh-treated R6 / 2 mice (30% improvement 1 week after implantation, p<0.01; and 19% improvement 3 weeks after implantation, p<0.05) (Figure 1A). In the pole test, comparing the time taken to descend a vertical beam, R6 / 2 mice had a longer latency to descend compared to NT mice. A statistically significant improvement (p=0.02) between the R6 / 2-treated groups was observed 4 weeks after implantation (25% improvement, Figure 1B). Neuromuscular function and motor coordination were also evaluated using a grip strength meter, and hNSC treatment resulted in significant improvement 4 weeks after implantation (p=0.02, 16% improvement, Figure 1C).

[0162] ESI-017 hNSC survival, migration, and differentiation Mice were euthanized four weeks after implantation, and their brains were harvested. Half were post-fixed for histology, and the other half were rapidly frozen for biochemistry. hNSCs were mainly clustered around the needletrack and remained in the striatum (Figure 1D), but some were in the cortex, and a few migrated to the microenvironment between cortical and striatal regions (corpus callosum / white matter tract) (Figure 10). Cells were stained using the human marker SC121 (cytoplasm) or Ku80 (nucleus), and mainly using the early neuron marker doublecortin (DCX) (SC121, mixed yellow in Figure 2A; Ku80, Figures 2B and 2C). Some cells were capable of differentiating into the astrocytic cell phenotype (glial fibrillary acidic protein [GFAP]) (Figure 2B). Non-human GFAP-positive immunostaining, potentially representing mouse glial cell scarring, was also present around the implantation site (Figures 2A and 2B). Differentiation of hNSCs into neuronal-limited precursor cells was confirmed using βIII-tubulin (Figures 2D and 10B) and microtubule-associated protein 2 (MAP-2) (Figures 2E and 10C), but the lack of co-localization with NeuN (Figure 2F) suggests the absence of postmittal neurons. Using stereochemical evaluation of Ku80-positive cells in one hemisphere, the number of implanted hNSCs survived averaged 41,323 cells (n=6, 3 males and 3 females), which represented approximately 41% of the initial 100,000 transplanted cells.

[0163] Implantation of ESI-017 hNSC prevents increased excitability of the corticostriate in R6 / 2 mice. The applicants then evaluated electrophysiological activity. 100,000 hNSCs (n=18) or veh (n=16) were implanted in the striatum of male and female mice over 5 weeks. The applicants recorded from hNSCs in acute brain slices 4–6 weeks after implantation (Figures 3A and 3B). hNSCs exhibited basic neuronal characteristics characteristic of immature cells, namely significantly lower membrane capacitance than host MSNs (hNSC 22.0±1.8pF, n=31 vs. MSN 71.3±3.5pF, n=44; p<0.001, Student's t-test) and significantly higher membrane input resistance (hNSC 2804.8±203.0MU vs. MSN 163.8±15.1MU; p<0.001, Student's t-test). hNSCs exhibited spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs), indicating that they received synaptic input from host tissue or other implanted hNSCs. However, their frequencies were much lower compared to MSNs. Some hNSCs also spontaneously generated action potentials, suggesting that these could affect host neurons and adjacent hNSCs (Figure 3B).

[0164] Electrophysiological changes occur in MSNs from symptomatic R6 / 2 mice compared to NT mice, including changes in endogenous membrane properties and reduced excitatory synaptic activity (Cepeda et al., 2003, 2007). hNSC implantation also affects the membrane properties of MSNs in R6 / 2 mice, as well as mean sE Neither the PSC frequency (1.1±0.1Hz vs. 1.4±0.2Hz) nor the mean SIPSC frequency were significantly altered. R6 / 2 mice also exhibit increased cortical pyramidal excitability, as well as a tendency to produce epileptic discharges and seizures (Cummings et al., 2009). Increased cortical excitability is evident in the striatal MSN by the appearance of large-amplitude EPSCs and high-frequency bursts, particularly after widespread blockade of GABAA receptors, which occur concurrently with increased sEPSC frequency (Cepedaetal., 2003, Cummings et al., 2009). In hNSC-implanted mice (20.5%, 9 / 44), a smaller proportion (not statistically significant) of MSNs exhibited increased corticostriate excitability compared to veh mice (28.6%, 16 / 56). However, this increase in sEPSC frequency within this population was not observed in hNSC-implanted R6 / 2 mice. A rightward shift occurred in the cumulative probability distribution of the inter-event interval plot (p<0.001), indicating that hNSCs can reduce the excitatory input from the cortex to the striatum when GABAA receptors are blocked (Figures 3E and 3F).

