GENERATING FUNCTIONAL OLIGODENDROCYTE PROGENITOR CELLS IN

SUFFICIENT NUMBER AND PURITY FOR CLINICAL CELL REPLACEMENT

THERAPY

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent application 62/638,355, filed March 5, 2018, which is hereby incorporated by reference for all purposes as if fully set forth herein.

BACKGROUND OF THE INVENTION

Demyelination (loss of myelin) or dysmyelination (abnormal myelination) is associated with multiple CNS disease such as spinal cord injury and multiple sclerosis (MS) and X- linked adrenoleukodystrophy (X-ALD). Oligodenrocyte progenitor cells (OPCs) as the predominant source of myelinating oligodendrocytes in the CNS have shown promise as a cellular therapeutic in animal demyelination models. However, primary OPCs are not readily available. Several approaches have been established to generate induced oligodendrocyte progenitor cells (iOPCs) from the fetal brain or human embryonic stem cells (hESCs) as well as from human induced pluripotent cells (hiPSCs). However, these approaches are time- consuming (75-l20days), and inefficient (20-75%), generating heterogeneous cells

(oligodendrocyte and astrocyte) and tumorigenic potential (undifferented pluripotent cells, using lentivirus-based gene delivery), and thus have limitations for regenerative medicine. Therefore, methods based on combination specific transcription factor cocktail (a mixture comprising two or more, three or more, or four or more transcription factor mRNA), with specific oligodendrocyte signaling for the rapid generation of oligodendrocyte progenitor cells from human iPSCs and somatic are greatly important as they would enable large-scale drug screening and cell-based regenerative medicine. For example, cell transplantation therapy using oligodendrocyte progenitor cells (OPCs) for treating dysmyelinating and demyelinating CNS disorder is largely limited by the time required to derived autologous OPCs, low efficiency of OPC derivation, and the

heterogeneous cell population using current state-of the art protocol. Optimized

differentiation strategies for generating functional OLs in sufficient numbers and purity for cell replacement therapy are urgently needed. Recent studies revealed that iOPCs can be directly derived from mouse and rat fibroblasts by defined sets of lineage-specific transcription factors (TFs). However, none of these approaches has been successful used to generate iOPCs from human fibroblasts.

SUMMARY OF THE INVENTION

The inventors have established a robust and efficient protocol that generate expandable iOPCs from hiPSCs within 7-14 days. Moreover, the inventors demonstrated that this protocol with some modifications can also be applied to generate iOPCs from human fibroblasts within 30-40 days. The protocol of the present invention is able to provide a source of safe and functional iOPCs without host genome modifications by using innovative non-viral delivery transcription factor mRNA cocktail combined with the oligodendrocyte specification signals while circumventing tumor formation concerns previously associated with ESCs or hiPSCs- derived OPCs by using lentiviral constructs in generation of patient- specific OPCs. The term‘ transcription factor cocktail” refers to a mixture comprising two or more, three or more, or four or more transcription factor mRNAs. Moreover, the present invention is sufficient to generate homogeneous, self-renewing iOPCs which can be expanded for at least 20 passages, and facilitates future clinical cell replacement therapeutic interventions for the treatment of demyelination central nervous system disorders. One embodiment of the present invention is a method of producing oligodendrocyte progenitor cells (OPCs). The method steps include plating mammalian pluripotent stem cells on a surface and incubating the cells with a cell culture media. Two or more first transcription factor mRNAs selected from the group consisting of a first basic helix-loop- helix (bHLH) transcriptional factor, a first Sox E transcriptional factor, or a combination thereof are added. The cells are incubated forming oligodendrocyte progenitor cells. A suitable cell culture media used is an OPC induction media that comprises Dulbeco Modified Eagle Medium (DMEM-F12) with N2 supplement, B27 supplement lacking vitamin A, GSK- 3 inhibitor such as CHIR99021, BMP pathway inhibitor such as LDN193189, Retinoic Acid, TGF-beta/Smad inhibitor such as SB431542, platelet-derived growth factor (PDGF), neurotrophin 3 (NT3), insulin-like growth factor (IGF-I), and fibroblast growth factor (FGF). The OPC induction media may also comprise an antibiotic such as in the range of 0.1 to 5%, 0.5 to 2% or 1% of the OPC induction media. Examples of suitable antibiotics include penicillin, streptomycin, as examples. Examples of suitable first basic helix-loop-helix (bHLH) transcriptional factors used in the present invention include of ASCL1, Olig2, or a combination thereof. Examples of suitable first Sox E transcriptional factor included Sox8, Sox9, or SoxlO. Suitable mammalian pluripotent stem cells are mammalian induced pluripotent stem cells (iPSCs) and human pluripotent stem cells, as examples. In some embodiments of the present invention the first SoxE transcription factor mRNA comprises a soxlO transcription factor mRNA, and the first bHLH transcriptional factor comprises an ASCL1 transcription factor mRNA, an olig2 transcription factor mRNA, or a combination thereof. In some embodiments of the present invention the SoxE transcriptional factor encodes a SoxlO protein or functional part thereof, and the first bHLH transcriptional factor mRNA encodes an Oligo2 protein or functional part thereof, an ASCL1 protein or functional part thereof, or a combination thereof. In some embodiments of the present invention, the two or more first transcription factor mRNA are transfected every day for 2- 4, 1-5, 2-3, 1-6, 2-6, 1-7, 2-7, 2-20, or 2-25 days after the plating of the mammalian pluripotent stem cells. A suitable surface for plating the cells, includes a laminin or a matrigel, for example.

