WO2016207300A1

WO2016207300A1 - Enhanced cell reprogramming by mrna

Info

Publication number: WO2016207300A1
Application number: PCT/EP2016/064580
Authority: WO
Inventors: Bronwen CONNOR; Erin FIRMIN; Kathryn Jones; Carsten Rudolph
Original assignee: Ethris Gmbh
Priority date: 2015-06-23
Filing date: 2016-06-23
Publication date: 2016-12-29

Abstract

Described is a method of reprogramming a somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell), said method comprising the steps of: a) introducing one or more mRNAs encoding a transcription factor into said somatic cell; b) culturing said somatic cell under conditions permissive to the culture of said iNP cell. Moreover, there is also described a reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method as described. Further, described is an mRNA encoding a transcription factor wherein said transcription factor is Sox2, Pax6 or Lmx1a and wherein said mRNA contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.

Description

Enhanced cell reprogramming by mRNA

The present invention relates to a method of reprogramming a somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell), said method comprising the steps of: a) introducing one or more mRNAs encoding a transcription factor into said somatic cell; b) culturing said somatic cell under conditions permissive to the culture of said iNP cell. The present invention also relates to a reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the invention. Further, the present invention relates to an mRNA encoding a transcription factor wherein said transcription factor is Sox2, Pax6 or Lmxl a and wherein said mRNA contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.

Reprogramming technology has recently provided the capability to directly generate specific neuronal lineages, such as dopaminergic neurons or motor neurons. The ability to directly reprogram mature somatic cells to an alternative cell lineage without requirement of a pluripotent state was first demonstrated by Weintraub and colleagues^{1 ,2} with the conversion of fibroblast cells to myoblasts by activation of MYOD. This initiated a number of studies investigating the existence of 'master regulator' genes that act as molecular switches to induce cell lineage changes between distantly related somatic cell types^3"6. Direct reprogramming of somatic cells holds great potential for the generation of patient-specific disease models, drug development, or the possibility of an autologous cell source for transplantation therapy. Given the limited ability to access and culture live human neural cells, a number of studies have focused on the direct lineage conversion of functional neural cells from non-ectodermal cells.

Neurons were the first cells demonstrated to be directly converted from mouse fibroblasts by forced expression of the neural lineage-specific transcription factors ASCL1, BRN2 and MYT1L ³, illustrating that direct lineage conversion is possible even between cell types representing different germ layers. This was extended to the direct conversion of human fibroblasts to neurons with the addition of NEUROD1 ¹. However, while direct conversion of human neurons is an interesting tool for investigating neurological disorders affecting neurons, this technology has limitations for diseases affecting glia. Further, induced neurons are post-mitotic and therefore cannot be expanded on mass for drug screening assays or used for cell replacement therapies.

To address this, direct reprogramming strategies have recently been identified to generate populations of induced neural stem/precursor (iNP) cells from fibroblasts. The objective is to provide a direct source of non-pluripotent, expandable iNP cells with the capability of generating multiple neural lineages. To date, iNP cells have been generated using two main approaches; either transient expression of the four pluripotent factors OCT3/4, KLF4, SOX2, and C-MYC ^{8 "14}, or forced expression of neural-specific transcription factors^15"21. Regardless of the strategy or transcription factor combination employed, each protocol results in the production of either bi- or tri-potent neural precursor cells with capability for prolonged expansion.

Recently, an efficient system has been developed utilizing the neural promoting transcription factors SOX2 and PAX6 to directly reprogram adult human fibroblasts to a neural stem/precursor cell-like state^17,21. SOX2 and PAX6 were identified based on their prominent roles in human neural development ^22-3 , and forced expression in adult human fibroblasts results in the generation of bipotent iNP colonies with the capability to differentiate into GFAP-positive astrocytes and mature region-specific neurons ¹⁷'²¹. A key feature of this protocol was the utilization of non-viral plasmid cDNA transfection to transiently over-express SOX2 and PAX6 in adult human dermal fibroblasts, without requirement of oncogenic-promoting transcription factors (such as c-myc) ^17,21.

Although DNA transfection-based methodologies, instead of employing viral gene transfer vectors, potentially reduces the risk of genomic recombination or insertional mutagenesis, considerable limitations accompany non-integrative cell reprogramming strategies^32"36. In particular, while the use of plasmid cDNA provided a robust and transient mechanism by which to generate human iNPs ^17,21 , the major limitation associated with the use of plasmid cDNA was the intrinsic risk of generating insertion mutations in the cells' genome and the extraordinary low level of transfection efficiency. Moreover, in addition to direct plasmid cDNA transfection it has recently been described that recombinant Sox2 and Pax6 can be transduced into adult fibroblast cells; WO 2011/096825. This procedure is, however, also accompanied by limitations since it requires multiple transduction events in order to accomplish a desired reprogramming.

The use of chemically modified mRNA has recently been described in general terms³⁹,⁴⁰; WO 2011/012316. However, the use of non-viral mRNA gene delivery has a number of technical challenges including immunogenicity and stability of mRNA expression³⁷. To combat these issues, delivery mechanisms and/or chemical modification of the mRNA is required. Previous work by Warren and colleagues ³⁸ demonstrated that, by using a combination of RNA modifications and a soluble interferon inhibitor to overcome innate antiviral responses, mature human cells can be reprogrammed to pluripotency with conversion efficiencies and kinetics substantially superior to established viral protocols. However, a combination of RNA modifications and a soluble interferon inhibitor is not always desirable.

Accordingly, although in the prior art there are already described means and methods to generate neural stem/precursor cells (iNPs) from mature somatic cells, there is still a need for improvements, in particular as regards the safety, the transfection efficiency and the rate of iNP reprogramming.

The present application addresses this need by providing the embodiments as defined in the claims and the present invention is based on the surprising finding that direct iNP reprogramming can be achieved with high efficiency by using an mRNA gene delivery system. The present invention surprisingly demonstrates that by using mRNA, it was possible to co-transfect somatic cells, such as adult human fibroblasts, with the reprogramming factors SOX2 and PAX6 with an efficiency of >80%, significantly higher than the 10-20% transfection efficiency obtained with plasmids. The use of a modified mRNA has the additional benefit that the mRNA is extremely stable and non-immunogenic. It is also surprisingly shown that cell survival was >85% post transfection, also significantly greater than 20-40% survival with plasmid transfection. Most importantly, co-transfection with SOX2 and PAX6 mRNA beneficially reduces the time for of iNP reprogramming to about 21 to 28 days compared with -45 days required using plasmid transfection.

Expression of neural positional genes is observed through qPCR and differentiation of mRNA-derived iNPs generated TuJ1 positive cells co-expressing phenotypic markers including GAD₆5/67, vGlut and tyrosine hydroxylase. SOX2/PAX6 RNA generated iNP cells that expressed a wide range of neural positional genes with differentiation resulting in neurons expressing either glutamateric, GABAergic or catecholaminergic phenotypes. Thus, the present invention provides for the first time an mRNA approach to directly reprogram somatic cells, such as adult human fibroblasts, to neural precursor cells.

Moreover, as observed by qPCR, SOX2/LMX1A RNA generated iNP cells that expressed a range of mesencephalic positional genes which are required for the generation of midbrain dopaminergic fate/dopamine neuron generation.

Thus, this finding leads to the provision of the embodiments characterized in the claims. Accordingly, the present invention relates to a method of reprogramming a somatic cell, preferably a mammalian somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell), said method comprising the steps of:

a) introducing one or more mRNAs encoding a transcription factor into said somatic cell;

b) culturing said somatic cell under conditions permissive to the culture of said iNP cell.

Moreover, in a preferred embodiment, the present invention relates to a method of reprogramming a mature somatic cell, preferably a mammalian mature somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell), said method comprising the steps of:

In a preferred embodiment, the somatic cell or the mature somatic cell is a mammalian somatic cell and a mammalian mature somatic cell, respectively. A somatic cell, preferably a mammalian somatic cell as used in accordance with the present invention relates to any cell other than germ cells, such as an egg, a sperm, or the like, which does not directly transfer its DNA to the next generation. Typically, somatic cells have limited or no pluripotency. "Pluripotency" refers to a (stem) cell that has the potential to differentiate into cells constituting one or more tissues or organs, or preferably, any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system).

Multipotent cells refer to cells which show a higher degree of differentiation than pluripotent cells. Multipotent cells have the ability to differentiate into other cell types, yet not into any cells of the three germ layers and, accordingly, into a lesser number of cell types than pluripotent cells. While pluripotent cells have a potential to differentiate into any cells of the three germ layers as outlined above, multipotency describes progenitor cells which have the gene activation potential to differentiate into multiple, but limited cell types.

The starting point for the reprogramming into an induced neural stem/precursor cell (iNP cell) in accordance with the present invention is a somatic cell, preferably a mammalian somatic cell. In another preferred embodiment, the starting point for the reprogramming into an induced neural stem/precursor cell (iNP cell) in accordance with the present invention is a mature somatic cell, preferably a mammalian mature somatic cell.

The term "mature" means that the somatic cell, in contrast to a pluripotent cell, exhibits some grade of differentiation. In a preferred embodiment, the term "mature" is to be construed to refer to cells which have reached their final differentiation state, i.e., cells which no longer have a potential to further differentiate. Such cells can be found at various developmental stages including embryonal, post natal or adult stages, but are most conveniently sourced from adults.

An induced neural stem/precursor cell (commonly abbreviated as iNP cell) as used in accordance with the present invention relates to a type of pluripotent, preferably multipotent, stem cell artificially prepared from a non-pluripotent cell, typically an mature somatic cell, or terminally differentiated cell, preferably a fibroblast, by reprogramming. "Reprogramming" is a process that confers on a cell a measurably increased capacity to form progeny of at least one new cell type, either in culture or in vivo, than it would have under the same conditions without reprogramming. More specifically, reprogramming is a process that confers on a (mature) somatic cell a pluripotent potential. This means that after sufficient proliferation, a measurable proportion of progeny having phenotypic characteristics of the new cell type if essentially no such progeny could form before reprogramming; in other words, the proportion having characteristics of the new cell type is measurably more than before reprogramming. Under certain conditions, the proportion of progeny with characteristics of the new cell type may be at least about 0.05%, 0.1%, 0.5%>, 1%, 5%, 25% or more in the order of increasing preference.

Preferably, the induced neural stem/precursor cell (iNP cell) is a cell of the nervous system or, more specifically, a cell of the neural cell lineage. The term "cell lineage" is a genealogic pedigree of cells related through mitotic division.

A precursor cell in accordance with the present invention relates to a cell which is capable of differentiating into a number of cell and/or tissue types of a neural cell lineage.

Thus, an induced neural stem/precursor cell as used in accordance with the present invention relates to a type of multi-potent precursor cell which has been artificially derived from a non-pluripotent or multi-potent source, typically a (mature) somatic cell, by inducing expression of certain genes characteristic for cells of the neural lineage. Therefore, induced neural stem/precursor cells (iNP) cells are only capable of differentiating into neural cell and/or tissue types.

A messenger ribonucleic acid (mRNA) molecule as used in accordance with the present invention relates to a polymeric molecule which is assembled as a chain of the nucleotides termed G, A, U, and C. Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1 ' through 5'. A nitrogenous base is attached to the 1' position, in general, adenine (A), cytosine (C), guanine (G), or uracil (U). In a polymeric mRNA molecule a phosphate group is attached to the 3' position of one ribose and the 5' position of the next. Thus, the nucleotides in a polymeric mRNA molecule are covalently linked to each other wherein the phosphate group from one nucleotide binds to the 3' carbon on the subsequent nucleotide, thereby forming a phosphodiester bond. Accordingly, an mRNA strand has a 5' end and a 3' end, so named for the carbons on the ribose ring. By convention, upstream and downstream relate to the 5' to 3' direction in which mRNA transcription takes place.

mRNA is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. Following transcription of primary transcript mRNA (known as pre-mRNA) by RNA polymerase, processed, mature mRNA is translated into a polymer of amino acids, i.e., a protein. As in DNA, mRNA genetic information is in the sequence of nucleotides, which are arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis.