[0165] In R6 / 2 mice, host tissue forms potential synaptic connections with the embedded ESI-017 hNSC. The applicants used immunohistochemistry (IHC) and electron microscopy (EM) to investigate whether host-derived nerve terminals form synaptic connections with hNSCs. The applicants found instances where unlabeled nerve terminals of host origin formed potential symmetric synaptic connections with embedded and immunolabeled hNSCs (Figure 4A). Several synaptic vesicles within the nerve terminal were extremely close to the presynaptic membrane, indicating a potential area for vesicle release (DAB labeling of hNSCs obscured the connections). In addition, the applicants found that unlabeled nerve terminals of host origin formed clearly asymmetric connections (Figure 4B), suggesting excitatory synaptic connections. Overall, the applicants found that 44.5% (n=71) of unlabeled nerve terminals of host origin formed asymmetric connections with labeled hNSCs, and 48.3% (n=69) formed symmetric connections. For the remaining 7.2% (n=11) of unlabeled host-origin nerve terminals juxtaposed with labeled hNSCs, the precise nature of their connections (asymmetric or symmetric) could not be determined.

[0166] ESI-017 hNSC rescues behavior, survives, and differentiates in Q140 knock-in mice. The applicants then determined whether hNSCs could also improve the defect in the slowly progressive full-length HD mouse model. Q140 mice express a modified mouse / human exon 1 in which 140 repeat sequences are inserted into the mouse huntingtin gene (Menalled et al., 2003). Homozygous mice exhibit early abnormalities in motor tests, including climbing deficiency at 1.5 months of age and cognitive impairment (Hickey et al., 2008; Simmons et al., 2009) and visible aggregates of HTT (Menalled et al., 2003) around 4 months of age. Striatal atrophy can be detected at 1 year. After 22 months, 35% of striatal cells were lost (Hickey et al., 2008). 24 two-month-old homozygous male and female mice per group were administered 100,000 hNSCs per hemisphere, delivered bilaterally to the striatum in a stereotactic, fixed manner (n=12 / sex), while control same-age Q140 (n=12 / sex) and wild-type (WT) (n=12 / sex) mice were injected with veh. All mice were immunosuppressed. Behavioral studies were initiated at 1.5 months of age (before cell transplantation), and mice were euthanized at 8 months, 6 months after transplantation. Behavioral studies were performed on all mice except for the running wheel, for which only males were used because the estrous cycle affects running activity (Hickeyetal., 2008). Early deficiency in walking motor activity in open field and reduced spontaneous motor activity in cage climbing tests were observed in Q140 mice, but hNSC treatment did not rescue the results (Figure 11).

[0167] In the Pall test, veh-treated Q140 mice took longer to turn compared to wild-type controls (p=0.004), while hNSC-treated Q140 mice performed significantly better than control Q140 mice (p=0.04), were no longer significantly different from wild-type mice, and showed beneficial effects 3 months after transplantation (Figure 5A). As reported by Hickey et al. (2008), 5.5-month-old male Q140 mice had a significant deficit in running speed (revolutions per 3 minutes) at 2 weeks (Figure 5B). Sustained improvement in running wheel deficit was observed post-treatment in hNSC-treated Q140 mice, showing a progressive increase in mean running wheel activity compared to veh-treated mice (Figures 5B and 5C). We conclude that hNSC administration mitigates some of the motor deficits observed in Q140 mice.

