Cell reprogramming

Abstract

The present invention relates generally to compositions for the cell reprogramming conversion of human cells into inducible neural precursor cells, methods of preparing inducible human neural precursor cells by cell reprogramming, and methods of treating diseases using reprogrammed inducible human neural precursor cells.

Description

[0001] 1. TECHNICAL FIELD

[0002] The present invention relates generally to compositions for converting human cells into induced neural precursor cells by cellular reprogramming, methods of making induced human neural precursor cells by cellular reprogramming, and methods of using reprogrammed induced human neural precursor cells to treat disease.

[0003] 2. BACKGROUND

[0004] Huntington's disease (HD) is a genetic neurological disorder caused by a trinucleotide (CAG) repeat expansion mutation in exon 1 of the IT15 gene, which encodes a 350 kDa protein called huntingtin (HTT). The disease is inherited in an autosomal dominant manner with a prevalence of approximately 1 in 15,000. HD is characterized by loss of neuronal cells in the caudate, putamen, and cerebral cortex. In later stages, regions such as the hippocampus and hypothalamus are also affected (Vonsattel et al., 1985). The primary degeneration of medium spiny striatal projection neurons (MSNs) leads to motor dysfunction, accompanied by cognitive and psychiatric disturbances. HTT (Huntingtin)

[0005] Current treatment options for Huntington's disease are extremely limited. Although some behavioral symptoms of HD respond to psychiatric treatment, and several medications are available to reduce the impact of chorea, other motor and cognitive symptoms of HD are currently untreatable (Caron et al., 1998). Cell transplantation is a viable option for treating HD, with the goal of reestablishing the damaged neural circuitry by replacing the cells lost during the disease process, with the expectation that the donor cells will re-connect to the remaining host neural network to repair connectivity. Early therapeutic intervention through genetic testing allows for the transplantation of replacement MSNs prior to extensive degeneration, to maintain the corticostriatal circuit. Both rodent and primate HD studies have shown that reestablishment of the corticostriatal circuit following cell transplantation can alleviate the motor and cognitive deficits observed in HD (Dunnett et al., 2000; Kendall et al., 1998; Palfi et al., 1998). Small open-label clinical trials have explored the transplantation of human fetal striatal tissue into HD patients, with preliminary validation of the principle that neural transplantation can benefit HD patients (Rosser and Bachoud-Levi, 2012). However, one of the major issues to be addressed for cell transplantation therapy to become a viable treatment option for HD patients is the identification of a source of donor cells that is both ethically and technically feasible, other than human fetal striatal tissue.

[0006] ​Attention has turned to the potential use of human stem cells, including human embryonic stem cells (hESCs) or human induced pluripotent stem cells (iPSCs), in the search for alternative donor cell sources (Connor, 2018). However, initial studies have shown that hESC-derived neural stem cells (NSCs) transplanted into a quinolinic acid (QA)-injured model of Huntington’s disease survive and generate new neurons, but transplanted human NSCs do not differentiate into region-specific neurons expressing MSN markers (Joannides et al., 2007; Reidling et al., 2018; Song et al., 2007; Vazey et al., 2010). To improve lineage specificity and promote differentiation toward MSNs, multiple research groups have differentiated hESCs into striatal precursor cells (Arber et al., 2015; Aubry et al., 2008; Delli Carri et al., 2013; Faedo et al., 2017; 2013). These studies report the generation of MSNs following transplantation of striatal precursor cells into a QA-injured striatum, indicating the need to direct hESCs to a lineage-specific precursor cell fate prior to transplantation. The potential of iPSC-derived NSCs as a source of cells for HD transplantation therapy has also been demonstrated by several studies (An et al., 2012; Jeon et al., 2012), with An and colleagues (An et al., 2012) demonstrating the ability to transplant genetically corrected HD patient-derived cells. However, the use of hESC- or hiPSC-derived NSCs for cell transplantation carries the potential risk of tumour formation and genetic mutation, which is associated with chromosomal abnormalities accumulation related to long-term passaging. Furthermore, hiPSCs carry the risk of generating genetic abnormalities and insertional mutagenic effects due to the oncogenic properties of the reprogramming factors and the gene delivery integration method used during the reprogramming process (González et al., 2011).

[0007] In view of the foregoing, there is a need in the art to develop alternative forms of cell transplantation therapy to avoid the problems identified with the use of hESC- or hiPSC-derived NSCs, particularly the risks associated with tumour formation, genetic mutation, generation of genetic abnormalities, and insertional mutagenic effects.

[0008] It is an object of the present invention to provide compositions and methods to support at least one such alternative form of cell transplantation therapy while avoiding at least some of the deficiencies identified in previous therapies, and / or to at least provide the public with a useful choice.

[0009] In this specification, a patent application, other external documents, or other sources of information are cited. The citation of such documents is not to be construed as an admission that it is prior art to the present invention. To the extent that any meaning or definition of a term in this document conflicts with the meaning or definition of the same term in a document incorporated by reference, the meaning or definition agreed to by the USPTO and the inventor controls.

[0010] 3. SUMMARY

[0011] The compositions and methods disclosed herein employ chemically modified mRNA to reprogram somatic cells into neural precursor cells. The present disclosure provides for the first time compositions and methods for reprogramming adult human somatic cells (aHS), particularly adult human fibroblasts (aHF), more particularly adult human dermal fibroblasts (aHDF), into human neural cells, particularly human lateral ganglionic eminence progenitor cells (hiLGEP).

[0012] The present inventors have also for the first time revealed that directly reprogrammed somatic cells generated using the compositions and methods described herein are able to survive and generate medium spiny striatal neurons (MSNs) upon transplantation into quinolinic acid (QA)-lesioned rat striatum, a well-established model of HD. These directly reprogrammed hiLGEPs restored motor dysfunction 14 weeks post-transplantation, indicating that directly reprogrammed hiLGEPs provide an effective and clinically feasible cell source for cell replacement therapy for the treatment of brain degenerative diseases, particularly Huntington’s disease (HD).

[0013] In one aspect, the present application relates to a composition comprising: a basal brain medium, and at least two active agents selected from the group consisting of Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

[0014] In another aspect, the present application relates to a method of preparing human induced lateral ganglionic eminence progenitor cells (hiLGEPs), comprising:

[0015] a) reprogramming human fibroblasts (HF) into hiLGEPs, comprising:

[0016] a. transfecting the HF with SOX2 cmRNA and PAX6 cmRNA;

[0017] b. culturing the transfected HF in a composition comprising: a basal brain medium, and at least two active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement;

[0018] c. passaging the HF in b. in a composition comprising: a basal brain medium, and at least three active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, an N-2 supplement, and Activin A (ActA); and

[0019] d. culturing the passaged HF.

[0020] In another aspect, the present application relates to a kit comprising:

[0021] i. A composition comprising: a basal brain medium, and at least two active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement; and

[0022] ii. A composition comprising: a basal brain medium, and at least three active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, an N-2 supplement, and an Activin A (ActA).

[0023] In another aspect, the present application relates to a human induced lateral ganglionic eminence progenitor cell (hiLGEP).

[0024] In another aspect, the present application relates to a composition comprising a human induced lateral ganglionic eminence progenitor cell (hiLGEP) and a carrier.

[0025] In another aspect, the present application relates to a composition comprising a basal brain medium, B27-RA, N2 supplement, a cyclic adenosine 3',5'-monophosphate (cAMP) activator, a pi 60 ROCK inhibitor, a brain-derived neurotrophic factor (BDNF), and an Activin A (ActA).

[0026] In another aspect, the present application relates to a composition comprising a basal brain medium, B27-RA, N2 supplement, a cyclic adenosine 3',5'-monophosphate (cAMP) activator, a pi 60 ROCK inhibitor, a brain-derived neurotrophic factor (BDNF), and dorsomorphin.

[0027] In another aspect, the present application relates to the use of a composition for inducing the expression of at least one lateral ganglionic eminence (LGE) transcription factor in reprogrammed HFs, the composition comprising a basal brain medium, and at least two active agents selected from the group consisting of an Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

[0028] In another aspect, the present application relates to the use of a composition for promoting the induction of a lateral ganglionic eminence (LGE) progenitor cell fate in fibroblasts, preferably human fibroblasts (HFs), the composition comprising a basal brain medium, and at least two active agents selected from the group consisting of an Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

[0029] In another aspect, the present application relates to the use of a composition comprising basal brain medium, B27-RA, N2 supplement, cAMP activator, pl60ROCK inhibitor, brain-derived neurotrophic factor (BDNF), and activin A for inducing expression of at least one biomarker associated with neuronal differentiation in reprogrammed HF LGE precursor cells.

[0030] In another aspect, the present application relates to the use of a composition comprising basal brain medium, B27-RA, N2 supplement, cAMP activator, pl60ROCK inhibitor, brain-derived neurotrophic factor (BDNF), and dorsomorphin for inducing expression of at least one biomarker associated with neuronal differentiation in reprogrammed HF LGE precursor cells.

[0031] In another aspect, the present application relates to the use of a composition comprising human induced lateral ganglionic eminence progenitor cells (hiLGEPs) or reprogrammed HFs expressing at least one lateral ganglionic eminence (LGE) transcription factor and a carrier for the preparation of medium spiny striatal projection neurons cells (MSNs).

[0032] In another aspect, the present application relates to the use of a composition comprising human induced lateral ganglionic eminence progenitor cells (hiLGEPs) and a carrier for the treatment of Huntington’s disease.

[0033] In another aspect, the present application relates to the use of a composition comprising human induced lateral ganglionic eminence progenitor cells (hiLGEPs) and a carrier for reducing the severity of Huntington’s disease.

[0034] In another aspect, the present application relates to the use of a composition comprising human induced lateral ganglionic eminence progenitor cells (hiLGEPs) and a carrier for delaying the onset of Huntington’s disease.

[0035] In another aspect, the present application relates to a method of treating Huntington’s disease comprising transplanting human induced lateral ganglionic eminence progenitor cells (hiLGEPs) into the striatum of a subject having or suspected of having Huntington’s disease.

[0036] In another aspect, the present application relates to a method of delaying the onset of Huntington’s disease comprising transplanting human induced lateral ganglionic eminence progenitor cells (hiLGEPs) into the striatum of a subject suspected of having Huntington’s disease or having at least one symptom of Huntington’s disease.

[0037] In another aspect, the present application relates to a method of reducing the severity of Huntington's disease, the method comprising transplanting human induced lateral ganglionic eminence precursor cells (hiLGEPs) into the striatum of a subject suspected of having Huntington's disease or having at least one symptom of Huntington's disease.

[0038] 4. BRIEF DESCRIPTION OF DRAWINGS

[0039] The present application will now be described, by way of example only, and with reference to the accompanying drawings in which:

[0040] Figure 1 Direct reprogramming and differentiation protocols disclosed herein are shown.

[0041] Figure 2 Reprogrammed HDFs express lateral ganglionic eminence precursor cell biomarkers are shown. (A) and (B) show the additive effects of ActA, Gö6983, Y-27632, and N-2 (GYN) in Neurobasal A (NBA) or BrainPhys-based reprogramming media on the expression of key biomarkers by hiLGEPs. CTIP2 Combined positive effects of LGE fate acquisition assessed by expression. (C) shows representative images of hiLGEPs expressing key biomarkers at the gene level (D) and protein level (E-H), respectively.

[0042] Figure 3 hiLGEPs differentiate into medium spiny striatal neurons are shown. hiLGEPs reprogrammed in BrainPhys-based media with the addition of ActA and GYN produced high yields of striatal neurons expressing (A) TUJ1 and (B) DARPP32. The addition of dorsomorphin to the striatal differentiation media caused hiLGEPs to generate a more stable and consistent number of DARPP32-positive striatal neurons. Reprogramming with GYN alone, or in combination with ActA, whether or not all three GYN compounds were included, produced hiLGEPs that could be differentiated into striatal neurons expressing (D) TUJ1 and (E) DARPP32, with the highest yields of striatal neurons produced using GYN + ActA during reprogramming. p < 0.05; p < 0.01; p < 0.001.

[0043] Figure 4 hiLGEPs express medium spiny striatal neuron biomarkers after differentiation are shown. (A) shows representative images of striatal neurons generated by the differentiation of hiLGEPs expressing key biomarkers (B) TUJ1, (C) DARPP32, (D) GABA, and (E) GAD 65 / 67hiLGEP-derived striatal neurons have calcium flux capacity (G; brighter staining indicates more intracellular calcium), a measure of cell function in response to increasing concentrations of glutamate (F).

[0044] Figure 5 (A) shows the cell transplantation protocol and timeline in the QA rat model disclosed herein. (B) shows that transplantation of hiLGEP into the Huntington’s disease QA rat model significantly reduced forelimb motor dysfunction, as evidenced by decreased ipsilateral forelimb use over time. # p < 0.05; ## p < 0.01 (compared to baseline); p < 0.05 (compared to post-QA).

[0045] Figure 6 shows key biomarkers expressed by transplanted hiLGEP-derived striatal neurons 14 weeks post-transplantation. hiLGEP generated human medium spiny neurons expressing human marker STEM121 (A) and co-expressed STEM121 (B1, C1, D1, and E1) with (B2) MAP2, (C2) DARPP32, (D2) GAD 65 / 67 and (E2) GABA. A’ is a high magnification image of A.

