Patent Application
20230287411
This invention relates to cellular and developmental biology and regenerative medicine. The invention provides compositions and in vivo, ex vivo and in vitro methods for trans-differentiation of, re-differentiating or re-programming mammalian cells to functional neurons. In particular, the invention provides methods for engineering non-neuronal cells into neurons, and methods for engineering non-neuronal cells into neurons in the brain to treat a neurodegenerative disease. In alternative embodiments, the invention provides compositions comprising re-differentiated or re-programmed mammalian cells of the invention. The invention also provides compositions and methods for direct reprogramming of cells to a second phenotype or differentiated phenotype, such as a neuron. The invention also provides formulations, products of manufacture, implants, artificial organs or tissues, or kits, comprising a trans-differentiated or re-programmed cell of the invention.
Accompanying this filing is a Sequence Listing entitled “00015-239US3.xml”, created on Oct. 4, 2022 and having 226,511 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated herein by reference in its entirety for all purposes.
Neuronal differentiation is a well-studied paradigm as a consequence of transcription reprogramming. Recent studies have shown that a set of neuronal lineage-specific transcription factors is sufficient to trans-differentiate fibroblasts into functional neurons. Neuronal differentiation is subject to additional layers of control, such as regulated RNA processing.
The Polypyrimidine Tract Binding protein, PTB and its homolog “neuronal PTB” or nPTB, undergo a programmed switch during neuronal differentiation. Homeostatic expression of PTB in non-neuronal cells is maintained through splicing auto-regulation. When PTB is down regulated by miR-124, an internal alternative exon is included, rendering the transcript sensitive to nonsense mediated RNA decay, thereby re-enforcing PTB down-regulation. Reduced PTB also results in increased nPTB expression and forced expression of PTB blocks miR-124 induced neuronal differentiation. However, it has been unclear whether the PTB/nPTB switch is sufficient to initiate neuronal differentiation and which specific PTB/nPTB-regulated splicing events contribute to the cell fate switch.
In alternative embodiments, the invention provides in vitro, ex vivo or in vivo methods for trans-differentiating, re-differentiating or re-programming a mammalian cell to a neuronal cell, comprising:
In alternative embodiments, the mammalian cell is: a human cell, a non-human primate cell, a monkey cell, a mouse cell, a rat cell, a guinea pig cell, a rabbit cell, a hamster cell, a goat cell, a bovine cell, an equine cell, an ovine cell, a canine cell or a feline cell; or a fibroblast, or a glial cell.
In alternative embodiments, the composition or compound comprises a or is formulation in or as a liquid or aqueous formulation, a vesicle, liposome, nanoparticle or nanolipid particle, and optionally the in vitro or ex vivo contacting is on mammalian cells embedded in a gel, or the in vitro or ex vivo contacting is on a mammalian cell that is adherent on (to) a plate or a fixed or gel structure.
In alternative embodiments, the mammalian cell is contacted with the composition, or the liquid or aqueous formulation, or the vesicle, liposome, nanoparticle or nanolipid particle, in an amount effective to cause the trans-differentiation or re-programming of the mammalian cell to a neuronal cell.
In alternative embodiments, the mammalian cell before trans-differentiation or re-programming, is an adult stem cell, an embryonic stem cell, a somatic stem cell, an adipose-derived stem cell (ASC), a stem cell derived from an epithelial cell or tissue, a hematopoietic stem cell, a mammary stem cell, a mesenchymal stem cell, a neural stem cell, an olfactory adult stem cell, a spermatogonial progenitor cell, a dental pulp-derived stem cell, or a cancer stem cell, or an adult somatic cell or an adult germ cell, or is a hematopoietic cell, a lymphocyte, a macrophage, a T cell, a B cell, a nerve cell, a neural cell, a glial cell, an astrocyte, a muscle cell, a cardiac cell, a liver cell, a hepatocyte, a pancreatic cell, a fibroblast cell, a connective tissue cell, a skin cell, a melanocyte, an adipose cell, an exocrine cell, a dermal cell, a keratinocyte, a retinal cell, a Muller cell, a mucosal cell, an esophageal cell, an epidermal cell, a bone cell, a chondrocyte, an osteoblast, an osteocyte, a prostate cell, an embryoid body cell, an ovary cell, a testis cell, an adipose tissue (fat) cell, or a cancer cell.
In alternative embodiments, the invention provides the mammalian cell is cultured for between about one to 24 hours, or between about one to two days. In alternative embodiments, the mammalian cell is cultured for between about one to 10 days after the contacting; or, the mammalian cell is cultured before, during and/or after the contacting.
In alternative embodiments, the mammalian cell is also contacted with a cytokine that has a trans-differentiation or re-programming effect on the mammalian cell, wherein optionally the cytokine comprises a transforming growth factor-beta (TGF-beta), interleukin-18 (IL-18, or interferon-γ-inducing factor), adipose complement-related protein or interferon-γ.
In alternative embodiments, the nucleic acid that is inhibitory comprises an miRNA, an siRNA, a ribozyme and/or an antisense nucleic acid.
In alternative embodiments, the identifying and/or isolating the trans-differentiated or re-programmed cell is by a negative selection of cells still expressing a non-neuronal cell marker, or the trans-differentiated or re-programmed cell is identified and/or isolated by fluorescent activated cell sorting (FACS) or affinity column chromatography, or by identification and/or isolation of plasma membrane proteins by mass spectography or chromatography, or by determining the presence or absence of a message (mRNA, transcript) determinative of an undifferentiated or neuronal cell phenotype.
In alternative embodiments, the methods of the invention further comprise implanting the trans-differentiated or re-programmed mammalian cell in or into a vessel, tissue or organ, wherein optionally the trans-differentiated or re-programmed mammalian cell is implanted in or into a vessel, tissue or organ ex vivo or in vivo. In alternative embodiments, the methods of the invention further comprise implanting the trans-differentiated or re-programmed mammalian cell in or into an individual in need thereof, wherein optionally the individual in need thereof has a neurodegenerative disease or an injury to the CNS, brain or spinal cord.
In alternative embodiments, the invention provides trans-differentiated or re-programmed cells made by practicing any method of the invention, wherein the trans-differentiated or re-differentiated or re-programmed cell is: a neuronal mammalian cell, or a fibroblast, or optionally a functional human cell or functional human neuronal cell, and optionally a cell having both the PTB and nPTB gene knocked out. In alternative embodiments, the mammalian cell is a human cell, a non-human primate cell, a monkey cell, a mouse cell, a rat cell, a guinea pig cell, a rabbit cell, a hamster cell, a goat cell, a bovine cell, an equine cell, an ovine cell, a canine cell or a feline cell.
In alternative embodiments, the invention provides methods for treating or ameliorating a neurodegenerative disease or an injury or neurodegenerative condition, comprising:
In alternative embodiments, the composition is administered in vivo in or in proximity to the diseased, injured or affected tissue.
In alternative embodiments, the neurodegenerative disease or injury, or neurodegenerative condition, is Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), a Polyglutamine (PolyQ) Disease, Amyotrophic lateral sclerosis (ALS), traumatic brain injury (TBI), Chronic traumatic encephalopathy (CTE), a paralysis, a stroke or an ischemic injury.
In alternative embodiments, the invention provides formulations, products of manufacture (e.g., implants, artificial organs or tissues), or kits comprising trans-differentiated or re-programmed cells of the invention.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications, and NCBI or PubMed sequences cited herein are hereby expressly incorporated by reference for all purposes.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Like reference symbols in the various drawings indicate like elements.
The invention provides compositions and in vivo, ex vivo and in vitro methods for trans-differentiation of, re-differentiating or re-programming mammalian cells to functional neurons. In alternative embodiments, the invention provides compositions capable of inactivating RNA polypyrimidine tract binding protein (PTB) for de-differentiating, re-differentiating or re-programming mammalian cells. The invention also provides compositions and methods for direct reprogramming, or trans-differentiation, of a first differentiated phenotype of a cell to a second differentiated phenotype, or to a functioning neuron.
This invention for the first time demonstrates that inactivation of a single RNA polypyrimidine tract binding protein (PTB) is sufficient to induce the expression of a specific set of transcription factors, which act together to trigger trans-differentiation of diverse cell types into functional neurons. The inventors identified a key gene that acts to regulate these factors. The invention demonstrates that PTB, which is naturally down regulated during brain development, is involved in regulating RNA metabolism at both the transcript splicing and microRNA (miRNA) levels. In alternative embodiments, the invention provides compositions and methods for engineering non-neuronal cells into neurons.
