FIELD OF THE INVENTION

The invention in general relates to methods and compositions for transdifferentiation of a somatic cell, e.g., a fibroblast to a dopaminergic precursor. Particularly, the invention relates to an induced dopaminergic precursor (iDP) cell, master transcription factors (TFs) therefor, methods for making iDP cells, and methods and compositions for using them in, e.g., treating neurodegenerative diseases such as Parkinson's disease.

BACKGROUND OF THE INVENTION

Selective degeneration of functional neurons is a key pathogenic event in many neurodegenerative disorders. Cell replacement through transplantation of stem/progenitor cells represents a particularly promising therapeutic strategy for these diseases. One of the most sought after diseases for cell replacement is Parkinson's disease (PD), which is a prototypical illness characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and decreased DA innervation in the striatum1,2. Recent breakthroughs in stem cell biology have established the feasibility of directly reprogramming cells of one lineage into another, e.g., neurons, by introducing crucial cell-fate determinants3,4. As a result of those advances, induced neurons5-9and induced neural stem cells (iNPCs)10-13have been successfully generated, and studies show that these cells hold therapeutic benefits. Direct reprogramming represents an important direction to obtain safe and less controversial cell sources for PD treatment. However, the low yield and non-proliferative nature of dopaminergic neurons derived from direct reprogramming limits broad application. Multipotent neural stem/progenitor cells (NSC/NPC), including iNPCs that give rise to all types of neural cells, may increase the yield of engraftable cells; however, specific and efficient induction of homogeneous DA neurons from NPCs/iNPCs remains a significant challenge. NSCs/NPCs often respond poorly to pre-patterning morphogens with low differentiation efficiency for specific neuronal subtypes, and are prone to a glial-restricted state14. Moreover, grafted NSCs/NPCs are more likely to terminally differentiate into astrocytes rather than functional neurons in response to injury15,16. Therefore, dopaminergic neuronal-lineage restricted precursors that hold great potential for DA neuronal differentiation are highly desirable for experimental PD treatment.

Thus, a need exists for dopaminergic precursors, in particularly in vitro induced dopaminergic precursors (iDP). Studies have previously revealed that Brn2/Brn4 and Sox2 are critical for the direct conversion of fibroblasts into induced neural progenitors9,10,13,17. Strategically, various groups have been working on the direct reprogramming of somatic cells into region-specific iNPCs as well as subtype-specific iNPCs by expressing defined transcript factors in addition to Brn2/Brn4 and Sox210,18. The factors that directly reprogram somatic cells into neuronal lineage-restricted progenitors have been expanded to the combination of Brn2/Brn4 and Sox2 with c-Myc19. In addition, in the presence of Brn2 and Sox2, FoxG1, a transcription factor that is predominantly expressed in the forebrain, contributes to the acquisition of forebrain identity in iNPCs10. In contrast, the midbrain DA neurons that originated from the floor plate cells in the mesencephalon are critically regulated by transcriptional determinants such as Foxa220-22. Furthermore, the development of DA neurons depends not only on initial Sonic hedgehog (SHH)/Fibroblast growth factor 8 (FGF8) and Wnt1 signaling pathway for the dopaminergic progenitors, but also on the cooperation of SHH-Foxa2 and Wnt1-Lmx1α pathways23. However, before the discovery herein, no complete panel of transcription factors have been identified to be able to transdifferentiate somatic cells into iDPs in vitro. In contrast, the derivation of engraftable DPs from human pluripotent stem cells has proven difficult due to low differentiation efficiency of DA neurons and primary DPs are not readily available.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an induced dopaminergic precursor (iDP), comprising ectopically expressed genes and/or proteins selected from: (1) one or both of Brn2 and Brn4, (2) Sox2, and (3) one or both of Foxa2 and Lmx1a, or a variant of any one or more of the foregoing, in a somatic cell that is not dopaminergic precursor. The gene or its variant can be the full-length gene, a fragment thereof or the cDNA, optionally under the control of a constitutive or conditional promoter.

In some embodiments, the iDP includes ectopically expressed Brn2, Sox2 and Foxa2, or a variant of any one or more of the foregoing. The ectopically expressed genes can be expressed from one or more vectors carrying them, or the ectopically expressed proteins can be expressed from one or more mRNA encoding them. In various embodiments, the iDP is a dopaminergic neuronal lineage-restricted progenitors and does not differentiate into a glial cell. In certain embodiments, differentiation of the iDP is independent of morphogens selected from SHH and FGF8. The iDP in some examples can further include ectopically expressed L-Myc, which is optionally conditionally expressed under the control of doxycycline. In some embodiments, the somatic cell is a fibroblast. The somatic cell can be present in vitro.

The iDP disclosed and claimed herein can be used in a treatment of a neurodegenerative disease selected from Parkinson's disease, depression, dementia and schizophrenia.

In another aspect, provided herein is a population of the iDPs disclosed herein, wherein more than 80% or more than 90% of the iDPs are capable of differentiating into dopaminergic neurons.

In yet another aspect, a method of transdifferentiating a somatic cell to an iDP is provided, comprising ectopically expressing genes selected from: (1) one or both of Brn2 and Brn4, (2) Sox2 and (3) one or both of Foxa2 and Lmx1a, or a variant of any one or more of the foregoing, in a somatic cell that is not dopaminergic precursor.

In some embodiments, the method includes ectopically expressing Brn2, Sox2 and Foxa2, or a variant of any one or more of the foregoing, in the somatic cell. The somatic cell may be a fibroblast. The somatic cell can be present in vitro. The somatic cell can be obtained from a patient in need of cell or tissue replacement therapy with iDP. The patient can have Parkinson's disease, depression, dementia or schizophrenia.

Also provided herein is a method of treating a neurodegenerative disease, comprising administering to a patient in need thereof the iDP disclosed herein.

A further aspect relates to a method of identifying an agent that regulates dopaminergic neuron development, comprising subjecting a population of the iDPs disclosed herein to a test agent, wherein a change in differentiation of the iDPs to dopaminergic neuron is indicative of a regulatory role of the test agent in dopaminergic neuron development. In some embodiments, the change is one or more of: cell growth, cell survival, or gene expression change in one or more of tyrosine hydroxylase (TH), Sox1, PAX6, ZBTB16, Sox3, CD133, Nestin, Aldh1A1, Corin (Lrp4), Lmx1a, Msx1, Ngn2, Otx2, Mash1, Pitx3 and Nkx6.1, ploysialic acid-neural cell adhesion molecule (PSA-NCAM), and/or doublecortin (DCX). In certain embodiments, the iDPs are transdifferentiated from a patient's somatic cells, wherein the patient has Parkinson's disease, depression, dementia or schizophrenia, and wherein the method identifies agents for the treatment of Parkinson's disease, depression, dementia or schizophrenia.

Another aspect relates to a composition comprising one or more vectors carrying isolated genes selected from: (1) one or both of Brn2 and Brn4, (2) Sox2 and (3) one or both of Foxa2 and Lmx1a, or a variant of any one or more of the foregoing. In some embodiments, the one or more vectors can carry Brn2, Sox2 and Foxa2, or a variant of any one or more of the foregoing. The vector is a viral vector.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive or limiting the scope of the invention described and claimed herein.