In the present invention, the mRNA encodes a transcription factor which is capable of reprogramming a mature somatic cell, preferably a mature mammalian somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP).

In general, a transcription factor (sometimes called a sequence-specific DNA-binding factor) is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA. Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes.

A defining feature of transcription factors is that they contain one or more DNA- binding domains (DBDs), which attach to specific sequences of DNA adjacent to the genes that they regulate. Transcription factors are essential for the regulation of gene expression and are, as a consequence, found in all living organisms. Transcription factors bind to either enhancer or promoter regions of DNA adjacent to the genes that they regulate. Depending on the transcription factor, the transcription of the adjacent gene is either up- or down-regulated. Transcription factors use a variety of mechanisms for the regulation of gene expression. These mechanisms include: stabilize or block the binding of RNA polymerase to DNA, catalyze the acetylation or deacetylation of histone proteins. The transcription factor can either do this directly or recruit other proteins with this catalytic activity. Many transcription factors use one or the other of two opposing mechanisms to regulate transcription: histone acetyltransferase (HAT) activity - acetylates histone proteins, which weakens the association of DNA with histones, which make the DNA more accessible to transcription, thereby up-regulating transcription; histone deacetylase (HDAC) activity - deacetylates histone proteins, which strengthens the association of DNA with histones, which make the DNA less accessible to transcription, thereby down- regulating transcription; recruit coactivator or corepressor proteins to the transcription factor DNA complex.

Many transcription factors in multicellular organisms are involved in development. Responding to cues (stimuli), these transcription factors turn on/off the transcription of the appropriate genes, which, in turn, allows for changes in cell morphology or activities needed for cell fate determination and cellular differentiation.

Transcription factors (like all proteins) are transcribed from a gene on a chromosome into an mRNA, and then the mRNA is translated into protein. Any of these steps can be regulated to affect the production (and thus activity) of a transcription factor.

In eukaryotes, transcription factors (like most proteins) are transcribed in the nucleus, the mRNA is then translated in the cell's cytoplasm and the protein of the transcription factor is then shuttled into the nucleus. Many proteins that are active in the nucleus contain nuclear localization signals that direct them to the nucleus.

In the context of the present invention, the transcription factor is a protein which is capable of reprogramming a mature somatic cell, preferably a mature mammalian somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP).

In the method according to the invention, the mRNA(s) encoding (a) transcription factor(s) are introduced into a somatic cell, preferably into a mature somatic cell. Generally, this step of introducing or delivering the mRNA(s) encoding (a) transcription factor(s) into a (mature) somatic cell is performed by standard delivery/introduction techniques. These delivery/introduction techniques are known in the art and can, e.g., be performed as outlined in the appended examples by transfection methods known in the art. These standard techniques have been described in, e.g., WO2011/012316 and by, e.g., Kim and Eberwine (Anal Bioanal Chem. 397(8):3173-8 (2010)). WO2011/012316 describes, e.g., a method for the transfection of lung cells with mRNA using Lipofectamin 2000 (Invitrogen). Kim and Eberwine summarize and describe the three major classes of transfection which are widely used to deliver nucleic acids into cells, i.e., biological, chemical and physical transfection methods. In the method according to the invention, in a subsequent step (i.e., the above step (b)), the somatic cells are cultured under conditions permissive to the culture of said iNP cells after one or more mRNAs encoding a transcription factor into said somatic cells have been introduced (i.e., after the above step (a)). The step of culturing the somatic cells under conditions permissive to the culture of said iNP cell includes culturing the cells in any medium capable of supporting growth of neural stem/precursor cells known in the art such as, for example, stem cell medium. Preferably the medium is supplemented with a chromatin modifying agent capable of facilitating the reprogramming of the somatic cell. The chromatin modifying agent may be selected from agents promoting acetylation of chromatin, inhibiting deacetylation of chromatin, altering histone methylation states within chromatin or leading to DNA demethylation within chromatin. Preferably the chromatin modifying agent is valproic acid. Even more preferable, it is valproic acid at a concentration of 1 μΜ.

Preferably, after the induction step (a) and prior to the culturing step (b), the method of the present invention may further comprise a selecting step wherein the thus produced induced neural stem/precursor cell (iNP cell) are selected and/or purified, for example, based on one or more embryonic cell characteristics of induced neural stem/precursor cells (iNP cells) as described further below by techniques known in the art and as, e.g., outlined in the appended examples, such as an ES cell-like morphology, more specifically a neural stem-like morphology in the present case. Subsequently, in another embodiment, the methods may comprise culturing the selected induced neural stem/precursor cell (iNPs) cells in an expansion medium under conditions permissive to the culture of said iNP cells.

The successfully generated induced neural stem/precursor cells (iNP cell) from the methods disclosed in this invention could be selected based on one or more of the following neural lineage stem cell characteristics, i.e., by selecting the cells based on the expression of at least one neural lineage marker selected from the group consisting of Pax6, Sox2, Lmxla, Nurrl , Pitx3, AscM , FoxG1 , Gsx2, Lhx2, Ngn2, Otx2, Six3, Hes1 , Hes5, Sox1 , Sox3, Mash 1 /Ash! 1 , HoxB9, Irx3, FOXA2, GLI1 , LMX1 B and neurogenin 2 as described in more detail further below by applying techniques known in the art and as, e.g., outlined in the appended examples. In general, in the method according to the invention, the somatic cells may be cultured under conditions permissive to the culture of said iNP cell. The culturing conditions according to the present invention will be appropriately defined depending on the medium and induced neural stem/precursor cells (iNP cells) used/produced. The medium according to certain aspects of the present invention can be prepared using a medium used for culturing animal cells as its basal medium, such as any of TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, MEM, DMEM, Ham, RPMI 1640, and Fischer's media, as well as any combinations thereof, but the medium is not particularly limited thereto as far as it can be used for culturing animal cells. The medium for the culturing of said somatic cells under conditions permissive to the culture of said iNP cells according to the present invention can be a serum-containing or serum-free medium. The serum- free medium refers to media with no unprocessed or unpurified serum, and accordingly can include media with purified blood-derived components or animal tissue-derived components (such as growth factors). From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s). The medium for the culturing of said somatic cells under conditions permissive to the culture of said iNP cells according to the present invention may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2- mercaptoethanol, 3'-thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98/30679, for example. Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

The medium for the culturing of said somatic cells under conditions permissive to the culture of said iNP cells can also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and inorganic salts. The concentration of 2-mercaptoethanol can be, for example, about 0.05 to 1.0 mM, and particularly about 0.1 to 0.5 mM, but the concentration is particularly not limited thereto as long as it is appropriate for culturing the stem cell(s).

The culture of the somatic cells under conditions permissive to the culture of said iNP cells can be performed in a culture vessel, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers, culture bag, and roller bottle, as long as it is capable of culturing the stem cells therein. The stem cells may be cultured in a volume of at least or about 0.2, 0.5, 1 , 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In a certain embodiment, the culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The bioreactor may have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1 , 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein. The culture vessel can be cellular adhesive or non-adhesive and selected depending on the purpose. The cellular adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells. The substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). The substrate for cell adhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, and fibronectin and mixtures thereof for example Matrigel™, and lysed cell membrane preparations.

Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 30 to 40°C, for example, at least or about 31 , 32, 33, 34, 35, 36, 37, 38, 39°C but particularly not limited to them. The C0₂ concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen tension can be at least or about 1 , 5, 8, 10, 20%, or any range derivable therein.

The culturing of the somatic cells under conditions permissive to the culture of said iNP cells can be an adhesion culture. In this case, the cells can be cultured in the presence of feeder cells. In the case where the feeder cells are used in the methods of the present invention, stromal cells such as fetal fibroblasts can be used as feeder cells (for example, refer to; Hogan et al, Manipulating the Mouse Embryo, A Laboratory Manual (1994); Gene Targeting, A Practical Approach (1993); Martin (1981 ); Evans and Kaufman (1981 ); Jainchill et al, (1969); Nakano et al. (1996); Kodama et al. (1982); and International Publication Nos. 01/088100 and 2005/080554).

The somatic cells under conditions permissive to the culture of said iNP cells can also be cultured in a suspension culture, including suspension culture on carriers or gel/biopolymer encapsulation. The term suspension culture of the stem cells means that the stem cells are cultured under non-adherent condition with respect to the culture vessel or feeder cells (if used) in a medium.

After somatic cells are introduced with the one or more mRNAs encoding a transcription factor in accordance with the method of the present invention, these cells may be cultured in a medium sufficient to maintain the pluripotency, preferably the multipotency, and the undifferentiated state. Culturing of induced neural stem/precursor cells (iNP cells) generated in this invention can use various medium and techniques developed to culture primate pluripotent stem cells, more specially, embryonic stem cells, as described in U.S. Pat. Publication 20070238170 and U.S. Pat. Publication 20030211603, and U.S. Pat. Publication 20080171385. It is appreciated that additional methods for the culture and maintenance of pluripotent stem cells, as would be known to one of skill, may be used with the present invention for the culturing of the induced neural stem/precursor cells (iNP cells).

The starting cells, i.e., the somatic cells, preferably the mature somatic cells, and the end, reprogrammed cells may generally have differing requirements for culture medium and conditions which are defined above for the reprogrammed cell (i.e., the induced neural stem/precursor cell (iNP cell)). Requirements for culture medium and conditions for mature somatic cells are known to the person skilled in the art.

In a preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein the somatic cell, preferably the mature somatic cell, is a mature fibroblast cell, preferably a mammalian mature fibroblast cell. In general, fibroblasts are found within fibrous connective tissue and are associated with the formation of collagen fibres and ground substance of connective tissue. While mammalian fibroblasts from any source such as, for example lung fibroblasts, kidney fibroblasts, cardiac fibroblasts, stromal fibroblasts, foreskin fibroblasts and the like, may be used in the methods of the present invention, mature mammalian dermal fibroblasts provide a convenient source of somatic cells. Such fibroblasts can be conveniently obtained from a commercial source or, if desired, may be isolated from various tissues using well established and documented techniques. Fibroblasts can be obtained from any source such as, for example lung fibroblasts, kidney fibroblasts, cardiac fibroblasts, stromal fibroblasts, foreskin fibroblasts and the like, when used in the methods of the present invention. However, as will be appreciated, mature human dermal fibroblasts provide a convenient source of somatic cells. Fibroblasts may be conveniently obtained from a commercial source or, if desired, isolated from tissue sources using well established and documented laboratory techniques and equipment.

In another preferred embodiment and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein the somatic cell, preferably the mature somatic cell, is a cell derived from the mammalian immune system, preferably from a component of peripheral mononuclear blood cells. Preferably, said cell derived from the mammalian immune system is a leukocyte, more preferably a lymphocyte.

