Neural stem cell compositions and methods for treating neurodegenerative diseases

文档序号：589488 发布日期：2021-05-25 浏览：25次中文

阅读说明：本技术 治疗神经退行性疾病的神经干细胞组合物和方法 (Neural stem cell compositions and methods for treating neurodegenerative diseases ) 是由约翰·莱德林布莱恩·弗瑞莱斯利·米歇尔斯·汤普森格哈德·鲍尔戴恩·科利尔-贝尔库姆于 2018-06-06 设计创作，主要内容包括：本发明提供了基于干细胞的疗法,用于治疗神经退行性疾病和CNS病症,例如亨廷顿氏病。该疗法改善了运动缺陷和挽救突触改变。该细胞示出具有电生理学活性并且其改善了运动和晚期认知损害。(The present invention provides stem cell-based therapies for treating neurodegenerative diseases and CNS disorders, such as huntington's disease. The therapy improves motor deficits and rescues synaptic changes. The cell was shown to have electrophysiological activity and it improved motor and late cognitive impairment.)

1. A method for preparing human neuronal stem cells (hNSC) from human embryonic stem cells (hESC), said method comprising the steps of:

a) isolating at least one stem cell rosette from a population of Embryoid Bodies (EBs) cultured in a differentiation medium;

b) incubating the at least one individual unit isolated from the rosette of step a) for an amount of time and under conditions provided for the generation of the at least one rosette until the at least one rosette is generated;

c) isolating the individual units of the rosette from step b) into individual cells; and

d) culturing the at least one single cell isolated from step c) for an amount of time and under conditions provided for the production of the fused population of hnscs until the fused population of hnscs is produced.

2. The method of claim 1, wherein separating the at least one individual unit from the rosette is performed manually.

3. The method of claim 1, wherein isolating the at least one single unit from the rosette is performed enzymatically.

4. The method of claim 1, wherein separating the at least one individual unit from the rosette of step a) is performed manually.

5. The method of claim 1 or 4, wherein the isolation of at least one individual cell of step c) is performed enzymatically.

6. The method of claim 1, wherein one or more of steps a) through c) are performed 2 or more times.

7. The method of claim 1, wherein at least one of steps a) through d) is performed manually.

8. The method of claim 1, wherein at least one of steps a) through d) is performed mechanically.

9. The method of claim 1, wherein the separation of the rosettes is performed digitally.

10. The method of claim 1, further comprising generating an embryoid body from ESI-017.

11. The method of claim 9 or 10, further comprising culturing Embryoid Bodies (EBs) in EB media on an ultra-low attachment surface.

12. The method of claim 11, further comprising replacing EB media with N2 media after EBs have been cultured on the ornithine/laminin coated surface for an effective amount of time at step a).

13. The method of claim 12, further comprising replacing EB media with N2 media after EBs have been cultured in EB media for an amount of time effective to produce at least one EB of step a).

14. The method of claim 1, wherein the at least one individual cell isolated in step c) is cultured in N2 medium on a guanine/laminin coated plate for an effective amount of time to generate a fused cell population of hnscs.

15. The method of claim 14, further comprising culturing the fused population of hnscs with an effective amount of N2 culture medium.

16. The method of claim 15, further comprising expanding the cell population.

17. The method of claim 1, further comprising genetically modifying the cell.

18. The method of claim 17, wherein the cell is genetically modified by insertion of a transgene or by modification of the CRISPR.

19. The method of claim 18, wherein the transgene is ApiCCT1, a fragment thereof, or an equivalent of each thereof, and optionally wherein the transgene is overexpressed in a cell.

20. An hNSC prepared by the method of any one of claims 15 to 19, and optionally wherein the cell expresses BNDF.

21. An hNSC prepared by the method of claim 10, wherein said hNSC expresses BNDF upon cell differentiation.

22. The hNSC of claim 21, wherein the cell is genetically modified by insertion of a transgene or by CRISPR.

23. A population of cells according to claim 18.

24. A composition comprising the isolated cell of claim 20.

25. A composition comprising the population of claim 24 and a carrier.

26. The composition of claim 24 or 25, further comprising a preservative and/or a cryoprotectant.

27. A method of delivering a transgene to a subject or a gene editing cell in a subject in need thereof, comprising administering an effective amount of the cell of any one of claims 20 or 21.

28. The method of claim 27, wherein the subject is a mammal.

29. The method of claim 28, wherein the subject is a human.

30. A method of treating a neurodegenerative disease or enhancing synaptic connectivity in a subject in need thereof, comprising administering to the subject an effective amount of the isolated cell of claim 20 or 21.

31. The method of claim 30, wherein the neurodegenerative disease is selected from huntington's disease, stroke, alzheimer's disease, parkinson's disease, traumatic brain injury, brain inflammation, stroke, autoimmune diseases (e.g., multiple sclerosis, primary or secondary progressive multiple sclerosis, relapsing and remitting multiple sclerosis), chronic spinal cord injury, bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumors, peripheral neuropathy, guillain-barre syndrome, spinal muscular atrophy, fredrich's ataxia, amyotrophic lateral sclerosis, and huntington's chorea.

32. The method of claim 30 or 31, wherein the subject is a mammal.

33. The method of claim 32, wherein the subject is a human.

34. A kit comprising hescs and instructions for performing the method of any one of claims 1-17.

35. A kit comprising the hESC of claim 20 or 21 and instructions for performing the method of any one of claims 1-17.

36. A non-human animal having the hESC of claim 20 or 21 transplanted into the animal.

37. The non-human animal of claim 36, wherein the animal is a mouse or a sheep.

Background

There are no disease modifying therapies currently available for many neurodegenerative diseases affecting the central or peripheral nervous system. Some have suggested that human stem cells offer potential therapeutic strategies for certain neurodegenerative diseases (reviewed in Drouin-oeullet, 2014, Golas and Sander,2016, Kirkeby et al, 2017).

For example, Huntington's Disease (HD) is an autosomal dominant neurodegenerative Disease caused by amplified CAG repeats (CAG repeat) encoding polyglutamine repeats within Huntington's protein (HTT) (The Huntington's Disease colletive Research Group, 1993). Involuntary movements, progressive intellectual decline and psychiatric disorders occur (Ross and Tabrizi,2011), and neuropathology is mainly related to degeneration of Medium Spiny Neurons (MSNs) in the striatum and atrophy of the cortex (vonstatel and difoglia, 1998). There is a need in the art to find therapies for neurodegenerative diseases and disorders (e.g., HD). The present invention fulfills this need and provides related advantages as well.

Disclosure of Invention

The present invention provides a method of preparing human neuronal stem cells (hNSC) from human embryonic stem cells (hESC), the method comprising, consisting essentially of, or consisting further of the steps of:

a) isolating at least one stem cell rosette (rosette) from a population of Embryoid Bodies (EBs) cultured in a differentiation medium;

b) incubating the at least one individual unit (individual cell) isolated from the rosette of step a) for an amount of time and under conditions provided for the generation of the at least one rosette until the at least one rosette is generated;

c) separating the single units of the rosette from step b) into single cells (individual cells); and

In some embodiments, the separating the at least one individual unit from the rosette is performed manually. In another aspect, isolating at least one individual unit/cell from the rosette is performed enzymatically. In another aspect, the separation of at least one individual unit from the rosette of step a) is performed digitally, optionally using digital two-dimensional or three-dimensional image recognition techniques. In another aspect, the isolation of at least one single cell of step c) is performed enzymatically.

In some embodiments, one or more of steps a) to c) may be performed manually or mechanically in a high-throughput manner (optionally using digital two-dimensional or three-dimensional image recognition techniques) 2 or more times.

In some embodiments, the method further comprises generating an embryoid body from ESI-017. In some embodiments, the method further comprises culturing Embryoid Bodies (EBs) in EB media on the ultra-low attachment surface. In some embodiments, the method further comprises replacing EB media with N2 media after the EB has been cultured on the ornithine/laminin coated surface for an effective amount of time at step a). In some embodiments, the method further comprises replacing EB media with N2 media after the EBs have been cultured in EB media for an amount of time effective to produce at least one EB of step a).

In some embodiments, the at least one individual cell isolated in step c) is cultured in N2 medium on a guanine/laminin coated plate for an effective amount of time to generate a fused cell population of hnscs. In some embodiments, the method further comprises culturing the fused population of hnscs with an effective amount of N2 medium. In some embodiments, the method further comprises expanding the cell population.

In some embodiments, the method further comprises genetically modifying the cell. In some embodiments, the cell is genetically modified by insertion of a transgene or by modification of the CRISPR. In some embodiments, the transgene is ApiCCT1, a fragment thereof, or an equivalent of each thereof, and optionally wherein the transgene is overexpressed in the cell.

In some aspects, the present invention provides an hNSC prepared by a method comprising, consisting essentially of, or consisting of:

a) isolating at least one stem cell rosette from a population of Embryoid Bodies (EBs) cultured in a differentiation medium;

b) incubating at least one individual unit isolated from the rosette of step a) for an amount of time and under conditions provided for the generation of at least one rosette until at least one rosette is generated;

c) isolating the individual units of the rosette from step b) into individual cells; and

In some embodiments, the hNSC expresses BNDF. In some embodiments, the hNSC expresses BNDF upon differentiation of the cell. In some embodiments, the cell is genetically modified by insertion of a transgene or by CRISPR.

In some aspects, the invention provides a population of cells prepared according to the methods described herein. Also provided are compositions comprising isolated cells prepared according to the methods described herein. In some embodiments, the composition further comprises a carrier. In some embodiments, the carrier is a preservative and/or a cryoprotectant.

In some aspects, the invention provides a method of delivering a transgene to a subject or a method of gene editing cells in a subject in need thereof comprising administering an effective amount of an isolated cell 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 aspects, the invention provides a method of treating a neurodegenerative disease or enhancing synaptic connectivity in a subject in need thereof, comprising administering an effective amount of an isolated cell 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 disease is selected from huntington's disease, stroke, alzheimer's disease, parkinson's disease, traumatic brain injury, brain inflammation, stroke, autoimmune diseases (e.g., multiple sclerosis, primary or secondary progressive multiple sclerosis, relapsing and remitting multiple sclerosis), chronic spinal cord injury, Bell's palsy (Bell's palsy), cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumors, peripheral neuropathy, Guillain-Barre syndrome (Guillain-Barre syndrome), spinal muscular atrophy, fredrich's ataxia, amyotrophic lateral sclerosis, and huntington's chorea.

In some aspects, the invention provides kits comprising hescs and instructions for performing the methods described herein.

In some aspects, the invention provides a non-human animal having an hNSC prepared according to the methods described herein and transplanted into the animal. In some embodiments, the animal is a mouse or a sheep.

Drawings

FIGS. 1A-1D: ESI-017hNSC implanted in R6/2 mice improved behavior and showed evidence of differentiation into immature neurons and astrocytes. (A) The rotarod task demonstrated a defect in R6/2 mice compared to non-transgenic littermates (NTs), and the mean latency increased at 1 week (black bars) and 3 weeks (grey bars) post-implantation in hNSC-treated R6/2 mice compared to vehicle-treated (Veh) mice. (B) Climbing pole tests demonstrated that R6/2 mice were deficient compared to NT. hNSC-treated R6/2 mice dropped faster than Veh mice 4 weeks post-implantation (grey bars), but not 2 weeks post-implantation (black bars). (C) The grip demonstrated a defect in R6/2 mice compared to NT. Compared to Veh mice (black bars), hNSC-treated R6/2 mice had greater force (grams) after 4 weeks, but not after 2 weeks (grey bars). (D) Immunohistochemistry (IHC). The hNSC (human marker SC121) implanted into the striatum of R6/2 mice co-localized with markers for neuronal restricted progenitor cells (double cortin [ DCX ]) and astrocytes (SC121 and GFAP). One-way anova followed by Tukey's HSD test and post hoc multiple comparative calculations of Scheffe', Bonferroni and Holm. P <0.05, p <0.01(n 15). Mean ± SEM are shown.

FIGS. 2A-2F: IHC showed differentiation of ESI-017hNSC implanted in R6/2 mice. (A) Hnscs (SC121) implanted in R6/2 mice differentiated into neuronal restricted progenitor cells (biscortin [ DCX ]) and astrocytes (SC121 and GFAP). (B) High magnification (633) shows differentiation: hnscs (human nuclear marker Ku80) implanted in R6/2 mice differentiated into neuronal restricted progenitor cells (DCX) and some astrocytes (Ku80 and GFAP). (C) hNSC (Ku80) and neuronal restricted progenitor cells (DCX). (D) hNSC (Ku80) and neuronal restricted progenitor cells (β III-tubulin); mouse nuclei are shown as DAPI. (E) hNSC (Ku80) and neuronal restricted progenitor cells (MAP-2); mouse nuclei are shown as DAPI. (F) hNSC (Ku80) is not co-localized with a differentiated post-mitotic neuronal cell marker (NeuN).

FIGS. 3A-3F: implantation of ESI-017hNSC reduced corticobasal hyperexcitability in R6/2 mice. (A) Hnscs filled (arrows) with biocytin recorded SC121 in striatum and IHC. Scale bar, 20 mm. (B) Top track: cell attachment of spontaneously discharged (spontaneousring) hNSC was recorded. Bottom track: sEPSC and sIPSC from hNSC. Recordings demonstrate spontaneous inward and outward synaptic currents in hnscs. (C) sEPSC and sIPSC recorded in MSN. (D) Biocytin-filled MSNs in the vicinity of hNSC cluster (SC 121). Scale bar, 20 mm. (E) The record of sEPSC in the R6/2MSN subgroup after addition of the GABAA receptor antagonist bicuculline (10mM) showed "epileptiform" activity (first trace). These large amplitude excitatory events are typically followed by high frequency small amplitude sepscs. In mice with hNSC implants, these events were significantly reduced in frequency (second trace). (F) In cells with "epileptiform" activity (6-8 min after BIC), the inter-event cumulative probability distribution of the R6/2 group implanted with hnscs was shifted to the right compared to the vehicle, corresponding to a significant decrease in high frequency spontaneous events (p <0.001, two replicates of anova followed by Bonferroni post hoc analysis;. p < 0.05).

FIGS. 4A-4B: the nerve endings from the host link the synapse to the implanted hNSC. (A) Unlabeled nerve terminal (U-NT), which contains synaptic vesicles, forms synapse-like associations with the underlying labeled (SC121) hNSC dendrite (L-DEND) (arrows). The association may be symmetrical. (B) Unlabeled nerve terminal (U-NT), which contains a synaptic vesicle, forms an asymmetric synaptic connection (arrow) with the underlying labeled (SC121) hNSC dendrite (L-DEND). This asymmetric association indicates excitatory synaptic association.

FIGS. 5A-5G: ESI-017hNSC implanted in Q140 mice improved the behaviours and showed evidence of differentiation into immature neurons and astrocytes. (A) Temporary improvement in motor coordination (pole climbing task) 3 months after cell injection. WT Veh (n ═ 20), Q140 Veh (n ═ 18), Q140 hNSC (n ═ 18). One-way analysis of variance with Bonferroni post hoc tests: p <0.05, p < 0.01. (B-D) continued to improve the runner defect 5.5 months after treatment (n-5 per group). (B) A graph showing the mean number of runner rotations per 3 min/night over 2 weeks in 7.5 month old male WT or Q140 mice at 5.5 months post-treatment. Comparison by two-factor analysis of variance: effect of group F52.93, p < 0.0001; the effect of night number in the wheel F is 17, p < 0.0001. Bonferroni post-hoc test: p <0.01, p <0.001 and p <0.0001 compared to Q140 Veh. (C) Total average number of wheel revolutions at night in2 weeks. Two-way analysis of variance with Bonferroni post hoc tests: p <0.01, p < 0.001. (D) The motion learning slopes between the three groups are not significant. (E and F) identification of new objects. hNSCs prevented deficiency in Q140 mice at 5 months post-treatment, but did not have a discrimination index at 3 months for sniffing time (E) or number of rounds (F). WT Veh n 18, Q140 hNSC n 19. One-way analysis of variance for Bonferroni post hoc tests: p <0.05, p < 0.01. (G) Survival and differentiation of hnscs in Q140 mice by staining human specific antibodies (HNA; a and d) co-expressed with astrocytes (GFAP; b and c) or neuronal restricted progenitor cells (DCX; e and f). Scale bar, 20 mm. All figures show mean ± SEM.

FIGS. 6A-6D: ESI-017hNSC implanted in HD mice increased the expression of BDNF. (A) ESI-017hNSC (Ku80) shows co-localization with BDNF; astrocytes were shown to be GFAP positive. (B) Veh treated mice did not show BDNF or hNSC, but had GFAP. (C) BDNF levels of ELISA in 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 for each group). Bars represent the percentage of cells per diameter, and grey parts represent confidence intervals. Significant striatal microglial activation was observed in Q140 Veh compared to WT Veh. Q140 hNSC mice showed significantly reduced microglial activation in the striatum compared to Q140 Veh mice. P <0.05 and p <0.01 by one-way analysis of variance with Bonferroni post-hoc tests. Mean ± SEM are shown.

FIGS. 7A-7F: ESI-017hNSC implanted in R6/2 mice resulted in a reduction of diffuse aggregates and inclusion bodies, and reduced huntington aggregates in Q140 mice. (A and B) ESI-017hNSC resulted in a reduction of diffuse aggregates and inclusion bodies (arrows in A) in R6/2 mice. (A) Images of Ku80 with nickel, HTT marker EM48, and cresol purple for non-hNSC nuclear staining. Stereological evaluation using StereoInvesticator. Traced in outline under 53 objectives (dashed line, example in the left panel) and counted at 1003. For 6 slices across the entire striatum, count every 3 slices (40mm coronal slices), where Ku80 can be seen between the top bregma 0.5mm and bregma — 0.34 mm. (B) The percentage of cells with aggregates or inclusion bodies (n 4/group) is shown, by one-way analysis of variance using Bonferroni post-hoc tests p < 0.01. (C and D) ESI-017hNSC reduced Henbuton aggregates in Q140 mice. (C) Image of HTT marker EM48 (arrows indicate inclusion bodies). (D) HTT stained nuclei and aggregates were analyzed with a stereoinvestor for quantification of aggregate type/section. Data are shown as mean ± SEM (n-5/panel). One-way anova by Bonferroni post-hoc test, # p < 0.05. (E and F) hNSC transplantation regulates the accumulation of insoluble proteins in R6/2 mice. The striatal lysates of the Western blots were separated into detergent soluble and detergent insoluble fractions. (E) Compared with NT, R6/2 is enriched in insoluble accumulated mHTT. Transplantation of hNSC in R6/2 resulted in a significant reduction in the HTT of insoluble HMW accumulation compared to veh treated animals. Compared to NT, R6/2 striatum is also enriched for insoluble ubiquitin conjugated proteins. Transplantation of hNSC in R6/2 mice resulted in a significant reduction of ubiquitin-modified insoluble conjugated proteins compared to veh treatment; whereas there was no significant effect in NT compared to veh control. (F) Quantification of relative protein expression of mHTT and ubiquitin. Values represent mean ± SEM. Statistical significance of the expression of the relatively insoluble accumulated mHTT and ubiquitin conjugated proteins in R6/2 was determined by one-way anova followed by Bonferroni post hoc test (n-3/treatment). P <0.05, p <0.01, p < 0.001. Mean ± SEM are shown.