[0046] Figure 7 shows that hiLGEP and medium spiny striatal neurons derived therefrom can be generated regardless of the cmRNA transfection method used. (A) HDFs were transduced with SOX2 and PAX6 cmRNA SNIM for 5 hours per day for 4 consecutive days (4 x 5-hour SNIM), or with SOX2 and PAX6 lipid nanoparticle for a single transfection of 24 hours (1 x 24-hour LNP) and exhibited identical morphological changes during reprogramming. HDFs reprogrammed with 4 x 5-hour SNIM or 1 x 24-hour LNP showed (B) SOX2 and (C) PAX6 upregulation of (D) and striatal lineage markers CTIP2 upregulation of (D) and striatal lineage markers

[0047] 5. DETAILED DESCRIPTION

[0048] 5.1 Definitions and Abbreviations

[0049] As used herein, the term "cmRNA" refers to the abbreviation for chemically modified mRNA, which contains a combination of modified and unmodified nucleotides, and refers to stabilized non-immunogenic mRNA (known commercially as SNIM RNA), as disclosed in WO 2011 / 012316, the entire contents of which are incorporated herein by reference. The chemical modifications of the cmRNA structural elements enable these molecules to evade the innate immune recognition and instability of native mRNA. cmRNA can be reused, i.e., in separate and / or consecutive transfection events, thereby enabling cells to produce a certain level of a protein product of interest on a sustained basis. The use of cmRNA is known to include enhancing or replacing missing / nonfunctional proteins, and / or introducing new proteins. In some embodiments, cmRNA can be transfected into cells using lipid nanoparticle technology (LNP), as described in the Examples.

[0050] As used herein, the term "Gö6983" refers to Gö6983 with CAS Number 133053-19-7.

[0051] As used herein, the term "Y27632" refers to Y-27632 dihydrochloride with CAS Number 129830-38-2.

[0052] As used herein, the term "N-2 supplement" refers to Bottenstein's N-2 formula (1), a chemically defined supplement consisting of human transferrin (holo-iron binding form), recombinant insulin holos, progesterone, putrescine, and sodium selenite (Bottenstein, J.E. (1985) Cell Culture in the Neurosciences, Bottenstein, J.E. and Harvey, A.L. eds., p. 3, Plenum Press: New York and London).

[0053] In this document, the combination of Gö6983, Y27632, and N-2 is abbreviated as "GYN".

[0054] As used herein, the term "cyclic adenosine 3', 5'-monophosphate (cAMP) activator" or "cAMP activator" refers to a molecule that activates the cAMP pathway. In one embodiment, the cAMP activator is dcAMP, forskolin (FSK), 8-bromo-cAMP, cAMPS-Sp.

[0055] As used herein, the term "active agent" refers to an agent that is a key component in the compositions, kits, methods, or uses described herein for driving the reprogramming of human fibroblasts (particularly adult human fibroblasts, more particularly adult human dermal fibroblasts) into human lateral ganglionic eminence progenitor cells (hiLGEPs). The compositions, kits, methods, and / or uses described herein can also comprise other agents that aid in and / or allow for cell culture and passaging during the process of cell reprogramming. Other agents can include valproic acid, penicillin-streptomycin-glutamine, B-27 without retinoic acid, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), heparin, and retinoic acid, including any combination thereof.

[0056] The active agents contemplated herein are selected from the group consisting of Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement. In a preferred embodiment, the active agents are Activin A, Gö6983, Y27632, and an N-2 supplement.

[0057] In some embodiments, according to the compositions, kits, methods, and / or uses described herein, at least two active agents (preferably three active agents, i.e., a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement) are used to culture fibroblasts to be reprogrammed into hiLGEPs. In a preferred embodiment, the three active agents are Gö6983, Y27632, and an N-2 supplement.

[0058] In some embodiments, according to the compositions, kits, methods, and / or uses described herein, at least three active agents (preferably four active agents, i.e., a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, an N-2 supplement, and Activin A (ActA)) are used to passage fibroblasts to be reprogrammed into hiLGEPs. In a preferred embodiment, the four active agents are Gö6983, Y27632, an N-2 supplement, and ActA.

[0059] As used herein, "basal brain medium" refers to a general medium suitable for in vitro culture of neural cells.

[0060] In one embodiment, the basal brain culture medium is Neurobasal A or Brain Phys, supplemented with at least one active agent, preferably at least two, three, or all four active agents selected from the group consisting of: activin A (ActA), protein kinase C (PKC) inhibitors, p160ROCK inhibitors, and N-2 supplements. In some embodiments, the basal brain culture medium is supplemented with at least one of activin A, Gö6983, Y27632, and N-2 supplements, preferably at least two, three, or all four.

[0061] The basal brain culture medium may also be supplemented with other reagents that are not “activators” and are known to those skilled in the art for human cell culture and passage.

[0062] In some embodiments, the other reagents are any combination and / or all of the following components: valproic acid, penicillin-streptomycin-glutamine, retinoic acid-free B-27, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), heparin, and retinoic acid. As used herein, the term "..." SOX2 "Refers to SRY-box transcription factor 2 [( Homo sapiens [(Person)], Gene ID: 6657. Encoding SOX2 The gene is an intronless gene and belongs to the SRY-associated HMG-box ( SOX This gene is a member of the family of transcription factors and is involved in regulating embryonic development and cell fate determination. Its product is crucial for the maintenance of stem cells in the central nervous system and regulates gene expression in the stomach. Mutations in this gene are closely associated with optic nerve dysplasia and microphthalmia syndrome (a severe structural eye malformation). Furthermore, this gene is located in another gene... SOX2 Within the intron region of the overlapping transcript (SOX2OT) (https: / / www.ncbi.nlm.nih.gov / gene / 6657).

[0063] The article uses SOX2 The specific sequence of the cmRNA is shown in SEQ ID NO: 1.

[0064]

[0065] As used in this article, the term " PAX6 "Refers to the paired box 6" Homo sapiens [(human)], NIH Gene ID: 5080. The gene encoding the pairing box protein Pax-6 is from the Drosophila melanogaster (…). Drosophila melanogaster The gene *prd* is one of many human homologs. In addition to a conserved pairing cassette domain (a hallmark of this gene family), the encoded protein also contains a homeobox domain. Both domains bind to DNA and regulate gene transcription. The activity of this protein plays a crucial role in the development of neural tissue, particularly the eye. This gene is regulated by multiple enhancers located hundreds of kilobases from the gene locus. Mutations in this gene or enhancer regions can lead to eye developmental abnormalities such as aniridia and Peter's anomaly. Using alternative promoters and alternative splicing, various transcriptomorphs can be generated, encoding different protein isoforms. Notably, it has been shown that the introduction of specific alternative coding exons lengthens the pairing cassette domain and alters its DNA-binding specificity. Therefore, isoforms carrying shorter pairing cassette domains regulate different gene profiles than isoforms carrying longer pairing cassette domains (https: / / www.ncbi.nlm.nih.gov / gene / 5080).

[0066] The article uses PAX6 The specific sequence of the cmRNA is shown in SEQ ID NO: 2.

[0067]

[0068] As used herein, the term "B27-RA" refers to a neuron cell culture medium B27 supplement without retinoic acid. As known in the art, the B27 supplement comprises biotin, DL-a-tocopheryl acetate, DL-a-tocopherol, vitamin A, biotin, fatty acid-free bovine serum albumin fraction V, catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine HCL, glutathione (reduced), L-carnitine HCL, linoleic acid, linolenic acid, progesterone, putrescine dihydrochloride, sodium selenite, and T3 (triiodothyronine).

[0069] As used herein, the term "adult" with respect to somatic cells, particularly fibroblasts, refers to cells obtained from an adult organism, preferably a human adult.

[0070] In one embodiment, the adult human fibroblast is a fibroblast obtained from a human at any non-embryonic or fetal stage.

[0071] As used herein, the term "mature" with respect to somatic cells, particularly fibroblasts, refers to a cell that has completed differentiation and has acquired a specific, rather than generalized, function. This is in contrast to an immature or stem cell, which still has pluripotency and can differentiate into any cell type in the body.

[0072] In one embodiment, the term "mature human somatic cell" refers to a human somatic cell that has reached a final state of differentiation. Such cells no longer have further differentiation potential. Such cells can be found at different stages of development, including embryonic, postnatal, or adult, but are typically obtained from an adult human.

[0073] As used herein, the term "therapeutically effective amount" is a suitable dose determined by a person of skill in the art based on known factors. Such dose can be administered as part of a dosing regimen formulated by an attending physician based on a variety of known clinical factors. Such factors would include, but are not limited to, the size, weight, age, sex, administration time and route of the subject, other drugs administered to the subject, and the overall health of the subject. A therapeutically effective amount would be an amount sufficient to produce a therapeutic effect on the disease or disorder being treated. In some embodiments, the disease or disorder being treated is Huntington's disease.

[0074] As used herein, the term "treatment" refers to obtaining an intended or desired result, generally an intended or desired pharmacological and / or physiological response or effect. In this context, the term "treatment" refers to a beneficial therapeutic result in the form of partial or complete cure of a disease and / or adverse effects and / or symptoms caused by the disease.

[0075] For example, a subject treated for Huntington’s disease can be treated at any stage of Huntington’s disease, including the acute stage of the disease. In the context of the present disclosure, “treatment” also includes taking steps to reduce the severity of the disease and / or delay onset, e.g., encompassing partial or complete treatment of the disease (or its symptoms). As used herein, the term “delaying onset” (and grammatical variations thereof) in the therapeutic context refers to shortening the time interval between the initial appearance of a preliminary indication that a subject has, or is suspected of having, Huntington’s disease, and the onset of the “acute” condition. As described herein, a subject is considered to be in the “acute” stage of Huntington’s disease if they exhibit some and / or all symptoms of the disease. Such subjects are in need of treatment after the onset of the disease, e.g., to reduce some and / or all symptoms.

[0076] The term “delaying onset” (including grammatical variations thereof) refers to delaying the appearance of at least one clinical symptom of Huntington’s disease in a subject. These will be determined by assessing the change in baseline of UHDRS-TMS. The UHDRS (Unified Huntington’s Disease Rating Scale) is a research tool known to those skilled in the art for providing a uniform assessment of the clinical features and course of Huntington’s disease. The components of the full UHDRS assess motor function, cognition, behavior, functional capacity, independence scale, and overall functional capacity. The motor function assessment includes the total motor score (TMS) and total functional capacity (TFC) score. The UHDRS TMS assesses all motor features of HD, including maximal chorea, maximal dystonia, eye tracking, saccadic initiation and velocity, dysarthria, tongue protrusion, finger tapping, hand pronation and supination, Luria, rigidity, bradykinesia, gait, tandem walking, and pull test. Each is scored on a scale of 0 (normal motor function) to 4 (severe motor dysfunction). The TMS score is the sum of the individual scores, ranging from 0 (normal motor function) to 124 (severe motor dysfunction). A lower TMS score indicates better motor function.

[0077] As used herein, the term “comprising” means “at least partially consisting of.” When interpreting each statement in this specification that includes the term “comprising,” features other than that or those specifically identified also can be present. Related terms such as “comprise” and “comprises” are interpreted in the same manner.

[0078] The term “about” as used herein means a reasonable amount of deviation from the stated amount, such that the end result is not significantly changed. For example, when applied to a value, the term is to be interpreted as including a ±5% deviation from that value.

[0079] References to numerical ranges of values (e.g., 1 to 10) disclosed herein are intended to include references to all rational numbers (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) within the range and any rational range of values within the range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus all subranges of all ranges expressly disclosed herein are expressly incorporated herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values between the lowest and highest values enumerated are to be considered to be expressly stated in this application in a similar manner.

[0080] Whenever a range is given in the specification, e.g., a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the present disclosure and claims, “and / or” means additionally or alternatively.

[0081] 5.1 Specific Description

[0082] Disclosed herein are the inventors’ research efforts that first demonstrated that human fibroblasts (HF), particularly human dermal fibroblasts (HDF), can be directly reprogrammed by SOX2 and PAX6 cmRNA into human induced lateral ganglionic eminence progenitor (hiLGEP) phenotype that, upon transplantation into the striatum of QA-injured rats, generates DARPP32 and GABA positive neurons and restores motor function. This work exemplifies adult human dermal fibroblasts, but is not limited thereto. Based on the present disclosure, the inventors believe that the cell reprogramming compositions and methods described herein can be successfully applied to the reprogramming of different types of human fibroblasts with reasonable expectation of success.

[0083] In one example, the compositions and methods described herein utilize cmRNA to generate hiLGEPs for transplantation. cmRNA provides an ideal non-viral, non-integrating delivery system for cell reprogramming in terms of safety and efficiency. The cmRNA system described herein allows for mRNA transfection without suppression of the immune response, reducing activation of the innate immune response and increasing mRNA stability by replacing uridine and cytidine residues with chemically modified uridine and cytidine analogs, respectively. Thus, the use of cmRNA to generate reprogrammed donor cells for cell replacement therapy is highly attractive as it provides a highly efficient and stable gene delivery system without the risk of genomic integration and insertional mutagenesis inherent to all DNA-based approaches and allows for no trace of transgene remnant upon cell reprogramming. These features make cmRNA an excellent choice for the clinical translation of reprogramming-based cell replacement therapies.