In a preferred embodiment, the somatic cell, preferably the mature somatic cell, more preferably the mature mammalian somatic cell, is a cell from a patient suffering from a neurological disease or disorder or injury in which tissue regeneration and/or restored functionality is a component of therapy or healing. Preferably, in this embodiment, the reprogrammed multi-potent lineage-specific precursor cell is a disease-specific reprogrammed multi-potent lineage-specific precursor cell. More preferably, the mammalian somatic cell, preferably the mature mammalian somatic cell is a cell from a patient suffering from a neurological disease or disorder wherein the neurological disease or disorder is Parkinon's disease or Huntington's disease. The term "restored functionality" refers to a component of healing or therapy wherein cells are employed which secrete beneficial components or substances (like, e.g., dopamine) into the environment and/or to a component of healing or therapy wherein new replacement lineage-specific tissues are generated. In a preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein said reprogrammed induced neural stem/precursor cell (iNP cell) expresses elevated or reduced levels of at least one neural lineage marker selected from the group consisting of Pax6, Sox2, Lmxla, Ascl1 , FoxG1 , Gsx2, Lhx2, Ngn2, Otx2, Six3, Hes1 , Hes5, Sox1 , Sox3, Mash1/Ashl1 , Nurrl , Pitx3, HoxB9, Irx3, FOXA2, GLI1 , LMX1 B and neurogenin 2 compared to a control cell.

As a control cell, a cell may be used which is transfected with modified mRNA coding eGFP (enhanced green fluorescent protein). Alternatively, for control experiments, protein co-transductions may be carried out using a mix of Sox2-TAT protein (PeproTech) and Pax6 protein (Abnova) or, as a control, using PE control protein (OzBioscience) with ProDeliverln (OzBioscience) in a ratio 1 :1 and a total protein amount of 5pg per transduction cycle. Control cells, i.e., the eGFP- and PE-treated control cells show either no mRNA expression or relatively lower levels of these markers. In contrast, in accordance with the appended examples, as shown by quantitative PCR, in a reprogrammed induced neural stem/precursor cell (iNP cell), the expression of other markers, e.g., the expression of Sox2 and Pax6 is elevated while Hes1 and Irx3 mRNA levels are reduced in iNP colonies generated by Sox2/Pax6 protein transduction compared to control cells.

Whether the expression level of at least one neural lineage marker selected from the group consisting of Pax6, Sox2, Lmxla, AscH , FoxG1 , Gsx2, Lhx2, Ngn2, Otx2, Six3, Hes1 , Hes5, Sox1 , Sox3, Mash1/Ashl1 , Nurrl , Pitx3, HoxB9, Irx3, FOXA2, GLI1 , LMX1 B and neurogenin 2 compared to a control cell has changed and, accordingly, is classified as a reprogrammed induced neural stem/precursor cell (iNP cell), can be determined by the skilled person by methods known in the art and as outlined in the appended examples. Thus, the skilled person can determine whether a mature somatic cell has been reprogrammed into an induced neural stem/precursor cell (iNP cell) with the method of the present invention by applying methods known in the art. Accordingly, by assessing whether a somatic cell into which one or more mRNAs encoding a transcription factor has been introduced has been reprogrammed into an induced neural stem/precursor cell (iNP cell) can be assessed by determining the expression level of at least one neural lineage marker selected from the group consisting of Pax6, Sox2, Lmxla, AscH , FoxG1 , Gsx2, Lhx2, Ngn2, Otx2, Six3, Hes1 , Hes5, Sox1 , Sox3, Mash1/Ashl1 , Nurrl , Pitx3, HoxB9, Irx3, FOXA2, GLI1 , LMX1B and neurogenin 2.

In a preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein a cell is classified as a reprogrammed induced neural stem/precursor cell (iNP cell) provided that the expression of one or more marker selected from the group consisting of Ngn2, Sox1 , HoxB9 and Six3 is/are elevated and the expression of one or more marker selected from the group consisting of Hes1 and Irx3 is/are reduced compared to control cells.

The latter expression profile is observed when iNPs cells, in accordance with the method of the present invention, are generated by the expression of the transcription factors Sox2 and Pax6 and/or LMX1A.

In a preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein said mRNA(s) encoding a transcription factor encode(s) one or more transcription factors selected from the group consisting of Sox2, Lmxla and Pax6.

The Sox family of genes is associated with maintaining pluripotency similar to Oct- 3/4, although it is associated with multipotent and unipotent stem cells in contrast with Oct-3/4, which is exclusively expressed in pluripotent stem cells. While Sox2 was the initial gene identified for induction and is the preferred one, other genes in the Sox family have been found to work as well in the induction process. Sox1 yields iPS cells with a similar efficiency as Sox2, and genes Sox3, Soxl 5, and Sox 18 also generate iPS cells, although with decreased efficiency.

Lmxla (LIM homeobox transcription factor 1 , alpha, also known as LMX1A) is a protein which is encoded by the LMX A gene. Lmx1 , a LIM homeobox transcription factor, is known to bind an A T -rich sequence in the insulin promoter and stimulates transcription of insulin. Insulin is produced exclusively by the beta cells in the islets of Langerhans in the pancreas. The level and beta-cell specificity of insulin gene expression are regulated by a set of nuclear genes that bind to specific sequences within the promoter of the insulin gene INS and interact with RNA polymerase to activate or repress transcription.

Pax6, i.e., paired box protein Pax-6 also known as aniridia type II protein (AN2) or oculorhombin is a protein that in humans is encoded by the Pax6 gene. Pax6 is a transcription factor present during embryonic development. The encoded protein contains two different binding sites that are known to bind DNA and function as regulators of gene transcription. It is a key regulatory gene of eye and brain development. Within the brain, the protein is involved in development of the specialized cells that process smell. As a transcription factor, Pax6 activates and/or deactivates gene expression patterns to ensure for proper development of the tissue. Mutations of the Pax6 gene are known to cause various disorders of the eyes. Two common disorders associated with a mutation are: aniridia, the absence of the iris, and Peter's anomaly, thinning and clouding of the cornea. A "knockout" model has been created using mice which do not express Pax6. The "knockout" model is eyeless or has very underdeveloped eyes further indicating Pax6 is required for proper eye development. More specifically, Pax6 is a member of the Pax gene family. It acts as a "master control" gene for the development of eyes and other sensory organs, certain neural and epidermal tissues as well as other homologous structures, usually derived from ectodermal tissues. However it has been recognized that a suite of genes is necessary for eye development, and therefore the term of "master control" gene may be inaccurate. This transcription factor is most noted for its use in the interspecifically induced expression of ectopic eyes and is of medical importance because heterozygous mutants produce a wide spectrum of ocular defects such as Aniridia in humans.

Pax6 serves as a regulator in the coordination and pattern formation required for differentiation and proliferation to successfully take place, ensuring that the processes of neurogenesis and oculogenesis are carried out successfully. As a transcription factor, Pax6 acts at the molecular level in the signaling and formation of the central nervous system. The characteristic paired DNA binding domain of Pax6 utilizes two DNA-binding domains, the paired domain (PD), and the paired-type homeodomain (HD). These domains function separately via utilization by Pax6 to carry out molecular signaling that regulates specific functions of Pax6. An example of this lies in HD's regulatory involvement in the formation of the lens and retina throughout oculogenesis contrasted by the molecular mechanisms of control exhibited on the patterns of neurogenesis in brain development by PD. The HD and PD domains act in close coordination, giving Pax6 its multifunctional nature in directing molecular signaling in formation of the CNS. Although many functions of Pax6 are known, the molecular mechanisms of these functions remain largely unresolved. The vertebrate PAX6 locus encodes at least three different protein isoforms, these being the canonical PAX6, PAX6(5a), and ΡΑΧ6(ΔΡϋ). The canonical PAX6 protein contains an N-terminal paired domain, connected by a linker region to a paired-type homeodomain, and a proline/serine/threonine (P/S/T)-rich C- terminal domain. The paired domain and paired-type homeodomain each have DNA binding activities, while the P/S/T-rich domain possesses a transactivation function. PAX6(5a) is a product of the alternatively spliced exon 5a resulting in a 14 residue insertion in the paired domain which alters the specificity of this DNA binding activity. The nucleotide sequence corresponding to the linker region encodes a set of three alternative translation start codons from which the third PAX6 isoform originates. Collectively known as the PAX6(APD) or pairedless isoforms, these three gene products all lack a paired domain. The pairedless proteins possess molecular weights of 43, 33, or 32kDa, depending on the particular start codon used. PAX6 transactivation function is attributed to the variable length C-terminal P/S T-rich domain which stretches to 153 residues in human and mouse proteins.

It is known that a combination of Sox2 and Pax6 is capable of producing iNP cells while a combination of Sox2 and Lmxla is speculated to produce dopamine- producing neuron precursor cells via iPS cells, more specifically iNP cells.

Accordingly, in a preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein said mRNA(s) encoding a transcription factor encode the transcription factors Sox2 and Pax6 or Sox2 and Lmxl a.

In a preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein said mRNA(s) encoding a transcription factor encode the transcription factors Sox2 and Lmxla. In a more preferred embodiment, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell, into a reprogrammed induced neural stem/precursor cell (iNP cell) having midbrain dopaminergic neuron characteristics (dopaminergic neuron fate, i.e., dopamine-producing neuron precursor cells) wherein said mRNA(s) encoding a transcription factor encode the transcription factors Sox2 and Lmxla. In a preferred embodiment, and in accordance with the foregoing, said reprogrammed induced neural stem/precursor cell (iNP cell) having midbrain dopaminergic neuron characteristics (dopaminergic neuron fate, i.e., dopamine-producing neuron precursor cells) expresses elevated or reduced levels of at least one neural lineage marker (more specifically mesencephalic genes) selected from the group consisting of FOXA2, GLI1 , LMX1 B, Nurrl and Pitx3 compared to a control cell as defined above.

In a preferred embodiment the mRNA molecule of the present invention contains a combination of modified and unmodified nucleotides. Preferably, the mRNA molecule of the present invention contains a combination of modified and unmodified nucleotides as described in WO 2011/012316. Such RNA molecules are also known and commercialized as "SNIM^®-RNA". The RNA molecule described in WO 2011/012316 is reported to show an increased stability and diminished immunogenicity. In a preferred embodiment, in such a modified mRNA molecule 5 to 50% of the cytidine nucleotides and 5 to 50% of the uridine nucleotides are modified. The adenosine- and guanosine-containing nucleotides can be unmodified.

Thus, in a preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein said mRNA(s) encoding said transcription factor(s) contain(s) a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides. The adenosine and guanosine nucleotides can be unmodified or partially modified, and they are preferably present in unmodified form. Preferably 10 to 35% of the cytidine and uridine nucleotides are modified and particularly preferably the content of the modified cytidine nucleotides lies in a range from 7.5 to 25% and the content of the modified uridine nucleotides in a range from 7.5 to 25%. It has been found that in fact a relatively low content, e.g. only 10% each, of modified cytidine and uridine nucleotides can achieve the desired properties. It is particularly preferred that the modified cytidine nucleotides are 5-methylcytidin residues and the modified uridine nucleotides are 2-thiouridin residues. Most preferably, the content of modified cytidine nucleotides and the content of the modified uridine nucleotides is 25%, respectively.

In another preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein said mRNA(s) encoding said transcription factor(s) is an mRNA wherein 5 to 30%, preferably 7.5 to 25%, of the uridine nucleotides and 5 to 30%, preferably 7.5 to 25%, of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.

The mRNA(s) encoding a transcription factor as used in the method of the present invention may be recombinantly (e.g., in an in vivo or an in vitro system) or synthetically generated/synthesized by methods known to the person skilled in the art.

More specifically, the mRNA(s) encoding a transcription factor of the present invention may be produced recombinantly in in vivo systems by methods known to the person skilled in the art.