The inventors found that a single RNA binding protein PTB, which is naturally down regulated during brain development, is involved in regulating RNA metabolism at both the splicing and microRNA levels. The function of PTB in regulating microRNA targeting in the human genome was first demonstrated in this study. These functions cause a series of molecular switches, a most important one being the inactivation of the RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex. This leads to the induction of a series of neuronal specific genes in non-neuronal cells. In the presence of other neural trophic factors, the morphologically transformed cells become functional neurons.
The inventors identified a key gene, the PTB gene, that acts to regulate transcription factors controlling trans-differentiation of diverse cell types into functional neurons. As a result, the invention for the first time demonstrates that altered expression of the PTB gene is sufficient to induce all morphological and functional changes towards the neural lineage. In one embodiment, methods of the invention inactivates the PTB gene to regulate transcription factors to trans-differentiate diverse cell types into functional neurons; this embodiment inactivates a gene, as compared to overexpressing a number of genes together, to switch a cell fate, e.g., into functional neurons.
In alternative embodiments, the invention provides compositions and methods for engineering non-neuronal cells in vivo or ex vivo into neurons in the central nervous system (CNS), e.g., the brain or spinal cord, to treat an injury, condition or disease, e.g., a neurodegenerative disease, a spinal injury, a paralysis due to an injury or disease, and the like.
The present invention demonstrates that regulated PTB expression is able to induce massive reprogramming at both the splicing and microRNA levels to drive the cell fate decision towards the neuronal lineage. Thus, the invention provides compositions and methods for manipulating, e.g., trans-differentiating or re-programming, mammalian cell phenotypes, e.g., human or animal cell phenotypes, comprising use of compositions or compounds, e.g., proteins (e.g., antibodies, aptamers), nucleic acids (e.g., antisense or miRNA), small molecules and the like, to inactivation of an RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex or inactivate the Polypyrimidine Tract Binding protein (PTB) gene.
Antibodies, Therapeutic and Humanized Antibodies
In alternative embodiments, the invention provides antibodies that specifically bind to and inhibit: an RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex, or, a Polypyrimidine Tract Binding protein (PTB) gene or protein.
In alternative embodiments, the invention uses isolated, synthetic or recombinant antibodies that specifically bind to and inhibit or activate a PTB gene or protein.
In alternative aspects, an antibody for practicing the invention can comprise a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. In alternative aspects, an antibody for practicing the invention includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.”
Methods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N Y (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
In alternative embodiments, the invention uses “humanized” antibodies, including forms of non-human (e.g., murine) antibodies that are chimeric antibodies comprising minimal sequence (e.g., the antigen binding fragment) derived from non-human immunoglobulin. In alternative embodiments, humanized antibodies are human immunoglobulins in which residues from a hypervariable region (HVR) of a recipient (e.g., a human antibody sequence) are replaced by residues from a hypervariable region (HVR) of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In alternative embodiments, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues to improve antigen binding affinity.
In alternative embodiments, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to improve antibody affinity or functional activity. In alternative embodiments, the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of Ab framework regions are those of a human immunoglobulin sequence.
In alternative embodiments, a humanized antibody used to practice this invention can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of or derived from a human immunoglobulin.
However, in alternative embodiments, completely human antibodies also can be used to practice this invention, including human antibodies comprising amino acid sequence which corresponds to that of an antibody produced by a human. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.
In alternative embodiments, antibodies used to practice this invention comprise “affinity matured” antibodies, e.g., antibodies comprising with one or more alterations in one or more hypervariable regions which result in an improvement in the affinity of the antibody for antigen; e.g., a targeted transcriptional activating factor, compared to a parent antibody which does not possess those alteration(s). In alternative embodiments, antibodies used to practice this invention are matured antibodies having nanomolar or even picomolar affinities for the target antigen, e.g., a targeted transcriptional activating factor. Affinity matured antibodies can be produced by procedures known in the art.
Generating and Manipulating Nucleic Acids
In alternative aspects, composition and methods of the invention comprise use of nucleic acids for inactivating an RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex, or, inactivating a Polypyrimidine Tract Binding protein (PTB) gene or protein.
In alternative embodiments, nucleic acids of the invention are made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like.
The nucleic acids used to practice this invention, whether RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Any recombinant expression system can be used, including e.g. bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
Alternatively, nucleic acids used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
Techniques for the manipulation of nucleic acids used to practice this invention, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages or plasmids.
Nucleic acids or nucleic acid sequences used to practice this invention can be an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. Compounds use to practice this invention include “nucleic acids” or “nucleic acid sequences” including oligonucleotide, nucleotide, polynucleotide, or any fragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or double-stranded; and can be a sense or antisense strand, or a peptide nucleic acid (PNA), or any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g., iRNPs). Compounds use to practice this invention include nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. Compounds use to practice this invention include nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156. Compounds use to practice this invention include “oligonucleotides” including a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that may be chemically synthesized. Compounds use to practice this invention include synthetic oligonucleotides having no 5′ phosphate, and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.
Antisense Inhibitory Nucleic Acid Molecules
In alternative embodiments, the invention provides antisense or otherwise inhibitory nucleic acid molecules capable of decreasing or inhibiting expression of: an RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex; a Polypyrimidine Tract Binding protein (PTB) gene or protein, e.g., a neuronal-specific miR-124; and/or a nPTB. In alternative embodiments, methods of the invention comprise use of molecules that can generate a PTB and a nPTB knockdown, or abrogation or significant decrease in PTB and nPTB expression. In alternative embodiments, methods of the invention comprise use of these molecules to sequentially knockout first PTB, then nPTB, thus efficiently converting a human cell (e.g., a fibroblast) to a functional neuronal cell with mature neuronal marks, such as MAP2. It was demonstrated that nPTB has to be knocked down 4 days or later to achieve this phenotype. Accordingly, this exemplary embodiment provides methods for converting non-neuronal human cells to functional neurons for regenerative medicine.
The sequences of PTB and nPTB are known (see e.g., Romanelli et al. (2005) Gene, August 15:356:11-8; Robinson et al., PLoS One. 2008 Mar. 12; 3(3):e1801. doi: 10.1371/journal.pone.0001801; Makeyev et al., Mol. Cell (2007) August 3; 27(3):435-48); thus, one of skill in the art can design and construct antisense, miRNA, siRNA molecules and the like to modulate, e.g., to decrease or inhibit, the expression of PTB and/or nPTB; to practice the methods of this invention.
Naturally occurring or synthetic nucleic acids can be used as antisense oligonucleotides. The antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening. The antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholino carbamate nucleic acids.
RNA Interference (RNAi)
In alternative embodiments, the invention uses RNAi inhibitory nucleic acid molecules capable of decreasing or inhibiting expression of: an RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex, or, a Polypyrimidine Tract Binding protein (PTB) or nPTB gene, message or protein.
In one aspect, the RNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAi molecule can comprise a double-stranded RNA (dsRNA) molecule, e.g., siRNA, miRNA (microRNA) and/or short hairpin RNA (shRNA) molecules. For example, in one embodiment, the invention uses inhibitory, e.g., siRNA, miRNA or shRNA, nucleic acids that inhibit or suppress the activity of a tumor suppressor gene retinoblastoma-1 (RB1) and/or a p53 tumor suppressor gene (TP53).
In alternative aspects, the RNAi is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. While the invention is not limited by any particular mechanism of action, the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). A possible basic mechanism behind RNAi, e.g., siRNA for inhibiting transcription and/or miRNA to inhibit translation, is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In one aspect, the RNAi's of the invention are used in gene-silencing therapeutics, e.g., to silence one or a set of transcription factors responsible for maintaining the differentiated phenotype of the differentiated cell; see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one aspect, the invention provides methods to selectively degrade an RNA using the RNAi's of the invention. In one aspect, the RNAi molecules of the invention can be used to generate a loss-of-function mutation in a cell. These processes may be practiced in vitro, ex vivo or in vivo.