FIGS. 8A-8D: characterization by ESI-017hNSC of a monochrome flow cytometer. (A) ESI-017hNSC stained positive for CD24, SOX1, SOX2, Nestin, and Pax6 NSC markers. ESI-017hNSC stained negatively for the pluripotent marker SSEA 4. Karyotyping was performed on ESI-017hNSC and metaphase was visualized by Giemsa staining of agglutinated chromosomes. The final karyotype showed a high mitotic index with a normal 46XX profile. (B) Flow chart of NSC manufacturing process: hnscs were generated by forming Embryoid Bodies (EBs) and then plating the generated EBs into polyornithine-laminin (Poly-O) -coated plates followed by formation of neural rosettes. Rosettes were manually cut and transferred to fresh Poly-O plates where they were attached. The amplified neural rosettes were then enzymatically cleaved and then plated onto fresh Poly-O plates. Where cells can be grown to confluence and enzymatically passaged to larger numbers of Poly-O plates. After sufficient expansion, the resultant hnscs are subjected to final harvest and cryopreservation. (C) Cultured ESI-017hNSC immunocytochemistry showed positive NSC staining and DAPI nuclear staining for the neuroectodermal stem cell marker nestin. The scale bar is equal to 30 μm. (D) Is a picture of a rosette.

FIG. 9: and (4) fastening action: r6/2 mice treated with ESI-017hNSC (n ═ 15) showed delayed fastening behavior after implantation. Non-transgenic (NT) mice did not display this phenotype. Mice were tested daily for this phenotype and the percentage of each group that buckled over the course of the study is plotted. The significance of the fastening assay was determined by Fisher's exact probability test.

FIGS. 10A-10E: ESI-017 low magnification immunohistochemistry. R6/2 mice implanted with hNSC: the hNSC (human marker SC121) implanted in R6/2 mice co-localized with the marker for neuronal restricted progenitor cells (biscortin DCX). For screening hNSC, IHC was performed on sections 34, 37, 40, 43, 46 and 49 (corresponding to Bregma 0.38mm, 0.26mm, 0.14mm, 0.02mm, -0.10mm and-0.22 mm, respectively). S2 is a reuse of the image as shown in fig. 1D for comparison with other coronal slices. Immunohistochemistry in ESI-017hNSC implant R6/2 mice: (A) the hNSC (human marker Ku80) implanted in R6/2 mice was not co-localized with the oligodendrocyte marker (Olig2) mouse nucleus shown with DAPI. High magnification (63x) shows differentiation: (B) hNSC (human nuclear marker Ku80 and cytoplasmic marker SC121 blue) showed co-localization with neuronal restricted progenitor cells (BIII-tubulin) (lt. blue). (C) hNSC (human nuclear marker Ku80 and cytoplasmic marker SC121) showed co-localization with neuronal restricted progenitor cells (MAP-2). (D) hNSC (human nuclear marker Ku80) was not co-localized with the huntington marker (EM 48). (E) S1-6 shows the collected coronal sections, 40um each, immunostained starting from bregma 1.70mm.

FIGS. 11A-11B: ESI-017hNSC implanted in the striatum did not improve the defects in open field or caging experiments in Q140 mice. Mice were tested in open field for 15 minutes (a) and 5 minutes (B) at 0.5 months prior to implantation or 3 and 5 months post implantation. Data are presented as mean ± SEM; wt Veh (n ═ 18), Q140 Veh (n ═ 18), and Q140 hNSC (n ═ 17). Two-way anova with Bonferroni post-hoc test was compared to vehicle-treated Wt mice at the same time points, # p <0.05, # p <0.01, # p < 0.001.

FIGS. 12A-12C: in vitro expression of ESI-017hNSC BDNF. ESI-017hNSC were cultured in neural stem cell medium (A) or differentiated (B), followed by staining for BDNF, the human nuclear marker Ku80, and the biscortin DCX. (C) qPCR comparing RNA levels from cultured ESI-017hNSC showed increased BDNF expression with differentiation. In comparison, the stem cell marker nestin decreased with differentiation, while DCX increased.

FIGS. 13A-13E: (A & B) increased levels of synaptophysin in the striatum of Q140 mice with ESI-017 hNSC. (A) Images were collected with a microarray scanner and fluorescence intensity was quantified. The white scale bar is equal to 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, with n-5 mice per group. hNSC treatment in R6/2 mice did not alter microglial activation. Data are presented as mean + 95% confidence interval (n-5 for each group). Bars represent the percentage of each diameter of the cells, and colored parts represent confidence intervals. (C) Significant striatal microglial activation was observed in R6/2 mice treated with vector (R6/2Veh) compared to non-transgenic control (NT Veh). (D) Comparison of NT + vector to NT + hNSC. (E) R6/2 mice treated with hNSC (R6/2NSC) showed no significant reduction in microglial activation in the striatum compared to R6/2Veh mice.

FIG. 14: real-time PCR of human HTT transgene expression in R6/2 mice. The RPLPO (large ribosomal protein) endogenous control was used to normalize gene expression differences in cDNA samples. No significance was observed as determined by one-way anova with Bonferroni post-hoc tests.

FIGS. 15A-15F: bilateral intrastriatal injection of expression sApicCT1 at 5 weeks of age in R6/1 miceOr mCherry-controlled AAV. In two separate experiments, mice were injected with 12x10⁹A genomic copy of AAV2/1 was obtained and harvested at 17 weeks of age. (A) Schematic representation. (B, C) quantification by agarose gel electrophoresis followed by Western blotting showed a significant reduction in oligomeric mHTT in the animals. (D) Immunohistochemistry showed expression of sapictc 1 (anti-HA). (E) Mice injected with sapictc rt 1 showed by stereology a reduction of approximately 40% in visualized mHTT inclusion bodies (anti-EM 48). (F) Mice injected with sApicCT1-AAV2/1 showed improvement in the rotarod motor task<0.05，**p>0.01。

FIGS. 16A-16D: ESI-017hNSC produces ApiCCT. (A) Following transduction, ESI-017 hnscs transduced with sapictc lentivirus at MOI 0, 5, 10 or 15 were incubated for 48 hours, lysed and Western blotted with HA antibody, then cleaved and re-probed with α -tubulin antibody for loading control. (B) ApiCCT secreted from hNSC entered PC12 Htt14A2.6 cells. Conditioned media from ESI-017hNSC transduced with sapictc lentivirus was applied to 14a2.6 cells induced by EtOH solution of ponasterone to express HTT-GFP, or to controls treated with EtOH alone. Detection of ApiCCT1 in cell lysates supports the feasibility of engineered hnscs to express a secreted form of ApiCCT1, which ApiCCT1 can be taken up by neighboring cells after transplantation. Western blots are shown using HA antibodies. With higher MOI, higher amounts of ApiCCT1 were detected in the treated PC12 cell lysate. (C) ApiCCT1 secreted from hNSC did not alter monomeric HTT in PC12 Htt14A2.6 cells. Conditioned media from ESI-017hNSC transduced with sapictc lentivirus was applied to 14a2.6 cells induced by sterone or to controls treated with EtOH alone. Treatment with secreted ApiCCT1 did not result in an alteration of the monomeric mHTT-GFP transgene. Western blots were shown using GFP antibody, which was then cleaved and reprobed for a-tubulin as a loading control. (D) ApiCCT1 secreted from hnscs altered oligomeric HTT species in PC12 httt14a2.6 cells. Conditioned media from ESI-017hNSC transduced with sapictc 1 lentivirus and applied to pinsterone-induced 14a2.6 cells or control treated with EtOH alone resulted in a reduction of oligomeric HTT at the highest MOI (red box). Western blot of representative samples shown using GFP antibody.

Fig. 17A and 17B: IHC showed that ESI-017hNSC, transduced by a virus of ApiCCT and implanted into the striatum of R6/2 mice, expressed ApiCCT. (A) Hnscs (human nuclear antigen [ HNA ]) implanted in R6/2 mice differentiated into neuronal restricted progenitor cells (biscortin [ DCX ]) and expressed HA-labeled apicct (HA). (B) High magnification (95 ×), taken from the white box region indicated by a, shows differentiation and ApiCCT expression: hnsc (hna) implanted in R6/2 mice differentiated into neuronal restricted progenitor cells (DCX) and expressed HA-labeled apicct (HA).

Detailed Description

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. All technical and patent publications are herein incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Throughout this application and within this application, technical and patent documents are cited by reference. For some of these references, an identifying citation may be found at the end of the application immediately preceding the claims. All publications are herein incorporated by reference to describe more fully the state of the art to which this invention pertains.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds (2001) Molecular Cloning A Laboratory Manual,3^rdedition; (2007) Current Protocols in Molecular Biology series; methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al (1991) PCR 1: A Practical Approach (IRL Press a)t Oxford University Press)；MacPherson et al.(1995)PCR 2:A Practical Approach；Harlow and Lane eds.(1999)Antibodies,A Laboratory Manual；Freshney(2005)Culture of Animal Cells:A Manual of Basic Technique,5^thedition; gait ed (1984) Oligonucleotide Synthesis; U.S. Pat. nos. 4,683,195; hames and Higgins eds (1984) Nucleic Acid Hybridization; anderson (1999) Nucleic Acid Hybridization; hames and Higgins eds (1984) transformation and transformation; immobilized Cells and Enzymes (IRL Press (1986)); perbal (1984) A Practical Guide to Molecular Cloning; miller and Calos eds (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); makrides ed (2003) Gene Transfer and Expression in Mammarian Cells; mayer and Walker eds (1987) biochemical Methods in Cell and Molecular Biology (Academic Press, London); herzenberg et al.eds (1996) Weir's Handbook of Experimental Immunology; a Laboratory Manual,3^rd edition(Cold Spring Harbor Laboratory Press(2002))；Sohail(ed.)(2004)Gene Silencing by RNA Interference:Technology and Application(CRC Press)。

All numerical designations of ranges (e.g., pH, temperature, time, concentration, and molecular weight) are approximate, varying (+) or (-) by increments of 0.1 or 1.0, as appropriate. It should be understood that all numerical designations are preceded by the term "about," although this is not always explicitly stated. It is also to be understood that, although not always explicitly indicated, the reagents described herein are exemplary only, and equivalents thereof are known in the art.

As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not exclude other elements. When used to define compositions and methods, "consisting essentially of … …" is meant to exclude other elements having any substantial meaning to the combination for the purpose. Thus, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants from the isolation and purification process as well as pharmaceutically acceptable carriers (e.g., phosphate buffered saline, preservatives, etc.). "consisting of … …" means excluding more than trace amounts of other elements and essential method steps for applying the compositions of the invention or process steps to produce the compositions or to achieve the intended results. Embodiments defined by each of these transitional terms are within the scope of the present invention.

As used herein with respect to nucleic acids (e.g., DNA or RNA), the term "isolated" refers to molecules that are separated from other DNA or RNA, respectively, that are present in macromolecules of natural origin. The term "isolated nucleic acid" is meant to include nucleic acid fragments which do not naturally occur as fragments and which are not found in the natural state. The term "isolated" is also used herein to refer to polypeptides, proteins and/or host cells that are isolated from other cellular proteins, and is meant to include both purified and recombinant polypeptides. In other embodiments, the term "isolated" refers to separation from a component, cell, or the like, wherein the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof is typically naturally associated. For example, an isolated cell is a cell that is isolated from a tissue or cell of dissimilar phenotype or genotype. It will be apparent to those skilled in the art that a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof need not be "isolated" to distinguish it from its naturally occurring counterpart.

The term "separating" is intended to mean a process of separating from other compositions or components an immediately adjacent other component or component. The cells/units may be separated manually (e.g., manually using a pipette or other tool), enzymatically by using chemical reagents, or digitally by digital techniques based on cell/unit or rosette morphology. See, e.g., cell vision, com/en/introducing-digital-cell-morphology-by-cell vision, visited on day 5-22 in 2018.

"differentiation medium" refers to a cell culture medium containing factors, such as certain growth factors, that promote the differentiation of immature cells to a more mature phenotype, such as differentiation from embryonic stem cells to neural cells.

As used herein, the term "fused population" refers to a population of cells that are in continuous contact with adjacent cells.

By "ultra-low attachment surface" is meant a cell or tissue culture surface that, in certain aspects, comprises a covalently bonded hydrogel layer of hydrophilic and neutral charges. Since proteins and other biomolecules are passively adsorbed to the polystyrene surface through hydrophobic or ionic interactions, the hydrogel surface naturally inhibits non-specific immobilization by these forces, thus inhibiting subsequent cell attachment. These surfaces are commercially available from a number of suppliers, such as Millipore-Sigma, Fisher-Scientific, and S-bio. Methods for making cell culture plates and surfaces are known in the art.

"transgenic" refers to a polynucleotide that has been added to a cell, tissue or organism. An example of a transgene is ApiCCT 1.

"ApiCCT 1" refers to the apical domain of CCT1 and/or a polynucleotide encoding the apical domain of CCT1 (Sontag, E.Proc Natl Acad Sci U S.2013Feb 19; 110(8):3077-82, incorporated herein by reference). CCT1 is a molecular chaperone, a member of chaperone proteins containing the TCP1 complex (CCT), also known as the TCP1 loop complex (try). The complex consists of two identical stacked loops, each loop containing eight different proteins. The unfolded polypeptide enters the central cavity of the complex and folds in an ATP-dependent manner. The complex folds a variety of proteins, including actin and tubulin. In some embodiments, ApiCCT1 is 20kDa in size. In humans, the TCP1 loop complex is encoded by the TCP1 gene (Entrez gene 6950). Herein as SEQ ID NO: 1-4 provide non-limiting examples of sequences of TCP1 mRNA and protein. The apical domain is involved in substrate binding. (Pappenberger, G.et al.J Mol biol.2002May 17; 318(5):1367-79, incorporated herein by reference). Non-limiting examples of sequences for ApiCCT1 are provided below (SEQ ID NO: 7):

MVPGYALNCTVASQAMPKRIAGGNVKIACLDLNLQKARMAMGVQINIDDPEQLEQIRKREAGIVLERVKKIIDAGAQWLTIKGIDDLCLKEFVEAKlMGVRRCKKEDLRRIARATGATLVSSMSNLEGEETFESSYLGLCDEWQAKFSDDECILIKGTSKAAAAALE。

"sapictc 1" refers to a secreted version of ApiCCT 1. Non-limiting examples of nucleic acid sequences and amino acid sequences for sapictc are provided below. The underlined sequence corresponds to the HA tag. In some embodiments, sapictc 1 does not comprise a tag.

sApiCCT1 mRNA(SEQ ID NO：8)

ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCTATCAGTGGCTATGCACTCAACTGTGTGGTGGGATCCCAGGGCATGCCCAAGAGAATCGTAAATGCAAAAATTGCTTGCCTTGACTTCAGCCTGCAAAAAACAAAAATGAAGCTTGGTGTACAGGTGGTCATTACAGACCCTGAAAAACTGGACCAAATTAGACAGAGAGAATCAGATATCACCAAGGAGAGAATTCAGAAGATCCTGGCAACTGGTGCCAATGTTATTCTAACCACTGGTGGAATTGATGATATGTGTCTGAAGTATTTTGTGGAGGCTGGTGCTATGGCAGTTAGAAGAGTTTTAAAAAGGGACCTTAAACGCATTGCCAAAGCTTCTGGAGCAACTATTCTGTCAACCCTGGCCAATTTGGAAGGTGAAGAAACTTTTGAAGCTGCAATGTTGGGACAGGCAGAAGAAGTGGTACAGGAGAGAATTTGTGATGATGAGCTGATCTTAATCAAAAATACTAAGGCTGCTGCGGCTGCGGGTGGACACTACCCTTACGACGTGCCTGACTACGCCTGA

sApicCT1 peptide (SEQ ID NO: 9)

MYRMQLLSCIALSLALVTNSISGYALNCVVGSQGMPKRIVNAKIACLDFSLQKTKMKLGVQVVITDPEKLDQIRQRESDITKERIQKILATGANVILTTGGIDDMCLKYFVEAGAMAVRRVLKRDLKRIAKASGATILSTLANLEGEETFEAAMLGQAEEVVQERICDDELILIKNTKAAAAAGGHYPYDVPDYA

As used herein, "BDNF" refers to brain-derived neurotrophic factor (BDNF) and equivalents thereof and/or to BDNF-encoding polynucleotides or equivalents thereof. BDNF acts on neurons of the central and peripheral nervous systems, helping to support the survival of existing neurons, and promoting the growth and differentiation of new neurons and synapses. BDNF is also active in the hippocampus, cortex and basal forebrain, regions that are critical for learning, memory and high-intelligence quotient. It is also expressed in the retina, motor neurons, kidney, 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, NM-001143805). Non-limiting examples of BDNF mRNA and protein sequences are set forth herein as SEQ ID NO: 5-6.

As used herein, the term "CRISPR" refers to a sequence-specific gene manipulation technique that relies on an aggregated regularly spaced short palindromic repeat pathway. CRISPRs can be used to perform gene editing and/or gene regulation, as well as simply target proteins to specific genomic locations. Gene editing refers to a genetic engineering in which the nucleotide sequence of a target polynucleotide sequence is altered by introducing deletions, insertions or base substitutions into the polynucleotide sequence. In certain aspects, CRISPR-mediated gene editing utilizes pathways of non-homologous end joining (NHEJ) or homologous recombination for editing. Gene regulation refers to increasing or decreasing the production of a particular gene product (e.g., a protein or RNA).

As used herein, the term "gRNA" or "guide RNA" refers to a guide RNA sequence used to target a particular gene for correction using CRISPR techniques. Techniques for designing grnas and donor therapeutic polynucleotides for target specificity are well known in the art. Such as Doench, j., et al, nature biotechnology 2014; 32(12) 1262-7, Mohr, S.et al (2016) FEBS Journal 283:3232-38, and Graham, D.et al genome biol.2015; 16:260. The gRNA comprises, consists essentially of, or consists further of: a fusion polynucleotide comprising CRISPR RNA (crRNA) and transactivation CRIPSPR RNA (tracrRNA); or a polynucleotide comprising CRISPR RNA (crRNA) and transactivation CRIPSPR RNA (tracrRNA). In some aspects, the gRNA is synthetic (Kelley, m.et al. (2016) J of Biotechnology 233(2016) 74-83). As used herein, biological equivalents of grnas include, but are not limited to, polynucleotides or targeting molecules that can direct Cas9 or its equivalent to a particular nucleotide sequence (e.g., a particular region of a cell genome).

Expression of CRISPR in cells can be achieved using conventional CRISPR/Cas systems and directs target gene-specific RNA in cells. Suitable expression systems, such as lentiviral or adenoviral expression systems, are known in the art. It is understood that CRISPR editing constructs can be used to knock out endogenous genes or knock in genes. Thus, it can be appreciated that CRISPR systems can be designed to achieve one or both of these objectives.

As known to those skilled in the art, there are six classes of viruses. DNA viruses constitute class I and class II. RNA viruses and retroviruses constitute the remaining categories. Class III viruses have a double-stranded RNA genome. Class IV viruses have a positive single-stranded RNA genome, which itself functions as mRNA. Class V viruses have a negative single-stranded RNA genome, which serves as a template for mRNA synthesis. Class VI viruses have a positive single-stranded RNA genome, but have DNA intermediates not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse transcribed into a DNA form and integrated into the genomic DNA of the infected cell. The integrated DNA form is called provirus.

The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Nucleotide structural modifications, if present, may be imparted before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the invention as a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form.

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (a), cytosine (C), guanine (G), thymine (T); when the polynucleotide is RNA, it is uracil (U) for thymine. Thus, the term "polynucleotide sequence" is a letter representation of a polynucleotide molecule. The alphabetical representation can be entered into a database in a computer having a central processing unit and used in bioinformatics applications such as functional genomics and homology searches.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then 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. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or less than 25% identity, with one of the sequences of the invention.