In some embodiment of the present invention, the two or more transcription factor mRNAs are attached to a nanoparticle or other transfection reagent such as Lipofectamine stem, 2000, or 3000 teal. The nanoparticle may be made of any suitable polymers such as Poly (ethylene oxide)-modified poly (beta-amino ester) PBAE polymer.

The methods of the present invention may include an additional step of adding two or more second transcriptional factor mRNA selected from the group consisting of a second basic helix-loop-helix (bHLH) transcriptional factors, a second Sox E transcriptional factor, or a combination thereof. Suitable second basic helix-loop-helix (bHLH) transcriptional factors include ASCL1, Olig2, or a combination thereof, as examples. Suitable second Sox E transcriptional factors include Sox8, Sox9, SoxlO, or a combination thereof. In some embodiments of the present invention the SoxE transcriptional factor mRNA encodes a

SoxlO protein, or functional part thereof, and the first bHLH transcriptional factor mRNA encodes an Olig2 protein or functional part thereof, an ASCL1 protein or functional part thereof, or a combination thereof. In some embodiments of the present invention the second SoxE transcriptional mRNA encodes a SoxlO protein, or functional part thereof, and the second bHLH transcriptional mRNA encodes an Olig2 protein, or functional part thereof. In some embodiments of the present invention the second SoxE transcription factor mRNA comprises a Sox 10 transcriptional factor mRNA and the second bHLH transcriptional factor mRNA comprises an Olig2 transcriptional factor mRNA. In some embodiments of the present invention the two or more second transcriptional factor mRNA are added 5, 6, 7, 8, 9, 10 days after the plating of the mammalian pluripotent stem cells. The mammalian pluripotent stem cells used in the methods of the present invention may come from a subject and the method of the present invention may include an additional step of placing the oligodendrocyte progenitor cells (OPCs) formed back into the subject. For example, mammalian pluripotent stem cells of a subject having a central nervous system (CNS) disorder may be used in the methods of the present invention to produce and oligodendrocyte progenitor cells that prevent or treat the central nervous system disorder of the subject when administered to the subject.

Another embodiment of the present invention is an OPC induction media comprising Dulbeco Modified Eagle Medium (DMEM-F12) with N2 supplement, B27 supplement lacking vitamin A, GSK-3 inhibitor such as CHIR99021, BMP pathway inhibitor such as LDN193189, Retinoic Acid, TGF-beta/Smad inhibitor such as SB431542, platelet-derived growth factor (PDGF), neurotrophin 3 (NT3), insulin-like growth factor (IGF-I), and fibroblast growth factor (FGF). The OPC induction media may also comprise an antibiotic such as in the range of 0.1 to 5%, 0.5 to 2% or 1% of the OPC induction media. Examples of suitable antibiotics include penicillin, streptomycin, as examples.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The term“activity” refers to the ability of a gene to perform its function such as Sox 10 transcription factor 1 catalyzing transcription.