Alternatively, the mRNA(s) encoding a transcription factor of the present invention may be produced in an in vitro system using, for example, an in vitro transcription system. In vitro transcription systems are commonly known and usually require a purified linear DNA template containing a DNA sequence "encoding" the mRNA(s) encoding a transcription factor wherein said DNA sequence is under the control of an appropriate promoter. Moreover, an in vitro transcription system also commonly requires ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate RNA polymerase which provides the enzymatic activity for the in vitro transcription of the DNA sequence into RNA. Methods which are commonly used to produce RNA molecules using in vitro transcription are well- known to the person skilled in the art and are, e.g., described in Methods Mol. Biol. 703 (2011 ):29-41.

Furthermore, the mRNA(s) encoding a transcription factor of the present invention can be chemically synthesized, e.g., by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid-phase support and standard techniques.

Methods to recover/purify the mRNA(s) which are either produced in an in vivo system or an vitro reaction are known to the person skilled in the art.

Thus, in a preferred embodiment, and in accordance with the foregoing, the present invention relates to a method of reprogramming a somatic cell, preferably a mature somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell) wherein said mRNA(s) encoding said transcription factor(s) is/are in vitro transcribed mRNA(s) (IVT mRNA(s)).

In a preferred embodiment, and in accordance with the foregoing, the somatic cell or the mature somatic cell is a mammalian somatic cell and a mammalian mature somatic cell, respectively.

The present invention also relates to a method of reprogramming a somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell) as described herein above, wherein said method further comprises the step of formulating the thus produced iNPs cells into a pharmaceutical composition.

In another preferred embodiment, the present invention relates to a reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method in accordance with the present invention and as described herein above. The thus produced reprogrammed induced neural stem/precursor cell (iNP cell) shows elevated or reduced expression levels of least one neural lineage marker selected from the group consisting of Pax6, Sox2, Lmxl a, AscH , FoxG1 , Gsx2, Lhx2, Ngn2, Otx2, Six3, Hes1 , Hes5, Sox1 , Sox3, Mash1/Ashl1 , Nun , Pitx3, HoxB9, Irx3, FOXA2, GLI1 , LMX1 B and neurogenin 2 compared to control cells as described herein above. As mentioned above, whether a cell shows elevated or reduced expression levels of at least one neural lineage marker selected from the group consisting of Pax6, Sox2, Lmxla, AscM , FoxG1 , Gsx2, Lhx2, Ngn2, Otx2, Six3, Hes1 , Hes5, Sox1 , Sox3, Mash1/Ashl1 , Nurrl , Pitx3, HoxB9, Irx3, FOXA2, GLI1 , LMX1 B and neurogenin 2 and, accordingly, is classified as a reprogrammed induced neural stem/precursor cell (iNP cell), can be determined by the skilled person by methods known in the art and as outlined in the appended examples. Thus, the skilled person can determine whether a somatic cell, preferably a mature somatic cell, has been reprogrammed into an induced neural stem/precursor cell (iNP cell) with the method of the present invention by applying methods known in the art. Accordingly, by assessing whether a somatic cell into which one or more mRNAs encoding a transcription factor has been introduced has been reprogrammed into an induced neural stem/precursor cell (iNP cell) can be assessed by determining whether said cell shows elevated or reduced expression levels of at least one neural lineage marker selected from the group consisting of Pax6, Sox2, Lmxla, AscH , FoxG1 , Gsx2, Lhx2, Ngn2, Otx2, Six3, Hes1 , Hes5, Sox1 , Sox3, Mash1/Ashl1 , Nurrl , Pitx3, , HoxB9, Irx3, FOXA2, GLI1 , LMX1 B and neurogenin 2.

In a preferred embodiment, and in accordance with the foregoing, the present invention relates to a reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method in accordance with the present invention wherein said reprogrammed induced neural stem/precursor cell (iNP cell) is characterized that the expression of one or more marker selected from the group consisting of Ngn2, Sox1 , HoxB9 and Six3 is/are elevated in said cell and the expression of one or more marker selected from the group consisting of Hes1 and Irx3 is/are reduced in said cell compared to control cells. The latter expression profile is observed when iNPs cells, in accordance with the method of the present invention, are generated by the expression of the transcription factors Sox2 and Pax6.

In another embodiment, the present invention relates to one or more mRNA molecules encoding a transcription factor wherein said transcription factor(s) is/are selected from the group consisting of Sox2 or Pax6 or Lmxla and wherein said mRNA contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides. As regards the preferred embodiments of the mRNA encoding a transcription factor wherein said transcription factor is Sox2 or Pax6 or Lmxla and wherein said mRNA contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides, the same applies, mutatis mutandis, as has been set forth above in the context of the mRNA molecule as defined above in the context of the method of the present invention.

In a preferred embodiment, the present invention relates to one or more mRNA molecules encoding a transcription factor wherein said transcription factor(s) is/are selected from the group consisting of Sox2 or Pax6 or Lmxla and wherein said mRNA contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides, wherein said mRNA is in vitro transcribed mRNA (IVT mRNA).

In another preferred embodiment and in accordance with the foregoing, the present invention relates to one or more mRNA molecules encoding a transcription factor wherein said transcription factor(s) is/are selected from the group consisting of Sox2 or Pax6 or Lmxla, wherein said polynucleotide is an mRNA wherein 5 to 30%, preferably 7.5 to 25%, of the uridine nucleotides and 5 to 30%, preferably 7.5 to 25%, of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.

The present invention also relates to the use of mRNA encoding a transcription factor for reprogramming a somatic cell, preferably a mature somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell) as defined above. As regards the preferred embodiments of the use the same applies, mutatis mutandis, as has been set forth above in the context of the method of the present invention. The reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be used in a cell transplantation therapy. Preferably, the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be used in the treatment or prevention of a neurological disorder. Cell transplantation therapy using stem cells offers a viable treatment strategy for patients with brain disease or injury, such as Parkinson's disease, Huntington's disease, stroke or spinal cord injury, by providing new cells to replace those lost through disease or accident. Cell transplantation therapy using stem cells also offers a treatment strategy for other neurological disorders or diseases like, e.g., Batten disease, Amyotrophic Lateral Sclerosis (ALS), Brain damage, Brain dysfunction, Spinal cord pathology, inflammation, injury, peripheral neuropathy, Cranial nerve disorder (Trigeminal neuralgia), epilepsy, essential tremor, Tourette's syndrome, multiple sclerosis and stroke.

Thus, the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be used in a cell transplantation therapy, preferably, in the treatment or prevention of a neurological disorder, wherein the neurological disorder is selected from the group consisting of brain disease or injury, Parkinson's disease, Huntington's disease, stroke or spinal cord injury, Batten disease, Amyotrophic Lateral Sclerosis (ALS), brain damage, brain dysfunction, spinal cord pathology, inflammation, injury, peripheral neuropathy, cranial nerve disorder (Trigeminal neuralgia), epilepsy, essential tremor, Tourette's syndrome, multiple sclerosis and stroke.

As mentioned above, induced neural stem/precursor cell (iNP cell) have the capability to grow indefinitely while maintaining the ability to generate all cell types of the neural lineage in the body. These properties may be utilized to treat patients with various diseases or injuries, including brain injury and disease, thereby revolutionising regenerative medicine with the induced neural stem/precursor cell (iNP cell) as produced by the method of the present invention. Thus, the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention provide an optimal cell source for regenerative medicine since, preferably, the induced neural stem/precursor cell (iNP cell) may be produced by a method according to the present invention from mature somatic cells which are directly obtained from the patient as described herein above. Accordingly, the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention are particularly useful in medical settings and in the treatment of a certain disease.

Therefore, the present invention also relates to a pharmaceutical composition comprising the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention and optionally a pharmaceutically acceptable carrier.

The term "treatment" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. Accordingly, the treatment of the present invention may relate to the treatment of (acute) states of a certain disease but may also relate to the prophylactic treatment in terms of completely or partially preventing a disease or symptom thereof. Preferably, the term "treatment" is to be understood as being therapeutic in terms of partially or completely curing a disease and/or adverse effect and/or symptoms attributed to the disease. "Acute" in this respect means that the subject shows symptoms of the disease. In other words, the subject to be treated is in actual need of a treatment and the term "acute treatment" in the context of the present invention relates to the measures taken to actually treat the disease after the onset of the disease or the breakout of the disease. The treatment may also be prophylactic or preventive treatment, i.e., measures taken for disease prevention, e.g., in order to prevent an infection and/or the onset of the disease.

The pharmaceutical composition of the present invention comprising the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be administered via a large range of classes of forms of transplantation. Methods to transplant cells for transplantation therapy are known to the person skilled in the art. Without being bound to theory, the cells to be transplanted may be transplanted by a local injection, intracranial injection, stereotaxic injection or by an injection into the vertebral channel. The methods to transplant cells for transplantation therapy may preferably be supported by "homing". "Homing" is a phenomenon where cells migrate autonomously to the place of their origin, a process which is mediated by chemokines. Thus, via "homing", the transplanted iNP cells produced by a method according to the present invention travel to the bone marrow or the brain and engraft or establish residence in the bone marrow or in the brain. It is known that iNP cells do not easily thrive when engrafted. Therefore, in a preferred embodiment of the present invention, the addition of a biomaterial scaffold is favourable. Preferred scaffolds may harbour extracellular matrix molecules, growth factors and/or immobilizing drugs to support cell replacement and restauration of neurological functions by iNP cells as it is, e.g., described in a review article by Skop et al. ⁴⁴.

As mentioned, the present invention relates to a pharmaceutical composition, comprising an effective amount of the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention in accordance with the above and at least one pharmaceutically acceptable excipient or carrier.

An excipient or carrier is an inactive substance formulated alongside the active ingredient, i.e., the iNPs produced in accordance with the present invention, for the purpose of bulking-up formulations that contain potent active ingredients. Excipients are often referred to as "bulking agents," "fillers," or "diluents". Bulking up allows convenient and accurate dispensation of a drug substance when producing a dosage form. They also can serve various therapeutic-enhancing purposes, such as facilitating drug absorption or solubility, or other pharmacokinetic considerations. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. The selection of appropriate excipients also depends upon the route of administration and the dosage form, as well as the active ingredient and other factors.

Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions comprising the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention can be administered to the subject at a suitable dose, i.e., in "an effective amount" which can easily be determined by the skilled person by methods known in the art. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's or subject's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

Thus, preferably, the iNPs produced in accordance with the present invention are included in the pharmaceutical composition in an effective amount. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition comprising the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention is to be administered. In accordance with the above, the content of the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention in the pharmaceutical composition is not limited as far as it is useful for treatment as described above, but preferably contains 1 ,000 to 100,000 50,000 cells/μΙ, more preferably about 50,000 cells/μΙ while, e.g., 750,000 cells per injection site may be injected. Further, the pharmaceutical composition described herein is preferably employed in a carrier. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to render the composition isotonic. Examples of the carrier include but are not limited to saline, Ringer's solution and dextrose solution. Preferably, acceptable excipients, carriers, or stabilisers are non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt- forming counter-ions, e.g. sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g. serum albumin, or gelatine; hydrophilic polymers, e.g. polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrins; monosaccharides; disaccharides; other sugars, e.g. sucrose, mannitol, trehalose or sorbitol; chelating agents, e.g. EDTA; non-ionic surfactants, e.g. Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and/or preservatives, e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g. methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Suitable carriers and their formulations are described in greater detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co. Progress can be monitored by periodic assessment.

In accordance with this invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient.