A polynucleotide or polynucleotide region (or polypeptide region) has a percentage (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that when aligned, the percentage of bases (or amino acids) are the same when the two sequences are compared. Such alignments and percent homology or sequence identity can be determined using software programs known in the art, such as those described by Ausubel et al. Preferably, default parameters are used for alignment. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: the genetic code is standard; no filter; the strand is the two; the critical value is 60; desirably 10; BLOSUM 62; describe 50 sequences; the sorting mode is high score; database-not redundant-GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet addresses: ncbi.nlm.nih.gov/cgi-bin/BLAST.

An equivalent or biologically equivalent nucleic acid, polynucleotide or oligonucleotide or peptide is a nucleic acid, polynucleotide or oligonucleotide or peptide that has at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 92% sequence identity, or at least 95% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity to a reference nucleic acid, polynucleotide, oligonucleotide or peptide.

The term "amplification of a polynucleotide" includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR) and amplification methods. These methods are known in the art and widely practiced. See, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al, 1990 (for PCR) and Wu et al (1989) Genomics 4: 560-. In general, the PCR procedure describes a method of gene amplification that includes (i) sequence-specific hybridization of primers to specific genes in a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, extension, and denaturation using a 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 provide polymerization initiation, i.e., each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

Reagents and hardware for performing PCR are commercially available. Primers that can be used to amplify sequences from a particular gene region are preferably complementary to and specifically hybridize to sequences in the target region or flanking regions thereof. The nucleic acid sequence generated by amplification can be sequenced directly. Alternatively, the amplified sequences can be cloned prior to sequence analysis. Methods for direct cloning and sequence analysis of enzymatically amplified genomic segments are known in the art.

"Gene" refers to a polynucleotide comprising at least one Open Reading Frame (ORF) that, upon transcription and translation, is capable of encoding a particular polypeptide or protein.

The term "expression" refers to the production of a gene product.

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 is derived from genomic DNA, expression may comprise splicing of the mRNA in a eukaryotic cell.

"Gene product" or "gene expression product" refers to an amino acid (e.g., a peptide or polypeptide) that is produced when a gene is transcribed and translated.

"under transcriptional control" is a term known in the art and indicates that transcription of a polynucleotide sequence (typically a DNA sequence) is dependent on being operably linked to elements that help initiate transcription or promote transcription. "operably linked" means that the polynucleotides are arranged in a manner such that they can function in a cell. In one aspect, the invention provides a promoter operably linked to a downstream sequence (e.g., a suicide gene, a polynucleotide encoding ApiCCT1, a fragment thereof such as sapictc 1, or an equivalent of each thereof).

The term "encoding" when applied to a polynucleotide means that the polynucleotide, if in its native state, is said to "encode" a polypeptide; or it may be transduced and/or translated to produce mRNA for the polypeptide and/or fragments thereof when manipulated by methods known to those skilled in the art. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

When used in the context of polynucleotide manipulation, "probe" refers to an oligonucleotide provided as a reagent that detects a target that may be present in a sample of interest by hybridization to the target. Typically, the probe comprises a detectable label or means by which the label can be attached before or after the hybridization reaction. Alternatively, a "probe" may be a biological compound, such as a polypeptide, antibody or fragment thereof, capable of binding to a target that may be present in a sample of interest.

"detectable label" or "label" includes but is not limited to radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes and includes enzyme proteins. A detectable label may also be attached to a polynucleotide, polypeptide, antibody, or composition described herein.

A "primer" is a short polynucleotide, usually with a free 3' -OH group, that binds to a target or a "template" that may be present in a sample of interest by hybridizing to the target and subsequently promoting polymerization of the polynucleotide complementary to the target. The "polymerase chain reaction" ("PCR") is a reaction in which a "pair of primers" or "set of primers" consisting of an "upstream" and a "downstream" primer, and a catalyst for polymerization (e.g., a DNA polymerase), and a polymerase, which is typically thermostable, are used to make duplicate copies of a target polynucleotide. Methods of PCR are 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 that produce duplicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as "replication". Primers can also be used as probes for hybridization reactions (e.g., Southern or Northern blot analysis). Sambrook and Russell (2001), see below.

"hybridization" refers to a reaction in which one or more polynucleotides react to form a complex, wherein the complex is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. Hybridization reactions can form a step in a wider process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

Hybridization reactions can be performed under different "stringency" conditions. Typically, low stringency hybridization reactions are performed in 10 XSSC at about 40 ℃ or in a plasma strength/temperature solution. Medium stringency hybridization is typically performed in 6 XSSC at about 50 ℃ and high stringency hybridization reactions are typically performed in 1 XSSC at about 60 ℃. Under stringent hybridization conditionsOther examples include: an incubation temperature of about 25 ℃ to about 37 ℃ of low stringency; a hybridization buffer concentration of about 6 XSSC to about 10 XSSC; formamide concentrations of about 0% to about 25%; and a wash solution from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40 ℃ to about 50 ℃; a buffer concentration of about 9 XSSC to about 2 XSSC; formamide concentrations of about 30% to about 50%; and a wash solution from about 5x SSC to about 2x SSC. Examples of high stringency conditions include: an incubation temperature of about 55 ℃ to about 68 ℃; a buffer concentration of about 1 XSSC to about 0.1 XSSC; formamide concentrations of about 55% to about 75%; and about 1x SSC, 0.1x SSC or deionized water. Typically, the hybridization incubation time is 5 minutes to 24 hours, with 1, 2, or more wash steps, and the wash incubation time is about 1, 2, or 15 minutes. SSC is 0.15M NaCl and 15mM citrate buffer. It is to be understood that equivalents of SSCs using other buffering systems can be employed. The hybridization reaction can also be carried out under "physiological conditions" known to the person skilled in the art. Non-limiting examples of physiological conditions are temperature, ionic strength, pH and Mg, which are typically found in cells²⁺The concentration of (c).

When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is referred to as "annealing" and those polynucleotides are described as "complementary". A double-stranded polynucleotide may be "complementary" or "homologous" to another polynucleotide if hybridization can occur between one of the first polynucleotide strands and one of the second polynucleotide strands. According to the generally accepted base pairing rules, "complementarity" or "homology" (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion of bases in the opposing strands that are expected to form hydrogen bonds with each other.

The term "propagation" or "expansion" refers to growing a cell or population of cells. The term "growth" also refers to the proliferation of cells in the presence of a support medium, nutrients, growth factors, support cells, or any chemical or biological compound required to obtain the desired number of cells or cell types.

The term "culturing" refers to the in vitro propagation of cells or organisms on or in various media. It is understood that progeny of a cell grown in culture may not be identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising an intact replicon, such that the vector may be replicated, e.g., by a transformation process, when placed in a cell. The vector may be viral or non-viral. Viral vectors include retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papuloviruses, or other modified naturally occurring viruses. Examples of non-viral vectors for delivering nucleic acids include naked DNA, DNA complexed with cationic lipids alone or in combination with cationic polymers, anionic and cationic liposomes, DNA-protein complexes and particles comprising DNA condensed with cationic polymers (e.g., heterogeneous polylysines, fixed length oligopeptides and polyethyleneimines), in some cases contained in liposomes, and the use of ternary complexes comprising virus and polylysine-DNA.

A "viral vector" is defined as a recombinantly produced virus or viral particle comprising a polynucleotide to be delivered to a host cell in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, and the like. Alphavirus vectors (e.g., Semliki Forest virus-based vectors and Sindbis virus-based vectors) have also been developed for gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) curr. Opin. Biotechnol.5: 434-.

In the context of lentiviral vector mediated gene transfer, a vector construct refers to a polynucleotide comprising a lentiviral genome or a portion thereof and a therapeutic gene. As used herein, "lentivirus-mediated gene transfer" or "lentivirus transduction" carries the same meaning and refers to the process of stably transferring a gene or nucleic acid sequence into a host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus may enter the host cell through its normal mechanism of infection, or it may be modified so that it binds to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA. However, once the virus infects a cell, the RNA is reverse transcribed into DNA form and integrated into the genomic DNA of the infected cell. The integrated DNA form is called provirus. As used herein, a lentiviral vector refers to a viral particle capable of introducing foreign nucleic acid into a cell via a viral or viroid entry mechanism. "Lentiviral vectors" are a type of retroviral vector known in the art that have certain advantages over other retroviral vectors in transducing non-dividing cells. See, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.

The lentiviral vectors of the invention are based on or derived from a karyotype virus (a subgroup of MLV-containing retroviruses) and a lentivirus (a subgroup of HIV-containing retroviruses). Examples include ASLV, SNV and RSV, all of which have been divided into packaging and vector components for lentiviral vector particle production systems. Lentiviral vector particles according to the invention can be based on genetically or otherwise (e.g., by specific selection of packaging cell systems) altered forms of a particular retrovirus.

The vector particle according to the invention is "based on" a particular retrovirus, meaning that the vector is derived from that particular retrovirus. The genome of the vector particle comprises components from the retrovirus as a backbone. The vector particles contain the essential vector components compatible with the RNA genome, including the reverse transcription and integration systems. Typically, these will include gag and pol proteins derived from a particular retrovirus. Thus, while most of the structural components of the vector particle may have been genetically or otherwise altered to provide the desired useful properties, they will generally be derived from the retrovirus. However, some structural components (especially the env protein) may be derived from different viruses. The host range and cell type of the vector infected or transduced can be varied by using different env genes in the vector particle production system to give the vector particles different specificities.

The term "promoter" refers to a region of DNA that turns on transcription of a particular gene. Promoters include the core promoter, which is the minimal part of the promoter necessary to properly turn on transcription, and may also include regulatory elements, such as transcription factor binding sites. The regulatory element may promote transcription or inhibit transcription. The regulatory element in the promoter may be a binding site for a transcriptional activator or transcriptional repressor. Promoters may be constitutive or inducible. A constitutive promoter is a promoter that is always active and/or directs transcription of a gene above basal transcription levels. Non-limiting examples of such include the phosphoglycerate kinase 1(PGK) promoter, SSFV, CMV, MNDU3, SV40, Ef1a, UBC, and CAGG. Inducible promoters are promoters that can be induced by molecules or factors that are added to or expressed in a cell. In the absence of induction, inducible promoters may still produce basal levels of transcription, but induction usually results in a significant increase in protein. Promoters may also be tissue specific. Tissue-specific promoters allow for the production of proteins in specific cell populations having appropriate transcription factors to activate the promoter.

Enhancers are regulatory elements that increase the expression of a target sequence. A "promoter/enhancer" is a polynucleotide containing sequences that provide promoter and enhancer functions. For example, the long terminal repeat of a retrovirus contains both promoter and enhancer functions. Enhancers/promoters may be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer/promoter is one that is naturally associated with a given gene in the genome. A "foreign" or "heterologous" enhancer/promoter is an enhancer/promoter that is placed in juxtaposition to a gene by means of gene manipulation (i.e., molecular biology techniques) such that transcription of the gene is directed by the linked enhancer/promoter.

As used herein, "stem cells" are defined as cells that have the ability to divide indefinitely in culture and produce specialized cells. In this case, stem cells are classified as somatic cells (adult) or embryonic cells for convenience. Somatic stem cells are undifferentiated cells found in differentiated tissues that can self-renew (clone) and (with certain limitations) differentiate to produce all specialized cell types of the tissue from which they originate. Embryonic stem cells are primitive (undifferentiated) cells from embryos with the potential to become a variety of specialized cell types. Embryonic stem cells are cells that have been cultured under in vitro conditions, which allow for months to years of proliferation without differentiation. Cloning refers to a line of cells that are genetically identical to the cell of origin (in this case, a stem cell).

"Stem cell rosette" refers to a cluster of stem cells that appear under magnification as a cluster of petals. See, e.g., fig. 8D.

A cell population refers to a collection of more than one cell that are phenotypically and/or genotypically identical (clonal) or different. A substantially homogeneous cell population is a cell population having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% of the same phenotype as measured by a preselected marker.

As used herein, "embryonic stem cells" refer to stem cells derived from tissue formed after fertilization but prior to the end of pregnancy, including pre-embryonic tissue (e.g., blastocyst), embryonic tissue, or fetal tissue, taken at a time during pregnancy, typically, but not necessarily, about 10-12 weeks prior to pregnancy. Most commonly, embryonic stem cells are pluripotent cells derived from early embryos or blastocysts. Embryonic stem cells can be obtained directly from suitable tissues, including but not limited to human tissues, or from established embryonic cell lines. "embryonic stem-like cell" refers to a cell that has one or more, but not all, of the characteristics of an embryonic stem cell.

Neural stem cells are cells that can be isolated from the adult central nervous system of mammals, including humans. They have been shown to produce neurons, migrate and give off axonal and dendritic projections, and integrate into preexisting neuronal circuits and contribute to normal brain function. 258-264 in Miller (2006), The premix of Stem Cells for Neural Repair, Brain Res.Vol.1091 (1); pluchino et al (2005) Neural Stem Cells and theory Use as Therapeutic tools in Neurological Disorders, Brain Res.brain Res.Rev., Vol.48(2): 211-219; and a review of this area can be found in Goh, et al, (2003) Adult Neural Stem Cells and Repair of the Adult Central Neural systems, J.Hematother.Stem Cell Res., Vol.12(6): 671-.

"differentiation" describes the process by which non-specialized cells acquire specialized cell characteristics such as heart, liver or muscle cells. By "directed differentiation" is meant the manipulation of stem cell culture conditions to induce differentiation into a particular cell type. "dedifferentiation" defines cells that can be reduced to a less directional location within the cell lineage. As used herein, the term "differentiated" defines cells that occupy more committed ("differentiated") locations within a cell lineage. As used herein, "cells that differentiate into a mesodermal (or ectodermal or endodermal) lineage" defines cells that are individually directed to a particular mesodermal, ectodermal or endodermal lineage. Examples of cells that differentiate into mesoderm lineages or give rise to specific mesoderm cells include, but are not limited to, adipogenic, myogenic, chondrogenic, cardiogenic, rawhide, hematopoietic, angiogenic (hematopoetic), myogenic, nephrogenic, urogenic, osteogenic, pericardial cells or matrices.

As used herein, the term "differentiated" defines cells that occupy more directional ("differentiated") locations within a cell lineage. "dedifferentiation" defines cells that can be reduced to a less directional location within the cell lineage. Induced pluripotent stem cells are examples of dedifferentiated cells.

As used herein, the "lineage" of a cell defines the inheritance of the cell, i.e., its ancestors and progeny. Cell lineages place cells into a genetic program of development and differentiation.

"Multi-lineage stem cell" or "pluripotent stem cell" refers to a stem cell that self-replicates as well as replicates at least two progeny cells from different developmental lineages that have further differentiated. These lineages may be from the same germ layer (i.e., mesoderm, ectoderm, or endoderm) or from different germ layers. Examples of two progeny cells with different developmental lineages that differentiate from a multi-lineage stem cell are myogenic cells and adipogenic cells (both of mesodermal origin, but giving rise to different tissues). Another example is a neural cell (of ectodermal origin) and a adipogenic cell (of mesodermal origin).

"precursor" or "progenitor" is intended to mean a cell that has the ability to differentiate into a particular type of cell. The progenitor cells may be stem cells. Progenitor cells may also be more specific than stem cells. Progenitor cells may be unipotent or pluripotent. Progenitor cells may be in a late stage of cell differentiation compared to adult stem cells. Examples of progenitor cells include, but are not limited to, progenitor neural cells.

"parthenogenetic stem cells" refer to stem cells resulting from parthenogenetic activation of an egg. Methods of generating parthenogenetic stem cells are known in the art. See, e.g., Cibelli et al (2002) Science 295(5556) 819 and Vrana et al (2003) Proc. Natl. Acad. Sci. USA 100(suppl.1) 11911-6.

As used herein, "pluripotent cell" defines a less differentiated cell that can give rise to at least two different (genotypically and/or phenotypically) progeny cells that are further differentiated. In another aspect, "pluripotent cells" include induced pluripotent stem cells (ipscs), which are stem cells artificially derived from non-pluripotent cells (typically adult somatic cells), which have historically been generated by inducing the expression of one or more stem cell-specific genes. Such stem cell specific genes include, but are not limited to: the octamer transcription factor family, i.e., Oct-3/4, Oct-3/4, Oct-3/4, Oct-3/4, Oct-3/4, Oct-3/4; the Sox gene family, namely Sox1, Sox2, Sox3, Sox 15 and Sox 18; the Klf gene family, i.e., Klf1, Klf2, Klf4, and Klf 5; the Myc gene family, i.e., c-Myc and L-Myc; the Nanog gene family, namely OCT4, Nanog, and REX 1; or LIN 28. Cell advance online publication 20November 2007 in Takahashi et al (2007); takahashi & Yamanaka (2006) Cell 126: 663-76; okita et al (2007) Nature 448: 260-; yu et al (2007) Science advance online publication 20November 2007; and examples of ipscs are described in Nakagawa et al, (2007) nat. biotechnol. advance only publication 30November 2007.

An "embryoid body or EB" is a three-dimensional (3D) aggregate of embryonic stem cells formed during culture that promotes subsequent differentiation. When grown in suspension culture, EB cells form small aggregates of cells surrounded by the visceral endodermal outer layer. After growth and differentiation, EBs develop into vesicular embryoid bodies with an inner layer filled with fluid cavities and ectoderm-like cells.

"composition" is intended to mean the active polypeptide, polynucleotide or antibody and inert (such as a detectable marker) or active (such as gene delivery vector) of another compound or composition combination.

"pharmaceutical composition" is intended to include the combination of an active polypeptide, polynucleotide or antibody with an inert or active carrier (e.g., a solid support) to render the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water and emulsions (e.g., oil/water or water/oil emulsions), as well as various types of wetting agents. The composition may also contain stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants are found in Martin (1975) Remington's pharm. sci.,15th Ed. (Mack pub. co., Easton).

"subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, rabbits, apes, bovines, ovines, porcines, canines, felines, farm animals, sport animals, pets, equines, and primates, particularly humans. In addition to being useful for human therapy, the present invention may also be useful for veterinary therapy of companion mammals, exotic animals, and domestic animals, including mammals, rodents, and the like, susceptible to neurodegenerative diseases. In one embodiment, the mammal includes horses, dogs, and cats. In another embodiment of the invention, the human is an adolescent or infant under the age of eighteen years.

"host cell" refers not only to a particular subject cell, but also to the progeny or potential progeny of such a cell. Certain modifications may occur in progeny due to mutation or environmental impact, and such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

"treating" diseases includes: (1) preventing a disease even if the clinical symptoms of the disease do not develop in a patient who may be predisposed to the disease but does not yet experience or exhibit symptoms of the disease; (2) inhibiting a disease, i.e., arresting or reducing the development of a disease or its clinical symptoms; or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.

The term "suffering from" in relation to the term "treatment" refers to a patient or individual who has been diagnosed as having or being susceptible to infection or disease. Patients may also be said to be "at risk" due to active or latent infection. The patient has not yet developed a characteristic disease pathology.