[0084] As disclosed herein, the inventors demonstrate that HF, in particular HDF, can be directly reprogrammed to a Lateral Ganglionic Eminence Progenitor (LGEP) fate. In this regard, the cell reprogramming compositions and methods described herein are not limited to lineage-specific neural progenitor cell reprogramming for transplantation. Rather, as described herein, cell reprogramming of various types of HF, in particular HDF, is performed in a variety of ways to ensure full differentiation to a striatal phenotype. In some embodiments, the HF, in particular HDF, is lineage-specific.

[0085] By transfecting HF, in particular HDF, with the combination of cmRNAs described herein SOX2 / PAX6 post-transfection, the inventors demonstrate that direct reprogramming can yield neural progenitor cells expressing striatal factors GSX2, DLX2, FOXP1, FOXP2, CTIP2 and MEIS2 Based on this expression profile, in particular CTIP2 the significant upregulation of expression of, the inventors determined that the use of Activin A can induce LGE fate (i.e., they can generate hiLGEPs), while the addition of Gö6983, Y27632, and N-2 can further promote the process. It is further described herein that the generation of hiLGEPs by direct reprogramming is further confirmed by the generation of DARPP32-positive neurons upon in vitro differentiation of hiLGEPs in a BrainPhys™-based striatal differentiation medium supplemented with Activin A and dorsomorphin.

[0086] Furthermore, described herein are the capabilities of hiLGEPs, which are described as surviving upon transplantation, differentiating into medium spiny striatal neurons (MSNs), and improving motor function in a QA-lesioned rat model of the brain degenerative disease, Huntington’s disease. Importantly, the inventors’ work disclosed herein demonstrates that transplantation of directly reprogrammed hiLGEPs into the QA-lesioned striatum restores motor dysfunction as measured by spontaneous forelimb use exploration, compared to saline-treated animals.

[0087] Accordingly, in one aspect, the present invention relates to a composition comprising: a basal brain medium, and at least two active agents selected from the group consisting of Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

[0088] In one embodiment, the composition comprises: a basal brain medium, and at least three active agents selected from the group consisting of Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

[0089] In one embodiment, the composition comprises: a basal brain medium and four active agents, the four active agents being Activin A (ActA), a Protein Kinase C (PKC) inhibitor, a pl60ROCK inhibitor, and an N-2 supplement.

[0090] In one embodiment, the composition is a medium.

[0091] In one embodiment, the PKC inhibitor is selected from the group consisting of Gö6983, Enzastaurin, Staurosporine, GF 109203X, Go6976, Ro 31-8220 mesylate, Ro 32-0432 hydrochloride, Sotrastaurin, and K252a. In one embodiment, the PKC inhibitor is Gö6983.

[0092] In one embodiment, the pl60ROCK inhibitor is selected from the group consisting of Y27632, Thiazovivin, HA 1100 hydrochloride, and GSK429286A. In one embodiment, the pl60ROCK inhibitor is Y27632.

[0093] In one embodiment, the “N-2 supplement” is Bottenstein’s N-2 formulation (1).

[0094] In one embodiment, the basal brain medium is Neurobasal-A (NBA) or BrainPhys, preferably BrainPhys.

[0095] In one embodiment, the basal brain medium further comprises at least one, at least two, three, four, at least five, six, or all seven of the additional agents selected from the group consisting of Valganciclovir, Penicillin-Streptomycin-Glutamine, B27-RA, FGF2, EGF, Retinoic Acid, and Heparin.

[0096] In one embodiment, the composition is used to reprogram fibroblasts, preferably human fibroblasts (HF), into hiLGEPs.

[0097] In one embodiment, the composition is used to reprogram fibroblasts, preferably human fibroblasts (HF), into hiLGEPs.

[0098] In one embodiment, the composition, when used, is used to reprogram fibroblasts, preferably human fibroblasts (HF), into hiLGEPs.

[0099] In one embodiment, the HF is a lineage-specific cell. In one embodiment, the HF is a non-lineage-specific cell. In one embodiment, the HF is a human dermal fibroblast (HDF). In one embodiment, the HF is an adult human fibroblast (aHF). In one embodiment, the HF is an adult human dermal fibroblast (aHDF).

[0100] In another aspect, the present application relates to a method of preparing a human induced lateral ganglionic eminence protuberance precursor cell (hiLGEP), comprising:

[0101] a) reprogramming a human fibroblast cell (HF) into a hiLGEP, comprising:

[0102] a. transfecting the HF with SOX2 cmRNA and PAX6 cmRNA;

[0103] b. culturing the transfected HF in a composition comprising basal brain medium, and at least two active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a p160 ROCK inhibitor, and N-2 supplement;

[0104] c. passaging the fibroblast cell in b. in a composition comprising basal brain medium, and at least three active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a p160 ROCK inhibitor, N-2 supplement, and Activin A (ActA); and

[0105] d. culturing the passaged HF.

[0106] In one embodiment, the transfection in a. comprises transfecting each of SOX2 cmRNA and PAX6 cmRNA with 0.5 to 5 pg, preferably 1.5 to 4 pg, preferably 2 to 3 pg, preferably 2.5 pg.

[0107] In one embodiment, the transfection in a. comprises at least one, preferably at least two, at least three, at least four, preferably five separate transfection events.

[0108] In one embodiment, the at least two separate transfection events are performed within a consecutive period of two to six days, preferably a consecutive period of three to five days, preferably a consecutive period of four days.

[0109] In one embodiment, the transfection event lasts for about 10 minutes, preferably for about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 400, 420, 480 minutes, or about 500 minutes, preferably for about 300 minutes.

[0110] In one embodiment, the transfection event lasts for about 100 to about 500 minutes, preferably about 150 to 450 minutes, 200 to 400 minutes, 250 to 350 minutes, preferably for about 300 minutes.

[0111] In one embodiment, SOX2 the cmRNA comprises SEQ ID NO: 1. In one embodiment, SOX2 the cmRNA consists essentially of or consists of SEQ ID NO: 1. In one embodiment, PAX6 the cmRNA comprises SEQ ID NO: 2. In one embodiment, PAX6 the cmRNA consists essentially of or consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0112] In one embodiment, the basal brain medium is Neurobasal A or BrainPhys, preferably BrainPhys. In one embodiment, the basal brain medium further comprises at least one, at least two, three, four, at least five, six, or all seven of the additional agents selected from the group consisting of valganciclovir, penicillin-streptomycin-glutamine, B27 without retinoic acid, FGF2, EGF, retinoic acid, and heparin.

[0113] In one embodiment, the culturing in b. lasts for about four to about ten days, preferably about five to about nine days, preferably about six to about eight days. In one embodiment, the culturing in b. lasts for about seven days.

[0114] In one embodiment, the PKC inhibitor in b. is selected from the group consisting of GÖ6983, enzastaurin, staurosporine, GF 109203X, Go6976, Ro 31-8220 methanesulfonate, Ro 32-0432 hydrochloride, sorbitol, and K252a. In one embodiment, the PKC inhibitor in b. is GÖ6983.

[0115] In one embodiment, the concentration of the PKC inhibitor in b. is about 0.5 nM to 50 µM, preferably about 1 nM to about 40 µM, about 10 nM to about 30 µM, about 100 nM to about 20 µM, about 500 nM to about 15 µM, about 1 µM to about 10 µM, about 3 µM to about 8 µM, about 5 µM to about 6 µM, preferably about 5 µM.

[0116] In one embodiment, the pl60ROCK inhibitor in b. is selected from the group consisting of Y27632, thiazovivin, HA 1100 hydrochloride, and GSK429286A. In one embodiment, the pl60ROCK inhibitor in b. is Y27632.

[0117] In one embodiment, the concentration of the pl60ROCK inhibitor in b. is about 0.1 nM to about 100 µM, preferably about 1 nM to about 75 µM, about 500 nM to about 50 µM, about 1 µM to about 25 µM, about 5 µM to about 15 µM, about 7 µM to about 13 µM, preferably about 10 µM.

[0118] In one embodiment, the passaging in c. is performed at about four to about ten days, preferably about five to nine days, preferably about six to eight days. In one embodiment, the passaging in c. is completed at about 7 days.

[0119] In one embodiment, the composition in c. comprises: a basal brain medium, and at least three active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pl60ROCK inhibitor, an N-2 supplement, and an Activin A (ActA).

[0120] In one embodiment, the composition in c. comprises: a basal brain medium, and all four active agents, the four active agents being a protein kinase C (PKC) inhibitor, a pl60ROCK inhibitor, an N-2 supplement, and an Activin A (ActA).

[0121] In one embodiment, the concentration of the N-2 supplement in c. is about 0.1% to about 10%, preferably about 0.3% to about 8%, about 0.5% to about 5%, about 0.7% to about 3%, about 0.9% to about 2%, preferably about 1%.

[0122] In one embodiment, the concentration of ActA in c. is about 25 pg / mL to about 25 pg / mL, preferably about 50 pg / mL to about 1 pg / mL, about 250 pg / mL to about 750 ng / mL, about 500 pg / mL to about 500 ng / mL, about 750 pg / mL to about 250 ng / mL, about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 75 ng / mL, about 10 ng / mL to about 65 ng / mL, about 15 ng / mL to about 50 ng / mL, about 20 ng / mL to about 30 ng / mL, about 22 ng / mL to about 28 ng / mL, preferably about 25 ng / mL.

[0123] In one embodiment, the PKC inhibitor in c. is selected from the group consisting of Gö 6983, enzastaurin, staurosporine, GF 109203X, Go 6976, Ro 31-8220 methanesulfonate, Ro 32-0432 hydrochloride, sorbitol and K252a. In one embodiment, the PKC inhibitor in b. is Gö 6983.

[0124] In one embodiment, the concentration of the PKC inhibitor in c. is about 0.5 nM to 50 pM, preferably about 1 nM to about 40 pM, about 10 nM to about 30 pM, about 100 nM to about 20 pM, about 500 nM to about 15 pM, about 1 pM to about 10 pM, about 3 pM to about 8 pM, about 5 pM to about 6 pM, preferably about 5 pM.

[0125] In one embodiment, the p160 ROCK inhibitor in c. is selected from the group consisting of Y27632, thiazovivin, HA 1100 hydrochloride and GSK429286A. In one embodiment, the p160 ROCK inhibitor in c. is Y27632.

[0126] In one embodiment, the concentration of the p160 ROCK inhibitor in c. is about 0.1 nM to about 100 pM, preferably about 1 nM to about 75 pM, about 500 nM to about 50 pM, about 1 pM to about 25 pM, about 5 pM to about 15 pM, about 7 pM to about 13 pM, preferably about 10 pM.

[0127] In one embodiment, the culturing in d. lasts for about four to about ten days, preferably about five to about nine days, preferably about six to about eight days. In one embodiment, the culturing in d. lasts for about seven days.

[0128] In one embodiment, the HF is a lineage-specific cell. In one embodiment, the HF is a non-lineage-specific cell.

[0129] In one embodiment, the HF is a human dermal fibroblast (HDF). In one embodiment, the HF is an adult human fibroblast (aHF). In one embodiment, the HF is an adult human dermal fibroblast (aHDF).

[0130] In one embodiment, the hiLGEP expresses at least one lateral ganglionic eminence protuberance (LGE) transcription factor. In one embodiment, the at least one LGE transcription factor is selected from the group consisting of: GSX2, FOXP1, FOXP2, MEIS and CTIP2.

[0131] In one embodiment, the hiLGEP expresses at least two, at least three, at least four, preferably five LGE transcription factors selected from the group consisting of GSX2, FOXP1, FOXP2, MEIS and CTIP2

[0132] In one embodiment, the method further comprises differentiating the hiLGEP in a striatal differentiation medium (STDM).

[0133] In one embodiment, the differentiation is for about four to about ten days, preferably about five to about nine days, preferably about six to about eight days. In one embodiment, the differentiation is for about seven days.

[0134] In one embodiment, the STDM comprises B27-RA, N-2 supplement, a cAMP activator, a pi 60 ROCK inhibitor, and brain-derived neurotrophic factor (BDNF), wherein the STDM is supplemented with dorsomorphin for about the first four to six days of the initial differentiation period and ActA for about the first six to eight days of the initial differentiation period.

[0135] In one embodiment, the cAMP activator is dcAMP or forskolin (FSK).

[0136] In one embodiment, the concentration of the cAMP activator is about 0.1 nM to about 10 mM, preferably about 1 nM to about 1 mM, about 10 nM to about 0.1 mM, about 100 nM to about 100 µM, about 1 µM to about 50 µM, about 5 µM to about 25 µM, about 7 µM to about 15 µM, preferably about 10 µM.

[0137] In one embodiment, the concentration of B27-RA is about 0.2% to about 20%, preferably about 0.5% to about 15%, about 0.7% to about 10%, about 0.9% to about 5%, about 1% to about 3%, preferably about 2%.

[0138] ​In one embodiment, the concentration of N2 supplement is about 0.1% to about 10%, preferably about 0.3% to about 8%, about 0.5% to about 5%, about 0.7% to about 3%, about 0.9% to about 2%, preferably about 1%.

[0139] In one embodiment, the concentration of BDNF is about 0.3 ng / mL to 3 ug / mL, preferably about 3 ng / mL to about 300 ng / mL, about 5 ng / mL to about 150 ng / mL, about 10 ng / mL to about 75 ng / mL, about 15 ng / mL to about 50 ng / mL, about 20 ng / mL to about 40 ng / mL, preferably about 30 ng / mL.