As mentioned, the pharmaceutical composition comprising the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be for use in cell transplantation therapies. Preferably, the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be used in the treatment or prevention of a neurological disorder. In a particular embodiment, the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be used in the treatment or prevention of patients with brain disease or injury, such as Parkinson's disease, Huntington's disease, stroke or spinal cord injury. In other embodiments, the pharmaceutical composition comprising the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be for the treatment or prevention of a neurological disorder, wherein the neurological disorder is selected from the group consisting of brain disease or injury, Parkinson's disease, Huntington's disease, stroke or spinal cord injury, Batten disease, Amyotrophic Lateral Sclerosis (ALS), brain damage, brain dysfunction, spinal cord pathology, inflammation, injury, peripheral neuropathy, cranial nerve disorder (Trigeminal neuralgia), epilepsy, essential tremor, Tourette's syndrome, multiple sclerosis and stroke. In preferred embodiments, the pharmaceutical composition comprising the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention may be for the treatment or prevention of Parkinson's disease, Huntington's disease.

In another embodiment, the invention also relates to a method of cell transplantation therapies making use of the iNP cells produced by a method of the present invention. Thus, the present invention relates to a method for the treatment of a neurological disorder or disease by a cell transplantation therapy making use of the iNP cells produced by a method of the present invention. As regards the preferred embodiments of the method for treatment the same applies, mutatis mutandis, as has been set forth above in the context of the pharmaceutical composition comprising the reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to the present invention as defined above.

In the present invention, the subject is in a preferred embodiment a mammal such as a dog, cat, pig, cow, sheep, horse, rodent, e.g., rat, mouse, and guinea pig, or a primate, e.g., gorilla, chimpanzee, and human. In a most preferable embodiment, the subject is a human.

Figure 1: Optimization of the amount of SNIM® to transfect with and the number of consecutive transfections by determining the transfection efficiency and the cell survival.

Figure 2: Time course of cell reprogramming following SOX2 and PAX6 SNIM®

RNA co-tranfection in adult HDFs.

Figure 3: shows qPCR-experiments determining the gene expression profile of

SOX2 and PAX6 SNIM® RNA derived iNP cells.

Figure 4: shows the differentiation of SOX2 and PAX6 SNIM®-derived iNPs for

21 days using a mixed neuronal differentiation media resulted in the generation of TUJ1+ neurons co-expressing either GAD65/67 or vGLUT. Scale bars = 50 μιτι.

Figure 5: Schematic diagram showing the induction of iNPs from adult HDFs using SOX2 and PAX6 SNIM® RNA transfection.

Figure 6: The determination of the optimal concentration of SNIM® RNA for effectively transfecting HDFs (A, C and D).

Analysis of the cell viability 3 days following transfection (B). Examination of the effect of co-transfecting SOX2 and PAX6 SNIM® RNA (E, G and H). SNIM® RNA transfection of either SOX2 alone, PAX6 alone or co- transfection of SOX2 and PAX6 at a concentration of either 0.25 or 0.5 pg SNIM® RNA maintained cell viability of at 80% or greater (F).

Figure 7: Comparison of cell viability following a single transfection (A).

Determination of the effect on cell viability of transfecting HDFs either once or four times for 5 hrs or 24 hrs duration for (B) SNIM® RNA GFP and for (C) SNIM® RNA SOX2 and/or PAX6.

Image demonstrating the co-expression of SOX2 (green) and PAX6 (red) protein (D). Nuclei are stained in blue by DAPI.

* P < 0.05; ** P < 0.01 ; **^* P < 0.001.

Figure 8: Time course of cell reprogramming following SOX2/PAX6 SNIM® RNA co-transfection in adult HDFs. Scale bar = 300 pm.

Figure 9: Gene expression profile of SOX2/PAX6 SNIM® RNA derived iNP cells.

(A) SOX2 and PAX6 transgene expression.

(B) SOX2/PAX6 SNIM® RNA derived iNP cells express the neuroectoderm markers LHX2, FOXG1 , OTX2 and SIX3.

(C) SOX2/PAX6 SNIM® RNA derived iNP cells express the pro-neural genes NGN2 and ASCL1 as well as region-specific neural transcription factors GSH2, GBX2, EN1 and PITX3.

(D) Image demonstrating the PAX6 (red) protein expression while SOX2 expression was not detected. Nuclei are stained in blue by DAPI.

(E) Image demonstrating the co-expression of Nestin (green) and FOXG1 (red) 28 days following transfection. Nuclei are stained in blue by DAPI.

Figure 10: Gene expression profile of SOX2/LMX1A SNIM® RNA derived iNP cells.

Figure 11: (A) Differentiation of SOX2/PAX6 SNIM®-derived iNPs. Scale bars

μητι. (B) Graph showing an increase in the generation of TUJ1 + neurons following differentiation on iNPs obtained at either Replate (RPL) 2, 3, or 4.

*** P < 0.001.

Figure 12: Schematic diagram showing the induction of iNPs from adult HDFs using SOX2 and PAX6 SNIM® RNA transfection.

Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.

Examples

I. Experimental design

An overview of the timeline and steps of the iNP reprogramming process is shown in Figures 5 and 12. In this protocol, the transcription factors SOX2 and PAX6 and/or LmxIA are transiently delivered into the cells by a non-viral method, using SNIM® RNA transfection.

For SNIM® RNA transfection, the GFP, SOX2 and PAX6 and LmxIA synthetic RNA was designed and manufactured by Ethris GmbH.

Primary adult human fibroblast cells were purchased from Cell Applications Ltd. Cells were proliferated and passaged in flasks until the required cell number was reached.

Cells were plated onto uncoated Nunclon plates at a density of -42,000 cells per cm²

(6-well plate format is a common choice). After overnight attachment, cells were co- transfected either with eGFP SNIM® RNA only, or with SOX2 and PAX6 SNIM®

RNA or with SOX2 and LMX1A SNIM® RNA, using Lipofectamine RNAiMAX transfection reagent (Life Technologies) for either 5 hr or 24hr. Cells were also transfected either once, or 2, 3, or 4 times (Figure 1 and Figures 6 and 7). After the transfection incubation, the cells received fresh neural reprogramming medium. If multiple RNA transfections were required, cells were given fresh transfection medium. Media was changed three times per week, and 3 days after the end of the final transfection, cells were replated. Replating occurred weekly thereafter and the cells were collected when iNP colony formation is optimal (see Figure 2B and Figure 8). The number of cells is greatly reduced during this process, hence it is recommended that around 5 times more fibroblasts are transfected than iNP cells required for downstream applications.

The iNP cells were characterized by analysis for the expression level of various neural stem and precursor genes, and positional markers within the neuroectodermal tube such as OCT3/4, SOX1, SOX2, NANOG, PAX6, BMI1, HES1, FOXG1, SIX3, GLI3, NGN2, EMX2, TBR2, DLX2, ASCL1, NCAM1, OLIG2 ¹⁷ , IRX3, HOXB9, NGN2, FOXA2, GLI1, NURR1, PITX3, OTX2 (Figure 3 and Figure 9). Total RNA was isolated from colonies of independent iNP cell lines and fibroblast control lines using the Nucleospin RNA kit (Macherey Nagel). cDNA was synthesized from total RNA using Superscript III reverse transcriptase (Life Technologies). Three independent duplex qPCR reactions were performed for each independent sample using the TaqMan® system (Applied Biosystems) with ribosomal 18S rRNA as the internal standard and an equivalent of 4-1 Ong mRNA per reaction. The fold change in gene expression was calculated using the ΔΔ-CT method⁴¹ and presented relative to the mean expression level in fibroblast control lines.

Fully formed iNP cultures were collected either for immunocytochemistry or for differentiation at 21 or 28 days post transfection. Immunocytochemical staining was on the iNP cultures to confirm expression of neural stem and precursor markers, using standard methods. Induced neural precursor cell cultures formed by plasmid DNA transfection have shown the ability to differentiate into neuronal and glial phenotypes after exposure to specific differentiation reagents ^{17 21}. Using a mixed neuronal differentiation media, SOX2/PAX6 SNIM® RNA derived iNP cells differentiate into neurons co-expressing the neuronal marker TUJIand the phenotype-specific markers GAD₆5/67 or vGlut after 14 - 21 days in culture (Figure 4 and Figure 11 ). A proportion of the SNIM® RNA derived iNP cells also express GFAP and exhibit an astrocytic morphology (Figure 4).

II. Materials

Reagents Alamar blue® cell viability reagent (Life Technologies; DAL1025)

Ascorbic acid (Sigma; A4403)

Astrocytic differentiation medium (see Reagent Setup)

B27 supplement with Vitamin A, 50x (Life Technologies; 17504-044)

B27 supplement without Vitamin A, 50x (Life Technologies; 12587-010)

BDNF (Peprotech; 450-02)DMEM (Life Technologies; 11965092)

Donkey serum (Sigma; D9663)Fibroblast cells, adult dermal (Cell Applications Ltd;

106-05a or the Coriell Repository)

Fibroblast growth medium (Cell Applications Ltd; 116-500)

Fibroblast proliferation medium (see Reagent Setup)

Fibronectin (BD Biosciences; 356008)

Foetal bovine serum (FBS) (Life Technologies; 10094142)

Glasgow minimum essential medium (Sigma; G5154)

Goat serum (Life Technologies; 16210-064)

Heparin sodium salt (Sigma; H3149)

K2 transfection system ® (Biontex Laboratories GmbH; T060-8.0)

Laminin, cone (Life Technologies; 23017-015)

Lipofectamine RNAiMAX (Life Technologies)

N2 supplement (Life Technologies; 17502-048)

Neural plating medium (see Reagent Setup)

Neural reprogramming medium (see Reagent Setup)

Neurobasal-A medium (Life Technologies; 10888022)

Neuronal Mixed differentiation medium (see Reagent Setup)

Opti-MEM (Life Technologies; 31985062)

Penicillin-Streptomycin-Glutamine (Life Technologies; 10378-016)

Poly-ornithine (Sigma; P3655)

Nucleospin RNA isolation kit (Macherey Nagel; 740955.50)

Recombinant human fibroblast growth factor (FGF2) (Peprotech; 100-18B)

Recombinant human epidermal growth factor (EGF) (Peprotech; AF-100-15)

Recombinant human Midkine (Peprotech; 450-16)

Recombinant human Sonic Hedgehog/Shh (Millipore; MPGF174)

Retinoic acid (Sigma; R2625)

Superscript® III First-Strand Synthesis System (Life Technologies; 18080-051 ) TaqMan® reagents for qPCR (Applied Biosystems/Life Technologies) - see Table 1 TaqMan® Gene Expression Master mix (Applied Biosystems; 4369514)

Triton-X-100 (Scharlau; TR0444005P)

Trypsin-EDTA (Life Technologies; 25300054)

Y27632 (Calbiochem; 688001 )

Valpromide (Sigma; V3640)

VPA - Valproic acid sodium salt (Sigma; P4543)

Reagent Setup

Human Dermal Fibroblast Proliferation Medium: DMEM + 2% FBS + 1 % glutamine.

Neural Reprogramming Medium: This medium consists of Neurobasal-A medium,

0.3% D-glucose, 1 % Penicillin/Streptomycin/Glutamine, 2% B27 supplement, 20 ng/mL EGF, 2 pg/mL Heparin, 20 ng/mL FGF2 and a final concentration of 1 mM VPA.

Neuronal Mixed Differentiation Medium: This medium consists of Neurobasal A medium, 0.3% D-glucose, 1 % Penicillin/Streptomycin/Glutamine, 2% B27 supplement, 25 ng/mL FGF2, 0.01 mM Retinoic acid,1 % N2 supplement, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dCAMP and 200 nM ascorbic acid.

III. Procedures

1. Expansion of human dermal fibroblasts

1.1. Loosen lid of ampoule and thaw quickly by placing the lower half of tube in a 37 °C water bath until a small piece of ice remains (approximately 1 min).