[0168] Novel Object Recognition (NOR) is a cortical-dependent cognitive test that requires both learning and memory (recognition), and utilizes the mouse's tendency to investigate novel objects rather than familiar ones. The veh-injected Q140 mouse was reported by Simmonset al. (2009). Furthermore, these mice exhibited significant functional impairment in the No-Oral Range (NOR) compared to veh-injected WT mice at 3 and 5 months after implantation (p=0.003 and p=0.03, respectively). Striatal implantation of hNSCs in Q140 mice rescued cognitive impairment at 5 months after implantation (p=0.03), but not earlier (Figures 5E and 5F).

[0169] A subset of veh and hNSC-transplanted Q140 male mice (n=5 for each group) were euthanized 6 months after treatment for IHC analysis. hNSCs identified by human nucleus-specific antibody (HNA) were present 6 months after transplantation, with the majority found along the injection route in the striatum (Figure 5 Ga, b). The number of HNA-positive cells was counted across the entire striatal region in six coronal sections, and double-labeled cells were calculated using DCX or GFAP (mean data from 5 mice per group ± SEM). Approximately 25% of 100,000 hNSCs survived, with the majority (84% ± 2%) being GFAP-positive (Figure 5 Gb, c) and a smaller proportion (16% ± 2%) being DCX-positive (Figure 5 Ge, f).

[0170] ESI-017 hNSC transplantation increases BDNF levels in HD mice. Increased levels of neurotrophic growth factor, and the subsequent increased synaptic connectivity, are associated with the behavioral improvements observed after NSC transplantation (Blurton-Joneset al., 2009). Furthermore, reduced BDNF levels have been demonstrated in multiple mouse models of HD and in human HD brains (Zuccato et al., 2011). Therefore, we believe that BDNF levels are linked to neuronal It was evaluated as a marker of nutritional effect. In R6 / 2 hNSC mice, IHC and confocal microscopy showed co-localization of BDNF with DCX-positive hNSCs, suggesting that differentiated cells produce BDNF (Figure 6A). In fact, in vitro-grown and differentiated hNSCs produce BDNF only after becoming DCX-positive. Q140 In hNSC mice, BDNF was quantified by ELISA in a subset of male mice (n=6 / group). Striatal BDNF decreased in Q140 mice compared to WT mice, but a significant increase in BDNF levels was observed in hNSC-treated mice compared to veh mice, restoring BDNF levels to WT levels (Figure 6C).

[0171] Considering that neurotrophic signaling can enhance synaptic activity, we investigated the levels of synaptic marker synaptophysin in the striatum of all perfused Q140 animals (n=5 / group) by IHC and quantification using a microarray scanner as previously described (Richter et al., 2017). Comparison of hNSC-treated Q140 mice with veh-treated Q140 mice revealed a significant increase in synaptophysin in hNSC mice (quantified in Figures 13A and 13B).

[0172] These results suggest that engrafted hNSCs may partially improve synaptic connectivity through increased neurotrophic effects, including BDNF.

[0173] Treatment with ESI-017 hNSC in Q140 mice reduced microglial activation. Striatal sections from Q140 mice (n=5 / group) were stained with an antibody against ionized calcium-binding adapter molecule 1 (Iba-1) to identify both resting and reactive microglia. Microglial cell body size correlated with activated state cell morphology (Watsonet al., 2012), and there was a significant increase in the diameter of Iba1-positive cells (strong microglial response). This was observed in the striatum of Q140 mice. This response was significantly reduced by hNSCs (Figure 6D). Similar analysis in hNSC-implanted R6 / 2 mice did not show significant changes in the striatum (Figure 13), suggesting a relatively localized effect or moderate levels of activated microglia.

[0174] ESI-017 hNSC transplantation reduces mHTT accumulation and aggregates. A characteristic feature of HD pathology is the presence of HTT inclusions, which can reflect altered protein homeostasis. Therefore, we performed unbiased stereochemical evaluations of brain sections from R6 / 2 and Q140 mice. For R6 / 2 mice, sections were stained first for Ku80 using nickel-enhanced DAB (black), then for HTT (EM48) using nickel-free DAB, and then with cresyl violet counterstaining for non-hNSC nuclei. Figure 7A shows the areas where stereochemical analysis was performed adjacent to the hNSC implantation, and areas away from the implantation did not show significant differences in mutant HTT (mHTT) accumulation or aggregates. The results show that R6 / 2 mice with implanted hNSCs have a reduced number of scattered stains and a reduced number of inclusions near the injection site compared to veh mice (Figures 7A and 7B).