In one aspect, intracellular introduction of the RNAi (e.g., miRNA or siRNA) is by internalization of a target cell specific ligand bonded to an RNA binding protein comprising an RNAi (e.g., microRNA) is adsorbed. The ligand can be specific to a unique target cell surface antigen. The ligand can be spontaneously internalized after binding to the cell surface antigen. If the unique cell surface antigen is not naturally internalized after binding to its ligand, internalization can be promoted by the incorporation of an arginine-rich peptide, or other membrane permeable peptide, into the structure of the ligand or RNA binding protein or attachment of such a peptide to the ligand or RNA binding protein. See, e.g., U.S. Patent App. Pub. Nos. 20060030003; 20060025361; 20060019286; 20060019258. In one aspect, the invention provides lipid-based formulations for delivering, e.g., introducing nucleic acids of the invention as nucleic acid-lipid particles comprising an RNAi molecule to a cell, see .g., U.S. Patent App. Pub. No. 20060008910.
Methods for making and using RNAi molecules, e.g., siRNA and/or miRNA, for selectively degrade RNA are well known in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.
Methods for making expression constructs, e.g., vectors or plasmids, from which an inhibitory polynucleotide (e.g., a duplex siRNA of the invention) is transcribed are well known and routine. A regulatory region (e.g., promoter, enhancer, silencer, splice donor, acceptor, etc.) can be used to transcribe an RNA strand or RNA strands of an inhibitory polynucleotide from an expression construct. When making a duplex siRNA inhibitory molecule, the sense and antisense strands of the targeted portion of the targeted IRES can be transcribed as two separate RNA strands that will anneal together, or as a single RNA strand that will form a hairpin loop and anneal with itself. For example, a construct targeting a portion of a gene, e.g., an NADPH oxidase enzyme coding sequence or transcriptional activation sequence, is inserted between two promoters (e.g., mammalian, viral, human, tissue specific, constitutive or other type of promoter) such that transcription occurs bidirectionally and will result in complementary RNA strands that may subsequently anneal to form an inhibitory siRNA of the invention.
Alternatively, a targeted portion of a gene, coding sequence, promoter or transcript can be designed as a first and second antisense binding region together on a single expression vector; for example, comprising a first coding region of a targeted gene in sense orientation relative to its controlling promoter, and wherein the second coding region of the gene is in antisense orientation relative to its controlling promoter. If transcription of the sense and antisense coding regions of the targeted portion of the targeted gene occurs from two separate promoters, the result may be two separate RNA strands that may subsequently anneal to form a gene-inhibitory siRNA used to practice this invention.
In another aspect, transcription of the sense and antisense targeted portion of the targeted gene is controlled by a single promoter, and the resulting transcript will be a single hairpin RNA strand that is self-complementary, i.e., forms a duplex by folding back on itself to create a gene-inhibitory siRNA molecule. In this configuration, a spacer, e.g., of nucleotides, between the sense and antisense coding regions of the targeted portion of the targeted gene can improve the ability of the single strand RNA to form a hairpin loop, wherein the hairpin loop comprises the spacer. In one embodiment, the spacer comprises a length of nucleotides of between about 5 to 50 nucleotides. In one aspect, the sense and antisense coding regions of the siRNA can each be on a separate expression vector and under the control of its own promoter.
Inhibitory Ribozymes
In alternative embodiments, the invention uses ribozymes capable of decreasing or inhibiting expression of: an RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex, or, a Polypyrimidine Tract Binding protein (PTB) or nPTB gene, message or protein.
These ribozymes can inhibit a gene's activity by, e.g., targeting a genomic DNA or an mRNA (a message, a transcript). Strategies for designing ribozymes and selecting a gene-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it can be released from that RNA to bind and cleave new targets repeatedly.
Kits and Instructions
The invention provides kits comprising compositions and methods of the invention, including instructions for use thereof. As such, kits, cells, vectors and the like can also be provided.
For example, in alternative embodiments, the invention provides kits comprising compositions capable of decreasing or inhibiting expression of: an RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex, or, a Polypyrimidine Tract Binding protein (PTB) or nPTB gene, message or protein, for e.g., trans-differentiating or re-programming a mammalian cell. In alternative embodiments, the kits comprise instruction for practicing methods of the invention.
Formulations
In alternative embodiments, the invention provides compositions and formulations for use in in vitro, ex vivo or in vivo methods of the invention for trans-differentiating, re-differentiating or re-programming a mammalian cell to a neuronal cell. In alternative embodiments, these compositions comprise a plurality of (a set of) proteins and/or nucleic acids formulated for these purposes (e.g., to decrease or inhibit expression of a PTB and nPTB gene, message or protein), e.g., formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a nanoparticle, in a nanolipoparticle and the like.
In alternative embodiments, the compositions can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vitro, ex vivo or in vivo conditions, a desired in vitro, ex vivo or in vivo method of administration and the like. Details on techniques for in vitro, ex vivo or in vivo formulations and administrations are well described in the scientific and patent literature.
Formulations and/or carriers used to practice this invention can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vitro, ex vivo or in vivo applications.
Compositions used to practice this invention can be in admixture with an aqueous and/or buffer solution or as an aqueous and/or buffered suspension, e.g., including a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate. Formulations can be adjusted for osmolarity, e.g., by use of an appropriate buffer.
In practicing this invention, the compounds (e.g., formulations) of the invention can comprise a solution of nucleic acids (e.g., a neuronal-specific miR-124) or other nucleic acids dissolved in a pharmaceutically acceptable carrier, e.g., acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, solutions and formulations used to practice the invention are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.
The solutions and formulations used to practice the invention can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent (e.g., a neuronal-specific miR-124) in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vitro, ex vivo or in vivo administration selected and the desired results, e.g., for trans-differentiating or re-programming a mammalian cell.
The solutions and formulations used to practice the invention can be lyophilized; for example, the invention provides a stable lyophilized formulation comprising a neuronal-specific miR-124. In one aspect, this formulation is made by lyophilizing a solution comprising an active agent used to practice the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. patent app. no. 20040028670.
The compositions and formulations of the invention can be delivered by the use of liposomes (see also discussion, below). By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the active agent into a target cells in an in vitro, ex vivo or in vivo application.
Nanoparticles, Nanolipoparticles and Liposomes
The invention also provides nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds used to practice methods of this invention (e.g., compounds to decrease or inhibit expression of a PTB or nPTB gene, message or protein), e.g., to deliver compositions of the invention to mammalian cells in vitro, ex vivo or in vivo. In alternative embodiments, these compositions are designed to target specific molecules, including biologic molecules, such as polypeptides, including cell surface polypeptides, e.g., for targeting a desired cell type, e.g., a mammalian cell targeted for trans-differentiation or re-programming.
The invention provides multilayered liposomes comprising compounds used to practice this invention, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice this invention (e.g., a neuronal-specific miR-124).
Liposomes can be made using any method, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., a composition used to practice this invention, e.g., a neuronal-specific miR-124), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
In one embodiment, liposome compositions used to practice this invention comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a composition used to practice this invention, e.g., a neuronal-specific miR-124, to a desired cell type, as described e.g., in U.S. Pat. Pub. No. 20070110798.
The invention also provides nanoparticles comprising a composition used to practice this invention, e.g., a neuronal-specific miR-124, in the form of active agent-containing nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment, the invention provides nanoparticles comprising a fat-soluble active agent of this invention or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.
In one embodiment, solid lipid suspensions can be used to formulate and to deliver a composition used to practice this invention, e.g., a neuronal-specific miR-124, to mammalian cells in vitro, ex vivo or in vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.
Peptide Delivery Vehicles
In alternative embodiments, any delivery vehicle can be used to practice the methods or compositions of this invention, e.g., to deliver a composition used to practice this invention (e.g., compounds to decrease or inhibit expression of a PTB or nPTB gene, message or protein), e.g., a neuronal-specific miR-124, to mammalian cells in vitro, ex vivo or in vivo. For example, delivery vehicles comprising polycations, cationic polymers and/or cationic peptides, such as polyethyleneimine derivatives, can be used e.g. as described, e.g., in U.S. Pat. Pub. No. 20060083737.
In one embodiment, a dried polypeptide-surfactant complex is used to formulate a composition used to practice this invention, wherein a surfactant is associated a composition used to practice this invention via a noncovalent bond e.g. as described, e.g., in U.S. Pat. Pub. No. 20040151766.
