WO2010009015A2 - Production of pluripotent cells through inhibition of bright/arid3a function - Google Patents
Production of pluripotent cells through inhibition of bright/arid3a function Download PDFInfo
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- WO2010009015A2 WO2010009015A2 PCT/US2009/050242 US2009050242W WO2010009015A2 WO 2010009015 A2 WO2010009015 A2 WO 2010009015A2 US 2009050242 W US2009050242 W US 2009050242W WO 2010009015 A2 WO2010009015 A2 WO 2010009015A2
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
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- C12N2510/00—Genetically modified cells
Definitions
- the present invention relates generally to the fields of developmental and molecular biology. More particularly, it concerns Bright/ ARID3 a function in the context of cell pluripotency. Specifically, the invention relates to the use inhibitors of Bright to regulate pluripotency, for example, in the context of de-differentiation and re-differentiation of cells.
- Embryonic stem cells are pluripotent and can ultimately lead to the generation of all tissue types. Therefore, these cells have great potential for tissue replacement therapy in degenerative diseases.
- ESCs Embryonic stem cells
- transplantation of tissues is best when major histocompatability antigens are identical and exact tissue matching has not been possible with currently utilized models.
- pluripotent cells can be generated from terminally differentiated cells, thereby opening up another possible source for ESCs.
- Somatic cell fusion technology and incubation of somatic cells with extracts from pluripotent cells have shown limited success, but more promising results have been obtained by focusing on four regulatory factors - Oct3/4, Sox2, c-Myc and Klf4. Yu et al. (2007) used these four genes to successfully reprogram fetal human fibroblasts.
- Takahashi et al. (2007) achieved similar results with adult human dermal fibroblasts.
- a method of rendering a differentiated cell pluripotent comprising (a) providing a differentiated cell;
- step (b) contacting the cell with a inhibitor of Bright/ ARID3 a function to induce de-differentiation in the cell, wherein de-differentiation renders the cell pluripotent.
- the cell of step (a) may be a bone marrow cell, fibroblast cell or a spleen cell, or a peripheral blood cell.
- Bright/ ARID3 a function may be an inhibitor of Bright/ ARID3 a expression, such as an interfering RNA.
- the inhibitor of Bright/ARID3a may be a dominant-negative
- the dominant-negative Bright/ARID3a molecule may be encoded by an expression vector, such as a viral expression vector.
- the Bright/ ARID3 a inhibitor may be a Bright/ARID3a peptide.
- the inhibition of Bright/ ARID3 a function may be reversible.
- the cell may be further treated, once pluripotent, with a signal that induces re-differentiation, such as a chemokine or growth factor.
- the cell, once treated, may express or or more markers of ectoderm, endoderm or mesoderm tissue.
- Re-differentiation may comprise developing one or more characteristics of a fat cell, a neuronal cell, a muscle cell or an endothelial cell, and Bright/ARID3a function in the cell may, once re-differentiated, be restored.
- the method may further comprise implanting the cell into a subject, and/or the subject may be the source of the cell in step (a).
- a method of reprogramming a differentiated cell comprising (a) providing a differentiated cell; (b) contacting the cell with an inhibitor of Bright/ ARID3a function to induce de-differentiation in the cell; (c) contacting the cell, following de-differentiation, with a signal selected to produce a re-differentiated cell phenotype; (d) culturing said cell with the signal for a period of time sufficient to produce the re-differentiated cell phenotype; and (e) identifying one or more aspects of the re- differentiated cell phenotype in said cell.
- the cell of step (a) may be a bone marrow cell, a spleen cell, or a peripheral blood cell.
- the method may further comprise restoring Bright/ARID3a function following step (d).
- the signal may be a chemokine.
- the re- differentiated cell phenotype may be a fat cell phenotype, a neuronal cell phenotype, a muscle cell phenotype, a pancreatic cell phenotype, a hematopoietic cell phenotype or an endothelial cell phenotype.
- a method of enhancing the growth of a stem cell comprising (a) providing a stem cell; (b) contacting the stem cell in culture with media conditioned by a Bright/ ARID3a-deficient cell; and (c) culturing the stem cell.
- the stem may be an embryonic stem cell or a chord blood stem cell.
- the conditioned media may be pre-conditioned by culture of Bright/ ARID3a-deficient cells media.
- the conditioned media may be conditioned by co-culturing of the stem cell and the Bright/ ARID3a-deficient cell.
- the Bright/ARID3a-deficient cell may contain an expression construct expressing a dominant-negative Bright/ ARID3 a, or an interfering RNA that blocks Bright/ ARID3 a expression.