[0175] A clear decrease in the number of aggregates was also observed in the striatum of Q140 mice (Figure 7C). Six months after treatment, hNSC-treated Q140 mice had fewer scattered stained nuclei (p=0.0102) and fewer reticular aggregates (p=0.0239) compared to veh-treated mice, but no decrease in nuclear inclusions or microaggregates (p=0.0753 and p=0.372, respectively) (Figure 7D). These results suggest that hNSC delivery modulated HD-related pathology. Inclusion acquisition was not observed in or near transplanted cells in either R6 / 2 (Figure 10D) or Q140 mice.

[0176] hNSC transplantation reduces the pathogenicity accumulation of mHTT and ubiquitinated proteins. The applicants then investigated the effects of hNSC treatment on high molecular weight (HMW) mHTT species and ubiquitin-modified proteins accumulating in the R6 / 2 brain. The reduction in these insoluble proteins corresponds to improved behavioral outcomes in R6 / 2 mice (Ochaba et al., 2016). Surfactant insolubility of NT and R6 / 2 striatum with and without hNSC transplantation. Fraction evaluation showed a significant increase in accumulated mHTT levels in the R6 / 2 striatum, and that treatment with hNSCs reduced insoluble HTT accumulation in the R6 / 2 striatum by approximately 70% compared to veh-treated mice (Figures 7E and 7F), which were not due to altered mHTT transgene mRNA expression (Figure 14). Accumulated ubiquitin conjugate protein was also significantly increased in the R6 / 2 striatum compared to NT mice, and hNSC treatment reduced insoluble ubiquitin conjugate protein in the R6 / 2 mouse striatum compared to veh-treated mice (Figures 7E and 7F). No significant difference was detected in treated NT mice.

[0177] CCT / TRiC (TCP1 ring complex) chaperonins are oligomeric chaperones that bind to newly translated polypeptides and fold them. CCT / TRiC expression prevents the aggregation of cleaved mHTT in multiple HD model systems (Tam, S., et al., The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nature CellBiol, 2006. 8(10): p.1155-1162). Overexpression of the bunit CCT1 is sufficient to inhibit aggregation in vitro and in cells and to mitigate mHTT-mediated cytotoxicity (Tam, S., et al., The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformal switch to aggregation. 2009. 16(12): p. 1279-1285). Surprisingly, the 20 kDa apical domain of yeast CCT1 (ApiCCT1) is sufficient to inhibit recombinant mHTT aggregation in vitro. Our data show that recombinant ApiCCT1, i.e., ApiCCT1r, can reduce the HD phenotype in cells (Sontag, EM, et al., Exogenous delivery of chaperonin subunit fragment ApiCCT1modulatesmutant Huntingtin cellular phenotypes. Proc Natl Acad Sci USA, 2013. 110(8):p. 3077-82) and can rescue BDNF transport deficiency in co-culture of primary HD mouse neurons (Zhao, X., et al., TRiCsubunits enhance BDNF axonal transport and rescue striatal atrophyin Huntington's disease. Proc Natl Acad Sci USA, 2016 This indicates that, importantly, this exogenously applied ApiCCT1r is taken up into the cytosol of cultured cells and primary neurons to exert its effects (Sontagetal, Zhao et al.), and if ApiCCT1 is able to deliver the protein to diseased tissue, then ApiCCT1 is able to act by cells. This suggests that it may be taken up and have beneficial effects. A single direct injection of ApiCCT1 into the R6 / 1 striatum reduced the levels of high molecular weight HTT and aggregated HTT, and was detectable even after two weeks. More recent preliminary data show that viral delivery of sApiCCT1, or delivery by mouse NSCs secreting ApiCCT1, provides in vivo improvements in HD mice. These data suggest that continuous delivery of ApiCCT1 may be neuroprotective.