An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount may be administered in one or more administrations, applications or doses. Such delivery depends on a number of variables, including the period of time over which the individual dosage units are used, the bioavailability of the therapeutic agent, the route of administration, and the like. However, it will be understood that the particular dosage level of a therapeutic agent of the invention for a particular subject will depend upon a variety of factors including: the activity of the particular compound employed, the age, body weight, sex and diet of the subject, the time of administration, the rate of excretion, the drug combination and the severity and form of the particular disease being treated. Therapeutic doses can generally be titrated to optimize safety and efficacy. Generally, the dose-effect relationship originally derived from in vitro and/or in vivo testing can provide useful guidance for the appropriate dose to be administered to a patient. In general, it is desirable to administer an amount of a compound effective to achieve serum levels commensurate with concentrations found to be effective in vitro. The determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and methods of administration, are known in the art and are described in standard texts. Consistent with this definition, the term "therapeutically effective amount" as used herein is an amount sufficient to inhibit the replication of an RNA virus ex vivo, in vitro or in vivo.

The term "administering" shall include, but is not limited to: suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles suitable for each route of administration may be prepared, either separately or together, by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant), by inhalation aerosol, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), intracranial, or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.). The present invention is not limited by the route of administration, formulation or regimen of administration.

Huntington's Disease (HD) is a genetic disease that leads to progressive failure (degeneration) of nerve cells in the brain. Huntington's disease has a wide range of effects on the functional abilities of humans, including loss of motor and cognitive function and psychosis. The purpose of treating or ameliorating the symptoms of HD is to improve the mental, cognitive or motor function of the patient, or to reduce the adverse effects of this genetic disorder. The symptoms and course of the disease are known to those skilled in the art and are described in mayocline, org/diseases-conditions/huntingtons-diseases/symptoms-consumers/syc-20356117, accessed on day 21, 5/2018.

Central Nervous System (CNS) diseases or disorders are intended to affect a group of neurological disorders that affect the functional structure of the brain or spinal cord and may lead to the degeneration of one or more portions of the brain or spinal cord. Non-limiting examples include HD, Alzheimer's disease, Parkinson's disease, traumatic brain injury, stroke, autoimmune diseases (e.g., multiple sclerosis, primary or secondary progressive multiple sclerosis, relapsing remitting multiple sclerosis), brain inflammation, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumors, peripheral neuropathy, Guillain-Barre syndrome, spinal muscular atrophy, Fradrich's ataxia, amyotrophic lateral sclerosis, and Huntington's chorea. Treating or ameliorating the symptoms of CNS injury is intended to improve neurological function in a patient, reduce the adverse effects of a genetic or acquired disease, injury or disorder. The symptoms and course of the disease are known to those skilled in the art, see hopkinsmedicine, org/healthcare/conditions/nervous _ system _ disorders/overview _ of _ nervous _ system _ tem _ disorders _85, P00799, accessed 5/21 days 2018.

A "neurodegenerative disease or disorder" is a disease or phenotype characterized by degeneration of neurons of the nervous system, particularly neurons in the CNS.

"enhancing synaptic connectivity" is intended to facilitate connections between neurons or neuronal receptors.

Synapses are connections between two nerve cells, consisting of a tiny gap through which a stimulus is transmitted by diffusion of a neurotransmitter.

Modes for carrying out the invention

a) isolating at least one stem cell rosette from a population of Embryoid Bodies (EBs) cultured in a differentiation medium;

b) incubating at least one individual unit isolated from the rosette ring structure of step a) for an amount of time and under conditions provided for the generation of at least one rosette until at least one rosette is generated;

c) isolating the individual units of the rosette from step b) into individual cells; and

In one aspect, separating at least one individual unit from the rosette is performed manually. In another aspect, isolating at least one individual unit/cell from the rosette is performed enzymatically. In a further aspect, the separation of at least one individual unit of the rosette from step a) is carried out by one or more of the following means: manual, enzymatic and/or digital. In another aspect, the isolation of at least one single cell of step c) is performed enzymatically. Methods and techniques for digitally identifying three-dimensional or two-dimensional images are known in the art, see, e.g., U.S. patent No. 7,689,043, U.S. patent No. 6,907,140, and U.S. patent No. 5,020,112.

In one embodiment, one or more of steps a) to c) are performed two or more times, which may be performed using one or both of manually or mechanically in a high-throughput manner. In another aspect, the separation of rosettes is performed digitally. Methods and techniques for digitally recognizing three-dimensional or two-dimensional images are known in the art, see, e.g., U.S. patent No. 7,689,043, U.S. patent No. 6,907,140, and U.S. patent No. 5,020,112.

In one aspect, the embryoid bodies were generated from an ESI-017 cell line available from BioTime (see esibi. com/ESI-017-human-organizing-stem-cell-line-46-xx, last visit 6/2018).

In one aspect of the invention, the method further comprises culturing Embryoid Bodies (EBs) on the ultra-low attachment surface in EB media. In another aspect, the method further comprises replacing EB media with N2 media after culturing the EBs on the ornithine/laminin coated surface for an effective amount of time at step a). Alternatively, the method further comprises replacing EB media with N2 media after culturing EBs in EB media for an amount of time effective to produce at least one EB of step a).

The method may be further modified by culturing the at least one individual cell isolated in step c) in N2 medium on a guanine/laminin coated plate for an effective amount of time to generate a fused cell population of hnscs. As known to those skilled in the art, a fused cell population is a population of cells in which a large number of cells are in contact with other cells in the population. This method can be further modified by culturing the fused population of hnscs with an effective amount of N2 medium.

The invention also provides a cell or population of cells prepared by a method as described herein. These neuronal cells and differentiated cells produce or overexpress BDNF.

The cell population may be amplified and/or genetically modified, for example by insertion of a transgene or by CRISPR. In one aspect, the transgene is ApiCCT1, a fragment thereof (e.g., sapictc), or an equivalent of each thereof. The cells and/or transgene may optionally be detectably labeled. The transgene can be inserted by inserting the transgene into a vector under the control of regulatory elements (e.g., promoter and optionally enhancer elements) using known conventional recombinant techniques. The cells and/or vectors containing the transgene may be detectably labeled. As described in detail below, a transgenic sapictc is inserted into a specific cell population of hnscs to provide further protection thereto when implanted as a therapeutic agent. The sapictc transgene may also be inserted into hescs or other stem cell derivatives, including but not limited to other embryonic cell lines, fetal-derived cell lines, mesenchymal-derived cell lines, neuronal-derived cell lines, and differentiated cell types.

Further provided are populations of these cells and non-human animals comprising these cells. The population may be substantially homogeneous, substantially heterogeneous or clonal. The population may be detectably labeled. The population can be combined with a carrier (e.g., a pharmaceutically acceptable carrier).

Further provided are compositions comprising the isolated cells and, for example, a carrier. In another aspect, the composition further comprises a preservative and/or a cryopreservative. Non-limiting examples of anti-freeze agents include commercially available DMSO, glycerol, see, e.g., streck. com/collection/streck-cell-preservative/, most recently visited on day 5/22 in 2018.

The cells are useful in therapeutic methods. In one aspect, there is provided a method of delivering a transgene to a subject or a method of gene editing a cell in a subject in need thereof by administering an effective amount of one or more of the cells, populations, or compositions as described herein. In another aspect, a method of treating a neurodegenerative disease or enhancing synaptic connectivity in a subject in need thereof is provided by administering to the subject an effective amount of one or more cells, populations, or compositions. In another aspect, there is provided a method of treating a neurodegenerative disease or enhancing synaptic connectivity or treating CNS injury in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more cells, populations, or compositions. Any suitable method of administration may be used, non-limiting examples of which are provided herein.

Non-limiting examples of neurodegenerative diseases are selected from the group consisting of huntington's disease, stroke, CNS injury, chronic spinal cord injury, aneurysm, surgery, arteriovenous malformations (AVM), radiation, spinal muscular atrophy, fredrich's ataxia, Amyotrophic Lateral Sclerosis (ALS), muscle sclerosis, primary or secondary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, vascular dementia, seizure, cerebral vasospasm, alzheimer's disease, acute or traumatic brain injury, brain inflammation, and any other CNS injury due to, for example, cardiopulmonary arrest or near drowning or resulting in acute physical injury to CNS tissue, and combinations thereof.

In certain embodiments, the CNS injury is an injury caused by stroke. By "stroke" is meant any condition that results in physical damage to the central nervous system due to the blood supply or distribution of oxygen to the brain. This may be due to ischemia (insufficient blood supply or oxygen) caused by thrombosis or embolism, or to bleeding.

Reagent kit

Kits are also provided. In one aspect, the kit comprises hescs and instructions to perform the methods as described herein. In another aspect, the kit comprises neuronal cells prepared using a method as described herein and instructions for use. The kit may further comprise compositions and reagents to carry out the instructions provided for the kit.

In some embodiments, the reagents described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. In one aspect, the kit comprises hescs and instructions for performing the methods as described herein. In another aspect, the kit comprises neuronal cells prepared using a method as described herein and instructions for use. The kit may further comprise compositions and reagents to carry out the instructions provided for the kit.

In some embodiments, the kit further comprises instructions for use. In particular, such kits can include one or more of the reagents described herein, as well as instructions describing the intended use and proper use of the reagents. For example, in one embodiment, a kit can include instructions for mixing one or more components of the kit and/or separating and mixing samples and applying to a subject. In certain embodiments, the agents in the kit are pharmaceutical formulations and dosages suitable for the particular application and method of administration of the agent. Kits for research purposes may contain components in appropriate concentrations or quantities for conducting various experiments.

Kits can be designed to facilitate the use of the methods described herein, and can take many forms. Each of the compositions of the kit, if applicable, may be provided in liquid form (e.g., as a solution) or in solid form (e.g., as a dry powder). In some cases, certain compositions may be composable or processable (e.g., in an active form), for example, by addition of a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit. In some embodiments, the composition may be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and(Stem Cell Technologies, Vancouver, Canada). In some embodiments, the preservation solution contains an amount of a metalloprotease inhibitor.

As used herein, "instructions" may define the components of the specification and/or promotion and generally relate to written instructions on or in connection with the packaging of the claimed methods or compositions. The instructions may also include any oral or electronic instructions provided in any manner so that the user will clearly recognize that the instructions are associated with the kit, such as audiovisual (e.g., videotape, DVD, etc.), internet and/or web-based communications, etc. In certain embodiments, the written instructions are in the form of a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for administration to an animal.

In some embodiments, the kit contains any one or more of the components described herein in one or more containers. Thus, in some embodiments, a kit can include a container holding a reagent described herein. The reagent may be in the form of a liquid, gel or solid (powder). The reagents may be prepared aseptically, packaged in syringes and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. The second container may have other reagents prepared aseptically. Alternatively, the kit may include the active agents pre-mixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more of the components required to administer the agent to a subject, such as a syringe, topical administration device, or IV syringe and bag.

The therapy as described herein may be combined with appropriate diagnostic techniques to identify and select patients for the therapy. For example, a genetic test can be provided that recognizes mutations in muscle atrophy genes. Thus, patients with mutations can be identified as suitable for treatment.

The following examples are intended to illustrate, but not limit, the present invention.

Test procedure

TABLE 1 ESI-017 hNSC-materials

TABLE 2 culture Medium formulation N2

TABLE 3 cryopreservation media formulation

TABLE 4 additional reagents

TABLE 5 preparation of solutions

Preparation of
	1. Stock poly-L-ornithine 1:3 was diluted in PBS-/- (1 part poly-L to 2 parts PBS)
2. Laminin was diluted, 10ul of stored laminin-/- (10ug/ml) per 1ml PBS
	3. 100ug vials of bFGF were diluted 10ug/ml or 10ng/ul in 10ml water

Step of generating NSC from ESC

Passage of ESCs for Embryoid Body (EB) formation

On day 1, differentiated colonies were manually cleared from ESC cultures using a P1000 tip. ESC medium was then changed from ESC culture to 2 mL/well of EB medium. The ESC collection was scraped in a back and forth motion (first horizontally through the hole and then vertically through) using a P1000 tip. The scraped colonies of each well were transferred to one well of an ultra low adhesion 6-well plate with a 10mL serum pipette. Using EB Medium, the wells of the ESC plates were cleaned with a P1000 micropipette and the washes were performedThe solution was added to each well of an ultra low adhesion 6-well plate to a final volume of 3 mL/well. EB plates were moved to 37 ℃ and 5% CO₂In an incubator. EB plates were plated at 37 ℃ and 5% CO for the entire 2 nd day₂And (4) incubating. On day 3, a half-change of EB medium was performed. The floating colonies were gently swirled to the center of their wells. Using a P1000 micropipette, 1ml of medium was removed from each well and discarded. 1.5mL of fresh EB media was gently added back to each well. The plate is then returned to the incubator. On day 4, no action was taken. At 37 ℃ and 5% CO₂While continuing stirring. On day 5, EBs derived from ESCs were plated onto laminin-coated plates. At room temperature, an appropriate number of wells in a 6-well plate were incubated at 1:3 poly-L-ornithine diluted in PBS was coated for 1 hour. N2 medium was prepared according to table 2 (N2 medium was good one week after preparation). After 1 hour, the poly-L-ornithine solution was removed and discarded. Wells were washed twice with PBS. The hole is used for 1:100 laminin diluted in PBS was coated for 1 hour at room temperature. A 10mL serological pipette was used to transfer the suspension of EB from each well into its own 15mL conical tube. EB was allowed to settle in suspension for 15 minutes at room temperature. For the coated wells of the 6-well plate, the laminin was aspirated and discarded. 1mL of N2 medium was added to each well. EB media was aspirated from each 15mL conical tube. The EBs were gently resuspended in 2mL of N2 medium using a 10mL serological pipette. EB was added to coated wells containing 1mL of N2 medium in a total volume of 3 mL. The plate was gently shaken back and forth to disperse the EBs and placed in an incubator. Rosette formation and isolation of plated EBs was performed from day 6 to day 14 (Ros1, round 1). Cells were examined for the next 4-6 days to check for rosette formation. The rosettes can be picked at any time according to the formed quality. If rosettes have not formed, the N2 medium was changed every other day until rosettes appeared. To pick rosettes, the rosettes were harvested at room temperature using a 1:3 Poly-L-ornithine diluted in PBS 12-well plates were coated for 1 hour. After 1 hour, the poly-L-ornithine solution was removed and discarded. Wells were washed 3 times with PBS. The wells were then aligned with a 1:100 layer viscosity diluted in PBSZonulin was coated for 1 hour at room temperature. If desired, a bottle of N2 medium was prepared according to Table 2. After 1 hour, laminin was removed and 1mL of N2 medium was added to each well to keep moist and stored in an incubator to cut rosettes. Around days 10-12, rosettes formed from previously plated EBs can be seen. The rosette was dissected under a dissecting microscope by tracing the rosette using an 18 gauge needle attached to a 1mL syringe. The mixture was then transferred to N2 medium in laminin-coated plates using a p200 micropipette. After transfer, the mixture was stored in an incubator overnight and labeled Ros 1. The medium was changed every other day during storage in the incubator. On days 15 to 18, the rosettes were dispersed into single cells. The cleanest rosettes were dispersed into single cells two to three days after the Ros1 isolation (round 1). N2 medium was aspirated from each desired well and 0.5mL of EDTA-containing 0.05% trypsin was added to each well. The mixture was heated at 37 ℃ and 5% CO₂Incubate for 90 seconds. After 90 seconds, 0.5mL DTI was added. Using a 1000. mu.L micropipette, the rosettes were dispersed in the wells, and then the volume of each well was added to its own 15mL conical tube. Each well or "clone" remains isolated throughout the passage. The conical tube was rapidly centrifuged at 1000RPM for 5 minutes. The supernatant was added and discarded. Each pellet was resuspended in 1mL of fresh N2 medium. Each 1mL suspension was plated in its own well on a poly L/laminin coated 12-well plate and the plate was labeled (passage 0 NSC). The plate was returned to the incubator and the culture was monitored, with fresh N2 medium being changed every other day. When the cells reached approximately 85% confluence, each "clone" was transferred to its own well of a 6-well plate using the same procedure as above, but using 1mL of 0.05% trypsin and 1mL of DTI, with the medium being changed every other day with 3mL of N2 medium. Cells were maintained at 10⁶The individual cell/well ratio is continued for passaging, or cryopreservation of the cells is undertaken (discussed below).

Cryopreservation of NSCs

A cryopreservation medium or a freezing medium was prepared according to Table 3 and was ensured inThe frozen medium was cooled to 4 ℃ all the time before use. The NSC plates were retrieved from the incubator and placed in a biosafety cabinet. Old media was aspirated and discarded into a waste container. To each well was added 1mL of 0.05% trypsin and incubated at 37 ℃ for 90 seconds. After 90 seconds, 1mL of DTI was added to each well to inactivate trypsin. Using a 1000. mu.L pipette tip, the mixture was pipetted up and down to wash the cells off the surface of each well, and then the mixture was transferred to a 15mL conical tube. A15 mL conical tube was rapidly centrifuged at 1000RPM for 3 minutes. The conical tube was returned to the biosafety cabinet and the supernatant aspirated. Cells were resuspended in 5mL of fresh N2 medium and cell counts were performed using 0.4% trypan blue and a hemocytometer. Cells were centrifuged at 1000rpm for 3 minutes in a bench top centrifuge. An appropriate volume of 4 ℃ freezing medium was added to the cells to give a cell concentration of 3.0x10⁶Individual cells/mL. 1mL of the cell suspension in freezing medium was added to each freezing vial using a 10mL serum pipette. A vial of cell-free freezing medium was prepared for use in the cryoprobe. The vials were capped tightly and immediately transferred to a pre-cooled controlled rate freezer-freezing rack. The probe was inserted into a vial containing only the freezing medium and then placed in the rack. The vials were transferred from the controlled rate refrigerator to a-80 ℃ pre-cooled fully labeled freezer and then immediately transferred to LN2 storage.

Mouse

All procedures were in accordance with the NIH guidelines for care and use of laboratory animals and animal study protocols approved by the institutional animal Care and use Committee at the UCI and UCLA, AAALAC accreditation agencies. R6/2 mice and their NT littermates (non-transgenic carrier C57B16/CBA) were obtained from UCI (strain 6494,CAG repeats) or UCLA (lines 2810,CAG repeats). Homozygous Q140 mice or WT (C57B16) littermatesMice were from breeding colonies at UCLA, where manipulations were performed. All mice were housed on an 12/12 hour light/dark schedule with free access to food and water. Mice were housed in groups of mixed treatment groups, and mice for running on a wheel were housed alone. The CAG repeat length of R6/2 mice (Laragen, Los Angeles, Calif.) was confirmed, while the frequency distribution of Q140 mice was not significantly different (Hickey et al, 2012 b). For the HD model, the assessment of the differences in the results was based on previous experience and published results (Hickey et al, 2005; Hockly et al, 2003) and efficacy analysis (G Power [ psych. uni-duesseldorf. de/abetilung/aap/gpower 3 |)]) For applicants behavioral minimum n-10, biochemical analysis minimum n-4.

Isolation of hNSC

The use of hNSC was approved by the human stem cell research supervision committee (hSCRO) of UCI, UCLA, and UC Davis, and cells were derived from biomime ESI-017 hESC. hESC colonies were transferred to EB media with Noggin, transitioned to NP media, and rosettes were excised, dispersed, and plated with hNSC media to generate hnscs (fig. 8B). Rosettes were manually cut out and plated on NSC medium in Matrigel-coated plates with reduced growth factor, and then dispersed and plated on polyornithine/laminin-coated plates with NSC medium using Accutase.