[0140] In one embodiment, the STDM supplements dorsomorphin about the first five days of initial differentiation. In one embodiment, the concentration of dorsomorphin is about 1 nM to about 1 mM, preferably about 100 nM to about 100 µM, about 500 nM to about 50 µM, about 750 nM to about 25 µM, about 800 nM to about 15 µM, about 900 nM to about 5 µM, about 950 nM to about 2.5 µM, preferably about 1 µM.

[0141] In one embodiment, the STDM supplements ActA about the first seven days of initial differentiation.

[0142] In one embodiment, the concentration of ActA is about 25 pg / mL to about 25 µg / mL, preferably about 50 pg / mL to about 1 µg / mL, about 250 pg / mL to about 750 ng / mL, about 500 pg / mL to about 500 ng / mL, about 750 pg / mL to about 250 ng / mL, about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 75 ng / mL, about 10 ng / mL to about 65 ng / mL, about 15 ng / mL to about 50 ng / mL, about 20 ng / mL to about 30 ng / mL, about 22 ng / mL to about 28 ng / mL, preferably about 25 ng / mL.

[0143] In one embodiment, the p160 ROCK inhibitor is selected from the group consisting of Y27632, thiazovivin, HA1100 hydrochloride, and GSK429286A. In one embodiment, the p160 ROCK inhibitor is Y27632.

[0144] In one embodiment, the concentration of p160 ROCK inhibitor is about 0.1 nM to about 100 µM, preferably about 1 nM to about 75 µM, about 500 nM to about 50 µM, about 1 µM to about 25 µM, about 5 µM to about 15 µM, about 7 µM to about 13 µM, preferably about 10 µM.

[0145] In one embodiment, the differentiating comprises differentiating the hiLGEPs into cells that express at least one biomarker associated with neuronal differentiation. In one embodiment, the differentiating comprises differentiating the hiLGEPs into medium spiny striatal neurons (MSNs).

[0146] In one embodiment, the hiLGEPs, after being differentiated in the STDM, express at least one of TUJ1, MAP2, DARPP32, GABA, and GAD 65 / 67 , preferably at least three of TUJ1, MAP2, DARPP32, GABA, and GAD 65 / 67 , preferably at least four, preferably all five.

[0147] In another aspect, the present application relates to a kit comprising:

[0148] i. a composition comprising: a basal brain medium, and at least two active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement; and

[0149] ii. a composition comprising: a basal brain medium, and at least three active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, an N-2 supplement, and Activin A (ActA).

[0150] In one embodiment, the composition in i. comprises a basal brain medium and all three active agents, which are a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

[0151] In one embodiment, the composition in ii. comprises a basal brain medium and all four active agents, which are a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, an N-2 supplement, and Activin A (ActA).

[0152] In one embodiment, the composition in i. or the composition in ii. or both are dry, substantially dry, lyophilized, liquid, or frozen compositions.

[0153] In one embodiment, i. and ii. are packaged separately in the kit and are to be used sequentially, with i. first and ii. second.

[0154] In one embodiment, the kit comprises iii., which is at least one cmRNA selected from SOX2 cmRNAs and PAX6 cmRNAs. In one embodiment, the kit comprises SOX2cmRNA and PAX6 cmRNA. In one embodiment, SOX2 cmRNA comprises SEQ ID NO: 1. In one embodiment, SOX2 cmRNA consists essentially of or consists of SEQ ID NO: 1. In one embodiment, PAX6 cmRNA comprises SEQ ID NO: 2. In one embodiment, PAX6 cmRNA consists essentially of or consists of SEQ ID NO: 2.

[0155] In one embodiment, iii. is provided in a composition comprising a carrier. In one embodiment, the composition is a dry, substantially dry, lyophilized, liquid or frozen composition.

[0156] In one embodiment, when the kit comprises iii., i., ii. and iii. are packaged separately in the kit and used sequentially in the order iii., then i., then ii.

[0157] In one embodiment, the kit is used to reprogram fibroblast cells (preferably human fibroblast cells (HF)) to hiLGEP.

[0158] In one embodiment, the kit is used to reprogram fibroblast cells (preferably human fibroblast cells (HF)) to hiLGEP.

[0159] In one embodiment, the kit, when used, is used to reprogram fibroblast cells (preferably human fibroblast cells (HF)) to hiLGEP.

[0160] In one embodiment, the HF is a lineage specific cell. In one embodiment, the HF is a non-lineage specific cell. In one embodiment, the HF is a human dermal fibroblast (HDF). In one embodiment, the HF is an adult human fibroblast (aHF). In one embodiment, the HF is an adult human dermal fibroblast (aHDF).

[0161] As a specific embodiment of this aspect of the application, specifically contemplated are all embodiments of the following as set forth in any other aspect as disclosed herein: basal brain medium, protein kinase (PKC) inhibitor, p160ROCK inhibitor, N-2 supplement, and ActA.

[0162] In another aspect, the present application relates to a human induced lateral ganglionic eminence progenitor cell (hiLGEP).

[0163] In one embodiment, the hiLGEP does not express ZNF503.

[0164] In one embodiment, the hiLGEP is prepared according to the methods described herein.

[0165] In another aspect, the present application relates to a composition comprising a human induced lateral ganglionic eminence progenitor cell (hiLGEP) and a carrier.

[0166] In one embodiment, the hiLGEP does not express ZNF503.

[0167] In one embodiment, the carrier is a buffer, a medium, or a pharmaceutically acceptable carrier. In one embodiment, the carrier is a pharmaceutically acceptable carrier.

[0168] In one embodiment, the composition comprises at least about 1,000,000 viable hiLGEPs, preferably at least about 2,000,000, at least about 3,000,000, at least about 4,000,000, preferably at least about 5,000,000 viable hiLGEPs.

[0169] In one embodiment, the hiLGEP is prepared according to the methods described herein.

[0170] In another aspect, the present application relates to a composition comprising basal brain medium, B27-RA, N-2 supplement, cyclic adenosine 3', 5'-monophosphate (cAMP) activator, p160 ROCK inhibitor, brain-derived neurotrophic factor (BDNF), and Activin A (ActA).

[0171] In one embodiment, the composition comprises dorsomorphin.

[0172] In another aspect, the present application relates to a composition comprising basal brain medium, B27-RA, N-2 supplement, cyclic adenosine 3', 5'-monophosphate (cAMP) activator, p160 ROCK inhibitor, brain-derived neurotrophic factor (BDNF), and dorsomorphin.

[0173] In one embodiment, the composition comprises Activin A.

[0174] In one embodiment, the composition comprises a hiLGEP.

[0175] In one embodiment, the hiLGEP is a reprogrammed human fibroblast (HF), human dermal fibroblast (HDF), adult human fibroblast (aHF), or adult human dermal fibroblast (aHDF).

[0176] In one embodiment, the hiLGEP is prepared according to the method described herein.

[0177] As a specific embodiment of this aspect of the application, it is specifically contemplated that all embodiments of the following are set forth as described in any other aspect disclosed herein: basal brain medium, B27-RA, N-2 supplement, cAMP activator, protein kinase (PKC) inhibitor, p160 ROCK inhibitor, BDNF, dorsomorphin, and ActA.

[0178] In another aspect, the application relates to the use of a composition for inducing the expression of at least one lateral ganglionic eminence (LGE) transcription factor in reprogrammed HF, the composition comprising basal brain medium, activin A (ActA), protein kinase C (PKC) inhibitor, p160 ROCK inhibitor, and N-2 supplement.

[0179] In one embodiment, the reprogrammed HF is a reprogrammed adult human fibroblast, human dermal fibroblast, or adult human dermal fibroblast.

[0180] In one embodiment, the LGE transcription factor induced is at least one of GSX2, FOXP1, FOXP2, MEIS, CTIP2 , preferably at least two of GSX2, FOXP1, FOXP2, MEIS , and CTIP2 , at least three, at least four, preferably all five.

[0181] As a specific embodiment of this aspect of the application, it is specifically contemplated that all embodiments of the following are set forth as described in any other aspect disclosed herein: basal brain medium, activin A (ActA), protein kinase C (PKC) inhibitor, p160 ROCK inhibitor, and N-2 supplement.

[0182] In another aspect, the application relates to the use of a composition for promoting the induction of lateral ganglionic eminence (LGE) precursor fate in fibroblasts, preferably human fibroblasts (HF), the composition comprising: basal brain medium, and at least three active agents selected from the group consisting of activin A (ActA), protein kinase C (PKC) inhibitor, p160 ROCK inhibitor, and N-2 supplement. In one embodiment, the composition comprises four active agents, namely activin A (ActA), protein kinase C (PKC) inhibitor, p160 ROCK inhibitor, and N-2 supplement.

[0183] In one embodiment, the HF is an adult human fibroblast, human dermal fibroblast, or adult human dermal fibroblast.

[0184] In one embodiment, the use is according to the method of reprogramming HF described herein.

[0185] In one embodiment, the application provides reprogrammed HFLGE precursors or hiLGEPs.

[0186] As a specific embodiment of this aspect of the application, specifically contemplated are all embodiments of the following: basal brain medium, ActA, protein kinase (PKC) inhibitor, p160ROCK inhibitor, and N-2 supplement, as set forth in any other aspect disclosed herein.

[0187] In another aspect, the application relates to the use of a composition for inducing expression of at least one biomarker associated with neuronal differentiation in reprogrammed HFLGE precursor cells, the composition comprising basal brain medium, B27-RA, N2 supplement, cAMP activator, p160ROCK inhibitor, brain-derived neurotrophic factor (BDNF), and Activin A.

[0188] In one embodiment, the reprogrammed HFLGE precursor cells are reprogrammed adult human fibroblasts, human dermal fibroblasts, or adult human dermal fibroblasts.

[0189] In one embodiment, the reprogrammed HF is a hiLGEP as described herein.

[0190] In one embodiment, the application is as a cell culture medium.

[0191] In one embodiment, the application lasts for about 13 to about 15 days, preferably about 14 days.

[0192] In one embodiment, the application comprises use of Activin A in the composition for about five to about nine days, preferably about six to about eight days, preferably about seven days.

[0193] In one embodiment, the composition comprises dorsomorphin.

[0194] In one embodiment, the application comprises use of dorsomorphin in the composition for about three to about seven days, preferably about four to about six days, preferably about five days.

[0195] In one embodiment, the biomarker is at least one of TUJ1, MAP2, DARPP32, GABA, and GAD 65 / 67 , preferably at least three, at least four, preferably all five of TUJ1, MAP2, DARPP32, GABA, and GAD 65 / 67 .

[0196] As a specific embodiment of this aspect of the application, specifically contemplated are all embodiments of the following as set forth in any other aspect disclosed herein: basal brain medium, B27-RA, N-2 supplement, cAMP activator, protein kinase (PKC) inhibitor, p160ROCK inhibitor, BDNF, dorsomorphin, and ActA.

[0197] In another aspect, the application relates to a use of a composition for inducing expression of at least one biomarker associated with neuronal differentiation in reprogrammed HF LGE precursor cells, the composition comprising basal brain medium, B27-RA, N2 supplement, cAMP activator, p160ROCK inhibitor, brain-derived neurotrophic factor (BDNF), and dorsomorphin.

[0198] In one embodiment, the reprogrammed HF LGE precursor cells are reprogrammed adult human fibroblasts, human dermal fibroblasts, or adult human dermal fibroblasts.

[0199] In one embodiment, the reprogrammed HF is a hiLGEP as described herein.

[0200] In one embodiment, the use is as a cell culture medium.

[0201] In one embodiment, the use is for about 13 to about 15 days, preferably about 14 days.

[0202] In one embodiment, the use comprises using dorsomorphin in the composition for about three to about seven days, preferably about four to about six days, preferably about five days.

[0203] In one embodiment, the composition comprises Activin A.

[0204] In one embodiment, the use comprises using Activin A in the composition for about five to about nine days, preferably about six to about eight days, preferably about seven days.

[0205] In one embodiment, the biomarker is at least one of TUJ1, MAP2, DARPP32, GABA, and GAD 65 / 67 , preferably at least three, at least four, preferably all five of TUJ1, MAP2, DARPP32, GABA, and GAD 65 / 67 .

[0206] As a specific embodiment of this aspect of the application, specifically contemplated are all embodiments of the following as set forth in any other aspect disclosed herein: basal brain medium, B27-RA, N-2 supplement, cAMP activator, protein kinase (PKC) inhibitor, p160ROCK inhibitor, BDNF, dorsomorphin, and ActA.

[0207] In another aspect, the application relates to the use of a composition comprising at least one induced human lateral ganglionic eminence progenitor cell (hiLGEP) and a carrier for the preparation of medium spiny neurons (MSNs).

[0208] In one embodiment, the carrier is a buffer, a medium, or a pharmaceutically acceptable carrier. In one embodiment, the carrier is a pharmaceutically acceptable carrier.

[0209] In one embodiment, the hiLGEP is a reprogrammed adult human fibroblast, human dermal fibroblast, or adult human dermal fibroblast. In one embodiment, the hiLGEP is prepared according to the methods described herein.

[0210] In one embodiment, the induced LGE transcription factors are at least one of GSX2, FOXP1, FOXP2, MEIS, CTIP2 , preferably at least two, at least three, at least four, preferably all five of GSX2, FOXP1, FOXP2, MEIS , and CTIP2 .