[0178] Viral delivery of ApiCCT1 is effective in vivo. To evaluate continuous sApiCCT1 delivery in vivo, AAV2 / 1-mediated delivery of sApiCCT1 was tested in a small pilot study for its effect on mHTT accumulation in R6 / 1 mice expressing human mHTT exon 1 with approximately 115 repeat sequences and exhibiting a slower disease progression than R6 / 2

[24] (construct in Figure 15A). Due to the rapid onset of the phenotype in R6 / 2 mice and the 2-3 week period during which AAV2 / 1 achieves full expression, earlier viral delivery in disease progression may be essential to achieve maximum modification of the pathological phenotype. Therefore, bilateral intrastriatal injection (12 × 10⁴) into R6 / 1 mice was tested. 9AAV2 / 1 animals expressing either sApiCCT1 or mCherry control of the genomic copy were injected at 5 weeks of age, and tissues were harvested at 17 weeks of age. Animals injected with sApiCCT1 showed an approximately 40% reduction in oligomeric mHTT (Figure 15B and C). Stereoanalysis also revealed an approximately 40% reduction in visible inclusions (Figure 15E), although this effect was not statistically significant, likely due to insufficient power due to sample size. These animals showed a significant improvement in clasping behavior at 16 weeks of age, and this assay is an indicator of motor dysfunction (data not shown). This study was repeated with a larger sample size (approximately 20 animals in each condition). AAV2 / 1-sApiCCT1 injected animals showed significant improvements in the rotarod task, measuring motor coordination and balance, at 10, 12, and 14 weeks (Figure 15F; data for 10 and 12 weeks not shown). These animals also showed improvements in clasping behavior consistent with previous studies (data not shown). Taken together, these studies suggest that continuous delivery of sApiCCT1 is sufficient to improve behavioral outcomes and suppress mHTT pathology in HD mice.

[0179] Viral transduced hNSCs invade Htt14A2.6 PC12 cells and produce secretory ApiCCT1, which affects the oligomeric mHTT species. The applicants conducted a small-scale pilot study to test the transduction of sApiCCT lentivirus into ESI-017 hNSCs, determine appropriate titers for transduction, and examine the effects of ApiCCT production and mutant HTT agglutination. Briefly, ESI-017 hNSCs were cultured in 6-well plates and then transduced with sApiCCT lentivirus at infection multiplicity (MOI) of 0, 5, 10, and 15. Cells were cultured for 48 hours, the medium was collected, and cells were harvested for protein analysis. Figure 16A shows Western blotting analysis of HA-tagged ApiCCT, demonstrating that transduced ESI-017 hNSCs produce ApiCCT, and that production increases with increasing viral MOI. The culture medium recovered from transduced hNSCs was added to Htt14A2.6 PC12 cell medium to determine that transduced secretory ApiCCT can invade adjacent cells, as previously described (SontagPNAS, 2013). In the presence of the factor ponasterone, these cells express a cleaved, elongated repeat HTT exon 1 protein (103Q) fused at the C-terminus with enhanced green fluorescent protein (GFP) within 48 hours. 48 hours after application of induction and conditioning media, the cells were washed, harvested, and subjected to Western blotting. The results showed that cell lysates from the treated 14A2.6 cells contained HA-tagged proteins of appropriate molecular weight for ApiCCT (Figure 16B). To assess whether ApiCCT delivery in conditioning media was effective against specific mutant huntingtin (mHTT) aggregate species, the applicants first evaluated changes in monomer-soluble HTT fragment levels. HTT monomer levels from the same experiment were examined by Western blotting using an antibody against GFP (Figure 16C). ApiCCT1 does not appear to alter monomeric-level mHTT expression, suggesting that ApiCCT does not alter steady-state monomeric mutant HTT (mHTT) and does not affect the gene expression of induced mHTT. Insoluble HTT aggregates and mHTT oligomers are characteristic of HD. In particular, oligomeric mHTT species can be a source of toxicity in affected neurons. Therefore, we measured mHTT oligomers to determine whether ApiCCT1 delivery affects the accumulation of these forms, as previously demonstrated with respect to direct delivery of purified ApiCCT1 protein. Since SDS-agarose gel electrophoresis (AGE) is thought to preferentially separate soluble fibrillary oligomers of mHTT, AGE was used to separate the oligomeric species. Equal amounts of protein from cell lysates were loaded onto SDS-AGE gels. Densitometry measurements were obtained using ImageJ, and ApiCCT1 caused a reduction in mHTT oligomer levels (over 10%) only at the highest MOI (Figure 16D). However, smear length decreased at both MOIs of 10 and 15. These data demonstrate that ApiCCT1 secretion from hNSCs can suppress oligomeric mHTT formation in adjacent cells, reproducing our published results regarding purified ApiCCT1.These results validate the method of introducing lentiviral traits into hNSCs for use in GMP production and establish the feasibility of hNSC delivery.