Transplant operation

Bilateral intrastriatal injections of hNSCs or veh were performed using a stereotaxic apparatus, and the coordinates relative to bregma were: front and rear bits, 0.00; middle and outer sides, ± 2.00; dorsoventral, -3.25. Mice were anesthetized, placed in a stereotactic frame, and injected with 100,000 hNSC per side (2 μ L/injection) or veh (2 μ L Hank's balanced salt solution with 20ng/mL human epidermal growth factor [ stem cell Technologies, #78003] and human fibroblast growth factor [ stem cell, #78006]) at an injection rate of 0.5 μ L/min using a 5 μ L hamilton micro-syringe (33 gauge). The wound was sealed and the mice were returned to their cages with the heating pad. The day before surgery, all mice were administered with immunosuppressive agents and continued throughout the procedure.

Behavioral assessment

R6/2

Mice were assigned in a semi-random fashion and behavioral testing was performed between 6 and 9 weeks. Researchers have employed blind methods for genotyping and processing for testing and data collection. To minimize experimenter variability, each test was conducted by a single researcher. Behavioral tasks were performed in R6/2 mice as previously described by Ochaba et al (2016).

Q140

In addition to runner running, males and females are used, where only males are used in runner running because the estrous cycle can affect the running activity. The genotype or treatment is unknown to the experimenter. All tests were performed during the light period, except for running in a wheel during the dark period. Behavioral tasks were performed in Q140 mice as previously described by Hickey et al (2008).

Electrophysiology in R6/2 brain sections

Using R6/2 (strain 2810, 150 ± 10CAG repeats) and NT littermates, a phenotype similar to strain 6494 used for behavioral experiments was shown (Cummings et al, 2012). The procedure was as described in Andre et al (2011) with modifications as detailed herein.

Immunohistochemistry and electron microscopy

Male R6/2 mice (n ═ 3) implanted with hNSC for 5 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 ]. Brains were then collected into EM fixative overnight at 4 ℃ and washed in PBS until serial sections (equivalent to from bregma +1.4 to +0.14mm) through the striatum containing hnscs at 60 mm by using a vibrating microtome (Leica Microsystems) (Franklin and Paxinos, 2007). IHC for pre-embedding striatum and tissue treatment for EM using Diaminobenzidine (DAB) (Sigma, St Louis, MO) and hNSC antibody (Stem121,1: 100; Stemcells) as previously described (Spinelli et al, 2014; Walker et al, 2012), and striatum sections were embedded in two pieces of ACLAR (Electron Microscopy Sciences, Hatfield, Pa.) between overnight placement in an oven at 60 ℃ overnight to polymerize the resin. The hNSC-containing regions were microdissected from the embedded sections and then super-adhered to a wax block for thin sectioning.

Photographs were taken on a JEOL 1400 transmission electron microscope (JEOL, Peabody, MA) of DAB-labeled structures (i.e. hNSC-labeled cells, dendrites) using a digital camera (AMT, Danvers, MA) at a final magnification of 346,200. Since the DAB mark is limited only to the front edge of the thin slice, only the area showing the DAB mark is photographed. Biochemical, molecular and immunohistochemical analyses in R6/2 mice.

Mice were euthanized by excess pentobarbital and perfused with 0.01M PBS. The striatum and cortex were excised from the left hemisphere and then snap frozen for isolation of RNA and protein in TRIzol using the manufacturer's (Life Technologies, Grand Island, NY) procedure or homogenized as described below. the other half was fixed in 4% paraformaldehyde, freeze protected in 30% sucrose and cut at 40 μm ON a slide shaker microtome for IHC. sections were rinsed 3 times and placed in blocking buffer for 1 hour (PBS, 0.02% Triton X-100, 5% goat serum) and placed primary antibodies blocked Overnight (ON) at 4 ℃.

Soluble/insoluble fractionation

Striatal tissue was treated as previously described (Ochaba et al, 2016). Antibody: anti-HTT (Millipore, # MAB 5492; RRID: AB-347723) and anti-ubiquitin (Santa Cruz Biotechnology, # sc-8017; RRID: AB-628423). Bands were quantified using software from NIH program ImageJ and densitometry application.

Confocal microscopy and quantitation

Sections were imaged by the Bio-Rad Radiance 2100 confocal system using a lambda-strobing mode. The images represent a single confocal z-slice or z-stack. All unbiased stereo assessments were performed using the StereoInvestimator software (MicroBrightField, Williston, VT). The number of average cells, diffuse aggregates and inclusions was estimated using an optical fractionator probe.

RNA isolation and real-time qPCR

The striatum was homogenized in TRIzol (Invitrogen) and then in RNEasy Mini kit (Qiagen). The RIN value for each sample was >9(Agilent Bioanalyzer). Using the SuperScript III first Strand Synthesis System (Invitrogen), RT used oligo (dT) primers and 1mg total RNA. qPCR was performed as described by vasishtha et al (2013).

Biochemical, molecular and immunohistochemical analysis in Q140 mice

Six months after treatment, Q140 males were euthanized by cervical dislocation (n-7 cryo) or paraformaldehyde perfusion (n-5 IHC).

IHC

Mice were perfused with 0.1M PBS and 4% paraformaldehyde. After overnight fixation in 4% paraformaldehyde, the brains were removed, cryopreserved in 30% sucrose, frozen, and cut into 40 μm coronal sections on a cryostat (Leica CM, 1850). Sections were blocked for 1 hour at room temperature, then primary-antibody ON was used. After several washes, sections were incubated in Alexa Fluor secondary antibody and counterstained with DAPI. The IHC of HTT aggregates and microglia was quantified as described in Menalled et al (2003) and Watson et al (2012), respectively.

HTT-stained nuclei and aggregates

The slices were analyzed using StereoInvestimator 5.00 software (Microbrillfield, Colchester, VT) (Hickey et al, 2012 a). For the striatal profile being drawn, the software sets a 200x 200 μm grid with a 20x 20 μm count box that is used to quantify each type of aggregate for each slice.

Quantification of IBA-1-positive cells in the striatum of Q140 mice

The analysis was performed using a Leica DM-LB microscope using the stereoinvestor software (MicroBrightField) for reflecting activated microglia diameters (Watson et al, 2012). For striatal contours drawn at 5x magnification, the software places a 200x 200 μm grid with a 20x 20 μm counting box in the upper left corner to allow unbiased sampling and quantification.

Biochemical analysis of Q140 mice

Frozen striatal treatments for ELISA were performed using the biosense BDNF rapid kit (biosense BEK-2211, SA, Australia) according to the manufacturer's instructions.

Statistical analysis

The results for R6/2 mice were from a single syngeneic group, but they were from different subgroups, in addition to electrophysiological and EM data; the same batch of cells was used for all. Using analysis prior to the study, it was determined that the numbers had sufficient intensity (as described above). Statistical significance was obtained as described using a rigorous analysis. All findings were reproducible. The various statistical methods are further detailed in the above legend. Significance level: p <0.05, p <0.01, p <0.001, p < 0.0001. Data are expressed as mean ± SEM in R6/2 mice; statistical testing of the behavioural task used one-way ANOVA followed by Tukey's Heff test and post hoc multiple comparisons of Scheffe', Bonferroni and Holm. The data met the assumptions of the statistical test used and a p-value of less than 0.05 was considered valid. All mice were randomly assigned and tasked in a random fashion with individuals of unknown 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 aggregate number comparison from EM48 stereology. Significance in the pinch is determined by the fisher exact probability. Behavioral and post hoc data were significantly different (p <0.05), and Q140 mice were statistically analyzed using one-way analysis of variance with Bonferroni post hoc tests, using GraphPad Prism 6.0(GraphPad Software, San Diego, CA). Two-way analysis of variance was used in the graph, which represents the average revolutions per 3 minute test, followed by Bonferroni post-hoc testing; and autonomous statistics using custom MATLAB functions were used for IBA-1 analysis. All error bars on the graph represent SEM.

And (4) separating the hNSC. The culture was carried out daily (D) as follows. D1: ESC colonies were enzymatically "dispersed" using collagenase IV until the colony edges began to rise. Colonies were scraped manually from wells, transferred to low attachment plates, and cultured overnight in EB media (ESC media minus bFGF). D2: EB medium was supplemented with 500ng/ml Noggin and 10. mu.M SB431542 and cultured for 2 days. D4: the medium was replaced. D5: in the same medium, EBs were plated onto low growth factor, matrigel coated 6-well plates. D6: medium was changed and NP medium was used to drive the differentiation of NPCs. The medium was changed every two days until the twelfth day. D12-14: rosettes were visually isolated under a dissecting microscope, manually cut with an 18-gauge needle, and plated into NSC medium in low growth factor matrigel-coated 6-well plates. After 2-3 days, rosettes were dispersed using Accutase and plated onto polyornithine/laminin coated plates with NSC medium and Y27632 compound. ESI-017hNSC cytogenetic analysis found that the karyotype was stable, no abnormalities were observed, and flow cytometry analyses were performed on CD271(Brilliant Violet 510- -BD horizons Cat. 563451), CD24(Brilliant Violet 711- -BD horizons Cat. 563401), Pax6(PE- -BD Pharmingen Cat. 561552), Nestin (Alexa Fluor 647- -BD Pharmingen Cat. 560341), SOX1(PerCP CY5.5- -BD Pharmingen Cat. 561549), SOX2(V450- -BD Horzon. 561610), CD44(APC-H7- -BD Pharmingen Cat. 560532), CD184(PE-CY7- -Biolend Cat. 306514), or SSEA4(lexa uor or 700- -SSRA 429).

And (5) transplanting operation. A stereotaxic instrument was used for a bilateral striatal in vivo injection of hNSC or vehicle, 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 on each side with 100,000 hNSCs (2. mu.l/injection) or vehicle treated as a control (2. mu.l HBSS with 20ng/ml hEGF and hFGF) using a 5. mu.l Hamilton microsyringe (No. 33) at an injection rate of 0.5. mu.l/min. R6/2 mice were anesthetized with isoflurane and Q140 mice were anesthetized with sodium pentobarbital (60mg/kg pentobarbital in sterile 0.9% saline, i.p.). For all mice; to maintain an anesthetic surgical plane, mice were administered isoflurane (1-2% in 100% oxygen, 0.5L/min) via a nose cone, given oxygen throughout the surgery, and maintained at a temperature of 15 degrees on an electronically controlled heating pad, monitored using a rectal probe thermometer (physiotemp). Accurate placement of the injectate to the target area was confirmed for all animals by visualizing the needle track within the brain area. The wound is sealed on the skull with bone wax and closed with a dermabond or suture. Mice were placed on a heating pad in a single cage post-surgery until they recovered from anesthesia. A single daily dose of the immunosuppressant CSA at a concentration of 10mg/kg was administered intraperitoneally starting the day before surgery on R6/2 and non-transgenic mice implanted with hNSC and vector. To further immunosuppresse the mice, an additional dosage regimen of CD4 antibody (BioXcell, Lebanon, NH) was administered at 10mg/kg i.p. once a week. Q140 mice or Wt littermates received immunosuppression of CSA (2 mg/kg/day) administered via a subcutaneous osmotic minipump (Alzet #1004) that was replaced once a month to ensure sustained delivery of CSA throughout the study. The procedure for removing and replacing the micropump is as follows. Mice were anesthetized with isoflurane (3% for induction and 1.75% for maintenance anesthesia in 100% oxygen). After the incision site is sterilized, the micro-pump is removed through a small incision in the back, and a new micro-pump is implanted before the incision is sutured.

R6/2: mice were assigned to each group in a semi-random fashion. The behavioral tests listed below were performed at 6, 7, 8, or 9 weeks of age, depending on the task. Mice were weighed daily and no significant difference was observed with treatment. During experimental testing and data collection, researchers used blinding methods for which mice were transplanted with hnscs. To minimize the variability of the experimenters, each behavioral test was conducted by a single researcher. Mice were obtained from colonies propagated at UCI using ovarian-transplanted female mice (Jackson labs).

Locomotor coordination and balance of forelimbs and hindlimbs were measured using a rotarod apparatus and mice were tested over 3 consecutive days using a 300s accelerated assay. The rotarod test was performed twice every other week at 6-week and 8-week ages. For the pole climbing test, the mouse was placed head down on the pole and then the head was lowered down to the length of the pole first. 16 total times of descent from the start of placement were measured. Pole climbing tests were performed twice every two weeks at 7 weeks of age and 9 weeks of age. The IITC life sciences instrument is used to measure forearm grip through a digital force sensor, the unit giving readings in 1 gram increments. Grip was measured twice every other week at 7 and 9 weeks of age.

Q140: climbing test and pole climbing test. To assess motor coordination and spontaneous activity during climbing, mice were placed on the bottom of a wire-mesh cage and spontaneous activity was videotaped. For the pole climbing test, each mouse was placed face up on top of the pole and timed to make a full turn to the down position and timed to drop from the pole into its respective home cage.

Electrophysiology in R6/2 mice

Briefly, mice were anesthetized, cardiac perfused with a high sucrose-based slice solution, and then coronal slices (300 μm) were transferred to culture chambers containing ACSF. MSN and NSC were visualized using an infrared luminometer with differential interferometric contrast optics. All recordings were made within or around the injection site (MSNs recorded between 150-. Biocytin was added to a patch pipette (patch pipette) for cell visualization. Spontaneous postsynaptic currents were recorded in a whole-cell configuration in standard ACSF. The membrane current was recorded in gapless mode. The cell voltage was clamped at +10mV and spontaneous inhibitory postsynaptic currents (ipscs) were recorded in ACSF. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded at-70 mV (baseline) in ACSF, and glutamatergic events were isolated in the presence of the GABAA receptor blocker bicuculine methobromide (Tocris, M innepanols, MN). Spontaneous synaptic currents were analyzed using MiniAnalysis software (version 6.0, synaptoft, Fort Lee, NJ). After recording, the sections were fixed and then transferred to 30% sucrose at 4 ℃ until IHC treatment. To identify biocytin-filled recording cells and hnscs, the fixed sections were washed, permeabilized and blocked for 4 hours, and then incubated with SC121 (1: 1000, StemCells, Inc.). After washing, sections were incubated in goat anti-mouse Alexa-488 (1: 1000, Life Technologies, Carlsbad, CA cat # A-11001) and streptavidin was conjugated to Alexa-594 (1: 1000, Life Technologies cat # S11227). Sections were washed, slides mounted and cells visualized with a Zeiss LSM510 confocal microscope.

Biochemical, molecular immunohistochemical analysis in R6/2 mice

Confocal microscopy and quantification. The sections were imaged with a Bio-Rad Radiance 2100 confocal system using a lambda-strobing mode. The images represent a single confocal Z-slice or Z-stack. All unbiased stereo assessments were performed using the StereoInvestimator software (MicroBrightField, Williston, VT). The average cell number, number of diffuse aggregates and inclusion body number were estimated using an optical fractionator probe. The protected zone was set at 3% of the measured thickness and the minimum optical dissector height was 14 μm. Contour tracking was performed with 5x targets and counting was performed with 100x targets. For each section, tracking was performed at about 70 μm from the edge of the stem cell patch. Counts were made in 6 sections per 3 rd section (40 μm coronal section) of the entire striatum, where Ku 80-labeled cells were visible between Bregma 0.5mm and Bregma-0.34 mm. All counts were performed in only one hemisphere using a 50x50 μm counting frame and a 250x250 μm sampling grid. CE values for each "individual" mouse were between 0.03 and 0.06. The sections were first stained with Ku80 using ABC kit with nickel and DAB substrate kit (Vector Laboratories), and then EM48 using ABC and DAB kit only. Sections were stained with cresyl violet for non-dry nuclear staining. The same stereological parameters were used to count aggregates and cells from the mice implanted with the carriers. Using this stereoscopic assessment of Ku80 positive cells in implanted R6/2 brain sections, the survival number of ESI-017NSC implants showed an average of 63,975 cells in male mice (n ═ 3) and an average of 18,673 cells in female mice (n ═ 3), corresponding to 64% (male) and 18.6% (female) of the originally transplanted cells. The average total number of 41,323 cells in mice (n-6, 3 males and 3 females) corresponds to-41% of the cells initially transplanted. The difference in the number of implanted cells between males and females may be due to technical difficulties in implanting smaller females at 5 weeks.

Primary antibody for IHC; GFAP (Abcam AB4674), NeuN (Millipore MAB377), SC121(STEM 121 human specific cytoplasmic marker, Clontech AB-121-U-050), Ku80(Abcam, Cambridge, United Kingdom AB80592), Doublecictin (Millipore AB2253), Olig2(R & D Systems AF2418), BIII tubulin (Abcam AB107216), MAP-2, (Abcam AB 2412), BDNF (Icelen, 329-100) and EM48(Millipore MAB 5374).

RNA isolation and real-time quantitative PCR. Brain tissue was homogenized in trizol (invitrogen) and total RNA was isolated using RNEasy Mini kit (QIAGEN). DNase treatment was incorporated into the RNeasy program to remove residual DNA. The RIN value for each sample was >9(Agilent Bioanalyzer). Reverse transcription was performed using SuperScript III first Strand Synthesis System (Invitrogen) using oligo (dT) primers and 1. mu.g total RNA. Quantitative pcr (qpcr) was performed as previously described (vasishtha, Ng et al 2013), ddCT values were quantified and analyzed for RPLPO. The primers used to amplify the R6/2-Htt transgene were: oIMR 1594: 5'-CCCCTCAGGTTCTGCTTTTA-3', oIMR 1596: 5'-TGGAAGGACTTGAGGGACTC-3', respectively; RPLPO forward: 5'-TGGTCATCCAGCAGGTGTTCGA-3', RPLPO reverse direction: 5'-ACAGACACTGGCAACATTGCGG-3' are provided. Other primers used were: nestin, F5'TCAAGATGTCCCTCAGCCTGGA3' R5 'AAGCTGAGGGAAGTCTTGGAGC 3'; BDNF 5'TATGCGCCGAAGCAAGTCTCCA3' R5 'CATCCAAGGACAGAGGCAGGTA 3'; and DCX, As 5'GTAAAGCCAACCCTGTGTCG3' S5 'TCCGCTCCAAAATCTGACTC 3'.

Immunohistochemical analysis in Q140 mice

Primary antibody for IHC: HNA (Millipore MAB1281), DCX (abcam ab18723), GFAP (Dako Z033401) or synaptophysin (Millipore 04-1019). IHC for HTT aggregate quantification used monoclonal antibody EM48(Millipore MAB5374) as described in (Menallend et al, 2003) and microglia used rabbit anti-Iba-1 (Wako 019-19741) as described in (Watson et al, 2012). For cell counting, HNA + cells were counted in the entire striatal region of 6 coronal sections. 2100 HNA-labeled cells 19 were quantified and the proportion of those cells was double-labeled with either a neuronal marker (DCX, Abcam ab18723) or a glial marker (GFAP, Dako Z033401). The final numbers are expressed as mean ± SEM of 5 mice per group.

ESI-017hNSC will modify behavior, survival and differentiation when transplanted into R6/2 mice

To assess the efficacy of hNSC transplantation in the HD transgenic model, applicants used exon-1 HTT R6/2 mice (rv120 CAG repeats) (Cummings et al, 2012) that exhibited rapid progressive motor and metabolic defects and early death (rv 12-14 weeks) (Mangiarini et al, 1996) and can provide a preliminary assessment of treatment paradigm in preclinical studies (Hickey and Chesselet, 2003; Hockly et al, 2003). ESI-017hNSC improved the behavioural characteristics.