1 .2. Dilute cells in 5 mL of Fibroblast proliferation media, centrifuge at 350 g for 5 min (to remove DMSO from the sample). Decant off supernatant then flick tube hard to break up cell pellet. Add half the appropriate volume of media for the flask size and add cells to flask, wash the tube with remaining media to meet the total volume for the flask (5 mL for T25, 10 mL for T75 flask).

1.3. Distribute cells evenly and incubate at 5% C0₂, 37 °C.

1.4. Do not disrupt culture for the first 24-48 hr. Change proliferation medium every second day.

Subculture ("passage") when human dermal fibroblasts (HDFs) are -70- 85% confluent. Doubling time of HDFs varies according to individual lines, but is typically 20-30 hours. Remove medium from the flask, being careful not to disturb the cell layer. Add warm PBS to rinse the cells.

Aspirate PBS and add warm Trypsin/0.05% EDTA to cover the cells (~1.5 mL T25, -2.5 ml_ T75, -3.5 mL T175 flask), rock flask until the whole surface is covered. Incubate at room temperature, tapping after 1-2 min to check for detachment. If cells are resistant to detachment, incubate at 37 °C for 1-2 minutes. Do not leave trypsin on longer than necessary. As soon as all cells are loose, add fibroblast proliferation media.

Collect cells into a 50 mL tube, and rinse flask with more media, until all cells are collected. Centrifuge (350 g; 5 min), decant off supernatant and discard, tap to loosen pellet then resuspend, and plate out as appropriate (a 1 :2 split usually means cells will need passaging again in 2-3 days), rinsing tube of remaining cells with fresh media.

Repeat until the desired number of cells has been reached. RNA Transfection of human dermal fibroblasts

Plate out cells. Trypsinize HDFs as above, collect and count cells. Plate out at a density of ~42,000/cm² per well (high density) on uncoated Nunclon plates (ie -400,000 cells in a 6-well format well, -75,000 cells in a 24-well format well, -14,000 in a 96-well format well).

Consider including 1 extra well for GFP transfection control (unless using fluorescently-tagged Sox2 and Pax6 plasmids), plus 1-2 wells for an HDF untransfected, non-reprogrammed control.

Transfect the cells using Lipofectamine RNAiMAX (Life Technologies). Switch the cells to 1.5 mL per well (for 6-well format) Neural reprogramming media (1.5 mL per 6-well format well, 400 pL per 24-well format well, 93 pL per 96-well format well), incubate at 37 °C. The following mix was made (1 reaction is calculated per well in a 24-well plate format; make enough for a well extra if making bulk mix. Scale for wells of other sizes, 6-well format is 5 reactions, 96-well format is -0.17 reactions per well ). 0.5 pg for each SNIM RNA, per reaction, for example:

0.5 pg SOX2 SNIM + 0.5 pg PAX6 SNIM RNA

Or 0.5 pg control GFP SNIM

was made up to 50 pL per reaction with Opti-MEM medium (Life Technologies)

A second mix with the transfection reagent was made; 1 pL RNAiMAX transfection reagent to 50 pL Opti-MEM for each reaction.

The RNA mix was gently added to the RNAiMAX mix, and mixed by inverting, swirling and tapping.

Incubated for 20 min at room temperature, to allow complex formation. 100 pL of mix was added to each well of cells (24-well format; use 500 pL or 17 pL for 6-well and 96-well formats respectively).

Incubate 5 hr or overnight at 37 °C, 5% CO₂.

After the incubation, remove medium and replace with fresh neural reprogramming medium.

If performing multiple transfections, give the cells fresh transfection mix 24hr after the initial transfection.

Proceed to Reprogramming of transfected HDF cells. ramminci of transfected human dermal fibroblasts

Check the cells under a microscope. Analysis of the survival of the cells post-transfection was performed using Alamar blue® Cell Viability Reagent (following the manufacturer's protocol). The transfection efficiency was verified using the fluorescently-expressing GFP SNIM and the number of fluorescent fibroblasts were quantified by performing FACs analysis (see Figure 1).

FACs analysis for determining transfection efficiency

. When planning for FACs analysis, allow for multiple replicates of each sample and minimum counts of 10,000 cells. Triplicate wells of a 24- well plate format are appropriate. Transfect cells with a fluorescently- expressing SNIM RNA, or alternatively, transfect with non-fluorescent RNA and stain with primary antibody for the transgene and then with fluorescent secondary antibody. 2. One to three days after transfection, cells were collected by trypsinization, centrifuged and the cell pellet resuspended in an appropriate volume of FACs buffer (e.g. 500 μΙ_ of PBS + 1 % FBS). Avoid diluting the cells too much or too little because dilute solution slows down FACs analysis while a concentrated one can block the machine.

3. Pass the sample through a cell strainer into a polystyrene FACs tube (BD Falcon) to ensure single cells in suspension.

4. Perform FACs analysis to count the number of fluorescently expressing cells as a percentage of the total number of living cells in the sample. The RNA degrades quickly and the resultant protein expression is transient, with timing of peak expression occurring ~24hr after the start of transfection^38,42.

The cells were replated - remove the medium (keep the medium to conserve any floating iNP cells) and wash the cells with warm PBS (also retaining this in case of floating cells). Add trypsin/0.05% EDTA to cover the cells and tap to detach. Add warm neural reprogramming medium to collect the cells, washing them off the plate and collecting into a centrifuge tube. It may be more advantageous to pool the wells of cells at this point, or to retain them separately as biological replicates. Centrifuge at 350 g for 5 min. Resuspend in fresh neural reprogramming medium, count the cells and replate at -31 ,250 cells/cm² (i.e., 300,000 per 6-well plate format well).

Continue thrice-weekly media changes and once-weekly passages until full iNP colony formation is achieved. There may be a mixed culture of adherent and floating cells, in this case, careful media changes are imperative; take just the top media leaving the low-lying floating cells and/or centrifuge the waste media to collect the floating cells and place these back into the dish with adherent cells and fresh media. When passaging a mixed culture, centrifuge the waste media along with the trypsinized cells to ensure all the cells are retained.

Cells were collected for downstream applications. aracterization of iNP cells by QPCR gene expression analysis . Collect iNP cells. Remove the medium (keep the medium to conserve any floating iNP cells) and wash the cells with warm PBS (also retaining this in case of floating cells). Add trypsin/0.05% EDTA to cover the cells and tap to detach. Add warm neural reprogramming medium to collect the cells, washing them off the plate and collecting into a centrifuge tube. Centrifuge at 350 g for 5 min to pellet the cells. Either freeze the cell pellet at -80 °C or proceed to Step 2.

. Isolate RNA from the cell pellet using an isolation kit such as the Macherey Nagel Nucleospin RNA kit, following the manufacturer's directions. Elute RNA in a very small volume (e.g. 30 μΙ_) to ensure a high concentration of RNA. Measure RNA concentration and quality (for example, utilizing a Nanodrop spectrophotometer).

. Synthesize cDNA from high-quality RNA, using a kit such as the Superscript III First Strand Synthesis kit and with either oligoDT and/or random hexamer primers, following the manufacturer's protocol. Include a RT negative sample to confirm specificity of RT reaction and ensure genomic DNA has been removed enzymatically. Dilute synthesized cDNA, for example to 4 ng/pL, in RNAse-free water.

Dispense qPCR reagents along with the cDNA template in triplicate, follows:

Seal the plate with film and keep in the dark until loading into the qPCR machine. Tap gently to mix components in the plate and centrifuge (2000 g for 1 min) prior to thermal cycling. Perform PCR with the following parameters:

. Analyze the cycle threshold (Ct) values for FAM-labelled samples and normalize to your chosen VIC-labelled housekeeping gene. Calculate fold changes in gene expression relative to HDF samples using the AAd method⁴¹. racterization of iNP cells by immunocytochemical analysis: immune-staining iNP cells

1 . For timepoints prior to replating, plate out cells at -42,000 cells/cm² on 96- well or 24-well format plates and transfect as normal, fixing at the desired timepoint. For timepoints after replating, plate out wells for immunocytochemistry, at -31 ,250 cells/cm² on the replate day prior to the timepoint of interest. Replating the cells appears to affect the gene expression.

2. Aspirate the medium and wash with room temperature PBS

3. Fix the cells with cold 4% (vol/vol) paraformaldehyde in PB for 10 min.

4. Wash each well with PBS.

5. Permeabilize the cells with PBS + 0.2% Triton-X for 2x 5 min.

6. Add primary antibodies in PBS with 3% serum (that your secondary antibodies are raised in) and incubate overnight at 4 °C.

7. Wash the wells with PBS twice for 5 min each.

8. Add the fluorescent secondary antibodies at 1 :500 in PBS + 3% serum.

Incubate for 1 hr at room temperature.

9. Wash the wells 2x 5 min with PBS. Add 1 :1000 DAPI in 0.1 M PB for 10 min. PB rinse and add fresh 0.1 M PB for imaging.

10. Image under a fluorescent microscope. Differentiation of the iNP cells · TIMING 14 days

The protocol for generating a mixed population of neuronal sub-types was adapted from that of Brennand and colleagues⁴³.

iNP cells were collected (via trypsinization or manual dissociation). Cells were plated out at high density (-100,000 cells/cm²) in differentiation medium onto poly-omithine/laminin coated glass chamber slides.

Neuronal mixed medium. The media was gently changed strictly every 2 days for 14-2 days. Cells were fixed with 4% paraformaldehyde for immunocytochemical analyses. Characterization of iNP-derived neurons bv immunocytochemical analysis: immunostaining for iNP-derived neurons

1. Fix the differentiated cells as in Step 4-6 as outlined above under item 5.

Be extremely gentle with the cells to prevent peeling.

2. Permeabilize the cells 3x 2 min with PBS + 0.2% Triton-X.

3. Follow the subsequent steps as outlined above under item 5.

4. Image under a fluorescent microscope. Tag Man® assays

FOXG1 Hs01850784_s1

GBX2 Hs00230965_m1

GSH2 Hs00370195_m1

LHX2 Hs00180351_m1

NGN2 Hs00702774_s1

NURR1 Hs00428691_m1

OCT3/4 Hs01654807_s1

OTX2 Hs00222238_m1

SIX3 Hs00193667_m1

GLI1 Hs00171790_m1

LMX1 B Hs00158750_m1

FOXA2 Hs00232764_m1

PITX3 Hs01013935_g1

The 18S normalisation gene is labelled with VIC dye, the genes of interest with FAM dye, to allow for duplex reactions. The normalization gene is also primer-limited.

9. Antibodies

vGlut 1 :250 MBL BMP078

FOXG1 1 :500 Abeam Ab18259

Nestin 1 :500 Chemicon MAB5326

IV. Results I

The optimal concentration of SNIM® RNA by which to effectively transfect HDFs was determined by transfecting normal HDFs once for 5 hrs with a concentration range of eGFP SNIM® RNA (0.01 - 2 pg) using Lipofectamine RNAiMAX (Figure 1 A, C and D). Cells were analysed by FACS 3 days following transfection. A concentration between 0.5 to 2 pg eGFP SNIM® RNA resulted in a transfection efficiency of 90% or greater. When cells were repeatedly transfected for 5 hrs either two or three times, a transfection efficiency of at least 85% with 0.5 pg SNIM® RNA was observed. Figure C shows FACS analysis of GFP SNIM® RNA. Figure D shows an image of GFP protein in HDFs 3 days following transfection.

Figure B shows the analysis of the cell viability by Alamar Blue 3 days following transfection. 0.5 or 1 pg of eGFP SNIM® RNA resulted in over 80% cell viability compared to control. When cells were repeatedly transfected for 5 hrs either two or three times, cell viability was maintained at 80% or greater for all concentrations examined.