[0180] Viral transdermal NSCs produce secretory ApiCCT1 after implantation into mice. ESI-017 hNSCs were cultured in UCI as described above. Lentivirus was transduced into the hNSCs at a MOI of 15 for 48 hours, and then the hNSCs were transplanted into 5-week-old mice as described above. Male and female R6 / 2 and non-transgenic same-age littermates, as well as vehicle controls, were included. Immunosuppression was administered to all mice. Mice were euthanized at 9 weeks of age, and their brains were collected. Half were post-fixed for histology, and the other half were rapidly frozen for biochemistry. hNSC-ApiCCT-embedded cells had IHC similar to that described for hNSCs (Figure 17). Cells were stained primarily with the early neuronal marker doublecortin (DCX, blue) using human nuclear antigen markers (HNA) (mixed pink in Figure 17A). Some cells expressed HA-tagged ApiCCT (Figure 17B).

[0181] Consideration Stem cell-based transplantation strategies are promising approaches for neurodegenerative disorders, based on their ability to modulate disease states through regenerative and regenerative mechanisms. In HD models, mouse-derived NSCs have shown promising results, while hNSC-based approaches have yielded mixed successes, exhibiting robust efficacy in toxic models and limited neuroprotection in genetically modified HD mice (El-Akabawy et al., 2012; Golas and Sander, 2016). Herein lies the inventors. We describe the transplantation of GMP-grade hNSCs that provide robust rescue for the deficiency and disease-modifying activity targeting mHTT protein accumulation. ESI-017 hNSCs were electrophysiologically active in R6 / 2 mice but had no significant effect on striatal MSN membrane properties or spontaneous synaptic activity. However, in a subset of MSNs, the increase in sEPSC frequency commonly observed after extensive blockade of GABAA receptors using bicuculin did not occur, suggesting that the graft helps reduce cortical hyperexcitability. Although we have not determined the mechanism underlying this effect, electrical stimulation into the graft induces IPSCs in adjacent cells, suggesting that the stimulation is inhibitory. Ultrastructural data indicate that the host potentially forms both symmetric (inhibitory) and asymmetric (excitatory) synaptic connections with the hNSCs. Our hypothesis is that the effect originates from the implanted cells, and in R6 / 2 mice, the implanted cells differentiate primarily along the neuronal lineage. However, other experiments, including those with Q140 mice, suggest the presence of a potential glial effect and that the driving factors for improvement are still not understood. Considering that neuronal loss does not occur in these mice until the very late stages of the disease, striatum-specific transplantation appears to act both through neuroprotection via trophic factors such as BDNF and by preventing the abnormal accumulation of mHTT species. However, findings of electrophysiological activity in the transplanted cells, and the binding of human cells to endogenous mouse cells which facilitates improved electrophysiological outcomes, suggest that there may also be opportunities for regenerative effects.