By“ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. "

By“ASCL1” is meant Achaete-scute homolog 1 a gene, or a functional part thereof, that encodes a member of the basic helix-loop-helix (BHLH) family of transcriptional factors. An example of an ASC11 sequence is shown in Figure 5.

By“disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include demyelination (loss of myelin) or dysmyelination (abnormal myelination) that is associated with multiple CNS disease such as spinal cord injury and multiple sclerosis (MS) and X-linked

adrenoleukodystrophy (X-ALD).

By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. The term“express” refers to the ability of a gene to express the gene product including for example its corresponding mRNA or protein sequence (s).

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

The term“Nkx2.2” refers to a protein, or functional part thereof, that in humans is encoded by the Nkx2-2 gene, or the nucleic acid sequences that code for the functional part of the Nkx2.2 protein. The encoded protein is likely to be a nuclear transcription factor.

The term“Olig2” refers to Oligodendrocyte transcription factor that is a basic helix- loop-helix (bHLH) transcription factor encoded by the Olig2 gene, or the functional part of the Olig2 protein, or the nucleic acid sequences that code for the functional part of the Nkx2.2 protein. An example of an Olig2 nucleic acid sequence is shown in Figure 6.

The terms“polypeptide,”“peptide” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms“polypeptide,”“peptide” and“protein” include glycoproteins, as well as non glycoproteins.

As used herein, the terms“prevent,”“preventing,”“prevention,”“prop hylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

By“reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or

100%.

A“reference” refers to a standard or control conditions such as a sample (human cells) or a subject that is a free, or substantially free, of transcriptional factor mRNA or OPCs produced by the method of the present invention.

A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or there between.

As used herein, the term“sensitivity” is the percentage of subjects with a particular disease. As used herein, the term“SoxlO” refers to Transcription factor SoxlO that is a protein that in humans is encoded by the SoxlO gene, or a functional part of the SoxlO protein or the nucleic acid sequences that code for the SoxlO protein. Figure 7 provides an example of a SOX- 10 nucleic acid sequence.

As used herein, the term“specificity” is the percentage of subjects correctly identified as having a particular disease i.e., normal or healthy subjects. For example, the specificity is calculated as the number of subjects with a particular disease as compared to non-cancer subjects (e.g., normal healthy subjects).

By "specifically binds" is meant a protein (such as one derived from a transcriptional factor mRNA) that recognizes and binds a target DNA sequence, but which does not substantially recognize and bind other molecules in a sample.

As used herein, the term "subject" is intended to refer to any individual or patient to which the method described herein is performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

As used herein, the term“transcriptional factor mRNA” refers to the basic helix- loop-helix (bHLH) transcription factors such as ASCL1 and Olig2, as examples, and the SoxE transcription factors including Sox8, Sox9, and SoxlO transcription factors, as examples. Please see Matthias Weider and Michael Wegner,“SoxE factors:

Transcriptional regulators of neural differentiation and nervous system development”, Seminats in Cell & Development Biology 63 (2017) 35-42. Transcriptional factor mRNAs encode transcriptional factor proteins, or functional parts thereof.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a transcriptional factor of the present invention. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.

Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.

By "hybridize" is meant pair to form a double-stranded molecule between

complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,

BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;

aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e ³ and e ¹⁰⁰ indicating a closely related sequence.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural. Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Such treatment (surgery and/or chemotherapy) will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for spinal cord injury, multiple sclerosis (MS) and X-linked adrenoleukodystrophy (X-ALD) or disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, a marker (as defined herein), family history, and the like).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A-1D. PBAE nanoparticles effectively transfect mRNA into hiPSCs. (A) The library of PBAE polymer were screened to deliver mRNA using a high-throughput 96-well transfection assay. 4 formulations were found to effectively deliver mRNA into hiPSCs; (B) Fluorescene microscope images show cells transfected with mRNA encoding GFP using PBAE-457 polymer; (C) immunoblot results show high expression of transcription factors SoxlO, NKX2.2, Olig2 and ASCL1 after 48 hrs transfection. (D) Fluorescene microscope images show cells transfected with mRNA encoding GFP using different transfection reagents. Figure 2A-2K. Differentiation of oligodendrocytes from hiPSCs. (A) A schematic protocol for directed differentiation of hiPSCs into iOPCs; Undifferentiated hiPSCs cultured in mTeSR(B); After 3 weeks transfection using poly(beta-amino esters(PBAEs) nanoparticles, pre-iOPCs were obvious(C); Within 40 days, 90% cells are bi-polar or tri-polar iOPCs morphology(D); maintain their self-renewal capacity and express OPC-specific markers NG2 (E), PDGFRa (F), SoxlO (G) and 04 (H). Moreover, these iOPCs can maintain high proliferation as shown 60% Ki67 ⁺ cells (I) (K). Immunofluorescence images of iOPCs at passage 3(p3) stained with NG2, soxlO, PDGFRa, 04 and Ki67. Cells were counterstained with DAPI. Scale bars, lOOpm.

Figure 3A-3D. Terminal differentiation of hiPSCc-derived iOPCs. Phase-contrast images of iOPCs cultured in OL differentiation medium (A); Immunofluorescence image of oligodendrocyte marker 04(B); qRT-PCR results show that myelin transcription factor 1 were significantly induced when iOPCs were cultured in OL differentiation medium for 7 days(C). After one week differentiation, the iOPCs became 04+ with multiple branches and express MBP as determined by qRT-PCR showing that MBP was highly expressed in the 04 cells compared with the parent cells(hiPSCs) (D).

Figure 4A-4H. Human fibroblasts can be directly converted into iOPCs. Phase-contrast images of human fibroblasts transfected with mRNA cocktail at day 0 (A), 10(B), 15(C) and 30(D). Immunofluorescence images of iOPC markers at passage 5(p5) show cells were stained with NG2, SoxlO, PDGFRa and 04. Cells were counterstained with DAPI. Scale bars, lOOpm. After consecutive passages, most of the cells can be maintained in a highly homogeneous monolayer culture and exhibit typical small bi-polar or tri-polar morphology. Immunostaining show that cells express OPC-specific markers NG2 (E), SoxlO (F), PDGFRa (G) and 04 (H). Figure 5. Example of an ASCL1-T7 Transcriptional Factor mRNA.

Figure 6. Example of an olig2-l-T7-F Transcriptional Factor mRNA.

Figure 7. Example of a soxlO-3-T7-F Transcriptional Factor mRNA.

DETAILED DESCRIPTION OF THE INVENTION PBAE polymer screen for mRNA cocktail transfection: The inventors have successfully established a platform to synthesize high yield and stable mRNA encoding transcription faetors(TFs) SoxlO, 01ig2, ASCL1 , NkX2.2 and control GFP using mMessage mMACHINE ^® T7 Ultra Kit (# AMI 345, life technol ogies)(Fig 1 A). Moreover, the inventors have identified and synthesized four nanoparticle formulations which have the potential to increase stability and transfection efficiency of mRNA cocktail into hiPSCs (> 50% transfection with less than 10% toxicity) as shown in Figl B, C). The inventors have established high transfection efficiency of mRNA cocktail using Lipofectamine stem or Lipofectamine 2000 as shown in Figl D.

Generation of hiPSCs-derived iOPCs: The inventors have established a protocol to generate iOPCs from hiPSCs using nanoparticle delivery system as shown in the Fig2A. Within 40 days after 5 time transfections, most of the cells (90%) exhibit typical small bi polar or tri-polar OPC-like morphologies (Fig2D), maintain their self-renewal capacity and express OPC-specific markers NG2 (Fig 2E), PDGFRa (Fig 2F), SoxlO (Fig 2G) and 04 (Fig 2H). Moreover, these iOPCs can maintain high proliferation as shown 60% Ki67 ⁺ cells (Fig