Based on these results, 0.5 pg SNIM® RNA were selected as the optimal concentration to effectively transfect HDFs with minimal loss of cell viability.

The effect of co-transfecting SOX2 and PAX6 SNIM® RNA was examined (Figure E, G and H). Transfection efficiency was examined at either 0.25 or 5 pg of SOX2 alone, PAX6 alone or co-transfection of SOX2 and PAX6. GFP SNIM® RNA at 0.5 pg was used as a positive control. Both, SOX2 alone and PAX6 alone at either 0.25 or 0.5 pg SNIM® RNA exhibited similar transfection efficiencies of ~80%. Further, co- transfection of SOX2 and PAX6 at either 0.25 or 0.5 pg SNIM® RNA resulted in at least 70% of transfected HDFs. FACS analysis of SOX2 and PAX6 SNIM® RNA (Figure 1 G). Image demonstrating the co-expression of SOX2 (green) and PAX6 (red) protein 24 hrs following SNIM® RNA transfected of HDFs (Figure H). Nuclei are stained in blue by DAPI. SNIM® RNA transfection of either SOX2 alone, PAX6 alone or co-transfection of SOX2 and PAX6 at a concentration of either 0.25 or 0.5 pg SNIM® RNA maintained cell viability of at 80% or greater (Figure 1 F).

Overall, a concentration of 0.5 pg each of SNIM® RNA was optimal for both maximal transfection efficiency and cell viability following co-transfection of SOX2 and PAX6. Further, HDFs could undergo multiple SNIM® RNA transfections without affecting efficiency or viability.

Time course of cell reprogramming following SOX2 and PAX6 SNIM® RNA co- transfection in adult HDFs was assessed (Figure 2). Day 3 represents the third of four consecutive days of SNIM® RNA transfection with no apparent difference in the morphology of the HDFs. From Day 6 and 7 populations of cells start to change to a rounded morphology and form small clusters. Day 7 represents the first replate following transfection. At days 10 and 19, the cells are continuing to proliferate and exhibit a change towards a more rounded, neural-like morphology. Days 10 and 19 represent a mid-stage between Replates 1-2 and Replates 2 -3, respectively. By Day 21 the cultures have become dense with the majority of the cells arranged in clusters or colonies. Day 21 represents cells prior to Replate 3 and the initiation of full iNP colony formation. iNP cells can be collected at this point or at Replate 4 (7 days later).

The Gene expression profile of SOX2 and PAX6 SNIM® RNA derived iNP cells was assessed. Adult human fibroblasts were co-transfected with 0.5 pg SOX2 and PAX6 SNIM® RNA 4x and incubated in the transfection mix for either 5 or 24 hrs. Cell samples were collected at 1 , 7, 14, 21 and 28 days post-transfection. Day 1 represents the day following the last transfection. Days 7, 14, 21 and 28 represent replate days. Figure 3 A shows that SOX2 and PAX6 transgene expression is highly expressed under both conditions with expression levels reducing at 21 or 28 days for 4x 24hr and 4x 5 hr incubation respectively. Figure 3 B shows that SOX2 and PAX6 SNIM® RNA derived iNP cells express the neuroectoderm markers LHX2, FOXG1 , OTX2 and SIX3. Interestingly, only the 4x 24 hr incubation protocol resulted in the induction of OTX2 with no change in expression seen in the 4x 5 hr incubation protocol when compare to aHDFs. The stem cell marker OCT3/4 is not expressed in SOX2 and PAX6 SNIM® RNA derived iNP cells. As shown in Figure 3 C, it was also observed that SOX2 and PAX6 SNIM® RNA derived iNP cells express the pro-neural genes NGN2 and ASCL1 as well as region-specific neural transcription factors GSH2, GBX2, EN1 and PITX3. Overall, these results demonstrate that SOX2 and PAX6 SNIM® RNA derived iNP cells express a heterogenous range of neural positioning genes.

As shown in Figure 4, the differentiation of SOX2 and PAX6 SNIM®-derived iNPs for 21 days using a mixed neuronal differentiation media resulted in the generation of TUJ1+ neurons co-expressing either GAD65/67 or vGLUT.

A schematic diagram showing the induction of iNPs from adult HDFs using SOX2 and PAX6 SNIM® RNA transfection is shown in Figure 5. The optimal protocol was shown to be 4x 5 hr incubations with each SNIM® RNA at a concentration of 0.5 pg. Full colony formation was observed 21 - 28 days (Replate 3 or 4) following transfection. Exposure of iNPs to a mixed neuronal differentiation media resulted in the formation of GAD₆5/67 and vGLUT neurons, as well as astrocytes after 14 - 21 days in culture.

V. Results II

The optimal concentration of SNIM® RNA by which to effectively transfect HDFs was determined by transfecting normal HDFs once for 5 hrs with a concentration range of eGFP SNIM® RNA (0.01 - 2 pg) using Lipofectamine RNAiMAX (Figure 6 A, C and D). Cells were analysed by FACS 3 days following transfection. A concentration between 0.5 to 2 pg eGFP SNIM® RNA resulted in a transfection efficiency of 90% or greater. When cells were repeatedly transfected for 5 hrs either two or three times, a transfection efficiency of at least 85% with 0.5 pg SNIM® RNA was observed. Figure 6C shows a FACS analysis of eGFP SNIM® RNA. Figure 6D shows the image of eGFP protein in HDFs 3 days following transfection.

The cell viability was analysed by Alamar Blue 3 days following transfection (Figure 6B). 0.5 or 1 pg of eGFP SNIM® RNA resulted in over 80% cell viability compared to control. When cells were repeatedly transfected for 5 hrs either two or three times, the cell viability was maintained at 80% or greater for all concentrations examined. Based on these results 0.5 pg SNIM® RNA was selected as the optimal concentration to effectively transfect HDFs with minimal loss of cell viability.

The effect of co-transfecting SOX2 and PAX6 SNIM® RNA was examined (Figure 6E, G and H). Transfection efficiency was examined at either 0.25 or 5 pg of SOX2 alone, PAX6 alone or co-transfection of SOX2 and PAX6. eGFP SNIM® RNA at 0.5 pg was used as a positive control. Both SOX2 alone and PAX6 alone at either 0.25 or 0.5 pg SNIM® RNA was observed to exhibit similar transfection efficiencies of -80%. Further, co-transfection of SOX2 and PAX6 at either 0.25 or 0.5 pg SNIM® RNA resulted in at least 70% of transfected HDFs.

FACS analysis of SOX2 (green+) and PAX6 (AF633+) SNIM® RNA is shown in Figure 6G. Figure 6H shows the image demonstrating the co-expression of SOX2 (green) and PAX6 (red) protein 20 hrs following SNIM® RNA transfected of HDFs. The nuclei are stained in blue by DAPI.

SNIM® RNA transfection of either SOX2 alone, PAX6 alone or co-transfection of SOX2 and PAX6 at a concentration of either 0.25 or 0.5 pg SNIM® RNA was observed to maintain cell viability of at 80% or greater (Figure 6F).

Overall, it has been shown that a concentration of 0.5 pg each of SNIM® RNA was optimal for both maximal transfection efficiency and cell viability following co- transfection of SOX2 and PAX6. Further, HDFs could undergo multiple SNIM® RNA transfections without affecting efficiency or viability.

A comparison of cell viability following a single transfection with either 0.5 pg of plasmid DNA (eGFP or SOX2/PAX6) or 0.5 pg of SNIM® RNA (eGFP or SOX2/PAX6) is shown in Figure 7A. Transfection of HDFs with SNIM® RNA significantly increased cell viability 3 days following transfection when compared to plasmid DNA.

The effect on cell viability of transfecting HDFs either once or four times for 5 hrs or 24 hrs duration was examined for (B) SNIM® RNA GFP and for (C) SNIM® RNA SOX2 and/or PAX6 (Figure 7B and C). It was observed that multiple transfections had no effect on cell viability. However, while cell viability was reduced following 24 hrs of transfection, the level of cell viability was still maintained above 70%.

Figure 7D shows an image demonstrating the co-expression of SOX2 (green) and PAX6 (red) protein 20 hrs following either a 4x 5hr or 4x 24 hr transfection with SNIM® RNA at a concentration of either 0.5 pg or 1 pg. The nuclei are stained in blue by DAPI.

Overall, SNIM® RNA has been demonstrated to significantly improve cell viability compared to plasmid DNA transfection and a maximum of 4 transfections (incubation of 5 - 24 hrs) with each SNIM® RNA at a concentration of 0.5 pg is optimal.

* P < 0.05; ** P < 0.01 ; *** P < 0.001.

A time course of cell reprogramming following SOX2/PAX6 SNIM® RNA co- transfection in adult HDFs is shown in Figure 8. Day 5 represents the day following four consecutive days of SNIM® RNA transfection. By Day 10 populations of cells start to change to a rounded morphology and form small clusters (first replate following transfection occurs at Day 7). From days 14 to 23 the cells continue to proliferate and exhibit a more rounded, neural-like morphology. By Day 28 the majority of the cells are arranged in clusters or colonies with neural-like morphologies extending from the centre colony. Day 28 represents cells prior to Replate 4 and the initiation of full iNP colony formation. iNP cells can be collected at this point. Scale bar = 300 pm.

Gene expression profile of SOX2/PAX6 SNIM® RNA derived iNP cells is shown in Figure 9. Adult human fibroblasts were co-transfected with 0.5 pg SOX2/PAX6 SNIM® RNA 4x and incubated in the transfection mix for either 5 or 24 hrs. Cell samples were collected at 1 , 7, 14, 21 and 28 days post-transfection. Day 1 represents the day following the last transfection. Days 7, 14, 21 and 28 represent replate days.

SOX2 and PAX6 transgene expression was found to be highly expressed in both conditions with expression levels reducing at 21 or 28 days for 4x 24hr and 4x 5 hr incubation respectively (Figure 9A).

SOX2/PAX6 SNIM® RNA derived iNP cells express the neuroectoderm markers LHX2, FOXG1 , OTX2 and SIX3 (Figure 9B). Interestingly, only the 4x 24 hr incubation protocol resulted in the induction of OTX2 with no change in expression seen in the 4x 5 hr incubation protocol when compare to aHDFs. The stem cell marker OCT3/4 is not expressed in SOX2/PAX6 SNIM® RNA derived iNP cells. It was also observed that SOX2/PAX6 SNIM® RNA derived iNP cells express the pro-neural genes NGN2 and ASCL1 as well as region-specific neural transcription factors GSH2, GBX2, EN1 and PITX3 (Figure 9C).

Figure 9D shows an image demonstrating the PAX6 (red) protein expression 28 days following transfection. SOX2 expression was not detected. The nuclei are stained in blue by DAPI.

Figure 9E shows an image demonstrating the co-expression of Nestin (green) and FOXG1 (red) 28 days following transfection. The nuclei are stained in blue by DAPI. Overall these results demonstrate that SOX2 and PAX6 SNIM® RNA derived iNP cells express a heterogenous range of neural positioning genes.

A gene expression profile of SOX2/LMX1A SNIM® RNA derived iNP cells has been established (Figure 10). Adult human fibroblasts were co-transfected with 0.5 pg SOX2/LMX1A SNIM® RNA 4x and incubated in the transfection mix for 24 hrs. Cell samples were collected at 7, 14, 21 and 28 days post-transfection. Over time, an increase in expression of the mesencephalic genes GLI1 , LMX1B, FOXA2, NURR1 and PITX3 was observed. In addition, the neural gene MASH1 increased at 28 days post transfection. Overall these results demonstrate that SOX2/LMX1A SNIM® RNA derived iNP cells express a range of mesencephalic position genes required for the generation of a midbrain dopaminergic fate.