In one embodiment, a covalent conjugate between a poly(alkylene oxide) and a glycosylated or non-glycosylated composition used to practice this invention is used, where a poly(alkylene oxide) can be conjugated to the composition via a glycosyl linking group, and a glycosyl linking group can be interposed between a composition used to practice this invention and a poly(alkylene oxide). A covalent conjugate can be formed by contacting a composition used to practice this invention with a glycosyltransferase and a modified sugar donor; the glycosyltransferase transfers the modified sugar moiety to the composition to form a covalent conjugate; the modified sugar moiety can be a poly(alkylene oxide). See e.g., U.S. Pat. No. 7,416,858.
In one embodiment, a composition used to practice this invention can be applied to cells as polymeric hydrogels or water-soluble copolymers, e.g., as described in U.S. Pat. No. 7,413,739; for example, a composition can be polymerized through a reaction between a strong nucleophile and a conjugated unsaturated bond or a conjugated unsaturated group, by nucleophilic addition, wherein each precursor component comprises at least two strong nucleophiles or at least two conjugated unsaturated bonds or conjugated unsaturated groups.
In one embodiment, a composition used to practice this invention, e.g., a neuronal-specific miR-124, can be applied to cells using vehicles with cell membrane-permeant peptide conjugates, e.g., as described in U.S. Pat. Nos. 7,306,783; 6,589,503. In one aspect, the composition itself is conjugated to a cell membrane-permeant peptide. In one embodiment, a composition and/or the delivery vehicle are conjugated to a transport-mediating peptide, e.g., as described in U.S. Pat. No. 5,846,743, describing transport-mediating peptides that are highly basic and bind to poly-phosphoinositides.
In one embodiment, electro-permeabilization is used as a primary or adjunctive means to deliver a composition of the invention to a cell, e.g., using any electroporation system as described e.g. in U.S. Pat. Nos. 7,109,034; 6,261,815; 5,874,268.
Products of Manufacture, Implants and Artificial Organs
The invention also provides products of manufacture comprising cells of the invention, and use of cells made by methods of this invention, including for example implants and artificial organs, bioreactor systems, cell culture systems, plates, dishes, tubes, bottles and flasks comprising cells of this invention, e.g., human cells generated by practicing a method of this invention. Any implant, artificial organ, bioreactor systems, cell culture system, cell culture plate, dish (e.g., petri dish), cell culture tube and/or cell culture flask (e.g., a roller bottle) can be used to practice this invention.
In alternative embodiments the invention provides a bioreactor, implant, stent, artificial organ or similar device comprising a cell of the invention, or cells made by a method of this invention; for example, including implants as described in U.S. Pat. Nos. 7,388,042; 7,381,418; 7,379,765; 7,361,332; 7,351,423; 6,886,568; 5,270,192; and U.S. Pat. App. Pub. Nos. 20040127987; 20080119909 (describing auricular implants); 20080118549 (describing ocular implants); 20080020015 (describing a bioactive wound dressing); 20070254005 (describing heart valve bio-prostheses, vascular grafts, meniscus implants); 20070059335; 20060128015 (describing liver implants).
Implanting Cells In Vivo
In alternative embodiments, the methods of the invention also comprise implanting or engrafting the trans-differentiated re-programmed cells (of the invention, or made by a method of this invention), or re-programmed differentiated cells (of the invention, or made by a method of this invention) in a vessel, tissue or organ; and in one aspect, comprise implanting or engrafting the re-programmed differentiated cell in a vessel, tissue or organ ex vivo or in vivo, or implanting or engrafting the re-programmed differentiated cell in an individual in need thereof.
Cells can be removed from an individual, treated using the compositions and/or methods of this invention, and reinserted (e.g., injected or engrafted) into a tissue, organ or into the individual, using any known technique or protocol. For example, trans-differentiated re-programmed cells, or re-programmed differentiated cells, can be re-implanted (e.g., injected or engrafted) using microspheres e.g., as described in U.S. Pat. No. 7,442,389; e.g., in one aspect, the cell carrier comprises a bulking agent comprising a plurality of round and smooth polymethylmethacrylate microparticles preloaded within a mixing and delivery system and an autologous carrier comprising these cells. In another embodiment, the cells are readministered to a tissue, an organ and/or an individual in need thereof in a biocompatible crosslinked matrix, as described e.g., in U.S. Pat. App. Pub. No. 20050027070.
In another embodiment, the cells of the invention (e.g., cells made by practicing the methods of this invention) are readministered (e.g., injected or engrafted) to a tissue, an organ and/or an individual in need thereof within, or protected by, a biocompatible, nonimmunogenic coating, e.g., as on the surface of a synthetic implant, e.g., as described in U.S. Pat. No. 6,969,400, describing e.g., a protocol where a composition can be conjugated to a polyethylene glycol that has been modified to contain multiple nucleophilic groups, such as primary amino or thiol group.
In one embodiment, the cells of the invention (e.g., cells made by practicing the methods of this invention) are readministered (e.g., injected or engrafted) to a tissue, an organ and/or an individual in need thereof using grafting methods as described e.g. by U.S. Pat. Nos. 7,442,390; 5,733,542.
The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.
This invention for the first time demonstrates that inactivation of a single RNA polypyrimidine tract binding protein (PTB) is sufficient to induce the expression of a specific set of transcription factors, which act together to trigger trans-differentiation of diverse cell types into functional neurons. The inventors found that a single RNA binding protein PTB, which is naturally down regulated during brain development, is involved in regulating RNA metabolism at both the splicing and microRNA levels. The function of PTB in regulating microRNA targeting in the human genome was first demonstrated in this study. These functions cause a series of molecular switches, a most important one being the inactivation of the RE1-Silencing Transcription factor (REST; also known as Neuron-Restrictive Silencer Factor, or NRSF) complex. This leads to the induction of a series of neuronal specific genes in non-neuronal cells. In the presence of other neural trophic factors, the morphologically transformed cells become functional neurons.
Here we report that repression of a single RNA binding protein PTB, which occurs during normal brain development, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. In this RNA program, neuronal-specific miR-124 targets PTB for degradation, which in turn triggers gene expression reprogramming, leading to induced expression of all critical transcription factors known to be sufficient to cause trans-differentiation of fibroblasts to neurons. Besides its established role in regulated splicing, we show that PTB has a previously undocumented function in regulating microRNA targeting. A key event in this pathway is PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby de-repressing many neuronal genes, including miR-124, in non-neuronal cells. This creates and accelerates a potent feed-forward loop to elicit cellular reprogramming to the neuronal lineage.
In PTB-depleted cells, we unexpectedly observed conversion of diverse cell types into neuronal-like cells. In addition to induced alternative splicing events, we found an extensive involvement of PTB in the regulation of microRNA targeting either through direct competition or induced switch of local RNA secondary structure. A key event is the activation of the miR-124/REST loop in which PTB not only serves as a target, but also acts as a potent regulator. Consequently, regulated PTB expression induces massive reprogramming at both the splicing and microRNA levels to drive the cell fate decision towards the neuronal lineage.