[0211] In one embodiment, the MSNs are prepared by in vitro or in vivo means.

[0212] In one embodiment, the MSNs are prepared in vitro by culturing the hiLGEPs in the STDM described herein supplemented with dorsomorphin and ActA.

[0213] In one embodiment, the MSNs are prepared in vivo by transplanting the hiLGEPs into a subject, preferably a human subject.

[0214] In one embodiment, the transplanting comprises inserting or implanting the hiLGEPs into the subject. In one embodiment, the inserting is by injection.

[0215] In another aspect, the application relates to the use of a composition comprising a human induced lateral ganglionic eminence progenitor cell (hiLGEP) and a carrier for the treatment of Huntington’s disease.

[0216] In another aspect, the application relates to the use of a composition comprising a human induced lateral ganglionic eminence progenitor cell (hiLGEP) and a carrier for the reduction of the severity of Huntington’s disease.

[0217] In another aspect, the application relates to the use of a composition comprising human induced Lateral Ganglionic Eminence Progenitor cells (hiLGEPs) and a carrier for delaying the onset of Huntington's disease.

[0218] In another aspect, the application relates to the use of human induced Lateral Ganglionic Eminence Progenitor cells (hiLGEPs) for the manufacture of a medicament for the treatment of Huntington's disease.

[0219] In another aspect, the application relates to the use of human induced Lateral Ganglionic Eminence Progenitor cells (hiLGEPs) for the manufacture of a medicament for reducing the severity of Huntington's disease.

[0220] In another aspect, the application relates to the use of human induced Lateral Ganglionic Eminence Progenitor cells (hiLGEPs) for the manufacture of a medicament for delaying the onset of Huntington's disease.

[0221] In another aspect, the application relates to the use of human induced Lateral Ganglionic Eminence Progenitor cells (hiLGEPs) for the treatment of Huntington's disease.

[0222] In another aspect, the application relates to the use of human induced Lateral Ganglionic Eminence Progenitor cells (hiLGEPs) for reducing the severity of Huntington's disease.

[0223] In another aspect, the application relates to the use of human induced Lateral Ganglionic Eminence Progenitor cells (hiLGEPs) for delaying the onset of Huntington's disease.

[0224] The following embodiments specifically encompass any and / or all embodiments described herein relating to the use of a composition comprising human induced Lateral Ganglionic Eminence Progenitor cells (hiLGEPs), the use of hiLGEPs for the manufacture of a medicament and / or the use of hiLGEPs as described herein.

[0225] In one embodiment, the carrier is a pharmaceutically acceptable carrier. In one embodiment, the composition is a pharmaceutical composition.

[0226] In one embodiment, the hiLGEPs express at least one LGE transcription factor selected from the group consisting of: GSX2, DLX2, FOXP1, FOXP2, CTIP2 and MEIS2 In one embodiment, the hiLGEPs express GSX2, DLX2, FOXP1, FOXP2, CTIP2 and MEIS2 at least two, preferably at least three, at least four, preferably all five of the group consisting of

[0227] In one embodiment, the hiLGEPs express at least one neurotrophic factor, preferably NESTIN.

[0228] In one embodiment, the hiLGEP is a lineage-specific cell. In one embodiment, the hiLGEP is a non-lineage-specific cell.

[0229] In one embodiment, the hiLGEP is a reprogrammed adult human fibroblast, human dermal fibroblast, or adult human dermal fibroblast.

[0230] In one embodiment, the application comprises implanting the hiLGEP or pharmaceutical composition into the striatum of a human subject. In one embodiment, the implanting is by injection.

[0231] In one embodiment, the injection is into the striatum of a human subject suspected of having Huntington's Disease or having at least one symptom of Huntington's Disease.

[0232] In one embodiment, the injection comprises injecting a therapeutically effective amount of the hiLGEP or pharmaceutical composition.

[0233] In one embodiment, the therapeutically effective amount of the composition comprises at least about 1,000,000 live hiLGEPs, preferably at least 2,000,000, at least 3,000,000, at least 4,000,000, preferably at least about 5,000,000 live hiLGEPs per injection.

[0234] In one embodiment, the therapeutically effective amount of the hiLGEP is at least about 1,000,000 live hiLGEPs, preferably at least 2,000,000, at least 3,000,000, at least 4,000,000, preferably at least about 5,000,000 live hiLGEPs per injection.

[0235] In another aspect, the present application relates to a method of treating Huntington's Disease, the method comprising implanting human induced lateral ganglionic eminence progenitor cells (hiLGEPs) into the striatum of a subject having or suspected of having Huntington's Disease.

[0236] In another aspect, the present application relates to a method of ameliorating Huntington's Disease, the method comprising implanting human induced lateral ganglionic eminence progenitor cells (hiLGEPs) into the striatum of a subject suspected of having Huntington's Disease or having at least one symptom of Huntington's Disease.

[0237] In another aspect, the present application relates to a method of delaying the onset of Huntington's Disease, the method comprising implanting human induced lateral ganglionic eminence progenitor cells (hiLGEPs) into the striatum of a subject suspected of having Huntington's Disease or having at least one symptom of Huntington's Disease.

[0238] In another aspect, the present application relates to a method of reducing the severity of Huntington's disease, the method comprising transplanting human induced lateral ganglionic eminence progenitor cells (hiLGEPs) into the striatum of a subject suspected of having Huntington's disease or having at least one symptom of Huntington's disease.

[0239] The following embodiments specifically encompass any and / or all embodiments of the method aspect of the above application.

[0240] In one embodiment, transplanting comprises transplanting a therapeutically effective amount of hiLGEPs.

[0241] In one embodiment, a therapeutically effective amount of hiLGEPs comprises at least about 1,000,000 live hiLGEPs, preferably at least 2,000,000, at least 3,000,000, at least 4,000,000, preferably at least about 5,000,000 live hiLGEPs per injection.

[0242] In one embodiment, transplanting comprises inserting hiLGEPs into the striatum of the subject. In one embodiment, the inserting is by injection.

[0243] In one embodiment, the hiLGEPs express at least one LGE transcription factor selected from the group consisting of: GSX2, DLX2, FOXP1, FOXP2, CTIP2 and MEIS2 In one embodiment, the hiLGEPs express GSX2, DLX2, FOXP1, FOXP2, CTIP2 and MEIS2 at least two, preferably at least three, at least four, preferably all five of the group consisting of:

[0244] In one embodiment, the hiLGEPs express at least one neurotrophic factor, preferably NESTIN.

[0245] In one embodiment, the hiLGEPs are lineage specific cells. In one embodiment, the hiLGEPs are non-lineage specific cells.

[0246] In one embodiment, the hiLGEPs are obtained by reprogramming adult human fibroblasts, human dermal fibroblasts, or adult human dermal fibroblasts.

[0247] In one embodiment, the hiLGEPs are comprised in a pharmaceutical composition comprising a physiologically acceptable carrier.

[0248] In one embodiment, transplanting comprises inserting a pharmaceutical composition into the striatum of a human subject. In one embodiment, the inserting is by injection.

[0249] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of hiLGEPs. In one embodiment, the therapeutically effective amount of hiLGEPs is at least about 1,000,000 live hiLGEPs per injection, preferably at least 2,000,000, at least 3,000,000, at least 4,000,000, preferably at least 5,000,000 live hiLGEPs.

[0250] In one embodiment, ameliorating Huntington's disease comprises ameliorating at least one symptom of Huntington's disease. In one embodiment, delaying onset of Huntington's disease comprises lengthening the time during which a subject has not been observed to develop at least one symptom of Huntington's disease. In one embodiment, reducing severity comprises reducing the severity of at least one symptom of Huntington's disease.

[0251] In one embodiment, the at least one symptom is selected from the group consisting of motor dysfunction, including involuntary jerking or writhing movements (chorea); muscle problems, including rigidity or muscle contraction (dystonia); slow or irregular eye movements; gait, posture, and balance disorders; and difficulty with speech or swallowing.

[0252] The compositions and methods disclosed herein enable cellular reprogramming of human fibroblasts (HFs) to neural precursor cells. In a particular embodiment, compositions and methods are disclosed for reprogramming HFs to human induced lateral ganglionic eminence (LGE) precursor cells. As described herein, cellular reprogramming is performed using SOX2 and PAX6 cmRNA, such that the cellular progeny of the HFs that form have at least one new cellular phenotype compared to the un-reprogrammed sibling HF cells. This new phenotype can be observed in the reprogrammed cells in culture or in vivo. The cellular reprogramming described herein confers pluripotent potential to the HFs that are transfected with the cmRNA. In this context, "pluripotent potential" refers to a measurable proportion of the reprogrammed cellular progeny having the potential to differentiate into cells displaying the phenotypic characteristics of the new cell type, as compared to cells that are not reprogrammed and do not have this potential. In some embodiments, the proportion of progeny displaying the phenotypic characteristics of the new cell type will be significantly higher than before reprogramming. In some embodiments, the proportion of progeny displaying the phenotypic characteristics of the new cell type will be at least 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 25%, 30%, 40%, 50%, or higher than observed in an appropriate control as would be understood by one of skill in the art.

[0253] In some embodiments, the reprogrammed cells are cells displaying phenotypic characteristics of neural system cells and / or lineage-specific neural cells. The term "lineage-specific" is a cell lineage from a tissue type that is a related product of cell division.

[0254] As described herein, neural precursor cells, particularly human induced lateral ganglionic eminence precursor cells, are cells that are capable of differentiating into cells and / or tissue types of the lateral ganglionic eminence neural cell lineage. In this regard, lineage specific neural precursor cells described herein, particularly lineage specific induced human lateral ganglionic eminence precursor cells, are artificially created pluripotent precursor cells derived from a non-pluripotent and non-multipotent source. Human dermal fibroblasts (HDFs) reprogrammed to express lineage specific neural cell characteristic genes are used herein as an example of this source.

[0255] In some embodiments, reprogramming of HFs with cmRNA as described herein comprises transfecting cmRNA encoding SOX2 and PAX6 into HDFs. Transfecting comprises introducing or delivering cmRNA encoding SOX2 or PAX6 into HDFs using standard transfection techniques known and used by those skilled in the art. Transfection protocols are found in the Examples of the present specification and can be implemented as described. Standard transfection techniques are also known to those skilled in the art, such as the methods described in WO2011 / 012316, which discloses methods for transfecting mRNA into lung cells with Lipofectamine 2000 (Invitrogen). Additional transfection protocols are described by Kim and Eberwine (Anal Bioanal Chem. 397(8):3173-8 (2010)), which reviews biological, chemical, and physical transfection methods used in the art for delivering nucleic acids to cells.

[0256] As described herein, transfecting HDFs comprises transfecting with cmRNA. In some embodiments, transfecting comprises transfecting with separate cmRNAs encoding SOX2 and PAX6 respectively. In some embodiments, about 5% to 50% of the cytosine nucleotides and 5% to 50% of the uridine nucleotides in the cmRNA are modified. In some embodiments, about 10% to 35% of the cytosine and uridine nucleotides are modified. In some embodiments, the cmRNA can comprise about 7.5% to 25% modified cytosine nucleotides and about 7.5% to 25% modified uridine nucleotides. In a preferred embodiment, about 25% of the cytosine nucleotides and about 25% of the uridine nucleotides are modified. In some embodiments, the modified uridine nucleotides are 2-thiouridine. In some embodiments, the modified cytosine nucleotides are 5-methylcytosine residues. In some embodiments, the adenosine and guanosine containing nucleotides are unmodified or partially modified.

[0257] cmRNAs encoding SOX2 and PAX6 as described herein can be prepared by In vivo or In vitroThe cmRNAs can be produced recombinantly in a system, or by synthetic means (e.g., routine chemical synthesis using solid phase support and standard techniques on an automated nucleotide sequence synthesizer). Those skilled in the art can use known methods to produce the cmRNAs in a system In vivo or In vitro The cmRNAs can be produced recombinantly in a system, or by synthetic means. Regardless of the method of production, the cmRNAs can be purified and recovered using methods known to the skilled artisan.

[0258] In some embodiments, the HF is an adult human fibroblast (aHF), a human dermal fibroblast (HDF), or an adult human dermal fibroblast (aHDF). Preferably, the HF is an adult HDF. Fibroblasts are commonly found in connective tissue and are associated with collagen fiber formation and production of the extracellular matrix. The cell reprogramming described herein contemplates mammalian fibroblasts from any source tissue, including but not limited to fibroblasts from kidney, cardiac tissue, lung tissue, interstitial, and dermal tissue. In some embodiments, the fibroblasts reprogrammed using the compositions and methods described herein are adult mammalian dermal fibroblasts, preferably adult human dermal fibroblasts (aHDF). aHDFs can be obtained from commercial sources, or isolated from various tissues using methods known in the art using known laboratory equipment and techniques.

[0259] Following transfection with the cmRNAs, the fibroblasts are cultured to allow for expression of the transfected RNA and subsequent reprogramming. As described herein, the HDFs are cultured under permissive conditions in a brain-based reprogramming medium that is capable of supporting neural precursor cell growth. A preferred brain-based reprogramming medium is Neurobasal-A and BrainPhys medium, but is not necessarily limited thereto.

[0260] In some embodiments, the medium is supplemented with a variety of components that promote the remodeling process. As described herein, certain components are critical, and particularly preferred, for reprogramming the HF (including HDF) into a neural precursor cell, particularly a hiLGEP. These components are referred to herein as “active agents.”