A manufacturing process and quality control chart for the GMP-grade hNSC line is depicted in fig. 8A and 8B. Flow cytometry indicated appropriate staining for hNSC proliferation and pluripotency markers (fig. 8A). Immunocytochemistry confirmed staining of the neuroectodermal stem cell marker nestin (fig. 8C). ESI-017hNSC was obtained as a frozen aliquot (UC Davis), thawed and cultured without passage using the same media reagents as the GMP facility, and then administered. Five-week-old mice were dosed via intrastriatal stereotactic delivery of 100,000 hnscs per hemisphere. Male (M) and female (F) R6/2, as well as non-transgenic (NT) age-matched littermates and vector controls (veh) (n ═ 8R 6/2hNSC M, 6R 6/2hNSC F, 7 NT hNSC M, 7 NT hNSC F, 7R 6/2veh M, 6R 6/2veh F, 8 NT veh M, and 6 NT veh F) were included. Immunosuppression was administered to all mice. Behavioral analysis was performed and mice were euthanized at 9 weeks of age immediately after the behavioral testing.

Veh treated mice developed HD-related behavior as described previously (Mangiarini et al, 1996). In short, at 5 weeks of age, the behavior of R6/2 mice was indistinguishable from that of NT mice. By week 8, neurological abnormalities included progressive typical hind limb hair movement (hind limb combing movement), clasping (clasping), and irregular gait. When lifted by the tail, a normal mouse will open the hind and fore limbs and be considered "buckled" if the mouse grips the limbs tightly around its abdomen. A delay in the onset of R6/2 clasping was observed in all hNSC-treated mice; mice treated with veh were fastened 3 weeks after implantation. At this time point hNSC-treated mice were not fastened and at euthanization (4 weeks post-implantation), only 50% of hNSC-treated mice were fastened (fig. 9). Two motion measurements were performed. Rotarod (Rotarod) tests the ability to walk on an accelerated rotating rod. On rotarod performance, hNSC-treated R6/2 mice showed a statistically significant improvement over veh-treated R6/2 mice (30% improvement 1 week post-implantation, p < 0.01; 19% improvement 3 weeks post-implantation, p <0.05) (fig. 1A). Pole test (pole test) compares the time when falling on a vertical pole; the drop latency was longer in R6/2 mice compared to NT mice. A statistically significant improvement (p ═ 0.02) (25% improvement, fig. 1B) was observed between the R6/2 treatment groups at 4 weeks post-implantation. The grip apparatus was also used to assess neuromuscular function and motor coordination, with hNSC treatment producing a significant improvement 4 weeks post-implantation (p 0.02, 16% improvement, fig. 1C).

ESI-017hNSC survival, migration and differentiation

Mice were euthanized and brains collected 4 weeks after implantation, half of which were fixed for histology and the other half were flash frozen for biochemistry. hNSC mainly accumulated around the needle track and remained in the striatum (fig. 1D); some in the cortex, migrated a small amount to the microenvironment (niche) (brain corpus callosum/white matter tract) between the cortex and the striatal region (fig. 10). Cells were stained predominantly with the early neuronal marker biscortin (DCX) using the human markers SC121 (cytoplasm) or Ku80 (nucleus) (SC121, FIG. 2A combined in yellow; Ku80, FIGS. 2B and 2C). Some cells may differentiate into the astrocytic phenotype (glial fibrillary acidic protein [ GFAP ]) (fig. 2B). There was also a non-human GFAP positive immunostaining around the implant site (fig. 2A and 2B), which potentially represents a mouse glial cell scar. Differentiation of hNSC into neuron-restricted progenitor cells was confirmed with β III-tubulin (fig. 2D and 10B) and microtubule-associated protein 2(MAP-2) (fig. 2E and 10C), but lack of co-localization with NeuN (fig. 2F) indicates the absence of post-mitotic neurons. Using a stereological assessment of Ku80 positive cells in one hemisphere, the survival number of hNSC implants showed an average of 41,323 cells (n ═ 6, 3 males, 3 females), approximately 41% of the 100,000 initially transplanted.

Implantation of ESI-017hNSC prevented corticosome hyperexcitability in R6/2 mice

Applicants next evaluated electrophysiological activity. At week 5, 100,000 hnscs (n-18) or veh (n-16) were implanted in the striatum in male and female mice. Applicants recorded hnscs in acute brain sections 4-6 weeks after implantation (fig. 3A and 3B). hNSC show the basic neuronal properties of immature cells with significantly smaller membrane capacitance than host MSN (hNSC 22.0 ± 1.8pF, n ═ 31 compared to 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 compared to MSN 163.8 ± 15.1 MU; p <0.001, Student's t test). hnscs show spontaneous excitatory postsynaptic currents and spontaneous inhibitory postsynaptic currents (sepscs and sipscs), suggesting that they receive synaptic input from host tissues or other implanted hnscs. However, the frequency is much lower compared to MSN. Some hnscs also spontaneously produced action potentials, suggesting that they may affect host neurons and neighboring hnscs (fig. 3B).

Electrophysiological changes, including changes in the intrinsic membrane properties and reduced excitatory synaptic activity, occurred in MSN from symptomatic R6/2, compared to NT mice (Cepeda et al, 2003,2007). hNSC implantation did not significantly change membrane properties, mean sEPSC frequency (1.1 ± 0.1Hz vs. 1.4 ± 0.2Hz), or mean SIPSC frequency of MSN in R6/2 mice. R6/2 mice also exhibited an increase in cortical pyramidal cell excitability and a propensity to develop epileptic discharges and seizures (Cummings et al, 2009). By the occurrence of large EPSC and high frequency bursts, it is particularly evident that cortical hyperexcitability was shown in striatal MSNs after prolonged blockade of GABAA receptors consistent with an increase in the frequency of sepscs (Cepeda et al, 2003; Cummings et al, 2009). A smaller proportion (not statistically significant) of MSN exhibited increased cortical excitability in hNSC implanted mice (20.5%, 9/44) compared to veh mice (28.6%, 16/56). However, the increase in frequency of sEPSC within this population did not occur in R6/2 mice implanted with hNSC. The cumulative probability distribution of the inter-event interval time plot shifts to the right (p <0.001), indicating that hNSC can reduce hyperexcitatory input from the cortex to the striatum when the GABAA receptor is blocked (fig. 3E and 3F).

Host tissues were potentially synaptically associated with ESI-017hNSC implanted in R6/2 mice

Applicants used Immunohistochemistry (IHC) and Electron Microscopy (EM) to examine whether nerve endings from the host were in synaptic communication with hNSC. Applicants found examples of host-derived unlabeled nerve endings that have potential symmetric synaptic connections with implanted and immunologically labeled hnscs (fig. 4A). A small number of synaptic vesicles in the nerve terminal are very close to the presynaptic membrane, indicating a potential vesicle release region (DAB labeling by hNSC covers the association). In addition, applicants found that unlabeled nerve endings derived from the host were significantly asymmetrically associated (FIG. 4B), indicating an excitatory synaptic association. Overall, applicants found that 44.5% (n 71) of host-derived unlabeled nerve endings were asymmetrically associated with labeled hNSC, while 48.3% (n 69) were symmetrically associated with labeled hNSC. The exact nature of the association (asymmetric versus symmetric) cannot be determined in the remaining 7.2% (n ═ 11) unlabeled nerve endings derived from the host juxtaposed to the labeled hNSC.

ESI-017hNSC rescue of ethology, survival and differentiation in Q140 knock-in mice

Next, applicants determined whether hNSC can also improve the deficiencies of the slow-progressing full-length HD mouse model. Q140 mice express a modified mouse/human exon 1 with 140 repeats inserted into the mouse huntington gene (Menalled et al, 2003). Homozygous mice exhibit early abnormalities in motor testing, both climbing and cognitive deficits at 1.5 months of age (Hickey et al, 2008; Simmons et al, 2009), and visible HTT clustering for around 4 months (Menalled et al, 2003). Striatal atrophy was detected at 1 year and at 22 months striatal cells were lost 35% (Hickey et al, 2008). 24 homozygous male and female mice at 2 months of age per group were dosed with 100,000 hnscs per hemisphere, delivered bilaterally stereotactically to the striatum (n-12/sex), and age-matched Q140 (n-12/sex) and wild-type (WT) (n-12/sex) control mice were injected with vehicle. All mice were immunosuppressed. Behavioral testing began at 1.5 months of age (prior to cell transplantation) and mice were euthanized at 6 months, 8 months post-transplantation. In addition to running on wheels, behavioral testing was performed on all mice, using only males because the estrous cycle affects running activity (Hickey et al, 2008). Early defects in spontaneous activity in the open field and a reduction in spontaneous locomotion in the cage-climbing test were observed in Q140 mice; however, hNSC treatment did not rescue performance (fig. 11).

In the pole climbing test, veh treated Q140 mice turned longer (p ═ 0.004) compared to WT controls; in contrast, hNSC-treated Q140 mice were significantly better than control Q140 mice (p ═ 0.04) and no longer significantly different from WT, indicating a beneficial effect at 3 months post-transplantation (fig. 5A). As reported by Hickey et al (2008), 5.5 month old male Q140 mice had a tremendous deficit in running speed (turning every 3 minutes), with a duration of 2 weeks being significant (fig. 5B). Persistent improvement of runner running was observed after treatment in hNSC-treated Q140 mice, showing a progressive increase in mean runner running activity compared to veh-treated mice (fig. 5B and 5C). Applicants concluded that hNSC administration ameliorated some of the motor deficits observed in Q140 mice.

New Object Recognition (NOR) is a cortical-dependent cognitive test that requires learning and memory (recognition), and utilizes mice for studying the preference of new objects over familiar ones. As reported by Simmons et al (2009), Veh-injected Q140 mice exhibited significant impairment in NOR (p ═ 0.003 and p ═ 0.03, respectively) compared to Veh-injected WT mice within 3 and 5 months post-implantation. Striatal transplantation of hnscs in Q140 mice rescued cognitive impairment at 5 months post-implantation (p ═ 0.03), but not earlier (fig. 5E and 5F).

A subset of veh and hNSC transplanted Q140 male mice (n ═ 5 per group) were euthanized for IHC analysis 6 months after treatment. Hnscs recognized by human nuclear specific antibodies (HNA) were present 6 months after transplantation and were mostly confined to the injection track in the striatum (fig. 5G a, b). The number of HNA positive cells was counted in the entire striatal region of six coronal sections and cells double labeled with DCX or GFAP were counted (mean data from 5 mice per group ± SEM). About 25% of 100,000 hnscs survived, with the majority (84% ± 2%) being GFAP positive (fig. 5Gb, c) and the smaller fraction (16% ± 2%) being DCX positive (fig. 5Ge, f).

ESI-017hNSC transplantation to increase BDNF levels in HD mice

Increased levels of neurotrophic growth factor and subsequent increased synaptic connectivity are involved in the behavioral improvement observed following NSC transplantation (Blurton-Jones et al, 2009). Furthermore, a reduction in BDNF has been demonstrated for various HD mouse models and human HD brains (Zuccato et al, 2011). Therefore, we assessed BDNF levels as markers of neurotrophic effects. In R6/2hNSC mice, IHC and confocal microscopy indicated that BDNF co-localized with DCX-positive hNSC, indicating that differentiated cells produced BDNF (fig. 6A). Indeed, BDNF is produced by hsscs that grow in vitro and have differentiated only after becoming positive for DCX. BDNF was quantified by ELISA in a male subgroup of mice (n-6/group) in Q140 hNSC mice. Striatal BDNF was reduced in Q140 mice compared to WT, but a significant increase in BDNF levels was observed in hNSC treatment compared to veh, restoring it to WT levels (fig. 6C).

Given that neurotrophic signaling can enhance synaptic activity, we examined the level of synaptic marker synaptophysin in the striatum of all perfused Q140 animals (n-5/group) by IHC and quantification using microarray scanners, as described previously (Richter et al, 2017). Comparison of hNSC-treated Q140 mice with veh-treated Q140 mice revealed a significant increase in synaptophysin in hNSC mice (fig. 13A, quantitative in fig. 13B).

These results indicate that transplanted hnscs can partially improve synaptic connectivity by increasing neurotrophic effects (including BDNF).

Treatment of ESI-017hNSC in Q140 mice reduced microglial activation

Striatal sections from Q140 mice (n-5/group) were stained with ionized calcium binding adaptor 1(Iba-1) antibody, which recognizes resting and reactive microglia. The size of microglia (Microglial soma) is related to the morphology of activated cells (Watson et al, 2012), and a significant increase in Iba 1-positive cell diameter was observed in the Q140 mouse striatum (strong Microglial response). This response was significantly reduced by hNSC (fig. 6D). Similar analysis in hNSC implanted R6/2 mice showed no significant changes in the striatum (fig. 13), and may be due to relatively local effects or moderate levels of activated microglia.

ESI-017hNSC transplantation reduced mHTT accumulation and aggregation

HD pathology is characterized by the presence of HTT inclusion bodies, which may reflect altered protein homeostasis. Therefore, we performed unbiased stereological assessment on brain sections from R6/2 and Q140 mice. For R6/2 mice, sections were first stained with nickel-enhanced DAB (black) for Ku80, followed by HTT (EM48) with DAB without nickel, followed by cresyl violet staining of non-hNSC nuclei. Fig. 7A shows a region of stereology near an hNSC implant; regions distant from the implant showed no significant difference in mutant htt (mhtt) accumulation or aggregates. The results show that R6/2 mice implanted with hNSC had reduced diffuse staining and reduced inclusion body numbers near the injection site compared to veh (fig. 7A and 7B).

A significant reduction in aggregate numbers was also observed in the striatum of Q140 mice (fig. 7C). At 6 months post-treatment, hNSC-treated Q140 mice had fewer diffusely stained nuclei (p-0.0102) and fewer neurofibrillary aggregates (p-0.0239) than veh-treated mice, but no reduction in nuclear inclusion bodies and microaggregates (p-0.0753 and p-0.372, respectively) (fig. 7D). This result indicates that hNSC delivery modulates HD-related pathologies. In R6/2 (FIG. 10D) or Q140 mice, no acquisition of inclusion bodies was observed in or near the transplanted cells. hNSC transplantation to reduce pathogenic accumulation of mHTT and ubiquitinated proteins

Applicants next investigated the effect of hNSC treatment on High Molecular Weight (HMW) mHTT species and ubiquitin modifying proteins accumulated in R6/2 brain. A reduction in these insoluble proteins corresponds to improved behavioral results in R6/2 mice (Ochaba et al, 2016). Evaluation of NT and R6/2 striatal detergent insoluble fractions with and without hNSC transplantation showed that levels of accumulated mHTT were significantly increased in R6/2 striatum, whereas treatment with hNSC in R6/2 striatum reduced insoluble HTT accumulation by about 70% compared to veh treated mice (fig. 7E and 7F), which is not due to altered mHTT transgene mRNA expression (fig. 14). Accumulated ubiquitin conjugated protein was significantly increased in the R6/2 striatum compared to NT mice, and hNSC treatment decreased insoluble ubiquitin conjugated protein in the R6/2 mouse striatum compared to veh treated mice (fig. 7E and 7F). No significant differences were detected in the treated NT mice.

CCT/try (TCP1 circular complex) chaperones are oligomeric chaperones that can bind to and fold newly translated polypeptides. CCT/TRiC expression prevents truncated mHTT aggregates in multiple HD model systems (Tam, S., et al., The characterisonin TRiC controls polyglutamamine aggregation and toxicity of nuclear repair-specific interactions Nature Cell Biol,2006.8(10): p.1155-1162). Overexpression of one subunit, CCT1, is sufficient to inhibit aggregation in vitro and in cells and to reduce mHTT-mediated cytotoxicity (Tam, S., et al, The cosmetic try blocks a huntingtin sequence element at proteins, 2009.16(12): p.1279-1285). Surprisingly, the apical domain of the 20kDa yeast CCT1 (ApiCCT1) was sufficient to inhibit the aggregation of recombinant mHTT in vitro. Applicants 'data show that recombinant ApiCCT1 ApiCCT1r can reduce HD phenotype in cells (Sontag, e.m., et al, innovation delivery of performance repair fragment ApiCCT1 models mutant hunting in cells. proc. natural Acad Sci U a,2013.110(8): p.3077-82) and rescue BDNF trafficking defects in co-cultures of HD mouse primary neurons (Zhao, x., et al, try repair issues BDNF axxotransport and recovery structure repair in cloning' S disease. proc. natural Acad Sci U a, 2016). Importantly, this exogenously applied ApiCCT1r is taken up into the cytosol of cultured cells and primary neurons to function (Sontag et al, Zhao et al), suggesting that ApiCCT1 may be taken up by cells and have beneficial effects if the protein can be delivered to diseased tissues. Even after 2 weeks, a single direct injection of ApiCCT1 into the R6/1 striatum was detected, and the levels of high molecular weight and aggregated HTT were reduced. In more recent preliminary data, virus-mediated delivery of sapictc 1 or delivery of mouse NSCs secreting ApiCCT1 provided improvements in HD mice. These data indicate that delivery of continuous ApiCCT1 is likely to have a neuroprotective effect.

Viral-mediated delivery of ApiCCT1 is effective in vivo.

To evaluate the sustained delivery of sapictc 1 in vivo, AAV2/1 mediated sapictc 1 delivery was tested in a small pilot study for the effect of mHTT accumulation in R6/1 mice, sapictc 1 expressing exon 1 of human mHTT with-115 repeats and displaying a slower disease progression than R6/2 [24](constructs in FIG. 15A). Due to the rapid onset of phenotype in R6/2 mice and 2-3 weeks for AAV2/1 to reach full expression, delivery of virus earlier in the course of disease progression may be essential to achieve maximal correction of pathological phenotype. Thus, a bilateral striatal injection was performed at 5 weeks of age in R6/1 mice (AAV 2/112 x10 expressing sApicCT1 or mCherry controls⁹Individual genome copies) and tissues were harvested at 17 weeks of age. Animals injected with sapictc 1 showed about a 40% reduction in oligo mHTT (fig. 15B)&C) In that respect Analysis by stereology also revealed a visible inclusion body reduction of about 40% (fig. 15E), although this effect was not statistically significant, presumably due to insufficient sample size. These animals exhibited a significant improvement in fastening behavior at 16 weeks of age; the assay indicated dyskinesias (data not shown). The study was repeated using larger sample sizes (about-20 in each case). Animals injected with AAV2/1-sApicCT1 showed significant improvement in the rotarod task at 10, 12, and 14 weeks, which measures motor coordination and balance (FIG. 15F; week 10 and 12 data not shown). These animals also showed improvement in the fastening behavior consistent with previous studies (data not shown). Taken together, these studies indicate that continuous delivery of sapictc 1 is sufficient to improve behavioral outcomes in HD mice and reduce mHTT pathology.