Results
PTB Down-Regulation Switches Multiple Cell Types to Neuronal-Like Cells
We attempted to use specific shRNAs to stably knock down PTB in order to systematically analyze PTB-regulated splicing. As expected, shPTB induced nPTB expression in HeLa cells (
We extended this analysis to multiple cell types of diverse origin, including human embryonic carcinoma stem cells (NT2), mouse neural progenitor cells (N2A), human retinal epithelial cells (ARPE19), and primary mouse embryo fibroblasts (MEFs). Upon PTB knockdown (
We further characterized two of these cell lines (N2A and MEFs) by examining additional neural markers, including Synapsin 1 (SYN1), vGLUT1 and NeuN (
Both N2A cells and MEFs were efficiently converted by two distinct shRNAs against PTB to neuronal-like cells (
MEF-Derived Neurons are Functional with Synaptic Activities
To determine the functionality of differentiated cells, we patch-clamped both shPTB-induced neurons from N2A cells and MEFs. We observed that 11 out of 12 N2A cell-derived neurons exhibited fast inward Na+ currents and action potential upon membrane depolarization (
The detected postsynaptic currents likely reflect both glutamatergic and GABAergic responses, because CNQX+APV (antagonists of glutamatergic channel receptors) and Picrotoxin (PiTX, antagonist of GABAA channel receptors) could sequentially block the expected signals (
PTB Regulates the Expression of Many Neuronal Genes in Non-Neuronal Cells
Because of the induced neuronal morphology and the availability of the genome-wide PTB-RNA interaction map on HeLa cells (Xue et al., 2009), we initially took this cell type as a surrogate model to understand shPTB-induced cellular reprogramming. We identified by RNA-seq a large number of up- or down-regulated genes induced by shPTB (
We noted the induction of Brn2 and Myt11, which correspond to 2 out of 5 key transcription factors previously shown to be sufficient to induce trans-differentiation of fibroblasts into neurons (Vierbuchen et al., 2010). Because HeLa cells have a severely re-arranged genome, we performed a focused analysis on MEFs by RT-qPCR (
REST Activity Contributes a Key Part to the shPTB-Induced Neuronal Program
The REST complex is known to repress a large set of neuronal genes in non-neuronal cells (Johnson et al., 2007). Interestingly, we noted that all induced transcription factors examined in
To determine how the RSET complex was compromised, we examined the response of REST and REST co-factors to shPTB in HeLa cells. While REST expression was little affected from our RNA-seq analysis, we found that SCP1, a Pol II Ser5 phosphatase associated with the REST complex (Yeo et al., 2005), was significantly down regulated by shPTB in multiple cell types with induced neuronal morphology (
Recent studies suggest that REST is required for maintaining the population of neural stem cells (Gao et al., 2011) and genetic inactivation of REST does not efficiently turn fibroblasts into neurons, despite the induction of some neuronal genes (Aoki et al., 2012). However, a dominant negative SCP1 was able to efficiently drive neuronal differentiation on P19 cells (Yeo et al., 2005). We thus wished to directly test the contribution of SCP1 to shPTB-induced neurogenesis under our experimental conditions and we similarly tested REST for comparison. We found that both shSCP1 and shREST, but not control shRNA, were able to trigger neuronal differentiation on MEFs (
PTB-Regulated Splicing Likely Facilitates the Development of the Neural Program
PTB is best known for its role in regulated splicing (Makeyev et al., 2007), which is consistent with our RNA-seq data from HeLa cells (
During the course of this investigation, we detected induced alternative splicing of two key genes, LSD1 (a histone lysine demethylase, a component of the REST complex) and PHF21A (a component of the histone deacetylase HDAC1 complex) upon PTB knockdown in HeLa and N2A cells (
PTB is Involved in the RNA Stability Control of Key Neuronal Genes
Because many PTB-affected genes could not be explained by induced splicing, we searched for other potential mechanisms. PTB has been reported to regulate RNA stability in multiple cases through C/U-rich sequences in the 3′UTR, but the mechanism has remained elusive (Knoch et al., 2004; Kosinski et al., 2003; Pautz et al., 2006; Porter et al., 2008; Tillmar and Welsh, 2002; Woo et al., 2009). By examining the PTB-RNA map (Xue et al., 2009), we noted extensive PTB binding events in the 3′UTR of all of those reported genes (
Multiple PTB binding peaks are evident in the 3′UTR of CoREST and HDAC1 (
PTB Regulates RNA Stability in Conjunction with microRNA
From this point, we used HeLa cells to understand the mechanism underlying PTB-regulated gene expression mainly because of the experimental manipulability of the cell type, although it is important to emphasize that caution must be taken when extrapolating deduced molecular mechanism from one cell type to another. To determine how extensively PTB is involved in RNA stability control, we performed RNA-seq on mock-depleted and PTB-depleted cells before (T0) or after blocking transcription with Actinomycin D for 4 hours (T4). This allowed us to calculate mRNA decay [(T0−T4)/T0×100%] and determine how such decay might be influenced by PTB for each expressed gene in the human genome. We identified a total of 142 genes that showed significantly increased (red dots in
We next selected a panel of PTB-bound genes to determine whether these PTB-regulated events were dependent on the microRNA machinery (
The 3′UTR of SCP1 Contains Multiple microRNA Targeting Sites
We used SCP1 as a model to investigate the functional interplay between PTB and microRNA. We compiled PTB and Ago2 CLIP-seq signals (see below in
Previous studies showed that forced miR-124 expression could switch the gene expression profile towards that of brain in HeLa cells (Lim et al., 2005). Relevant to the present study, miR-124 has also been shown to subject to regulation by SCP1 during neurogenesis in vivo (Visvanathan et al., 2007). Collectively, these observations suggest an important pathway for neuronal differentiation that involves the functional interplay between miR-124, PTB and SCP1/REST.
PTB Directly Competes with microRNA Targeting on the 3′UTR of SCP1
Perturbation experiments confirmed the role of PTB in the regulation of microRNA function. For example, overexpression of miR-96 suppressed SCP1 expression and PTB knockdown enhanced the effect, whereas miR-96 antagomir showed the opposite response (
To determine the sequence requirement for both microRNA- and PTB-mediated actions, we carried out mutational analysis in the seed region of individual microRNA target sites and on the nearby PTB binding sites (
PTB can Also Boost microRNA Action on Specific Genes
Our RNA-seq experiments and luciferase-based assays revealed both up- and down-regulated genes in response to PTB knockdown. While many up-regulated genes likely resulted from de-repression, we detected multiple examples of up-regulated genes in PTB knockdown cells that appear to depend on their 3′UTRs (
To understand how PTB knockdown could induce gene expression, we took GNPDA1 as a model, which was up regulated by PTB via its 3′UTR (
PTB Facilitates microRNA Action by Changing Local RNA Secondary Structure
To uncover the mechanism for PTB-dependent enhancement of microRNA action, we determined the secondary structure in the 3′UTR of GNPDA1 gene using RNase T1 to cut single-stranded RNA after the nucleotide G, and RNase V1 to cleave double-stranded RNA (
We substantiated the increase of single-strandness by in-line probing, an approach widely used to detect riboswitches, which measures spontaneous RNA cleavage in solution with strong preference for U-rich residues (Regulski and Breaker, 2008). With increasing amounts of PTB, we found that the entire region gradually opened up, as indicated by enhanced cleavage on nearly all residues from 10G to 33G and the flanking U-rich PTB binding sites from 34C to 40U (
PTB Globally Regulates microRNA-mRNA Interactions in the Human Genome
To assess the global impact of PTB on both positive and negative modulation of microRNA targeting, we conducted CLIP-seq mapping of Ago2 before and after PTB knockdown in HeLa cells. As previously described (Chi et al., 2009), we detected Ago2-RNA crosslinking adducts IPed with anti-Ago2 above the position of the Ago2 protein on SDS-PAGE (
We next compared the relationship between PTB and Ago2 occupancies in the 3′UTR of protein-coding genes in response to PTB knockdown. The Ago2 binding profiles were similar in the protein-coding side (upstream of the stop codon) on both wild type (wt) and shPTB-treated cells, which provide important internal controls for our comparison. By separately analyzing PTB bound and unbound genes, we found that PTB depletion caused a dramatic increase in Ago2 binding in the 3′UTR of PTB bound targets, but had only a minor increase on PTB unbound targets (
PTB-Regulated Ago2 Binding Functionally Correlates to Induced Gene Expression
To determine how such changes in Ago2 binding might be related to altered gene expression, we took a strategy recently used to analyze the interplay between HuR and microRNA (Mukherjee et al., 2011) to segregate expressed genes into 5 groups based on mapped PTB and Ago2 binding events in their 3′UTRs: (1) −Ago2, −PTB, (2)+Ago2, −PTB, (3) −Ago2, +PTB, (4) +Ago2, +PTB, but no overlap, and (5) +Ago2, +PTB with at least one overlapping binding event within 10 nt. This allowed us to compare gene expression changes in different groups in response to PTB knockdown by plotting genes in each group against induced transcript changes in a cumulative fashion (
We found no significant differences between Groups 1-3, consistent with the lack of PTB and Ago2 actions on these genes. In comparison, relative to genes in Group 1 (black line), genes bound by both PTB and Ago2 but with little overlap (Group 4, green line) were linked to both repressed (right-shift at top) and enhanced gene expression (left-shift at bottom), consistent with changes in RNA secondary structure that caused increased or decreased microRNA targeting on different genes (
Discussion
We now report that the reduced expression of a single RNA binding PTB, which occurs during brain development, is able to potently induce differentiation or trans-differentiation of diverse cell types into neuronal-like cells or even functional neurons.