[0261] Additional components of the medium can include chromatin-modifying agents that promote reprogramming. The chromatin-modifying agent can be an agent that inhibits chromatin deacetylation, alters histone methylation status within chromatin, induces DNA demethylation within chromatin, or promotes chromatin acetylation. In one embodiment, valproic acid at an appropriate concentration (typically 1 mM) can be used as a chromatin-modifying agent.

[0262] The brain-based reprogramming media can also contain various combinations of some or all of the following components: amino acids (including non-essential amino acids), fatty acids, lipids, growth factors, vitamins, antioxidants, cytokines, inorganic salts, pyruvate (pyruvate salts), and reducing agents (such as 2-mercaptoethanol). In some embodiments, the listed components are selected and added to the media according to concentrations known in the art and used for neural cell culture.

[0263] In some embodiments, the permissive conditions for neural cell culture are those described in the examples of the disclosure, but are not limited thereto. Cell culture can also be performed under permissive conditions for neural cell culture known in the art and used. Culture can be grown in vessels including culture bags, test tubes, flasks, bottles, culture dishes (including petri dishes and culture trays), well plates (micro- and multi-well), trays, and slide chambers, but are not limited thereto. The skilled artisan can select an appropriate vessel for cell culture.

[0264] HFs (especially HDFs) being reprogrammed as disclosed herein can be cultured in any appropriate volume of media. For example, a volume of about 0.2 ml to about 2000 ml can be selected according to equipment and protocols available to the skilled artisan, and depending on the desired permissive conditions. In some embodiments, bioreactors known in the art and used can be employed.

[0265] Culture vessels can be selected according to the purpose, and can be adherent or non-adherent, as known in the art. Adherent culture vessels can include a coating that promotes and / or improves cell adhesion to the vessel. Vessels with interior walls coated to promote and / or improve cell adhesion can be coated with various components known in the art, including but not limited to fibronectin, gelatin, laminin, collagen, vitronectin, poly-L-lysine or poly-D-lysine, or mixtures thereof.

[0266] The skilled artisan can define further culture conditions based on the disclosure in combination with knowledge known in the art and used. Such conditions include selection of temperature, oxygen tension, and CO2concentration. By way of non-limiting example, culture temperature can range from about 30 to 40 °C, including all temperature points therebetween, but is not necessarily limited thereto; oxygen tension can range from about 1 to 20%, including all percentages therebetween, but is not necessarily limited thereto; CO2concentration can range from about 1 to 10%, again including any percentage therebetween, but is not necessarily limited thereto.

[0267] Components of the media / compositions

[0268] In addition to the general considerations provided regarding cell culture, the inventors have determined that certain culture conditions are required for effective reprogramming of HFs to hiLGEPs. These required conditions are listed as embodiments herein.

[0269] In one example, reprogramming of HF, in particular HDF, in particular aHDF, comprises: transfecting cells with cmRNA encoding transcription factors SOX2 and PAX6 Subsequently, the transfected cells are cultured in a brain-based reprogramming medium containing 1 mM valproic acid, 1% penicillin-streptomycin-glutamine, 2% B-27 without retinoic acid, 20 ng / ml EGF, 20 ng / ml FGF2, and 2 pg / ml heparin, with the broad-spectrum protein kinase C inhibitor Gö6983 (5 mM), the pl60ROCK inhibitor Y27632 (10 mM), 1% N-2 supplement (combined abbreviation GYN), 10 mM retinoic acid. The preferred brain-based reprogramming medium is BrainPhys. Cells are cultured for about 6 to 8 days, preferably about 7 days, then the culture is passaged and activin A (25 ng / ml) is added to the medium. The passaged cells are cultured for about 6 to 8 days, preferably about 7 days, for a total culture period of about 12 to about 16 days. The preferred total culture days are about 14 days.

[0270] The reprogrammed cells are tested by molecular and cellular techniques known in the art and described in the appendix examples to confirm that they have acquired a lateral ganglionic eminence progenitor fate.

[0271] A portion of the reprogrammed cells with LGEP fate are further differentiated in vitro to functional DARPP32-positive neurons as described in the appendix examples. To

[0272] The pharmaceutical compositions described herein are compositions suitable for administration to a subject, preferably a human subject.

[0273] The pharmaceutical compositions comprising hiLGEPs as described herein can be formulated into methods suitable for cell transplantation therapy. In some embodiments, such pharmaceutical compositions are used to treat Huntington’s disease, or at least to reduce the severity and / or delay the onset thereof.

[0274] In addition to comprising hiLGEPs as described herein, the pharmaceutical compositions described herein further comprise a pharmaceutically acceptable carrier, diluent, and / or excipient. As used herein, “pharmaceutically acceptable carrier” refers to a physiologically acceptable carrier.

[0275] ​Pharmaceutically acceptable carriers, diluents, and / or excipients include non-active substances as contained in the hiLGEP formulations as described herein. Such substances serve multiple purposes in the formulation. In one example, such substances are used to increase the volume of the formulation in order to increase the convenience and / or accuracy in producing the dosage form. Such substances can be referred to as diluents, fillers, or extenders. Pharmaceutically acceptable carriers, diluents, and / or excipients can also provide therapeutic enhancement properties to the formulation, including but not limited to facilitating dissolution or absorption of the active ingredients. Various excipients are also used to reduce the difficulty of handling the active ingredients during the production process, for example by providing flow or anti-sticking properties. In addition, different carriers, diluents, and / or excipients can provide stabilization properties, including protection from oxidation, crystallization, or denaturation, to extend the shelf life of the formulation. The selection of the appropriate pharmaceutically acceptable carrier, diluent, and / or excipient for any given formulation is considered to be within the ability of those in the art. Multiple factors are taken into account when designing such formulations, including the dosage form, the nature of the active ingredients, the route of administration, and other factors.

[0276] Several well-known pharmaceutically acceptable carriers, excipients, and / or diluents suitable for use in various formulations include water, physiological saline, phosphate buffered saline (PBS), wetting agents, and emulsifiers. Pharmaceutical compositions comprising various pharmaceutically acceptable carriers can be formulated by well-known conventional methods.

[0277] Pharmaceutical compositions comprising the hiLGEP as described herein are formulated with the intent to include an effective amount of the hiLGEP, along with the appropriate pharmaceutically acceptable carriers, diluents, and / or excipients. Such formulations can be readily determined by one of skill in the art based on the disclosure provided herein and methods known in the art. It will be appreciated that a "therapeutically effective amount" means an amount sufficient to elicit a detectable therapeutic response in a subject to whom the pharmaceutical composition is administered.

[0278] In some embodiments, the therapeutically effective amount is at least about 1,000,000 hiLGEP per injection dose, preferably 2,000,000, preferably 3,000,000, preferably 4,000,000, preferably at least about 5,000,000 hiLGEP per dose. In one embodiment, the dose is an injection dose.

[0279] Administration of a therapeutically effective amount of the hiLGEP as described herein, including administration of the hiLGEP itself or a pharmaceutical composition comprising the hiLGEP as described herein, can be performed by various cell transplantation techniques known to those of skill in the art as appropriate for cell therapy. It is believed that methods of cell transplantation for transplantation therapy are well known to those of skill in the art. By way of non-limiting example, the cells to be transplanted can be administered by intracerebral injection, stereotactic injection, local injection, or direct injection into the spinal canal.

[0280] The pharmaceutical compositions described herein can also include a suitable amount of carrier comprising a pharmaceutically acceptable salt or other pharmaceutically acceptable material. The presence of the pharmaceutically acceptable salt and / or other material is to render the pharmaceutical composition isotonic. As non-limiting examples, the carrier can include saline, Ringer's solution, and dextrose solution. The pharmaceutically acceptable carrier, diluent and / or carrier (including stabilizers) are nontoxic to the subject at the dosages and concentrations employed. Suitable carriers and their formulations are described in more detail in Remington's Pharmaceutical Sciences 17th Ed. (1985) Mack Publishing Co. Advances.

[0281] The pharmaceutically acceptable carrier, diluent and / or excipient can include, but are not limited to, buffers such as citrated, phosphated and other organic acid buffers; proteins, including serum albumin, gelatin and / or low molecular weight (>10 amino acid residues) polypeptides; chelating agents, including EDTA; nonionic surfactants such as Tween, pluronics or polyethylene glycol; salt-forming counterions, including sodium and potassium; hydrophilic polymers such as polyvinylpyrrolidone (PVP); amino acids, including histidine, glutamine, lysine, asparagine, arginine or glycine; antioxidants including methionine, ascorbic acid and tocopherol; carbohydrates such as glucose, mannose, dextrose or dextrin; various monosaccharides and disaccharides; various sugars including sucrose, mannitol, trehalose or sorbitol; and / or a variety of different preservatives, e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol.

[0282] 6. Examples

[0283] Materials and Methods

[0284] Direct reprogramming of adult human fibroblasts into induced lateral ganglionic eminence progenitor cells and differentiation.

[0285] Human induced lateral ganglionic eminence progenitor (hiLGEP) cells were generated from an adult human dermal fibroblast (aHDF) cell line (1507: male Caucasian, 50 years old, facial tissue; 1838: male Caucasian, 50 years old, facial tissue; 2116: female Caucasian, 35 years old, abdominal tissue; 2298: female Caucasian, 33 years old, abdominal tissue; Cell Applications Inc). aHDF cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific).

[0286] By using chemically modified mRNA (cmRNA; Ethris, Munich, Germany) (Connor et al., 2018), aHDF were induced into lateral ganglionic eminence progenitor (LGEP) fate using transient overexpression of the neurogenic genes SOX2 and PAX6 .

[0287] Several transfection protocols were used in this work, both of which achieved successful transfection and the same morphological changes were observed in the reprogrammed cells during the reprogramming process (Figure 7).

[0288] In the first protocol, aHDF were transfected with 2.5 pg of each SOX2 and PAX6 cmRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific) transfection reagent. Transfection was performed for 5 hours for four consecutive days.

[0289] In the second protocol, the neurogenic genes SOX2 and PAX6 were combined using lipid nanoparticle technology (LNP; Ethris, Munich, Germany) for transient overexpression to reprogram aHDF to induce hiLGEP. A one-time 24-hour transfection was performed using 2.5 pg of SOX2-PAX6 -LNP. Briefly, the SOX2-PAX6 -LNP was added to 500 pL Opti-MEM™ Reduced Serum Medium (Gibco) and gently mixed, after which the transfection mixture was added dropwise to the wells containing the reprogramming medium.

[0290] Cells transfected with both protocols were cultured and reprogrammed under normoxic conditions in Neurobasal-A (NBA; Thermo Fisher Scientific) or BrainPhys™ (Stem Cell Technologies)-based reprogramming medium containing 1 mM valproic acid (Sigma Aldrich), 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific), 2% B-27 without retinoic acid (Thermo Fisher Scientific), 20 ng / ml epidermal growth factor (EGF) (Prospec Bio), 20 ng / ml fibroblast growth factor 2 (FGF2) (Prospec Bio), 2 pg / ml heparin (Sigma Aldrich), 1% N-2 supplement (Thermo Fisher Scientific), 5 mM Gö6983 (Abcam), 10 mM Y27632 (Abcam), and 10 mM retinoic acid (Sigma Aldrich). Cells were passaged at day 7 of reprogramming and from day 7 to day 14 of reprogramming medium was supplemented with Activin A (25 ng / mL; Prospec Bio) Figure 1 ).

[0291] After 14 days of reprogramming, hiLGEPs were collected for transplantation into the striatum of rats with quinolinic acid (QA) lesions, a well-established model of Huntington’s disease.

[0292] At day 14 of reprogramming, hiLGEPs were collected for transplantation or processed for RT-qPCR and immunocytochemistry to confirm their acquisition of LGEP cell fate. A separate set of cells was plated at a density of 60,000 cells / well on GelTrex-coated glass coverslips for differentiation in NBA- or BrainPhys™-based striatal differentiation medium containing 1% penicillin-streptomycin-glutamine, 2% B-27 supplement without retinoic acid, 1% N-2 supplement, 10 mM Y-27632, 10 mM forskolin (Sigma Aldrich), 30 ng / mL BDNF (PeproTech), with 1 mM dorsomorphin (Sigma Aldrich) added for the first 5 days (with or without 5 mM Gö6983) and 25 ng / mL Activin A for the first 7 days. After 14 days of differentiation, cells were fixed with 4% paraformaldehyde at 4°C and processed for immunocytochemistry.

[0293] Quantitative RT-PCR

[0294] Total RNA was extracted from reprogrammed 14-day hiLGEPs and original aHDF cell lines using the Nucleospin RNA kit (Macherey Nagel). cDNA was synthesized from total RNA using Superscript IV Reverse Transcriptase (Thermo Fisher Scientific). Duplex qPCR reactions were performed using the TaqMan system (Applied Biosystems) with 4-10 ng of RNA per reaction, in triplicate, normalizing gene expression to the internal control ribosomal 18S rRNA. Gene expression was normalized to the internal control ribosomal 18S rRNA and expressed as fold change relative to aHDF using the ΔΔCt method.