21). These iOPCs can be stably expanded for more than 20 passages to continue to drive O lineage. The inventors cultured bipolar OPCs on PDL-laminin coated plate with reduced growth factor medium. After one week differentiation, the iOPCs became multipolar mature OLs (Fig 3A, B). Moreover, the inventors have established a protocol to generate iOPCs from hiPSCs using Lipo stem or Lipo 2000 delivery system as shown in the Fig3. Within one-week after 3-4 time transfections, most of the cells (90%) exhibit typical small bi-polar or tri-polar OPC-like morphologies (Fig3B), maintain their self-renewal capacity and express OPC- specific markers NG2, SoxlO, PDGFR and 04. The inventors cultured bipolar OPCs on PDL-laminin coated plate with reduced growth factor medium. After one week

differentiation, the iOPCs became 04+ with multiple branches and express MBP as determined by qRT-PCR showing that MBP was highly expressed in the 04 cells compared with the parent cells(hiPSCs) (Fig 3D).

Generation of human fibroblasts-derived iOPCs: The inventors used the protocol shown above with some modifications to induce OPCs from human fibroblasts (Fig 4A). Within 8-10 days, the inventors observed the cells morphological changes (Fig 4B), and cell aggregates (Fig 4C) were shown after 15 days. The inventors mechanically isolated the cell aggregates and plated them in OPC proliferation medium. Most of cells (90%) exhibits bi-polar morphology (Fig 4D). After consecutive passages, most of the cells can be maintained in a highly homogeneous monolayer culture and exhibit typical small bi-polar or tri-polar morphology. Immunostaining show that cells express OPC-specific markers NG2 (Fig 4E), SoxlO (Fig 4F), PDGFRa (Fig 4G) and 04 (Fig 4H). One of the main advantages of the present invention is the robustness to convert human hiPSCs and or fibroblasts with high efficiency (90% of reprogrammed cells express PDGFRa by 30d). The present invention describes the unexpected discovery of directed conversion of human fibroblast into induced OPCs for cell replacement therapies. The present invention is capable of generating patient-specific iOPC cell source for cell replacement therapies in patients with dysmyelinating and demyelinating central nervous system disorders. The present invention also provides a source of cells for screening for critical small molecules that could be useful in for treatment of disease or for determining the cell fate of OL. Further, the present invention will be beneficial to researchers interested in studying molecular mechanisms governing OL development and myelination in order to characterize key genes, mRNA transcriptions, and proteins relevant. The present invention includes unique OPC induction media and proliferation media, mRNA cocktails and iOPCs for research.

The cells generated by the present invention may be used in cell-based therapies for treatment of dysmyelination or demyelination disorders such as spinal cord injury. In particular, transplantation of oligodendrocyte precursor cells (OPCs) is a potentially promising therapeutic intervention for such disorders. The robust protocol of the present invention for the highly efficient generation of iOPCs from hiPSCs by using nanoparticle delivery transcription factor mRNA cocktail combined with the oligodendrocyte specification signals makes cell- based therapies possible. Moreover, the methods of the present invention may be modified and used to directly convert human fibroblasts into OPCs-like cells. The present invention allows for the establishment of precisely lineage reprogramming protocols to generate expandable, functional and safety OPCs from easily extractable fibroblasts for therapeutic purposes. Without being held to a particular theory, the inventors believe that OL-specific transcription factors enhance OL lineage reprogramming when combined with oligodendrocyte specification signals, and generate myelinating OLs. Recently established OPCs-specific differentiation protocol may be used to generate iOPCs derived from different labs and from different cell source, and for generating functional OLs. The present invention provides approaches for generating clinically relevant OPCs for regenerative medicine and drug discovery, and studying the molecular mechanisms underlying reprogramming processes and demyelination.

A protocol that can directly convert human fibroblasts into OPCs-like cells as shown in Fig 4. Based on these results, primary normal and X-ALD patient-derived fibroblasts will be transfected with mRNA cocktail combined with OPCs induction medium following the strategy shown in the Fig 4. Cell aggregates will be picked and plated in OPC proliferation medium after 15-20 days transfection.