Our data highlights the contribution of specific regulated splicing during the induction of neuronal differentiation. We discovered a PTB-regulated microRNA program responsible for dismantling of multiple components of REST. We provide further evidence that knockdown of SCP1 or the REST itself is sufficient to trigger trans-differentiation of MEFs into neuronal-like cells. The REST complex is well known for its role in suppressing many neuronal genes, including miR-124, in non-neuronal cells, while miR-124 and other neuronal specific microRNAs target various REST components, including SCP1 and CoREST. This creates a potent regulatory loop (
Strikingly, PTB down-regulation induced the expression of all critical transcription factors previously shown to be sufficient to induce trans-differentiation of MEFs into functional neurons. Our data provide a mechanism for the induction of these transcription factors because all of these transcription factors appear to be direct REST targets. The puzzle is why genetic ablation of REST or HADC1 impaired self-renewal of neural stem cells, thus preventing unintended neurogenesis in various cellular and animal models (Dovey et al., 2010; Gao et al., 2011; Lee et al., 2002). While the cellular context undoubtedly contributes to such restriction of neurogenesis in vivo, it is possible that PTB knockdown may mimic a gradual and sequential switch of a series of events during normal developmental processes by preventing abrupt induction of gene expression that may cause cell death before differentiation. We note that the PTB-regulated RNA program takes place in cells containing induced nPTB and our preliminary results indicate that simultaneous knockdown of PTB and nPTB greatly compromised the development of neuronal morphology. This may indeed represent critical sequential events during normal brain development (Zheng et al., 2012).
Mechanistically, our study joined PTB to a growing list of RNA binding proteins, including HuR, Dnd1, CRD-BP, and PUM1, that have been implicated in modulating microRNA targeting in mammalian cells (van Kouwenhove et al., 2011). In comparison with previous studies where specific RNA binding proteins appear to either positively or negatively regulate microRNA targeting, we found that PTB can function in both ways, competing with microRNA targeting on some genes, but promoting microRNA targeting on the others. These two modes of regulation may simultaneously occur on different locations in the same genes, and thus, the net effect of positive and negative regulation may dictate the final functional outcome. These working principles may be generally applicable to many other RNA binding proteins involved in the regulation of microRNA-mRNA interactions. Our global analysis of Ago2 binding in response to PTB knockdown also suggests that PTB binding may have some long-range effects on microRNA targeting in addition to local events. This may result from potential PTB-mediated RNA looping, as proposed earlier (Oberstrass et al., 2005), the action of other induced microRNAs, or synergy with other RNA binding proteins, all of which represent interesting regulatory paradigms to be investigated in future studies.
Experimental Procedures
Cell Culture, RNAi, Immunocytochemistry and Electrophysiological Analysis
Cell culture conditions, treatments with siRNA and shRNA, and immunocytochemistry are detailed in the supplemental experimental procedures. Glial cells were isolated from GFP-transgenic rat brain (Hakamata et al., 2001) and single cell patch clamp recordings were performed using an Axopatch 200B amplifier and pClamp 10.0 software (HEKA Elektronik, Lambrecht/Pfalz, Germany), as described in the supplemental information.
RT-qPCR, Western Blotting, and Luciferase Assays
qPCR was performed with Fast Start universal SYBR green master mix using gene specific primers listed in Table 1 (
RNA-Seq, CLIP-Seq, and Probing of RNA Secondary Structure
RNA-seq and CLIP-seq was performed as previously described (Xue et al., 2009). Normalized Ago2 tags are plotted relative to the stop codon at the 3′ end of genes as described (Chi et al., 2009). Two-sided Kolmogorov-Smimov statistics (in the R package, http://cran.r-project.org/) was used to determine the significance of the shift in pair-wise comparison. RNA foot-printing by RNase T1 and V1 was according to the manual from Ambion. The in-line probing assay was as previously described (Regulski and Breaker, 2008), which is also detailed in the supplemental information.
Accession Numbers
The RNA-seq and CLIP-seq data are available at the Gene Expression Omnibus (GEO), which is a public functional genomics data repository run by NCBI, NIH; see e.g., Barrett, et al. Methods Enzymol. 2006; 411:352-69.
For studies illustrated in supplemental figures, or
Cell Culture, RNAi, and Immunocytochemistry
HeLa cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (Omega Scientific) and 100 U of penicillin/streptomycin (Life Technology). NT2 cells were cultured in Minimum Essential Medium (MEMα, which contains ribonucleosides, deoxyribonucleosides and GlutaMAX™) plus 10% FBS and 100 U of penicillin/streptomycin. N2A cells were propagated in DMEM supplemented with 10% FBS, 100U of penicillin/streptomycin. Mouse Embryonic Fibroblasts (MEFs) were isolated from E14.5 C57/BL6 mouse embryos. Head, vertebral column, and all internal organs were removed and the remaining embryonic tissues were manually dissociated followed by incubation in 0.25% Trypsin (Life Technology) for 10 min. MEFs were cultured in DMEM plus 10% FBS, non-essential amino acids, sodium pyruvate, and penicillin/streptomycin. ARPE19 cells were cultured in DMEM/F12 plus 10% FBS, 1% non-essential amino acids, and 100 U of penicillin/streptomycin.
Lentiviral shRNAs against human PTBP1 (TRCN0000231420, TRCN0000001062), mouse PTBP1 (TRCN0000109272, TRCN0000109274), Mouse REST (TRCN0000321488, TRCN0000071346), mouse CoREST (TRCN0000071368, TRCN0000071371) and mouse CTDSP1 (which encodes for SCP1) were purchased from Thermo Scientific and cloned in the pLKO.1 vector. Individual shRNAs were packaged into replication-incompetent lentiviral particles in HEK293T cells by co-transfecting individual pLKO plasmids with the packaging mix (Sigma). Viral particles were collected twice 48 hrs and 72 hrs post-transfection. Cells were infected with individual lentiviral particles for 16 hrs followed by selection with 2 μg/ml Puromycin for 48 hrs.
Selected cells were switched to different media to allow further development of complex neuronal morphology: HeLa and NT2 cells were switched to N3 media (DMEM/F12 plus 25 μg/ml insulin, 50 □g/ml transferring, 30 nM sodium selenite, 20 nM progesterone, 100 nM putrescrine) or N3 media supplemented with a panel of neurotrophic factors, including BDNF, GDNF, NT3 and CNTF (Peprotech) and Ara-C (2 μM, Sigma). MEF and ARPE19 cells were first cultured in N3 media plus FGF2 (10 ng/ml) for 3 days, switched to N3 media for a week to 10 days, and then supplemented with BDNF, GDNF, NT3 and CNTF (Peprotech) for additional 6 days before immunocytochemical and electrophysiological analyses. N2A cells were maintained in DMEM supplemented with 10% FBS, 100U of penicillin/streptomycin and 1 μg/ml Puromycin (Clontech). The media were then supplemented with BDNF, GDNF, NT3 and CNTF for 3 days prior to electrophysiological analyses. It is important to emphasize that none of the cell types cultured under above described conditions exhibited neurite outgrowth when treated with a control shRNA.
Immunocytochemistry experiments were performed on cells seeded on coverslip that had been coated with poly-D-lysin (0.05 mg/ml) and laminin (0.005 mg/ml) overnight at 37° C. Cells were washed twice with PBS, fixed in 4% Paraformaldehyde (Wako) for 15 min at room temperature, and permeabilized with 0.1% Triton X-100 in PBS for 15 min on ice. After washing three times with PBS, cells were blocked in PBS containing 3% BSA for 1 hr at room temperature.