Viral transduced hnscs produce secreted ApiCCT1, which enters ht14a2.6 PC12 cells and affects oligomeric mHTT species

The applicants conducted a small preliminary study to test sapictc lentiviral transduction of ESI-017hNSC to determine appropriate transduction titers and to examine the generation of ApiCCT and the effect on mutant HTT aggregation. Briefly, ESI-017hNSC were cultured in 6-well plates and then transduced with sapictc lentivirus at a multiplicity of infection (MOI) of 0, 5, 10 and 15. The cells were cultured for 48 hours, the medium was collected and the cells were collected for protein analysis. Fig. 16A shows a Western analysis of HA-labeled ApiCCT and indicates that transduced ESI-017hNSC is producing ApiCCT and that yield increases with increasing virus MOI. The medium collected from the transduced hnscs was added to the httt14a2.6pc12 cell culture medium to confirm that the transduced and secreted ApiCCT could enter neighboring cells as previously described (Sontag PNAS, 2013). In the presence of the inducer, pinsterone, these cells expressed a truncated form of the expanded repeat HTT exon 1 protein (103Qs) fused at the C-terminus to the enhanced Green Fluorescent Protein (GFP) within 48 hours. 48 hours after induction and conditioned medium application, cells were washed, harvested and subjected to Western analysis. The results indicated that cell lysates from treated 14a2.6 cells contained HA-tagged protein of appropriate molecular weight, which was ApiCCT (fig. 16B). To assess whether conditioned medium delivery of ApiCCT has an effect on a particular mutant huntingtin (mhtt) aggregation species, applicants first assessed whether the level of monomeric soluble HTT fragment is altered. HTT monomer levels from the same experiment were examined by Western analysis using antibodies against GFP (fig. 16C). ApiCCT1 does not appear to alter expression of monomeric levels of mHTT, suggesting that ApiCCT does not alter steady-state levels of monomeric mutant htt (mHTT), and also does not appear to affect gene expression of induced mHTT. Insoluble HTT aggregates and mHTT oligomers are characteristic of HD. In particular, oligomeric mHTT species may represent a source of toxicity in affected neurons. Thus, applicants measured mHTT oligomers to determine whether delivery of ApiCCT1 affected accumulation of these forms of ApiCCT1 protein as previously demonstrated with direct delivery of purified ApiCCT1 protein. SDS Agarose Gel Electrophoresis (AGE) was used to resolve oligomeric species, as this method appears to resolve soluble fibrillar oligomers of mHTT preferentially. Equal amounts of protein from cell lysates were loaded onto SDS-AGE gels. Optical density measurements were obtained using ImageJ, ApiCCT1 caused a decrease (> 10%) in mHTT oligomer levels only at the highest MOI (fig. 16D). But at both MOI 10 and 15 smear length (smear length) was reduced. These data indicate that apnsc secreted ApiCCT1 was able to reduce the formation of oligomeric mHTT in neighboring cells, reproducing our published results for purification of ApiCCT 1. These results validate the methods employed in GMP production useful for lentiviral transduction of hNSC and establish the potential for hNSC delivery. Viral transduced hnscs produce secreted ApiCCT1 following implantation in mice

ESI-017hNSC was cultured in UCI as described above. Hnscs were transduced with lentiviruses at MOI 15 for 48 hours and then transplanted into five week old mice as described above. Male and female R6/2 and non-transgenic age-matched littermates and vehicle controls were included. Immunosuppression was administered to all mice. Mice were euthanized at 9 weeks of age and brains were collected, half of which were fixed for histology and the other half were flash frozen for biochemistry. As described for hNSC, hNSC-ApiCCT implanted cells had similar IHC (fig. 17). Cells were stained predominantly with the early neuronal marker, biscortin (DCX, blue) using the human nuclear antigen marker (HNA) (fig. 17A combined to pink). Some cells expressed HA-tagged ApiCCT (fig. 17B).

Discussion of the related Art

Stem cell-based transplantation strategies are promising approaches for neurodegenerative diseases based on their ability to modulate pathology through regenerative and repair mechanisms. Mouse-derived NSCs show promising results in the HD model, while hNSC-based approaches have met with varying success, with strong efficacy in toxin models, and limited neuroprotection in genetically HD mice (El-akacawy et al, 2012; Golas and Sander, 2016). Here we describe the transplantation of GMP-grade hnscs, which provides a strong rescue targeting the accumulated defects of mHTT proteins and disease modifying activity. ESI-017hNSC is electrophysiological active in R6/2 mice, but has no significant effect on striatal MSN membrane properties or on spontaneous synaptic activity. However, in the subset of MSNs, no increase in the frequency of sepscs occurred, usually after long-term blockade of GABAA receptors with bicuculline, indicating that the grafts contribute to a reduction of cortical hyperexcitability. The applicant has not determined the underlying mechanism of this effect, but electrical stimulation inside the graft induces IPSCs in neighboring cells, suggesting that they are inhibitory. Ultrastructural data suggests that the host may have an equal number of symmetric (inhibitory) and asymmetric (excitatory) synaptic connections to hNSC. Our hypothesis is that this effect originates from implanted cells, and that they differentiate predominantly along neuronal lineages in R6/2 mice. However, in other experiments, including Q140 mice, there was a potential glial effect, suggesting that the motivation for its improvement was not known. Given that these mice do not develop neuronal loss until very advanced stages of the disease, striatal-specific transplantation appears to work by neuroprotection (by trophic factors (e.g. BDNF)) as well as by preventing abnormal accumulation of mHTT species. However, the discovery of electrophysiological activity in transplanted cells and contact between human and endogenous mouse cells may promote improved electrophysiological results, suggesting that there may also be opportunities for regenerative effects.

The rationale for transplanting NSCs, in contrast to other progenitor cell types, is based on their ability to differentiate along multiple lineages. In R6/2 mice, cells exhibited evidence of early astrocyte or neuronal differentiation; most were co-labeled with neuronal restricted progenitor markers (DCX,. beta.III-tubulin and MAP-2). Since hnscs typically take several months to differentiate finally, we expect that only partial differentiation of the transplanted cells is observed at the 4-week time point. Interestingly, few ESI-017 hnscs were DCX positive prior to in vitro implantation. The results of cell fate in R6/2 mice are in contrast to our findings in the Q140 long-term HD model and in other studies using hnscs in parkinson's disease and Alzheimer's Disease (AD) models, in which more cells are becoming astrocytes (Goldberg et al, 2017), although the latter are derived from fetal NSCs which tend to be collagenous. These data suggest that depending on the disease microenvironment, there may be different responses, that the immunosuppressive paradigm may influence elucidation or developmental cues, and that the timing of cell specificity in humans and mice may influence outcome.

Decreased levels of BDNF in HD mice and human HD subjects (Strand et al, 2007; Zuccato et al, 2011), as well as many effective treatment methods in HD mice, have shown a concomitant increase in BDNF (Ross and Tabrizi, 2011). Consistent with the idea of supporting trophic factors by stem cell transplantation; ex vivo delivery of mouse NSCs expressing GDNF can maintain motor function and prevent neuronal loss in HD mice (Ebert et al, 2010), and BDNF is required to improve cognitive ability after transplantation of mouse NSCs into AD mice (Blurton-Jones et al, 2009) or lewy body dementia models (Goldberg et al, 2015). BDNF must be transported to the striatum via afferent pathways, including the cortical striatal pathway that is altered in HD (Laforet et al, 2001). It is possible that by providing nutritional support to the striatum, the cortical striatal pathway remains sufficient to indicate production of BDNF in the cortex, or retrograde transport of BDNF from the striatum back to the neuronal soma of the cortical striatum from the stem cells, resulting in improved electrophysiological activity after transplantation.

One mechanism of action of implanted hnscs may be by reducing aberrant mHTT accumulation and aggregation, possibly by preventing their formation or inducing selective clearance mechanisms (e.g., Chen et al, 2013). We have recently described the finding that a reduction in the mHTT species of a particular HMW insolubles is associated with improved behaviour in R6/2 mice and normalization of several molecular readings (Ochaba et al, 2016). It is reasonable that a reduction in pathogenic accumulation of mHTT and ubiquinated HMW insoluble species may prevent the neuronal dysfunction observed in HD mice.

It is important to note that in contrast to the observed results, aggregates could be obtained in fetal cell transplantation studies in human HD subjects (Cicchetti et al, 2014), and no evidence of the acquired HD phenotype (e.g., inclusion bodies) was observed for the transplantation process in either mouse model (fig. 10). The lack of significant protein transmission or acquisition pathology may be the result of increased trophic signaling by hnscs or the result of a decrease in mHTT species that may promote protein transmission into the transplanted cells. Alternatively, it may take years for cells to acquire pathology that is not manifested in mouse studies.

In summary, we show that hnscs transplanted into HD mice can survive, differentiate into neural cell populations, can protect or repair damaged tissues and delay disease progression, reduce pathology and increase production of protective molecules, and may come into contact with surrounding tissues, suggesting a prospective therapeutic strategy for HD. In view of the results of An et al (2012), which shows that patient-derived NSCs that are genetically corrected can form human neurons and DARPP-32 positive cells, and the results reported here, future applications using autologous transplantation of corrected patient cells may also be feasible.

Equivalents of the formula

It should be understood that while the invention has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate, but not 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 pertains.

In addition, where features or aspects of the invention are described in terms of markush groups, those skilled in the art will recognize that the invention is thereby also described in terms of any individual member or subgroup of members of the markush group.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety as if each had been individually incorporated by reference. In case of conflict, the present specification, including definitions, will control. Throughout the specification, technical literature is cited by author citation, and full bibliographic details are provided below.

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A sequence table:

NM-030752.2 and homo sapiens t-complex 1(TCP1), transcript variant 1, mRNA

(SEQ ID NO.:1)

GTCCTGTTTCTCTCCCTGTTGTCCCTGCCTCTTTTTCCTTCCCGCCGTGCCCCGCGGCCGGGCCGGGGCAGCCGGGAAGCGGGTGGGGTGGTGTGTTACCCAGTAGCTCCTGGGACATCGCTCGGGTACGCTCCACGCCGTCGCAGCCACTGCTGTGGTCGCCGGTCGGCCGAGGGGCCGCGATACTGGTTGCCCGCGGTGTAAGCAGAATTCGACGTGTATCGCTGCCGTCAAGATGGAGGGGCCTTTGTCCGTGTTCGGTGACCGCAGCACTGGGGAAACGATCCGCTCCCAAAACGTTATGGCTGCAGCTTCGATTGCCAATATTGTAAAAAGTTCTCTTGGTCCAGTTGGCTTGGATAAAATGTTGGTGGATGATATTGGTGATGTAACCATTACTAACGATGGTGCAACCATCCTGAAGTTACTGGAGGTAGAACATCCTGCAGCTAAAGTTCTTTGTGAGCTGGCTGATCTGCAAGACAAAGAAGTTGGAGATGGAACTACTTCAGTGGTTATTATTGCAGCAGAACTCCTAAAAAATGCAGATGAATTAGTCAAACAGAAAATTCATCCCACATCAGTTATTAGTGGCTATCGACTTGCTTGCAAGGAAGCAGTGCGTTATATCAATGAAAACCTAATTGTTAACACAGATGAACTGGGAAGAGATTGCCTGATTAATGCTGCTAAGACATCCATGTCTTCCAAAATCATTGGAATAAATGGTGATTTCTTTGCTAACATGGTAGTAGATGCTGTACTTGCTATTAAATACACAGACATAAGAGGCCAGCCACGCTATCCAGTCAACTCTGTTAATATTTTGAAAGCCCATGGGAGAAGTCAAATGGAGAGTATGCTCATCAGTGGCTATGCACTCAACTGTGTGGTGGGATCCCAGGGCATGCCCAAGAGAATCGTAAATGCAAAAATTGCTTGCCTTGACTTCAGCCTGCAAAAAACAAAAATGAAGCTTGGTGTACAGGTGGTCATTACAGACCCTGAAAAACTGGACCAAATTAGACAGAGAGAATCAGATATCACCAAGGAGAGAATTCAGAAGATCCTGGCAACTGGTGCCAATGTTATTCTAACCACTGGTGGAATTGATGATATGTGTCTGAAGTATTTTGTGGAGGCTGGTGCTATGGCAGTTAGAAGAGTTTTAAAAAGGGACCTTAAACGCATTGCCAAAGCTTCTGGAGCAACTATTCTGTCAACCCTGGCCAATTTGGAAGGTGAAGAAACTTTTGAAGCTGCAATGTTGGGACAGGCAGAAGAAGTGGTACAGGAGAGAATTTGTGATGATGAGCTGATCTTAATCAAAAATACTAAGGCTCGTACGTCTGCATCGATTATCTTACGTGGGGCAAATGATTTCATGTGTGATGAGATGGAGCGCTCTTTACATGATGCACTTTGTGTAGTGAAGAGAGTTTTGGAGTCAAAATCTGTGGTTCCCGGTGGGGGTGCTGTAGAAGCAGCCCTTTCCATATACCTTGAAAACTATGCAACCAGCATGGGGTCTCGGGAACAGCTTGCGATTGCAGAGTTTGCAAGATCACTTCTTGTTATTCCCAATACACTAGCAGTTAATGCTGCCCAGGACTCCACAGATCTGGTTGCAAAATTAAGAGCTTTTCATAATGAGGCCCAGGTTAACCCAGAACGTAAAAATCTAAAATGGATTGGTCTTGATTTGAGCAATGGTAAACCTCGAGACAACAAACAAGCAGGGGTGTTTGAACCAACCATAGTTAAAGTTAAGAGTTTGAAATTTGCAACAGAAGCTGCAATCACCATTCTTCGAATTGATGATCTTATTAAATTACATCCAGAAAGTAAAGATGATAAACATGGAAGTTATGAAGATGCTGTTCACTCTGGAGCCCTTAATGATTGATCTGATGTTCCTTTTATTTATAACAATGTTAAATGCAATTGTCTTGTACCTTGAGTTGAGTATTACACATTAAAGTAAAGTACAAGCTGTAAACTTGGGTTTTTGTGATGTAGGAAATGGTTTCCATCTGTACTTTGGTCCTCTGATTTCACATATTGCAACCTAGTACTTTATTAGTTTAAAAAGAAATTGAGGTTGTTCAAAGTTTAAGCAATTCATTCTCTCTGAACACACATTGCTATTCCCATCCCACCCCCAATGCACAGGGCTGCAACACCACGACTTCTGCCCATTCTCTCCAGTGTGTGTAACAGGGTCACAAGAATTCGACAGCCAGATGCTCCAAGAGGGTGGCCCAAGGCTATAGCCCCTCCTTCAATATTGACCTAACGGGGGAGAAAAGATTTAGATTGTTTATTCTTCTGTGGACACAGTTTAAAATCTTAAACTTGTCTTTTTCCTCTTAATGTATCAGCATGCTACCCTTTCAAACTCAAATTTTCATTTTAACTGCTTAGGAATAAATTTACACCTTTGTGAAAATTCAAAAAAAAAAA

Characteristic location/qualifier

Source 1..2463

Biological body ═ intelligence'

(ii) mol type mRNA "

Anddb _ xref ═ Classification Unit 9606"

Chromosome ═ 6"

(map ═ 6q 25.3) "

2463 Gene 1

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(Note: t-Complex 1) "

[ Gene ID:6950 ], [ db _ xref ] "

/db_xref＝"HGNC:HGNC:11655"

/db_xref＝"MIM:186980"

Exon 1..299

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

misc _ features 104..106

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

note "upstream in-frame stop codon"

CDS 236..1906

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

note that "isoform a is encoded by transcript variant 1;

t-complex protein 1, alpha subunit; a tailless complex polypeptide 1;

a T-complex protein 1 α subunit;

t-Complex 1 protein "

Codon _ start ═ 1

The product is ═ T-complex protein 1 alpha subunit isomer a "

Protein _ id ═ NP _110379.2"

/db_xref＝"CCDS:CCDS5269.1"

[ Gene ID:6950 ], [ db _ xref ] "

/db_xref＝"HGNC:HGNC:11655"

/db_xref＝"MIM:186980"

Translation ═ 2 (SEQ ID No.:2)

"MEGPLSVFGDRSTGETIRSQNVMAAASIANIVKSSLGPVGLDKMLVDDIGDVTITNDGATILKLLEVEHPAAKVLCELADLQDKEVGDGTTSVVIIAAELLKNADELVKQKIHPTSVISGYRLACKEAVRYINENLIVNTDELGRDCLINAAKTSMSSKIIGINGDFFANMVVDAVLAIKYTDIRGQPRYPVNSVNILKAHGRSQMESMLISGYALNCVVGSQGMPKRIVNAKIACLDFSLQKTKMKLGVQVVITDPEKLDQIRQRESDITKERIQKILATGANVILTTGGIDDMCLKYFVEAGAMAVRRVLKRDLKRIAKASGATILSTLANLEGEETFEAAMLGQAEEVVQERICDDELILIKNTKARTSASIILRGANDFMCDEMERSLHDALCVVKRVLESKSVVPGGGAVEAALSIYLENYATSMGSREQLAIAEFARSLLVIPNTLAVNAAQDSTDLVAKLRAFHNEAQVNPERKNLKWIGLDLSNGKPRDNKQAGVFEPTIVKVKSLKFATEAAITILRIDDLIKLHPESKDDKHGSYEDAVHSGALND"

misc _ features 236..238

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

And/note "N-acetylmethionine. { ECO 0000244| PubMed:19413330,

ECO:0000244|PubMed:22223895,ECO:0000244|PubMed:22814378,

ECO:0000269|PubMed:12665801}；

spread from UniProtKB/Swiss-Prot (P17987.1); acetylation site "

misc _ feature 251..253

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

And/note ═ phosphoserine. { ECO 0000244| PubMed:23186163 };

spread from UniProtKB/Swiss-Prot (P17987.1);

phosphorylation sites "

misc _ characteristics 776..778

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

And/note ═ phosphoserine. { ECO 0000244| PubMed:19690332 };

spread from UniProtKB/Swiss-Prot (P17987.1);

phosphorylation sites "

misc _ features 830..832

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

"N6-acetyl lysine. { ECO:0000244| PubMed:19608861 };

spread from UniProtKB/Swiss-Prot (P17987.1);

acetylation site "

misc _ features 1433..1435

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

"N6-acetyl lysine. { ECO:0000244| PubMed:19608861 };

spread from UniProtKB/Swiss-Prot (P17987.1);

acetylation site "

misc _ characteristics 1706..1708

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

"phosphoserine" { ECO:0000244| PubMed:23186163 };

spread from UniProtKB/Swiss-Prot (P17987.1);

phosphorylation sites "

misc_feature 1715..1717

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

"N6-acetyl lysine. { ECO:0000250| UniProtKB: P11983 };

spread from UniProtKB/Swiss-Prot (P17987.1);

acetylation site "

misc _ characteristics 1865..1867

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

{ ECO:0000244| PubMed:18669648,

ECO:0000244|PubMed:19690332,ECO:0000244|PubMed:20068231,

ECO:0000244|PubMed:21406692,ECO:0000244|PubMed:23186163,

ECO:0000244|PubMed:24275569}；

spread from UniProtKB/Swiss-Prot (P17987.1); phosphorylation sites "

misc _ characteristics 1886..1888

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

{ ECO:0000244| PubMed:20068231,

ECO:0000244|PubMed:21406692,ECO:0000244|PubMed:23186163}；

spread from UniProtKB/Swiss-Prot (P17987.1);

phosphorylation sites "

Exon 300..385

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 386..514

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 515..612

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 613..723

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 724..905

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 906..1032

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 1033..1208

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

STS 1073..1300

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

standard-name (GDB: 451649) "

/db_xref＝"UniSTS:157336"

Exons 1209..1332

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 1333..1525

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

STS 1493..1674

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

standard _ name ═ G06897"

/db_xref＝"UniSTS:35313"

Exon 1526..1689

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 1690..2453

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

STS 1857..1964

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

standard-name (SHGC-36020) "

/db_xref＝"UniSTS:22807"

1980 regulatory site 1975

Regulatory classification (poly a signal sequence) "

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

poly a _ site 1999

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

experiment, no other details are recorded "

STS 2009..2140

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

standard _ name ═ D6S1840"

/db_xref＝"UniSTS:58762"

Regulatory site 2426..2431

Regulatory classification (poly a signal sequence) "

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

poly a _ site 2452

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

NM-001008897.1 homo sapiens t-complex 1(TCP1), transcript variant 2, mRNA

(SEQ ID NO:.3)