Characterization of iOPCs: After two passages, the expression levels of pre-OPC marker (A2B5), OPC markers (PDGFaR, NG2 and Olig2), astrocytes (GFAP), neuron (Tuj l and NeuN) will be quantified by immunostaining at days 25 and 30. The percentage of these markers normalized to total nuclei will be used to compare the efficiency of OPC induction between different sources of fibroblasts. Cell proliferation will be quantified by Ki67 staining.

Differentiation: Terminal differentiation of fibroblasts-derived iOPCs will be accomplished by culturing the cells for 4-weeks in reduced growth factor media (no growth factors/ with T3 and cAMP) that supports mature OL as described in Fig 3. The following markers of the OL lineage will be use to characterize iOLs, OPC (PDGFaR), non-myelinating OLs (01 and 04), and mature OLs (MBP, Rip and APC-CC1).

Myelination: The inventors will examine the ability of iOLs to myelinate axons using an in vitro myelination assay. Briefly, mouse cortical neurons will be cultured on laminin for 10-14 days to allow phenotypic maturation and axonal growth. iOPCs (late stage) will be co cultured with neurons for 4 weeks in reduced growth factor media containing T3 and cAMP to support OL maturation. Myelination will be assessed by counting the number of tubular segments of MBP immunoreactivity that co-localized continuously with neurofilament (NF) over a distance of at least 10 pm according to a published protocol. Co-localization will be confirmed using confocal microscopy on 1 pm optical sections. Furthermore, the formation of ultrastrcturally mature myelin will be confirmed using electron microscope (EM) according to the published protocol. It is possible that fibroblasts from different source have low reprogramming efficiency or hard to reprogramming. In this case, we will 1) modify the ratio of TFs; 2) add pluripotency stem cell-specific miRNAs such as miRNA-302/367, which enhance reprogramming efficiency. After optimizing the protocol, we expect that combining TFs and miRNAs will efficiently convert fibroblast into OPCs.

Transcriptome analysis of iOPCs by RNA-seq.

The inventors will compare the transcriptome signature patterns of iOPCs and parental fibroblasts by using RNA-seq. The inventors will purify total RNA from different sources of fibroblast-derived iOPCs and parent cells using QIAGEN RNeasy kit. RNA (2pg) will be subjected to RNA-seq library preparation using ILLUMINA TruSeq RNA Sample Preparation Kit. Libraries quality will be checked by Agilent Bioanalyzer. Sequencing will be performed in Johns Hopkins High Throughput Sequencing Center using HiSeq 2000 system and 50bp single end read. The raw reads will be aligned to reference human genome and converted to gene expression levels using TopHat, Cufflinks and R bioconductor package GenomicFeatures. The differential gene expression analysis will be processed and analyzed by Biostat Center Core at Johns Hopkins Bloomberg School of Public Health by using R bioconductor package edgeR to calculate fold change and FDR. Fold changes will be computed for each gene and the greatest positive and negative results will be ranked and the top differentially expressed genes will be identified. The inventors will compare gene expression profile of iOPCs from fibroblasts with existing NPS-derived OPCs databases and hESCs-derived OLs by using heat map analysis and investigate the degree of similarity in molecular identity between these cells. Additionally, the top differentially expressed genes will be validated with qPCR, and their targets and functions will be predicted through Gene Ontology (GO) term enrichment profiling. Successful completion of this aim could help the inventors to identify new key genes involved in OPC-specific lineage reprogramming that could lead to improved OPC-specific lineage reprogramming strategies. Considering these cells are being used in clinical trial for treatment of CNS disorder, this data will be used as safety data in clinical trials with fibroblasts-derived iOPCs.