The following primary antibodies with indicated dilution in blocking buffer were used: Rabbit anti-Tuj1 (Covance, 1:1,000), Mouse anti-Tuj1 (Covance, 1:1,000), Rabbit anti-MAP2 (Cell Signaling Technology, 1:200), Mouse anti-NeuN (Milipore, 1:200), Rabbit anti-Synapsin I (Sigma, 1:1000), Rabbit anti-Synapsin I (Milipore, 1:500), Rabbit anti-VGLUT1 (Synaptic Systems, 1:200), Rabbit anti-GABA (Sigma, 1:1000), Mouse anti-PSD95 (NeuroMab, 1:100), Rabbit anti-NGF receptor P75 (Milipore, 1:100), Rabbit anti-Brn2/POU3F2 (Cell Signaling Technology, 1:200), goat anti-Sox2 (Santa cruz, 1:200), Mouse anti-Pax3 (DSHB, 1:250), Mouse anti-Pax6 (Covance, 1:100), Mouse anti-Pax7 (DSHB, 1:250), Mouse anti-NKX2.2 (DSHB, 1:100), Mouse anti-Olig1 (Neuromab, 1:100), Mouse anti-GFAP (Neuromab, 1:100), Mouse anti-CoREST (BD biosciences, 612146), Rabbit anti-CTDSP1 (Sigma, SAB4502550). After staining with corresponding secondary antibodies in PBS plus 1% BSA, coverslips were washed six times with PBS, each for 5 min, mounted with the mounting medium containing DAPI (Vector Labs) onto glass slides, and examined under Olympus FluoView FV1000.
Glial Cell Isolation and Electrophysiological Analysis
GFP-marked glial cells were prepared from GFP-transgenic rat brain that ubiquitously expresses GFP from a chicken β-actin promoter (Hakamata et al., 2001). In this published study, GFP was detected in all cell types in the brain. The procedure for glial cell isolation was according to a published protocol (Pang et al., 2011). Briefly, postnatal day 1 pups were anesthetized on ice. Heads were removed with surgical scissors and transferred into a fresh 10 cm plate. Brain tissues were dissected out with a curved-tip forceps and collected in a 10 cm dish containing 10 ml cold HBSS. Cortices were isolated under a dissecting microscope and placed in a fresh 10 cm dish. Cortical tissues were cut into small pieces, re-suspended in 2 ml HBSS, and transferred to a 50 ml centrifuge tube. The dissection of cortical tissues was repeated twice. Small tissue pieces in 6 ml of HBSS were combined, to which 750 μl 10× Trypsin/EDTA and 750 μl of 10 mg/ml DNase I were added. The sample was vigorously agitated for 15 min in a 37° C. water bath to favor enzymatic digestion of the tissue. The tube was let stand for 5 min and 5 ml dissociated cells collected in a new 50-ml centrifuge tube containing the MEF media. The remaining undissociated tissue was trypsinized one more time with another 6 ml of HBSS containing 750 μl of 10× trypsin/EDTA and 750 μl of 10 mg/ml DNase I. Dissociated cells were filtered through a 100-μm nylon cell strainer and collected in a fresh 50-ml centrifuge tube. Dissociated glial cells were collected by centrifugation at 200 g. Supernatant was removed and the cells were re-suspended in culture media and seeded on a 10 cm tissue culture dish (at the density of cells from 2-3 cortices per 10 cm dish). The media were replaced daily until cells become confluent. Cells were split three times at 1:2 ratio with 0.25% Trypsin in order to remove any remaining neurons from the culture. Before co-culturing with MEF-derived neuronal-like cells, Tuj1 staining was performed to ensure no contaminating neurons.
Single cell patch clamp recordings were performed using an Axopatch 200B amplifier and pClamp 10.0 software (HEKA Elektronik, Lambrecht/Pfalz, Germany), as described (Ouyang et al., 2005). Under whole-cell voltage clamp conditions, membrane voltage was held at −70 mV with the pipette resistance of 4-6 MΩ. Test pulses in 80-ms duration were applied from −60 mV to +80 mV every 2 s. Action potentials were elicited by injecting 20-ms depolarizing currents with graded stimulus amplitudes under current clamp conditions. Standard external solution contains 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES-pH 7.4 (pH adjusted with NaOH), and 10 mM glucose. Intracellular pipette solution contains 150 mM KCl, 5 mM NaCl, 1 mM MgCl2, 2 mM EGTA, 1 mM MgATP, and 10 mM HEPES-pH 7.2 (pH adjusted with KOH). All experiments were performed at room temperature (20-22° C.).
RT-qPCR, Western Blotting, and Luciferase Assays
RNA was isolated with Trizol (Life Technology) following manufacturer's instructions, treated with DNase I (Promega), and reverse transcribed with Superscript III (Life Technology). Quantitative PCR (qPCR) was performed with Fast Start universal SYBR green master mix (Roche) along with gene specific primers on a real-time PCR machine (Applied Biosystems). PCR primers used in the present study are listed in Table 1. Statistical significance was determined by Students t-test based on triplicated experiments.
For analyses by Western blotting, total protein in 1×SDS loading buffer was first normalized based on quantification on NanoDrop (Thermo Scientific), and then resolved by 10% SDS-PAGE. Antibodies used in this study include Rabbit anti-PTBP1 (NT), Mouse anti-PTBP1 (monoclonal BB7) and Rabbit anti-PTBP2 (IS2), all of which are gifts of Douglass Black, Rabbit anti-PTBP2, a gift of Robert Darnell, Mouse anti-ACTB (Sigma), Rabbit anti-SCP1, a gift of Samuel L. Pfaff. Mouse anti-HDAC1 (Active Motif) and Mouse anti-EIF2C2 were purchased from Abnova.
Luciferase reporters were constructed by cloning the 3′UTR region of PTB regulated genes PCR-amplified from HEK293T genomic DNA into the Psicheck-2 vector between XhoI and Not I restriction sites. PCR primers used for constructing individual luciferase reporters are listed in Table 1. For transfection, cells were seeded in 24-well plates for 16 hrs and transfected using Lipofectamine 2000 (Life Technology) with a mix containing 20 ng reporter plasmid, 20 μmol miRNA mimics (Qiagen) or siRNAs (Dharmacon). Luciferase activity was measured 24 hrs post-transfection using the dual-luciferase reporter assay kit (Promega) on Veritas microplate luminometer (Promega).
RNA-Seq, CLIP-Seq, ChIP and Statistical Analysis of Data
RNA-seq was typically done after shRNA treatment for 72 hrs. Trizol-isolated RNA was enriched in two rounds for Poly(A+) RNA with paramagnetic oligo(dT), fragmented into ˜200 nt in length, converted to cDNA with Superscript III (Invitrogen), and subjected to deep sequencing. RNA-seq tags were mapped to the human genome (hg18) by using Tophat with parameters (--mate-inner-dist 150--solexal.3-quals--max-multihits 10--microexon-search). The junction library was made from transcripts from UCSC RefGene and knownGene tables. RefGene transcripts were clustered by using NCBI Entrez GeneID, and treated as one gene to calculate gene expression. For each gene, only tags uniquely mapped and localized in exons or exon-exon junctions were counted.
Differential expressed genes were identified by using edgeR/DEGseq (Robinson et al., 2010; Wang et al., 2010) in combination with a fold-change cutoff as specified in the text. For example, at a threshold of FDR (Bonferroni corrected)<0.001 and >1.8-fold change, 538 down-regulated and 420 up-regulated genes were detected to be significantly differentially expressed upon PTB depletion in HeLa cells. Gene ontology category enrichment was assessed using GOrilla (http://cbl-gorilla.cs.technion.ac.il/) and DAVID online tools (http://david.abcc.ncifcrf gov/).
To determine PTB knockdown-induced switch of polyadenylation, we employed the MAPS technology as described (Fox-Walsh et al., 2011), which measures the tag count upstream of individual polyadenylation sites of expressed genes. For data analysis, we first removed sequences of adaptor and polyA tail from sequenced tags. To avoid false calls resulting from priming of internal A-rich regions, we scanned the genome for polyA-stretch defined as consecutive 8As, which were removed. We next adaptively clustered tags within a specific distance 30 nt along transcripts and sorted cluster intervals by the length and tag in a decreasing order. If a cluster contains a known 3′-end within a 300 nt window, we used the end and then counted the number of reads in each cluster. Only cleavage sites that are supported by at least 10 reads were considered significant polyadenylation sites and used for subsequent analyses. A total of 6166 poly(A) sites was identified in Hela cells in the current analysis. To statistically detect transcripts that showed significant switch in polyadenylation in response to PTB knockdown, we defined the polyA switch ratio in order to measure the relative usage of competing sites within a transcript and then computer ratio changes in response to PTB knockdown. This analysis revealed 324 transcripts that show alternative polyadenylation sites. 14 of these transcripts showed PTB knockdown-induced shift from the distal to the proximal site. For global analysis, cumulative distribution was determined in both PTB expressing and PTB depleted cells. The plot was generated using R (http://www.r-project.org/) and Matlab (http://www.mathworks.com/). Two-sided Kolmogorov-Smirnov statistics (in the R package, http://cran.r-project.org/) was used to determine the significance of the shift in pair-wise comparison (Conover, 1971).