[0182] The rationale for transplanting NSCs rather than other precursor cell types lies in the NSCs' ability to differentiate along multiple lineages. In R6 / 2 mice, cells exhibit evidence of early astrocyte or neuronal differentiation, and most are co-labeled with neuronal-specific precursor cell markers (DCX, βIII-tubulin, and MAP-2). Since hNSCs typically take several months to terminally differentiate, we expected to observe only partial differentiation of transplanted cells at 4 weeks. Interestingly, ESI-017 hNSCs were mostly DCX-negative in vitro before implantation. The cell fate results in R6 / 2 mice contrast with our findings in the Q140 long-term HD model, as well as other studies in Parkinson's disease and Alzheimer's disease (AD) models using hNSCs where more cells become astrocytes (Goldberg et al., 2017), but later... The cells were derived from fetal NSCs that were more gliogenic. These data suggest that different responses may exist depending on the disease niche, that immunosuppressive methodologies may influence specifications, or that developmental stimuli and timing specific to human cells rather than mouse cells influence outcomes.

[0183] Reduced BDNF levels were observed in HD mice and human HD subjects (Strandet). (al., 2007; Zuccato et al., 2011), many effective treatments in HD mice show an increase in associated BDNF (RossandTabrizi, 2011). Nutritional factor support via stem cell transplantation. Consistent with this view, ex vivo delivery of GDNF-expressing mouse NSCs maintained motor function and prevented neuronal loss in HD mice (Ebertetal, 2010), and BDNF was required for improved cognition after transplantation of mouse NSCs into either AD mice (Blurton-Jones et al., 2009) or a model of Lewy body dementia (Goldbergetal, 2015). BDNF is also involved in the altered corticostriate tract in HD (Laforetetal, 2001). It must be transported to the striatum via afferent pathways including ). It is thought that by supplying nutritional support to the striatum, the corticostriatal pathway is sufficiently conserved to signal BDNF production in the cortex, or that stem cell-derived BDNF is transported retrogradely from the striatum to the cell bodies of corticostriatal neurons, resulting in improved electrophysiological activity after transplantation.

[0184] One possible mechanism of action of embedded hNSCs may be through a reduction in abnormal mHTT accumulation and aggregates, either by potentially preventing aggregate formation or potentially inducing a selective clearance mechanism (e.g., Chenet al., 2013). The inventors have recently In 2016, we reported findings that a reduction in specific HMW-insoluble mHTT species was associated with improved behavior and normalization of several molecular readout outcomes in R6 / 2 mice. It is reasonable to assume that a reduction in the pathogenicity accumulation of mHTT and ubiquitinated HMW-insoluble species prevents the neuronal dysfunction observed in HD mice.

[0185] In contrast to the observation that aggregates can be obtained in studies of fetal cell transplants in human HD subjects (Cicchetti et al., 2014), evidence of acquired HD phenotypes such as inclusion bodies is not always obtained. It is important to note that this was not observed throughout the transplantation process in the mouse model either (Figure 10). The absence of apparent protein transfer or acquired pathogenesis may result from increased nutrient signaling by hNSCs, or from a reduction in mHTT species that could otherwise facilitate protein transfer to transplanted cells. Alternatively, although not demonstrated by mouse studies, it may take several years for cells to acquire pathogenesis.

[0186] In summary, we have shown that hNSCs transplanted into HD mice may survive, differentiate into neural populations, protect or repair damaged tissue, delay disease progression, mitigate pathology, increase the production of protective molecules, and potentially form connections with surrounding tissues, suggesting a promising treatment strategy for HD. Considering the results by Anet al. (2012) showing that genetically corrected patient-derived NSCs can form human neurons and DARPP-32-positive cells, as well as the results reported here, the future application of autologous transplantation using corrected patient cells may also be feasible.

[0187] Equal portions Although the present invention has been described in relation to the embodiments described above, it should be understood that the foregoing description and examples are intended to be illustrative and not intended to limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention belongs.

[0188] In addition, if any feature or aspect of the present invention is described by a Markush group, a person skilled in the art will recognize that the present invention is also described by any individual member or subgroup of a member of the Markush group.

[0189] All publications, patent applications, patents, and other references mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the specification, including definitions, will control. Throughout this specification, technical references are cited by author and the complete bibliographic details are provided below. [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula]

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Claims

[Claim 1] The invention as shown in the drawings.

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