Figure 11 A demonstrates that the differentiation of SOX2/PAX6 SNIM®-derived iNPs for 21 days using a mixed neuronal differentiation media resulted in the generation of TUJ1 + neurons co-expressing either GAD₆5/67 or vGLUT. Scale bars = 50 pm.

Figure 11B shows a graph showing an increase in the generation of TUJ1 + neurons following differentiation on iNPs obtained at either Replate (RPL) 2, 3, or 4. ^*** P < 0.001.

Overall, this demonstrates that optimal neuronal differentiation is obtained following Replate 4, and SOX2/PAX6 SNIM®-derived iNPs can generate both a GABAergic and glutamatergic phenotype.

A schematic diagram showing the induction of iNPs from adult HDFs using SOX2 and PAX6 SNIM® RNA transfection is shown in Figure 12. A maximum of 4 transfections (incubation of 5 - 24 hrs) with each SNIM® RNA at a concentration of 0.5 pg is optimal to generate iNPs. Full colony formation was observed between 21- 28 days (Replate 4) following transfection. Exposure of iNPs to a mixed neuronal differentiation media resulted in the formation of GAD₆5/67 and vGLUT neurons after 21 days in culture.

VI. Objectives

Functionally mature human neurons using SNIM® mRNA directing the reprogramming for cell replacement therapy to treat Parkinson's disease and Huntington's disease are produced.

Functionally mature patient-derived human neurons using SNIM® mRNA directing the reprogramming to model neurological diseases including Parkinson's disease and Huntington's disease are produced. These models are used to characterise disease phenotype and identify potential therapeutic targets.

Functionally SNIM® mRNA-derived human neurons are used to screen and develop new therapeutic agents.

With respect to the objective to produce functionally mature human neurons using SNIM® mRNA directing the reprogramming for cell replacement therapy to treat Parkinson's disease, the ability to generate functionally mature human dopamine neurons using SNIM® mRNA direct reprogramming is confirmed by ex vivo electrophysiology with optogenetics, ex vivo electrochemistry and in vivo electrochemistry with optogenetics and behavioural analysis.

Pre-clinical assessment of transplantation of SNIM® mRNA-derived human neurons in a rodent model of Parkinson's disease is performed by assessing the cell viability and integration, the generation of functional dopamine neurons, tumour formation and the improvement in motor and cognitive function.

With respect to the objective to produce functionally mature human neurons using SNIM® mRNA directing the reprogramming for cell replacement therapy to treat Huntington's disease, the ability to generate functionally mature GABA neurons using SNIM® mRNA direct reprogramming is confirmed by ex vivo electrophysiology.

Pre-clinical assessment of transplantation of SNIM® mRNA-derived human neurons in a rodent model of Huntington's disease is performed by assessing the cell viability and integration, the generation of functional GABA neurons, tumour formation and the improvement in motor and cognitive function.

References Lassar, A.B., Paterson, B.M. & Weintraub, H. Transfection of a DNA locus that mediates the conversion of 10T1 2 fibroblasts to myoblasts. Cell 47, 649-656 (1986).

Davis, R.L, Weintraub, H. & Lassar, A.B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51 , 987-1000 (1987).

Vierbuchen, T., et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035-1041 (2010).

leda, M., et al. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell 142, 375-386 (2010).

Sekiya, S. & Suzuki, A. Direct conversion of mouse fibroblasts to hepatocyte- like cells by defined factors. Nature 475, 390-U148 (2011 ).

Pereira, C.F., et al. Induction of a hemogenic program in mouse fibroblasts. Cell Stem Cell 13, 205-218 (2013).

Pang, Z.P., et al. Induction of human neuronal cells by defined transcription factors. Nature 467, 220-223 (2011 ).

Kim, J., et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proceedings of the National Academy of Sciences 108, 7838-7843 (2011 ). Thier, M., et al. Direct Conversion of Fibroblasts into Stably Expandable Neural Stem Cells. Cell Stem Cell 10, 473-479 (20 2).

Kumar, A., et al. Zic3 induces conversion of human fibroblasts to stable neural progenitor-like cells. Journal of Molecular Cell Biology 4, 252-255 (2012).

Matsui, T., et al. Neural Stem Cells Directly Differentiated from Partially Reprogrammed Fibroblasts Rapidly Acquire Gliogenic Competency. STEM CELLS, 1109-1119 (2012).

Lu, J., et al. Generation of integration-free and region-specific neural progenitors from primate fibroblasts. Cell Rep 3, 1580-1591 (2013).

Mitchell, R.R., et al. Activation of Neural Cell Fate Programs Toward Direct Conversion of Adult Human Fibroblasts into Tri-potent Neural Progenitors Using OCT-4. Stem Cells Dev (2014).

Liao, W., et al. Direct Conversion of Cord Blood CD34+ Cells Into Neural Stem Cells by OCT4. Stem Cells Transl Med (2015).

Lujan, E., Chanda, S., Ahlenius, H., Sudhof, T.C. & Wernig, M. Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells. P Natl Acad Sci USA 109, 2527-2532 (2012).

Tian, C, et al. Direct conversion of dermal fibroblasts into neural progenitor cells by a novel cocktail of defined factors. Current Molecular Medicine 12, 126-137 (2012).

Maucksch, C, et al. Non-Viral Generation of Neural Precursor-like Cells from Adult Human Fibroblasts. Journal of Stem Cells & Regenerative Medicine 8, 162-170 (2012).

Han, Dong W., et al. Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors. Cell Stem Cell 10, 465-472 (2012).

Ring, Karen L., et al. Direct Reprogramming of Mouse and Human Fibroblasts into Multipotent Neural Stem Cells with a Single Factor. Cell Stem Cell 11 , 100-109 (2012).

Yu, K.R., et al. Rapid and Efficient Direct Conversion of Human Adult Somatic Cells into Neural Stem Cells by HMGA2/let-7b. Cell Rep (2015). Connor, B., et al. Direct Conversion of Adult Human Fibroblasts into Induced Neural Precursor Cells by Non-Viral Transfection. Exchange Protocol (2015). Yu, J., et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917 - 1920 (2007).

Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131 , 861-872 (2007).

Bylund, M., Andersson, E., Novitch, B.G. & Muhr, J. Vertebrate neurogenesis is counteracted by Sox1-3 activity. Nature Neuroscience 6, 1162-1168 (2003). Graham, V., Khudyakov, J., Ellis, P. & Pevny, L. SOX2 functions to maintain neural progenitor identity. Neuron 39, 749-765 (2003).

Pevny, L. & Placzek, M. SOX genes and neural progenitor cell identity. Current Opinion in Neurobiology 15, 7-13 (2005).

Maucksch, C, Jones, K.S. & Connor, B. Concise Review: The Involvement of SOX2 in Direct Reprogramming of Induced Neural Stem/Precursor Cells. Stem Cells Translational Medicine 2, 579-583 (2013).

Sansom, S.N., et al. The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self-Renewal and Neurogenesis. PLoS Genet 5, e1000511 (2009).

Davidson, K.C., et al. Wnt3a regulates survival, expansion, and maintenance of neural progenitors derived from human embryonic stem cells. Molecular and Cellular Neuroscience 36, 408-415 (2007).

Gajovic, S., St-Onge, L, Yokota, Y. & Gruss, P. Retinoic acid mediated Pax6 expression during in vitro differentiation of embryonic stem cells. Differentiation 62, 187-192 (1997).

Zhang, X., et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell 7, 90-100 (2010).

Zhou, H., et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381-384 (2009).

Okita, K., Hong, H., Takahashi, K. & Yamanaka, S. Generation of mouse- induced pluripotent stem cells with plasmid vectors. Nat. Protocols 5, 418-428 (2010).

Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85, 348-362 (2009).

Narsinh, K.H., et al. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nature protocols 6, 78-88 (2011 ).

Okita, K., Hong, H., Takahashi, K. & Yamanaka, S. Generation of mouse- induced pluripotent stem cells with plasmid vectors. Nature protocols 5, 418- 428 (2010).

Yin, H., et al. Non-viral vectors for gene-based therapy. Nat Rev Genet 15, 541-555 (2014).

Warren, L, et al. Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. Cell Stem Cell 7, 618-630 (2010).

Simeonov, K.P. & Uppal, H. Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs. PLoS One 9, e100134 (2014).

Kormann, M.S.D., et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat Biotech 29, 154-157 (2011 ). Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.) 25, 402-408 (2001 ).

Yakubov, E., Rechavi, G., Rozenblatt, S. & Givol, D. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophys Res Commun 394, 189-193 (2010).

Brennand, K.J., et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature 473, 221-225 (2011 ).

Skop B.N., Calderon F., Cho C.H., Gandhi CD. and Levison S.W. Improvement in biomaterial matrices for neural precursor cell transplantation, Molecular and Cellular Therapies 2:19 (2014).

Claims

A method of reprogramming a somatic cell into a reprogrammed induced neural stem/precursor cell (iNP cell), said method comprising the steps of:

The method according to claim 1 , wherein said somatic cell is a fibroblast cell or a cell derived from the mammalian immune system.

The method according to claim 1 or 2, wherein said fibroblast cell is selected from the group consisting of lung fibroblasts, kidney fibroblasts, cardiac fibroblasts, stromal fibroblasts, foreskin fibroblasts and dermal fibroblasts.

The method according to claim 1 or 2, wherein said cell derived from the mammalian immune system is a leukocyte, preferably a lymphocyte.

The method according to any one of claims 1 to 4, wherein said somatic cell is a cell from a patient suffering from a neurological disorder or injury in which tissue regeneration and/or restored functionality is a component of therapy.

The method according to any one of claims 1 to 5, wherein said induced neural stem/precursor cell (iNP cell) shows elevated or reduced expression levels of at least one neural lineage marker selected from the group consisting of Pax6, Sox2, Lmxla, AscM , FoxG1 , Gsx2, Lhx2, Ngn2, Otx2, Six3, Hes1 , Hes5, Sox1 , Sox3, Mash1/Ashl1 , Nurrl , Pitx3, HoxB9, Irx3, FOXA2, GLI1 , LMX1 B and neurogenin 2 compared to a control cell.

7. The method according to any one of claims 1 to 6, wherein said mRNA(s) encoding a transcription factor encode(s) a transcription factor selected from the group consisting of Sox2, Lmxla, and Pax6.

8. The method according to any one of claims 1 to 7, wherein said mRNA(s) encoding said transcription factor(s) contain(s) a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.

9. The method according to any one of claims 1 to 8, wherein said mRNAs encoding said transcription factor(s) is/are in vitro transcribed mRNA (IVT mRNA).

10. The method according to any one of claims 1 to 9, wherein said mRNA(s) encoding said transcription factor(s) is an mRNA wherein 5 to 30%, preferably 7.5 to 25%, of the uridine nucleotides and 5 to 30%, preferably 7.5 to 25%, of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.

11. A reprogrammed induced neural stem/precursor cell (iNP cell) produced by a method according to any one of claims 1 to 10.

12. An mRNA encoding a transcription factor wherein said transcription factor is Sox2 or Pax6 or Lmxl a and wherein said mRNA contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.

13. The mRNA according to claim 12, wherein said mRNA is in vitro transcribed mRNA (IVT mRNA).

14. The mRNA according to claim 12 or 13, wherein said polynucleotide is an mRNA wherein 5 to 30%, preferably 7.5 to 25%, of the uridine nucleotides and 5 to 30%, preferably 7.5 to 25%, of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.