[0295] Immunocytochemistry

[0296] Cells were first permeabilized in phosphate-buffered saline containing 0.5% Triton X-100 for 5 min. The following human-specific primary antibodies were used: GSX2 (1 :500, Abeam), FOXP1 (1 :100, R&D), FOXP2 (1 :500, Abeam), MEIS2 (1 :500, Abeam), TUJ1 (1 :500, Abeam), DARPP32 (1 :500, Invitrogen), GABA (1 :500, Invitrogen), and GAD 65 / 67 (1 :500, AbCam). Primary antibodies were visualized using species-matched Alexa Fluor™-labeled secondary antibodies (1 :500; Invitrogen). Single nuclei were confirmed using DAPI included in Prolong Diamond antifade mountant (Thermo Fisher Scientific). Images were acquired using an inverted Nikon TE2000E fluorescence microscope equipped with a DS-Ri2 camera. The number of TUJ1+ or DARPP32+ hiLGEP-derived neurons was manually quantified in ImageJ software as the proportion of DAPI+ cells out of at least 500 DAPI+ cells.

[0297] Live cell calcium imaging

[0298] hiLGEPs were plated at a density of 80,000 cells / well on GelTrex-coated Greiner black-walled glass-bottomed plates for differentiation, cultured in the above BrainPhys™-based striatal differentiation medium for 14 days. Cells were loaded with 5 mM Cal-520 AM (Abeam) with 0.04% Pluronic F-127 (Thermo Fisher Scientific) for 1 hour at 37°C, followed by 30 minutes at room temperature. The Cal-520 AM dye working solution was replaced with phenol red-free Hank’s Balanced Salt Solution buffer (Thermo Fisher Scientific) containing 1 mM probenecid and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Thermo Fisher Scientific). Cells were imaged at Ex / Em = 420 / 525 nm, recording Cal-520 AM fluorescence intensity for a total of 180 seconds, with or without 12.5, 25, and 50 mM glutamate treatment. Time-lapse recordings were captured by a Nikon TE2000E inverted microscope equipped with a color DS-Ri2 camera using NIS Elements BR software. Live cell calcium imaging analysis was performed using the time series analysis plugin in FIJI software. Mean fluorescence intensity values were recorded for 100 regions of interest from 2 replicate experiments. Fluorescence was measured as percent increase in mean fluorescence intensity relative to baseline fluorescence at time 0 (DF / F0).

[0299] Animals

[0300] Adult male Sprague-Dawley rats (Vernon Jansen Unit, University of Auckland) weighing 250-350 g (8 weeks old) were used at the time of quinolinic acid (QA) lesion. All procedures were strictly in accordance with the University of Auckland Animal Ethics Guidelines, in compliance with the New Zealand Animal Welfare Act 1999 and international ethical guidelines. Every effort was made to reduce the number of animals used and their suffering. Rats were randomly assigned to the following treatment groups: hiLGEPs transplantation group, n = 15; sham transplantation group (0.9% sterile saline), n = 15. Rats were group-housed in a temperature and humidity-controlled room with a 12-hour light-dark cycle. Food and water were available ad libitum throughout the study period. Forty-eight hours prior to transplantation surgery, all rats received an intraperitoneal injection of Sandimmun (20 mg / kg cyclosporine; provided by the Vernon Jansen Unit, University of Auckland). After transplantation surgery, Sandimmun injections were given three times per week for the duration of the experiment. Free

[0301] Surgical procedure ​

[0302] All surgeries were performed under isoflurane anaesthesia (induction: 5% isoflurane, O2 flow rate 2 L / min; maintenance: 1.5% isoflurane, O2 flow rate 1.5 L / min). All rats received a unilateral intrastriatal infusion of QA (50 nmol, 400 nl; flow rate 100 nl / min, using a 32G Hamilton syringe controlled by a WPI UltraMicroPump II) with the following stereotaxic coordinates: +0.5 mm anteroposterior (AP), -2.7 mm mediolateral (ML), -5.0 mm dorsoventral (DV) from the dura surface (Vazey and Connor, 2010; Vazey et al., 2006; Vazey et al., 2010). Twenty-one days after the QA lesion, hiLGEPs (approximately 250,000 viable cells per animal) or 0.9% sterile saline were injected into two adjacent sites in the lesioned striatum (approximately 62,500 viable cells / μΙ: 2 μΙ per injection site; flow rate 400 nl / min, using a 26G Hamilton syringe) with the following stereotaxic coordinates: +0.3 mm AP, -2.5 mm ML, at -5.0 and -4.0 mm DV (Vazey and Connor, 2010; Vazey et al., 2006; Vazey et al., 2010).

[0303] Spontaneous forelimb use

[0304] Rats were placed in a Perspex cylinder (20 cm diameter) and their behaviour was video recorded for 5 minutes. Baseline test data for motor function were obtained prior to the QA lesion. Motor function was also assessed 2 weeks after the QA lesion. Following transplantation, rats were assessed 2, 4, 12 and 14 weeks after transplantation. Spontaneous forelimb use was scored using the forelimb asymmetry analysis (Schallert et al., 2000) by an experimenter blind to the animal's condition during slow motion playback of the video session. Forelimb use was assessed as an asymmetry score representing the total use of the ipsilateral forepaw for stance, wall placement and landing during exploratory stance over the 5 minute test period (Vazey and Connor, 2010; Vazey et al., 2006). Rats that did not respond to the QA lesion with a predominantly contralateral forelimb use were excluded from all analyses.

[0305] Immunohistochemical analysis

[0306] At 14 weeks post-transplantation, rats were sacrificed with sodium pentobarbital (120 mg / kg, i.p.) followed by transcardial perfusion with 0.9% saline and 4% paraformaldehyde. Brain tissue was coronally sectioned at 40 pm thickness using an HM450 sliding microtome (Microm International GmbH, Waldorf, Germany) after cryoprotection in 30% sucrose. Eight sets of sections (with 320 pm between consecutive sections in each set) were collected from each brain and stored at -20 °C.

[0307] Free-floating coronal sections from each animal were subjected to fluorescent immunohistochemistry using antibodies against STEM121 (1 :500, Takara), TUJ1 (1 :500, Biolegend), MAP2 (1 :500, Sigma Aldrich), DARPP32 (1 :500, Invitrogen), GAD 65 / 67 (1 :500, AbCam), and GABA (1 :500, Invitrogen). Primary antibodies were visualized using species-matched Alexa Fluor™-labelled secondary antibodies (1 :500; Invitrogen). Single nuclei were confirmed using DAPI (1 :1000, Thermo Fisher Scientific). Imaging was performed on a Nikon TE2000E inverted microscope (Nikon) equipped with a Nikon DS-Ri2 camera, or on a Zeiss LSM 710 inverted confocal scanning laser microscope (BioMedical Imaging Resource, University of Auckland).

[0308] Statistical analysis

[0309] Statistical analysis was performed using IBM SPSS Statistics v28 (IBM Corporation). Levene’s test was performed on all data. Single or two-way ANOVA was used to compare media composition and / or cell lines. Post-hoc analysis was performed using the Bonferroni test. Two-way mixed ANOVA (followed by simple main effect analysis with Bonferroni correction) was used to compare spontaneous forelimb use in exploration between hiLGEP-transplanted and sham animals over time. All data are presented as mean ± SEM. Results were considered significant if p < 0.05.

[0310] Results

[0311] Adult human dermal fibroblasts can be directly reprogrammed into lateral ganglionic eminence phenotype.

[0312] Figure 1This paper presents a direct reprogramming and differentiation scheme that promotes the generation of neural progenitor cells with the hiLGEP phenotype and enhances their differentiation into medium-sized multispinous striatal neurons (MSNs).

[0313] In the example provided herein, the inventors investigated the effects of adding the following components to a standard NBA-based reprogramming medium: a broad-spectrum protein kinase C inhibitor, Gö6983 (5 µM), a p160ROCK inhibitor, Y27632 (10 µM), 1% N-2 supplement (combination abbreviation GYN), and 10 µM retinoic acid. This standard NBA-based reprogramming medium contained 1 mM valproic acid, 1% penicillin-streptomycin-glutamine, 2% retinoic acid-free B-27, 20 ng / ml EGF, 20 ng / ml FGF2, and 2 µg / ml heparin. Crucially, the inventors added activator A (25 ng / ml) to the medium 7 days after reprogramming. After 14 days of reprogramming, the inventors observed that, compared to using NBA medium alone, the addition of activator A (with or without GYN) significantly improved striatal transcription factors. GSX2 or DLX2 The expression has very little impact ( Figure 2 A). In contrast, NBA culture medium supplemented with activin A upregulates lateral ganglion eminence (LGE) selective genes. CTIP2 The expression of CTIP2 was further upregulated after the addition of GYN. Figure 2 A).

[0314] The inventors then compared the effects of basal culture medium BrainPhys™ and NBA on hiLGEP gene expression 14 days after reprogramming. Figure 2 B). 14 days after reprogramming, no adverse effects were observed in cells reprogrammed in BrainPhys™ with only activin A added. CTIP2 Upregulation. However, when BrainPhys™ was supplemented with both GYN and activin A, we observed... CTIP2 Significantly increased ( Figure 2 B). In summary, these results indicate that activin A, in combination with Gö6983, Y27632, and N-2, can promote the key LGEP gene. CTIP2 The expression.

[0315] To confirm the ability of BrainPhys™ supplemented with GYN and activin A to induce LGE transcription factor expression, the inventors used... SOX2 / PAX6 Cells were collected 14 days after cmRNA reprogramming to assess gene and protein expression. The inventors observed widespread colony formation 14 days after reprogramming. Figure 2 C), and compared with the original aHDF, the neurotrophic factor NESTIN and LGE transcription factorsDLX2, FOXP2 and CTIP2 expression of both Figure 2 D). The inventors did not observe changes in expression of pro- glutamatergic neuronal factors NGN2 (Colasante et al., 2019). At the protein level, reprogrammed cells expressed GSX2, FOXP1, FOXP2 and MEIS2 Figure 2 E-H). Without wishing to be bound by theory, the inventors believe that the above findings support the proposition that BrainPhys™ medium supplemented with GYN and Activin A can promote induction of LGE precursor fate.

[0316] Directly reprogrammed human lateral ganglionic eminence progenitors differentiate in vitro into functional DARPP32 positive neurons.

[0317] To ensure optimal differentiation of hiLGEPs towards MSN fate, the inventors compared the effect of NBA and BrainPhys™ medium on neuronal differentiation. It was also investigated whether the addition of Activin A for the first 7 days of differentiation could enhance striatal differentiation Figure 1 hiLGEPs reprogrammed in NBA medium were subsequently differentiated in NBA, while hiLGEPs reprogrammed in BrainPhys™ medium were differentiated in BrainPhys™. It was observed that, regardless of the reprogramming protocol, the proportion of TUJ1 / DAPI positive cells was 1.2% ± 0.33% to 14.33% ± 3.4% when hiLGEPs were differentiated in striatal differentiation medium based on NBA with Activin A Figure 3 A). In contrast, hiLGEPs reprogrammed in BrainPhys™ supplemented with GYN and Activin A and differentiated in striatal differentiation medium based on BrainPhys™ with Activin A, had a significantly higher proportion of TUJ1 positive cells out of the total DAPI+ cell population, 63.8% ± 4.59%, while hiLGEPs reprogrammed in BrainPhys™ and Activin A only had 9.4% ± 1.74% TUJ1 / DAPI positive cells Figure 3 A). Similarly, after differentiation in standard striatal differentiation medium based on NBA, the number of DARPP32 positive cells was very low (0.15% ± 1.15% to 10.76% ± 2.2%; Figure 3 B). However, hiLGEPs reprogrammed in BrainPhys™ supplemented with GYN and Activin A and differentiated in striatal differentiation medium based on BrainPhys™ with Activin A, had a proportion of DARPP32 expressing cells out of the total DAPI+ cell population of 42.45% ± 2.72%, while hiLGEPs reprogrammed in BrainPhys™ and Activin A only had 3.77% ± 0.95% DARPP32 / DAPI positive cells Figure 3 B,Figure 4 A, B, C). Further validation of the reprogramming impact of GYN alone or in combination with Activin A is seen in Figure 3 D and E. These findings demonstrate that reprogramming of aHDFs transfected with cmRNA in BrainPhys™ supplemented with GYN and Activin A promotes the induction of hiLGEPs, while differentiation of hiLGEPs in BrainPhys™ medium supplemented with Activin A enhances the generation of DARPP32-positive neurons. SOX2 / PAX6 cmRNA-transfected aHDFs in BrainPhys™ supplemented with GYN and Activin A promotes the induction of hiLGEPs, while differentiation of hiLGEPs in BrainPhys™ medium supplemented with Activin A enhances the generation of DARPP32-positive neurons.