Characterization of human hiPSCs and fibroblast-derived OPC functions in vivo

a. Functionality of the iOPCs

The inventors have shown (data not provided) that engrafted hiPSCs-derived pre-iOPCs in rag2-/-shi/shi can survive, expand and proliferation (Fig). [PLEASE PROVIDE THIS DATA] The inventors will extensively examine the in vivo functionality and safety of the iOPCs from hiPSCs and fibroblasts by using the neonatal myelin deficient shi/shi immune deficient rag2- /2 mouse and adult demyelinated rat spinal cord injury. Using two different models in parallel ( rag2-/-shi/shi and adult rat spinal cord) will allow the inventors to evaluate and compare the capabilities of iOPCs in two environments that differ in their innate ability for new myelination. Importantly, the inventors will assess the in vivo myelination potential of different differentiation stage of iOPCs (i.e. early stage of iOPCs (PDGFR+), late stage of iOPCs (04+). Neonate mice: Postnatal day 1 rag2 -/-shi/shi mice will be transplanted with different stage of iOPCs according to their published protocol (total N=l6, two sites per each mice and 1*10 ⁵ / each site) [Glia, 59:499], 6-weeks after transplantation, mice (n=3)wii! be sacrificed, and double immunohistochemistry for human specific nuclear antigen (hSNA) and cell-type specific markers(GFAP, MBP and olig2) will perform to assess cell differentiation and distribution. 13 weeks after transplantation, mice (n=3) will be sacrificed and double immunohistochemistry for human specific nuclear antigen (hSNA) and myelination (MBP). Moreover, immunoEM sections will be used to measure g-ratios from axons that are myelinated by iOLs. Since shiverer mice have a reduced lifespan, the inventors also will determine if engrafted iOPCs can improve shiverer mice survival (h=10). Rat spinal cord injury: A focal demyelinated lesion in the spinal cord of rats will be induced according to our published protocol. One week after lesion induction, iOPCs (4X10 ⁵ ) will be transplanted into the epicenter of the demyelinated dorsal funiculus lesion of rat spinal cord (N=8). Animals will be sacrificed and spinal cord tissue will be collected at 3, 7, and 14 weeks after transplantation. H&E staining of coronal and sagittal section of rat spinal cord will be performed to examine the recovery of spinal cord in the shape and cavity size compared with PBS-injected control. Furthermore, double immunostainning for human specific nuclear antigen (hSNA) and myelination (MBP) will be perform to assess engrafted iOPCs distribution and myelination. b. Tumorigenicity of the iOPCs.

Tumorigenic potential of hiOPCs will be examined in 8-12 week old non-obese severely immunocompromised (NOD/SCID) mice. Teratoma formation will be examined 6-9 months after transplantation of FiOPCs (h=10 per time point). Under these conditions, mice will be monitored twice daily and sacrificed if showing pam or distress. All surgical interventions and postoperative animal care will be carried out m accordance with the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Care and Use Committee of Johns Hopkins University (Protocol number #M015M4Q7). The inventors recognize that human hiPSCs-derived iOPCs could form tumors. To address this issue, the inventors will transplant different stages of iOPCs (early iOPCs (A2B5A, late iOPCs (PDGFaR ⁺⁾ and early lOL (04 ⁺ ) to control proliferation and differentiation. The inventors will determine the safety (no tumor formation) and functionality (myelination) of iOPCs that can be used in a clinical setting.

Embodiments of the disclosure concern methods and/or compositions for treating and/or preventing central nervous system disorder by creating autologous oligodendrocytes for use in cell replacement therapy for treatment of spinal cord injury, multiple sclerosis (MS), and other central nervous system (CNS) disorders. In certain embodiments, individuals with a central nervous system disorder is giving cell replacement therapy using oligodendrocytes made by the methods of the present invention to teat of prevent demyelination (loss of myelin) or dysmyelination (abnormal myelination). .

An individual known to have a CNS disease , suspected of having a CNS disease, or at risk for having CNS disease may be provided an effective amount of the oligodendrocytes cells made by the methods of the present invention. Those at risk for a CNS disorder may be those individuals having one or more genetic factors, may be of advancing age, and/or may have a family history, for example.

In particular embodiments of the disclosure, an individual is given an agent for a CNS disorder in addition to the one or more oligodendrocytes of the present invention. When combination therapy is employed with one or more the oligodendrocytes of the present invention, the additional therapy may be given prior to, at the same time as, and/or subsequent to the cell replacement therapy using the oligodendrocytes of the present invention. Cell Replacement therapy includes administering the oligodendrocytes cells of the present invention to a subject resulting in enhanced myelination of a subject compared to subject who has not been administered the oligodendrocytes of the present invention.

Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise an effective amount of one or more oligodendrocytes of the present invention, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that comprises at least one of the oligodendrocytes cells of the present invention and/or an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21 ^st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference).

Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

The oligodendrocytes of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The oligodendrocytes cells of the present invention may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.

Further in accordance with the present disclosure, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art. In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of a

pharmaceutical lipid vehicle compositions that include oligodendrocytes or the present invention, one or more lipids, and an aqueous solvent. As used herein, the term“lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term“lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e.. designed or produced by man). However, a lipid is usually a biological substance.

Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the

oligodendrocytes of the present invention may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.

Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

In other non-limiting examples, a dose may also comprise from about 1

microgram/kg/body weight, about 5 microgram/kg/body weight, about 10

microgram/kg/body weight, about 50 microgram/kg/body weight, about 100

microgram/kg/body weight, about 200 microgram/kg/body weight, about 350

microgram/kg/body weight, about 500 microgram/kg/body weight, about 1

milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500

milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

Parenteral Compositions and Formulations

In further embodiments, oligodendrocytes of the present invention may be administered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat.

Nos. 6, 7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).

Solutions of the active compounds including oligodendrocytes of the present invention as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" l5th Edition, pages 1035-1038 and 1570- 1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.

Kits of the Disclosure

Any of the compositions described herein may be comprised in a kit. In a non-limiting example, one or more oligodendrocytes of the present invention may be comprised in a kit.

The kits may comprise a suitably aliquoted of one or more oligodendrocytes of the present invention and, in some cases, one or more additional agents. The component(s) of the kits may be packaged either in aqueous media. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the oligodendrocytes of the present invention and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The oligodendrocytes of the present invention may be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit. METHODS/EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation.

Primary human fibroblasts and X-ALD-derived fibroblasts: Primary human fibroblasts from individuals of different ages or gender (GM5565 and GM9503) were purchased from Coriell Biorepository. X-linked adrenoleukodystrophy (X-ALD) patient-derived fibroblasts (PDL- 35774, PDL-10527 and PDL-22627) are banked at the Kennedy Krieger institute. These cells are currently cultured and available for research.

Bench Supplies: Cell culture reagents including dium, N2 ,Nl and B27 Supplements, and growth factors FGF, PDGF, NT3, IGF, CNTF and purmorphamine; Molecular biological reagents including RNA, and DNA isolation kit, SYBR Green PCR Master Mix kit for qRT- PCR, TOPO-TA cloning kit, poly(beta-aminoester) nanoparticles, mMessage mMACFUNE ^® T7 Ultra Kit, precast polyacrylamide gels, membranes, and LI-COR blocking buffer; Immunologic reagents including primary antibodies and secondary antibodies will be required for immunohistochemistry, immunofluorescence and flow cytometry.

Animal Procurement and Housing: The experiments will use shiverer mice for studying myelination and tumor formation. The inventors already have shiverer mice (from Jackson lab, strain name: C3Fe.SWV-Mbpshi/J; stock number: 001428). The inventors will breed, and mice will house at the Johns Hopkins School of Medicine Animal Facility. Our experience with in vivo experiments indicates that the number of animals in each experimental group must be at least h=10 to obtain statistically meaningful data for each biological endpoint. Thus we are planning to use 40 !shiverer mice. Average length of stay in the housing facility is estimated to be 90 days for tumor formation analysis.

General lab supplies and Chemicals: General Supplies include commonly used buffers, chemicals, detergents, glassware, nonsterile plasticware, scintillation vials, disposable cuvettes, blotting paper, plastic wrap and aluminum foil, etc.

Flow Cytometry: Flow cytomtery will be conducted at the Johns Hopkins Flow Cytometry Core Facility which charges an hourly rate for the use of the cytometers. Cell sorting and the percent of positive cells will be analyzed by flow cytometry.

Microscopy & Imaging: Ultrastructural analysis will be done at John Hopkins Microscopy and Imaging Core Facility. The services fee will be charged by hour.

RNA-seqs & Analysis: microRNA expression array will be done in the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Microarray Core Facility. Several comparisons will be performed. The statistical and bioinformatics analysis will be done in the Biostat center Cores. The services fee will be charged by hour.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms“a” and“an” and“the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms“comprising,”“having,”“including,” and “containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.