CLIP-seq was performed as previously described (Xue et al., 2009) with a mouse monoclonal anti-Ago2 antibody (also called EIF2C2). To eliminate redundancies from PCR amplification, all tags mapped to identical locations in the human genome were compressed to singles. Individual Ago2 tags after normalization according to total density between samples are plotted relative to the stop codon at the 3′ end of genes as described (Chi et al., 2009). To determine the distribution/distance of Ago2 tags relative to PTB peaks, we plotted the distribution of Ago2 tags in a 1 kb window around distinct genomic regions of PTB binding clusters. To compensate for differences in the number of reads in different samples, the number of tags at each position was divided by the total number of mapped tags in the two libraries constructed on cells before and after PTB knockdown. Tag density heat maps were created by first using custom Python scripts to generate tag densities matrix by dividing each region into 5 nt bins for each PTB cluster in genic 3′UTR region and then visualized using Java TreeView (http://jtreeview.sourceforge.net), as described (Saldanha, 2004). The observed density in the heap map was ranked by tag counts in 1 kb windows around peak center (from bottom to top). The sum of Ago2 reads at each position was calculated and displayed as fraction value (dark line).
To determine the functional correlation between PTB/microRNA interplay and gene expression, we focused on PTB and Ago2 binding sites at the 3′UTR. Cumulative distribution was individually determined in each of 5 categories as described in the text. Plots were generated using R (http://www.r-project.org/) and Matlab (http://www.mathworks.com/). Two-sided Kolmogorov-Smirnov statistics (in the R package, http://cran.r-project.org/) was used to determine the significance of the shift in each pair-wise comparison (Conover, 1971).
ChIP was performed with a rabbit anti-REST antibody purchased from Milipore (07-579). Briefly, MEF cells were crosslinked with 1% formaldehyde for 10 min at room temperature, which was quenched with 100 mM Tris-Cl (pH 9.4), 10 mM DTT for 10 min on ice. Cell pellets were lysed with cell lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-Cl, pH8.1, 1× Protease Inhibitor cocktail) for 10 min on ice. The lysate was sonicated five times for 10 second each at the maximum setting. The sonicated chromatin was checked on 1% agarose gel to make sure sheared chromatin in a range of 200-300 bp. The sonicated lysate was centrifuged at 14000 rpm for 10 min at 4° C. Soluble chromatin was then 1:10 diluted in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-Cl, pH8.1, 1× Protease Inhibitor cocktail). Equal volumes of diluted chromatin were taken to two Eppendorf tubes to which 5 μg of rabbit normal IgG or rabbit anti-REST were added. The reaction was incubated with periodic shake overnight at 4° C. 35 μl protein G magnetic beads were then added to each tube and the reaction continued with periodic shake for another 4 hours at 4° C. At the end of the reaction, the beads were washed twice with TSE 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-Cl, pH8.1), with TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-Cl, pH8.1), and finally three times with TE buffer. Lastly, beads were eluted twice with TE buffer plus 1% SDS at 65° C. for 10 min. The eluents and input samples were reverse crosslinked overnight at 65° C. DNA fragments were purified with QIAquick spin gel extraction kit and qPCR was performed with gene specific primer pairs.
Analysis of RNA Secondary Structure by RNase Protection and In-Line Probing.
For RNA foot-printing assay, GNPDA1 RNA was generated by T7 in vitro transcription. RNA was 5′-labeled using T4 PNK with γ-32P ATP. The RNA was purified by cutting specific labeled band from 7M urea-8% polyacrylamide gel and eluted 3 hrs with G-50 buffer (300 mM NaoAC, 1 mM EDTA, 0.05% SDS). The RNA structure was probed with RNase T1 and RNase V1 following the manual of Ambion/Life Technology. Briefly, 20,000 cpm of end-labeled RNA and 3 μg yeast tRNA were incubated with 0.1 U RNase T1 or 0.01 U RNase V1 in 1×RNA structure probing buffer for 15 min at room temperature. After the addition of 20 μl of Inactivation/Precipitation buffer to the tube and incubation at −20° C. for 15 min, samples were centrifuged at 13,200 rpm for 15 min, supernatant aspirated, and pellet washed with 70% ethanol. The pellet was dissolved in 7 μl of acrylamide gel loading buffer, denatured at 95° C. for 5 min, and 3 μl was fractionated on 8% acrylamide/7M urea gel. For RNA sequencing reaction, the same amount of end-labeled RNA and tRNA were incubated with 0.1 U RNase T1 or 0.01 U RNase V1 in 1× sequencing buffer at 50° C. for 5 min. Single nucleotide RNA ladders were generated by incubating similar amounts of 5′-end labeled RNA and tRNA with RNA hydrolysis buffer (50 mM sodium carbonate pH-9.2, 1 mM EDTA) at 95° C. for 12 min. To probe PTB-RNA interactions, His-tagged PTB4 protein was added to the RNA structure buffer to a final concentration of 2 μM and the reaction was incubated at 30° C. for 10 min after which the same amounts of RNase T1 or Rnase V1 were added to probe structural changes.
For in-line probing, 30,000 cpm of 5′-labeled RNA and 1 μg yeast tRNA were first incubated with varying amounts of His-tagged PTB4 protein in 1× In-line reaction buffer (50 mM Tris-HCl, pH-8.3, 20 mM MgCl2, 100 mM KCl) at 30° C. for 10 min. The reaction was further incubated at 23° C. for 40 h. The reaction was quenched by adding 2× colorless gel-loading solution and 5 μl was fractionated on 8% acrylamide/7M urea gel.
Sequentially Knocking Down PTB and nPTB to Generate Functional Human Neuronal Cells
In alternative embodiments, the invention provides methods for generating a fully functional human mature neuron from non-neuronal cells, e.g., fibroblasts, or neuronal precursors, such as ectodermal or neuronal stem cells or undifferentiated cells, comprising the sequential knocking down of first Polypyrimidine Tract Binding protein (PTB) and then nPTB (the “neuronal PTB” homolog, or nPTB).
In mouse cells, it appears that PTB knockdown is sufficient to drive cells to fully functional mature neurons. However, this does not seem to be the case on human fibroblasts, especially those aged individuals. PTB knockdown can potentially induce the neuronal morphology and early neuronal marks, such Tuj1, but those human cell-derived neurons lack mature neuron marks. This may explain why human cells are much harder to reprogram into functional neurons.
PTB has a homolog known as nPTB in mammalian genomes (the “neuronal PTB” homolog, or nPTB). Published studies reveal a sequential switch in PTB and nPTB expression during neuronal induction and maturation: In neuroblasts, PTB but not nPTB is expressed; during early neuronal induction, PTB expression is diminished and nPTB is induced; in mature neurons, the expression of both PTB and nPTB is diminished. Based on this temporal pattern of PTB and nPTB expression, we hypothesized that PTB may function as a key barrier for initial neuronal induction, while nPTB may act as another key barrier for neuronal maturation.
We performed the experiment to test this hypothesis by sequentially knocking down PTB and nPTB. This is critical because simultaneous knockdown of PTB and nPTB will cause a cell lethal phenotype. As shown in the
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application is a continuation of U.S. patent application Ser. No. 16/030,022, filed Jul. 9, 2018, which is a continuation of U.S. patent application Ser. No. 14/439,125, having a filing date of Apr. 28, 2015, which is a U.S. National Stage Application filed under 35 U.S.C. § 371 claiming priority to International Application No. PCT/US2013/068005, filed Nov. 1, 2013, which application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/721,439, filed Nov. 1, 2012. The aforementioned applications are expressly incorporated herein by reference in their entirety and for all purposes.
This invention was made with government support under GM049369, HG004659, and GM052872, awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61721439 | Nov 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16030022 | Jul 2018 | US |
| Child | 17938008 | US | |
| Parent | 14439125 | Apr 2015 | US |
| Child | 16030022 | US |