GTCCTGTTTCTCTCCCTGTTGTCCCTGCCTCTTTTTCCTTCCCGCCGTGCCCCGCGGCCGGGCCGGGGCAGCCGGGAAGCGGGTGGGGTGGTGTGTTACCCAGTAGCTCCTGGGACATCGCTCGGGTACGCTCCACGCCGTCGCAGCCACTGCTGTGGTCGCCGGTCGGCCGAGGGGCCGCGATACTGGTTGCCCGCGGTGTAAGCAGAATTCGACGTGTATCGCTGCCGTCAAGATGGAGGGGCCTTTGTCCGTGTTCGGTGACCGCAGCACTGGGGAAACGATCCGCTCCCAAAACGGATGTAACCATTACTAACGATGGTGCAACCATCCTGAAGTTACTGGAGGTAGAACATCCTGCAGCTAAAGTTCTTTGTGAGCTGGCTGATCTGCAAGACAAAGAAGTTGGAGATGGAACTACTTCAGTGGTTATTATTGCAGCAGAACTCCTAAAAAATGCAGATGAATTAGTCAAACAGAAAATTCATCCCACATCAGTTATTAGTGGCTATCGACTTGCTTGCAAGGAAGCAGTGCGTTATATCAATGAAAACCTAATTGTTAACACAGATGAACTGGGAAGAGATTGCCTGATTAATGCTGCTAAGACATCCATGTCTTCCAAAATCATTGGAATAAATGGTGATTTCTTTGCTAACATGGTAGTAGATGCTGTACTTGCTATTAAATACACAGACATAAGAGGCCAGCCACGCTATCCAGTCAACTCTGTTAATATTTTGAAAGCCCATGGGAGAAGTCAAATGGAGAGTATGCTCATCAGTGGCTATGCACTCAACTGTGTGGTGGGATCCCAGGGCATGCCCAAGAGAATCGTAAATGCAAAAATTGCTTGCCTTGACTTCAGCCTGCAAAAAACAAAAATGAAGCTTGGTGTACAGGTGGTCATTACAGACCCTGAAAAACTGGACCAAATTAGACAGAGAGAATCAGATATCACCAAGGAGAGAATTCAGAAGATCCTGGCAACTGGTGCCAATGTTATTCTAACCACTGGTGGAATTGATGATATGTGTCTGAAGTATTTTGTGGAGGCTGGTGCTATGGCAGTTAGAAGAGTTTTAAAAAGGGACCTTAAACGCATTGCCAAAGCTTCTGGAGCAACTATTCTGTCAACCCTGGCCAATTTGGAAGGTGAAGAAACTTTTGAAGCTGCAATGTTGGGACAGGCAGAAGAAGTGGTACAGGAGAGAATTTGTGATGATGAGCTGATCTTAATCAAAAATACTAAGGCTCGTACGTCTGCATCGATTATCTTACGTGGGGCAAATGATTTCATGTGTGATGAGATGGAGCGCTCTTTACATGATGCACTTTGTGTAGTGAAGAGAGTTTTGGAGTCAAAATCTGTGGTTCCCGGTGGGGGTGCTGTAGAAGCAGCCCTTTCCATATACCTTGAAAACTATGCAACCAGCATGGGGTCTCGGGAACAGCTTGCGATTGCAGAGTTTGCAAGATCACTTCTTGTTATTCCCAATACACTAGCAGTTAATGCTGCCCAGGACTCCACAGATCTGGTTGCAAAATTAAGAGCTTTTCATAATGAGGCCCAGGTTAACCCAGAACGTAAAAATCTAAAATGGATTGGTCTTGATTTGAGCAATGGTAAACCTCGAGACAACAAACAAGCAGGGGTGTTTGAACCAACCATAGTTAAAGTTAAGAGTTTGAAATTTGCAACAGAAGCTGCAATCACCATTCTTCGAATTGATGATCTTATTAAATTACATCCAGAAAGTAAAGATGATAAACATGGAAGTTATGAAGATGCTGTTCACTCTGGAGCCCTTAATGATTGATCTGATGTTCCTTTTATTTATAACAATGTTAAATGCAATTGTCTTGTACCTTGAGTTGAGTATTACACATTAAAGTAAAGTACAAGCTGTAAACTTGGGTTTTTGTGATGTAGGAAATGGTTTCCATCTGTACTTTGGTCCTCTGATTTCACATATTGCAACCTAGTACTTTATTAGTTTAAAAAGAAATTGAGGTTGTTCAAAGTTTAAGCAATTCATTCTCTCTGAACACACATTGCTATTCCCATCCCACCCCCAATGCACAGGGCTGCAACACCACGACTTCTGCCCATTCTCTCCAGTGTGTGTAACAGGGTCACAAGAATTCGACAGCCAGATGCTCCAAGAGGGTGGCCCAAGGCTATAGCCCCTCCTTCAATATTGACCTAACGGGGGAGAAAAGATTTAGATTGTTTATTCTTCTGTGGACACAGTTTAAAATCTTAAACTTGTCTTTTTCCTCTTAATGTATCAGCATGCTACCCTTTCAAACTCAAATTTTCATTTTAACTGCTTAGGAATAAATTTACACCTTTGTGAAAATTCAAAAAAAAAAA

Characteristic location/qualifier

2377 sources

Biological body ═ intelligence'

(ii) mol type mRNA "

Anddb _ xref ═ Classification Unit 9606"

Chromosome ═ 6"

(map ═ 6q 25.3) "

2377 Gene 1

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(Note: t-Complex 1) "

[ Gene ID:6950 ], [ db _ xref ] "

/db_xref＝"HGNC:HGNC:11655"

/db_xref＝"MIM:186980"

Exon 1..299

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 300..428

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 429..526

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 527..637

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

CDS 615..1820

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

note that "isoform b is encoded by transcript variant 2;

t-complex protein 1, alpha subunit; a tailless complex polypeptide 1;

T-Complex protein 1 subunit α;

t-Complex 1 protein "

Codon _ start ═ 1

The product is ═ T-complex protein 1 subunit alpha isomer b "

Protein _ id ═ NP _001008897.1"

/db_xref＝"CCDS:CCDS43522.1"

[ Gene ID:6950 ], [ db _ xref ] "

/db_xref＝"HGNC:HGNC:11655"

/db_xref＝"MIM:186980"

Translation ═ 4 (SEQ ID NO: 4)

"MSSKIIGINGDFFANMVVDAVLAIKYTDIRGQPRYPVNSVNILKAHGRSQMESMLISGYALNCVVGSQGMPKRIVNAKIACLDFSLQKTKMKLGVQVVITDPEKLDQIRQRESDITKERIQKILATGANVILTTGGIDDMCLKYFVEAGAMAVRRVLKRDLKRIAKASGATILSTLANLEGEETFEAAMLGQAEEVVQERICDDELILIKNTKARTSASIILRGANDFMCDEMERSLHDALCVVKRVLESKSVVPGGGAVEAALSIYLENYATSMGSREQLAIAEFARSLLVIPNTLAVNAAQDSTDLVAKLRAFHNEAQVNPERKNLKWIGLDLSNGKPRDNKQAGVFEPTIVKVKSLKFATEAAITILRIDDLIKLHPESKDDKHGSYEDAVHSGALND"

Exon 638..819

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 820..946

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 947..1122

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

STS 987..1214

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

standard-name (GDB: 451649) "

/db_xref＝"UniSTS:157336"

Exon 1123..1246

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

Exon 1247..1439

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

STS 1407..1588

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

standard-name G06897"

/db_xref＝"UniSTS:35313"

Exon 1440..1603

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

2367 exon 1604

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

(ii) extrapolation-comparison: Splign:2.1.0"

STS 1771..1878

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

standard-name (SHGC-36020) "

/db_xref＝"UniSTS:22807"

Regulatory site 1889..1894

Regulatory classification (poly a signal sequence) "

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

poly a _ locus 1913

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

STS 1923..2054

gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

standard _ name ═ D6S1840"

/db_xref＝"UniSTS:58762"

2340..2345 regulatory site

Regulatory classification (poly a signal sequence) "

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

poly a _ site 2366

Gene ═ TCP1"

Gene _ synonym ═ CCT- α; CCT 1; CCTa; D6S 230E;

TCP-1-α"

NM-001143805.1 brain-derived neurotrophic factor (BDNF), transcript variant 7, mRNA

(SEQ ID NO:5)

AATATCAAGTATCACTTAATTAGAGATTTTTAAGCCTTTTCCTCCTGCTGTGCCGGGTGTGTAATCCGGGCGATAGGAGTCCATTCAGCACCTTGGACAGAGCCAACGGATTTGTCCGAGGTGGCGGTACCCCCAGTTCCACCAGGTGAGAAGAGTGATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTTGGTTGCATGAAGGCTGCCCCCATGAAAGAAGCAAACATCCGAGGACAAGGTGGCTTGGCCTACCCAGGTGTGCGGACCCATGGGACTCTGGAGAGCGTGAATGGGCCCAAGGCAGGTTCAAGAGGCTTGACATCATTGGCTGACACTTTCGAACACGTGATAGAAGAGCTGTTGGATGAGGACCAGAAAGTTCGGCCCAATGAAGAAAACAATAAGGACGCAGACTTGTACACGTCCAGGGTGATGCTCAGTAGTCAAGTGCCTTTGGAGCCTCCTCTTCTCTTTCTGCTGGAGGAATACAAAAATTACCTAGATGCTGCAAACATGTCCATGAGGGTCCGGCGCCACTCTGACCCTGCCCGCCGAGGGGAGCTGAGCGTGTGTGACAGTATTAGTGAGTGGGTAACGGCGGCAGACAAAAAGACTGCAGTGGACATGTCGGGCGGGACGGTCACAGTCCTTGAAAAGGTCCCTGTATCAAAAGGCCAACTGAAGCAATACTTCTACGAGACCAAGTGCAATCCCATGGGTTACACAAAAGAAGGCTGCAGGGGCATAGACAAAAGGCATTGGAACTCCCAGTGCCGAACTACCCAGTCGTACGTGCGGGCCCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACACTTCTTGTGTATGTACATTGACCATTAAAAGGGGAAGATAGTGGATTTATGTTGTATAGATTAGATTATATTGAGACAAAAATTATCTATTTGTATATATACATAACAGGGTAAATTATTCAGTTAAGAAAAAAATAATTTTATGAACTGCATGTATAAATGAAGTTTATACAGTACAGTGGTTCTACAATCTATTTATTGGACATGTCCATGACCAGAAGGGAAACAGTCATTTGCGCACAACTTAAAAAGTCTGCATTACATTCCTTGATAATGTTGTGGTTTGTTGCCGTTGCCAAGAACTGAAAACATAAAAAGTTAAAAAAAATAATAAATTGCATGCTGCTTTAATTGTGAATTGATAATAAACTGTCCTCTTTCAGAAAACAGAAAAAAACACACACACACACAACAAAAATTTGAACCAAAACATTCCGTTTACATTTTAGACAGTAAGTATCTTCGTTCTTGTTAGTACTATATCTGTTTTACTGCTTTTAACTTCTGATAGCGTTGGAATTAAAACAATGTCAAGGTGCTGTTGTCATTGCTTTACTGGCTTAGGGGATGGGGGATGGGGGGTATATTTTTGTTTGTTTTGTGTTTTTTTTTCGTTTGTTTGTTTTGTTTTTTAGTTCCCACAGGGAGTAGAGATGGGGAAAGAATTCCTACAATATATATTCTGGCTGATAAAAGATACATTTGTATGTTGTGAAGATGTTTGCAATATCGATCAGATGACTAGAAAGTGAATAAAAATTAAGGCAACTGAACAAAAAAATGCTCACACTCCACATCCCGTGATGCACCTCCCAGGCCCCGCTCATTCTTTGGGCGTTGGTCAGAGTAAGCTGCTTTTGACGGAAGGACCTATGTTTGCTCAGAACACATTCTTTCCCCCCCTCCCCCTCTGGTCTCCTCTTTGTTTTGTTTTAAGGAAGAAAAATCAGTTGCGCGTTCTGAAATATTTTACCACTGCTGTGAACAAGTGAACACATTGTGTCACATCATGACACTCGTATAAGCATGGAGAACAGTGATTTTTTTTTAGAACAGAAAACAACAAAAAATAACCCCAAAATGAAGATTATTTTTTATGAGGAGTGAACATTTGGGTAAATCATGGCTAAGCTTAAAAAAAACTCATGGTGAGGCTTAACAATGTCTTGTAAGCAAAAGGTAGAGCCCTGTATCAACCCAGAAACACCTAGATCAGAACAGGAATCCACATTGCCAGTGACATGAGACTGAACAGCCAAATGGAGGCTATGTGGAGTTGGCATTGCATTTACCGGCAGTGCGGGAGGAATTTCTGAGTGGCCATCCCAAGGTCTAGGTGGAGGTGGGGCATGGTATTTGAGACATTCCAAAACGAAGGCCTCTGAAGGACCCTTCAGAGGTGGCTCTGGAATGACATGTGTCAAGCTGCTTGGACCTCGTGCTTTAAGTGCCTACATTATCTAACTGTGCTCAAGAGGTTCTCGACTGGAGGACCACACTCAAGCCGACTTATGCCCACCATCCCACCTCTGGATAATTTTGCATAAAATTGGATTAGCCTGGAGCAGGTTGGGAGCCAAATGTGGCATTTGTGATCATGAGATTGATGCAATGAGATAGAAGATGTTTGCTACCTGAACACTTATTGCTTTGAAACTAGACTTGAGGAAACCAGGGTTTATCTTTTGAGAACTTTTGGTAAGGGAAAAGGGAACAGGAAAAGAAACCCCAAACTCAGGCCGAATGATCAAGGGGACCCATAGGAAATCTTGTCCAGAGACAAGACTTCGGGAAGGTGTCTGGACATTCAGAACACCAAGACTTGAAGGTGCCTTGCTCAATGGAAGAGGCCAGGACAGAGCTGACAAAATTTTGCTCCCCAGTGAAGGCCACAGCAACCTTCTGCCCATCCTGTCTGTTCATGGAGAGGGTCCCTGCCTCACCTCTGCCATTTTGGGTTAGGAGAAGTCAAGTTGGGAGCCTGAAATAGTGGTTCTTGGAAAAATGGATCCCCAGTGAAAACTAGAGCTCTAAGCCCATTCAGCCCATTTCACACCTGAAAATGTTAGTGATCACCACTTGGACCAGCATCCTTAAGTATCAGAAAGCCCCAAGCAATTGCTGCATCTTAGTAGGGTGAGGGATAAGCAAAAGAGGATGTTCACCATAACCCAGGAATGAAGATACCATCAGCAAAGAATTTCAATTTGTTCAGTCTTTCATTTAGAGCTAGTCTTTCACAGTACCATCTGAATACCTCTTTGAAAGAAGGAAGACTTTACGTAGTGTAGATTTGTTTTGTGTTGTTTGAAAATATTATCTTTGTAATTATTTTTAATATGTAAGGAATGCTTGGAATATCTGCTATATGTCAACTTTATGCAGCTTCCTTTTGAGGGACAAATTTAAAACAAACAACCCCCCATCACAAACTTAAAGGATTGCAAGGGCCAGATCTGTTAAGTGGTTTCATAGGAGACACATCCAGCAATTGTGTGGTCAGTGGCTCTTTTACCCAATAAGATACATCACAGTCACATGCTTGATGGTTTATGTTGACCTAAGATTTATTTTGTTAAAATCTCTCTCTGTTGTGTTCGTTCTTGTTCTGTTTTGTTTTGTTTTTTAAAGTCTTGCTGTGGTCTCTTTGTGGCAGAAGTGTTTCATGCATGGCAGCAGGCCTGTTGCTTTTTTATGGCGATTCCCATTGAAAATGTAAGTAAATGTCTGTGGCCTTGTTCTCTCTATGGTAAAGATATTATTCACCATGTAAAACAAAAAACAATATTTATTGTATTTTAGTATATTTATATAATTATGTTATTGAAAAAAATTGGCATTAAAACTTAACCGCATCAGAACCTATTGTAAATACAAGTTCTATTTAAGTGTACTAATTAACATATAATATATGTTTTAAATATAGAATTTTTAATGTTTTTAAATATATTTTCAAAGTACATAAAA

Characteristic location/qualifier

Source 1..3827

Biological body ═ intelligence'

(ii) mol type mRNA "

Anddb _ xref ═ Classification Unit 9606"

Chromosome ═ 11"

(map) '11 p 14.1'

Gene 1..3827

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

'brain-derived neurotrophic factor'

(db) — xref ═ Gene ID:627"

/db_xref＝"HGNC:HGNC:1033"

/db_xref＝"MIM:113505"

Exon 1..136

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

(ii) extrapolation-comparison: Splign:2.1.0"

misc _ characteristics 11

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Annotation for "alternative transcription initiation site"

misc _ characteristics 12

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Annotation for "alternative transcription initiation site"

misc _ characteristics 18

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Annotation for "alternative transcription initiation site"

misc _ characteristics 27

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Annotation for "alternative transcription initiation site"

misc _ characteristics 34

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Annotation for "alternative transcription initiation site"

misc _ feature 74..76

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Note "upstream in-frame stop codon"

3827 exon 137

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

(ii) extrapolation-comparison: Splign:2.1.0"

CDS 158..901

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Annotation ═ isoform a preproprotein is encoded by transcript variant 7;

a neurotrophic factor; alinenglin'

Codon _ start ═ 1

Product ═ brain-derived neurotrophic factor isomer a preproprotein "

Protein _ id ═ NP _001137277.1"

/db_xref＝"CCDS:CCDS7866.1"

(db) — xref ═ Gene ID:627"

/db_xref＝"HGNC:HGNC:1033"

/db_xref＝"MIM:113505"

Translation ═ 6 (SEQ ID No.:6)

"MTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR"

sig_peptide 158..211

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Coordinate de novo prediction method SignalP 4.0"

misc _ feature 326..331

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Experiment, no other details are recorded "

Note ═ cleavage by S1P;

spread from UniProtKB/Swiss-Prot (P23560.1); cleavage site "

mat_peptide 542..898

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Product of 'brain-derived neurotrophic factor'

Experimental evidence, no further details were recorded

"propagation from UniProtKB/Swiss-Prot (P23560.1)"

STS 163..771

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Standard-name "BDNF"

/db_xref＝"UniSTS:266531"

STS 514..796

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Standard-name (BDNF-1) "

/db_xref＝"UniSTS:253960"

STS 578..1460

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Standard _ name ═ BDNF _2411"

/db_xref＝"UniSTS:280459"

STS 1062..1163

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

Standard-name ═ D11S4429"

/db_xref＝"UniSTS:43225"

Poly A _ site 3827

Gene BDNF "

Gene _ synonym "ANON 2; BULN2"

ApiCCT1(SEQ ID NO:7):

MVPGYALNCTVASQAMPKRIAGGNVKIACLDLNLQKARMAMGVQINIDDPEQLEQIRKREAGIVLERVKKIIDAGAQWLTIKGIDDLCLKEFVEAKlMGVRRCKKEDLRRIARATGATLVSSMSNLEGEETFESSYLGLCDEWQAKFSDDECILIKGTSKAAAAALE.

sApiCCT1 mRNA(SEQ ID NO:8)

sApiCCT1(SEQ ID NO:9)

MYRMQLLSCIALSLALVTNSISGYALNCVVGSQGMPKRIVNAKIACLDFSLQKTKMKLGVQVVITDPEKLDQIRQRESDITKERIQKILATGANVILTTGGIDDMCLKYFVEAGAMAVRRVLKRDLKRIAKASGATILSTLANLEGEETFEAAMLGQAEEVVQERICDDELILIKNTKAAAAAGGHYPYDVPDYA

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