[0318] Finally, the inventors investigated whether the addition of the AMP-activated kinase inhibitor dorsomorphin for the first 5 days of differentiation, or the broad-spectrum protein kinase C inhibitor Gö6983 for the first 5 days followed by dorsomorphin for the subsequent 5 days, could further enhance the generation of DARPP32-positive neurons. The effect of dorsomorphin alone or in combination with Gö6983 was also compared in two independent cell lines (2116 and 1507) to assess the stability and consistency of DARPP32-positive neuron production. Interestingly, the addition of dorsomorphin (with or without Gö6983) did not significantly alter the proportion of DARPP32-positive neurons compared to standard BrainPhys™-based striatal differentiation medium (p = 0.999; Figure 3 C). There was a significant interaction between differentiation conditions and cell lines (two-way mixed ANOVA; p = 0.001), indicating that the effect of different medium conditions on DARPP32 production varied between cell lines. While for the 2116 cell line, there was a significant increase in DARPP32-positive cells when the striatal differentiation medium was supplemented with both Gö6983 and dorsomorphin compared to dorsomorphin alone (p = 0.007; Figure 3 C), further post-hoc analysis determined that BrainPhys™-based striatal differentiation medium supplemented with dorsomorphin alone produced the most consistent DARPP32-positive cell production across cell lines (striatal differentiation + dorsomorphin, 2116 vs. 1507, p = 0.088; striatal differentiation only, 2116 vs. 1507, p = 0.0008; striatal differentiation + Gö6983 + dorsomorphin, 2116 vs. 1507, p = 0.0003). Based on these findings, the inventors selected BrainPhys™-based striatal differentiation medium supplemented with dorsomorphin for the first 5 days of differentiation and Activin A for the first 7 days.

[0319] With these culture conditions, the inventors successfully generated TUJ1+ neurons co-expressing DARPP32, as well as GABA-positive and GAD65 / 67 Positive neurons, which exhibited extensive neurite growth and network formation Figure 4 A-E). Functionality of hiLGEP-derived neurons was confirmed by live cell calcium imaging (video data not shown, representative data see snapshots Figure 4 F and G). Cultures were loaded with the fluorescence-based calcium indicator Cal-520 and exposed to 0, 12.5, 25, or 50 mM glutamate. Cal-520 fluorescence was measured as percent increase in mean fluorescence intensity relative to the baseline at time 0. hiLGEP-derived neurons exhibited an increase in Cal-520 fluorescence with increasing glutamate concentration, with maximal average intensity at 25 mM glutamate, peaking at 90 seconds post glutamate administration, followed by a decrease in average intensity of -300% to -750% relative to baseline during 95-100 seconds post glutamate administration Figure 4 F).

[0320] Transplantation of directly reprogrammed human lateral ganglionic eminence progenitors can reduce spontaneous forelimb use impairment.

[0321] The spontaneous forelimb use test is a non-pharmacologically induced test of forelimb motor function that relies on the integrity of the intrinsic striatal neurons, the nigrostriatal dopamine system, and the sensorimotor cortex. Following unilateral striatal injury, rats preferentially use the ipsilateral forelimb to initiate and terminate the center of gravity shift movements when standing and exploring along a vertical surface (Vazey and Connor, 2010; Vazey et al., 2006). Using the spontaneous forelimb use test, the inventors investigated the effect of transplanting hiLGEPs into the QA-injured striatum on forelimb motor function at 2, 4, 12, and 14 weeks post-transplantation Figure 5 A).

[0322] Two-way mixed ANOVA followed by simple main effect analysis showed a significant interaction between time and treatment (F = 2.83, df = 5, p = 0.043). Both groups exhibited a significant preference for using the ipsilateral forelimb relative to baseline following QA injury (saline treatment, p = 0.017; hiLGEP treatment, p = 0.027, Figure 5 B). Animals receiving saline treatment continued to exhibit a significant ipsilateral forelimb use preference relative to baseline over time (2 weeks post-transplantation, p = 0.008; 12 weeks post-transplantation, p = 0.036; 14 weeks post-transplantation, p = 0.026; Figure 5 B). In contrast, hiLGEP-transplanted animals exhibited a significant ipsilateral forelimb use preference relative to baseline only at 2 weeks post-transplantation (p = 0.04; Figure 5 B). Extending this observation, hiLGEP-transplanted animals exhibited a significant decrease in ipsilateral forelimb use at 14 weeks post-transplantation compared to following QA injury ; p = 0.027; Figure 5B). These results indicate that striatal transplantation of hiLGEPs can alleviate the forelimb use impairment of spontaneous exploration caused by striatal QA injury.

[0323] Human induced lateral ganglionic eminence progenitors survive and differentiate into medium-sized spiny striatal neurons after transplantation into the QA lesioned striatum.

[0324] The inventors also investigated SOX2 / PAX6 the ability of directly reprogrammed hiLGEPs to survive and differentiate into medium spiny striatal neurons (MSNs) in the QA-injured HD rat model. hiLGEP-derived neurons were identified in the QA-injured rat striatum by expression of the human cytoplasmic marker STEM121 (14 weeks post-transplantation; Figure 6 A). STEM121 -positive cells were detected within defined boundaries in the anterior striatum of hiLGEP-transplanted animals 14 weeks post-transplantation Figure 6 A). STEM121 -positive cells exhibited a unique neuronal morphology with extensive neurite growth 14 weeks post-transplantation Figure 6 A and A'). The inventors did not observe STEM121 -positive cells exhibiting astrocytic morphology, and STEM121 did not co-localize with GFAP (data not shown).

[0325] To determine the final phenotype of hiLGEP-derived neurons 14 weeks post-transplantation into the QA-injured striatum, double-label immunofluorescence analysis for STEM121 and cell-type specific markers was performed.

[0326] STEM121 -positive cells co-expressed MAP2 14 weeks post-transplantation Figure 6 B1 and Figure 6 B2), and most STEM121 -positive cells co-expressed DARPP32 Figure 6 C1 and Figure 6 C2). A subset of STEM121 -positive cells also expressed GABA Figure 6 E1 and Figure 6 E2), and a small number of STEM121 -positive cells co-expressed the enzyme GAD 65 / 67 Figure 6 D1 and Figure 6 D2). We also observed GABA-positive / STEM121 -negative cells exhibiting astrocytic morphology Figure 6 E1 and Figure 6 ​E2). It has been demonstrated that astrocytes synthesize and uptake GABA through multiple pathways (Ishibashi et al., 2019; Jo et al., 2014). The lack of co-expression of STEM121 indicates that these astrocytes are host rat brain-derived in response to QA injury. Moreover, this observation in combination with the lack of co-expression of STEM121 with GFAP demonstrates that transplanted hiLGEPs do not generate astrocytes after transplantation.

[0327] These results demonstrate that, SOX2 / PAX6 cmRNA directly reprogrammed hiLGEPs survive and differentiate into medium spiny striatal neurons (MSNs) after transplantation in the striatum of QA-injured rats.

[0328] Discussion - Conclusions

[0329] The results of this study demonstrate that, in BrainPhys™-based reprogramming medium, HDFs can be directly reprogrammed into hiLGEPs by transient cmRNA-mediated SOX2 and PAX6 overexpression and exposure to Activin A, Gö6983, Y27632 and N-2, aHDFs. This study also demonstrates for the first time that transplantation of directly reprogrammed hiLGEPs into the striatum of QA-injured rats, an accepted animal model of brain degenerative diseases, specifically Huntington’s disease, generates high yields of medium spiny striatal neurons (MSNs). Moreover, transplantation of hiLGEPs into the striatum of QA-injured rats significantly reduces motor dysfunction as measured by spontaneous forelimb use exploration compared to saline-treated animals. Without wishing to be bound by theory, the inventors, based on these findings, believe that the use of cmRNA directly reprogrammed hiLGEPs provides an effective and clinically feasible strategy for cell replacement therapy for the treatment of brain degenerative diseases, specifically Huntington’s disease.

[0330] 7. Industrial applicability

[0331] The present application can be used to reprogram HDFs into neural precursor cells and has applications in research and medicine. It will be understood by those skilled in the art that the foregoing description is only illustrative of the present application and that the present application should not be limited thereto.

[0332] 8. References

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Claims

1. A composition comprising: a basal brain medium, and at least two active agents selected from the group consisting of Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

2. The composition of claim 1, comprising: a basal brain medium, and at least three active agents selected from the group consisting of Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

3. The composition of claim 1 or claim 2, comprising: a basal brain medium, and all four active agents of Activin A (ActA), a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

4. The composition according to any one of claims 1 to 3, wherein, the PKC inhibitor is selected from the group consisting of Gö6983, enzalutamide, staurosporine, GF 109203X, Go6976, Ro 31-8220 methanesulfonate, Ro 32-0432 hydrochloride, sorbitol, and K252a, preferably the PKC inhibitor is Gö6983.

5. The composition according to any one of claims 1 to 4, wherein, the pi 60 ROCK inhibitor is selected from the group consisting of Y27632, thiazovivin, HA 1100 hydrochloride, and GSK429286A, preferably the pi 60 ROCK inhibitor is Y27632.

6. A method of making human induced lateral ganglionic eminence precursor cells (hiLGEPs), comprising: a) reprogramming human fibroblasts (HFs) to hiLGEPs, comprising: a. with SOX2 cmRNA and PAX6 cmRNA transfection of HFs; b. culturing the transfected HFs in a composition comprising: a basal brain medium, and at least two active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement; c. passaging the HFs in b. in a composition comprising: a basal brain medium, and at least three active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pi 60 ROCK inhibitor, an N-2 supplement, and Activin A (ActA); and d. culturing the passaged HFs.

7. The method of claim 6, wherein, The SOX2 The cmRNA comprises SEQ ID NO:

1.

8. The method of claim 6 or claim 7, wherein, The PAX6 The cmRNA comprises SEQ ID NO:

2.

9. The method of any one of claims 6 to 8, wherein the composition in b. comprises a basal brain medium and all three active agents of a protein kinase (PKC) inhibitor, a pi 60 ROCK inhibitor, and an N-2 supplement.

10. The method of any one of claims 6-9, wherein, the composition in c. comprises a basal brain medium and all four active agents of a protein kinase (PKC) inhibitor, a pi 60 ROCK inhibitor, an N-2 supplement, and Activin A (ActA).

11. The method of any one of claims 6 to 10, wherein, the PKC inhibitor in b. and / or c. is selected from the group consisting of Gö6983, enzalutamide, staurosporine, GF 109203X, Go6976, Ro 31-8220 methanesulfonate, Ro 32-0432 hydrochloride, sorbitol, and K252a, preferably wherein the PKC inhibitor is Gö6983.

12. The method of any one of claims 6-11, wherein, The p160ROCK inhibitor in b. and / or c. is selected from the group consisting of Y27632, thiazovivin, HA 1100 hydrochloride and GSK429286A, preferably wherein the p160ROCK inhibitor is Y27632.

13. The method of any one of claims 6 to 12, wherein, The HF is a human dermal fibroblast (HDF).

14. The method of any one of claims 6-12, wherein, The HF is an adult human fibroblast (aHF), preferably an adult human dermal fibroblast (aHDF).

15. A kit comprising: i. a composition comprising: basal brain medium, and at least two active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pl60ROCK inhibitor and an N-2 supplement; and ii. a composition comprising: basal brain medium, and at least three active agents selected from the group consisting of a protein kinase C (PKC) inhibitor, a pl60ROCK inhibitor, an N-2 supplement and an Activin A (ActA).

16. The kit of claim 15, wherein, The composition in i. comprises all three active agents, which are a protein kinase (PKC) inhibitor, a pl60ROCK inhibitor and an N-2 supplement.

17. The kit of claim 15 or claim 16, wherein, The composition in ii. comprises all four active agents, which are a protein kinase (PKC) inhibitor, a pl60ROCK inhibitor, an N-2 supplement and an Activin A (ActA).

18. The kit of any one of claims 15 to 17, wherein, The PKC inhibitor in i. and / or ii. is selected from the group consisting of Gö6983, enzalutamide, staurosporine, GF 109203X, Go6976, Ro 31-8220 methanesulfonate, Ro 32-0432 hydrochloride, sorbitol and K252a, preferably wherein the PKC inhibitor is Gö6983.

19. The kit of any one of claims 15 to 18, wherein, The pl60ROCK inhibitor in i. and / or ii. is selected from the group consisting of Y27632, thiazovivin, HA 1100 hydrochloride and GSK429286A, preferably wherein the pl60ROCK inhibitor is Y27632.

20. A human induced lateral ganglionic eminence precursor cell (hiLGEP).

21. The hiLGEP of claim 20, prepared by the method of any one of claims 6 to 14.

22. A pharmaceutical composition comprising at least one hiLGEP according to claim 20 or claim 21.

23. Use of a composition as defined in any one of claims 1 to 5 in promoting the induction of fibroblast cells into a lateral ganglionic eminence (LGE) precursor fate, preferably wherein the fibroblast cells are human fibroblast cells, human dermal fibroblast cells, adult human fibroblast cells or adult human dermal fibroblast cells.

24. Use of a composition comprising human induced lateral ganglionic eminence precursor cells (hiLGEPs) and a carrier in the treatment of Huntington’s disease.

25. Use of a human induced lateral ganglionic eminence precursor cell (hiLGEP) in the manufacture of a medicament for the treatment of Huntington’s disease.

26. A method of treating Huntington’s disease, the method comprising transplanting human induced lateral ganglionic eminence progenitor cells (hiLGEPs) into the striatum of a subject having or suspected of having Huntington’s disease.

27. The use of any one of claims 23 to 25, or the method of claim 24, wherein, The hiLGEPs are reprogrammed from fibroblasts, preferably human fibroblasts, human dermal fibroblasts, or adult human dermal fibroblasts.

28. The use of any one of claims 23 to 25, or the method of claim 24, wherein, The hiLGEPs are as defined in claim 21.

29. Use of a composition according to any one of claims 1 to 5 or a kit according to any one of claims 15 to 19 for reprogramming fibroblasts into hiLGEPs, preferably wherein the fibroblasts are human fibroblasts, human dermal fibroblasts, adult human fibroblasts, or adult human dermal fibroblasts.

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