- the method may further comprise contacting the stem cell following step (c) with a signal that induces differentiation.
- the signal may be chemokine.
- Bright/ ARID3 a function may be restored in the Bright/ARID3a-deficient cell.
- kits comprising an inhibitor of Bright/ ARID3 a in a suitable receptacle.
- the receptacle may be a vial, syringe or tube.
- the kit may further comprise a pharmaceutically acceptable buffer, diluent or excipient, growth media, cytokines and/or growth factors, and/or may further comprise instructions for the preparation of the Bright/ARID3a inhibitor in a form suitable for administration to a subject.
- the Bright/ ARID3 a inhibitor may be selected from the group consisting of an interfering RNA, a dominant-negative Bright/ ARID3 a molecule, a Bright/ARID3a peptide or an expression vector coding therefor.
- FIG. 1 Formation of embryoid bodies from ARID3a-deficient mice spleen.
- FIGS. 2A-D - ARID3a-deficient cells differentiate into multiple cell types (nerve cells, endothelial cells, adipocytes).
- FIG. 2A Tube formation of endothelial-like cells from DN ARID3a spleen cultures.
- FIG. 2B Adherent cells from DN ARID3a cultures shown in FIG. 2 A.
- FIGS. 2C and 2D Neural-lineage cells derived from bone marrow cells grown in matrigel and shown at low (FIG. 2C) and high (FIG. 2D) magnifications.
- FIGS. 1 Formation of embryoid bodies from ARID3a-deficient mice spleen.
- FIGS. 2A-D ARID3a-deficient cells differentiate into multiple cell types (nerve cells, endothelial cells, adipocytes).
- FIG. 2A Tube formation of endothelial
- FIG. 3A-D Early stem cell gene markers are expressed in ARID3a-deficient cell cultures as shown by RT-PCR.
- FIG. 3A cDNA from LPS stimulated spleen cells from a BCL2 transgenic (negative control) and a splenic ARID3a-deficient culture (BrSES) were amplified for Sox2 expression.
- FIG. 3B Nanog and C-myc expression were measured in mRNA from a standard ES cell line (+ control), two dominant-negative ARID3a spleen cell cultures (SCDND*36 and 50) and a culture derived from a non-transgenic control spleen.
- FIGGS. 3 C and 3D The samples from (FIG.
- FIG. 4 Conditioned medium from ARID3a-deficient spleen cell cultures enhances growth and differentiation of standard ES cells. Standard ES cells were initiated as hanging drop cultures and were grown in either standard media or ES cell differentiation medium (top two panels). Parallel cultures were grown in conditioned media from ARID3a-deficient cells with enhanced endothelial cell growth (ENDO, bottom left) or with conditioned standard RPMI from ARID3a-deficient spleen cell cultures.
- FIGS. 5A-F Pluripotent stem cell production from B cells.
- Bone marrow pro-B cells from dominant-negative Bright transgenic mice were isolated by flow cytometry as CD43- IgM- B220+ cells (FIG. 5A) to >95% purity as shown by the post sort in (FIG. 5B). After 4 weeks of growth on irradiated murine embryonic fibroblasts with added LIF, the control C57B1/6 background pre-B cells still resembled pre-B cells as shown in (FIG. 5C). Multicellular stem cell-like colonies were observed in the dominant-negative cultures even in the absence of LIF (FIGS. 5D and 5E).
- FIGS. 6A-E - Bright deficient cells are spontaneously multipotent.
- FIG. 6B Bright ' ' ' spleen cells were grown in DMEM with 10% FCS and endothelial cell growth factors (magnification is 4x).
- FIG. 6C Flow cytometry reveals variable expression levels of the markers endothelial protein C receptor (EPCR), mouse thrombomodulin (MTM), and CD31 in a mouse hemangioma cell line (gift of C. Esmon, this institution) as well as the Bright ' ' ' cells.
- EPCR endothelial protein C receptor
- MTM mouse thrombomodulin
- FIG. 6D Bright ' bone marrow cells cultured in RPMI with 5% FCS were seeded into matrigel and grown for three weeks. Branched neuron-like projections developed from cells growing in clusters (left panel, 1Ox magnification, right panel, 2Ox).
- FIG. 6E Neuron-like cells isolated from matrigel were stained with DAPI, anti-nestin or an isotype control.
- FISG. 7A-B Bright deficient cells form iPS-like colonies and express key stem cell markers.
- FIG. 7A RT-PCR assays were performed with conventional ES cells, 6 week-old normal spleen (WTl) and 2 Bright '1' spleen cultures (BrSPSl and BrSPS2) greater than 6 months old.
- FIGS. 8A-E - DN Bright cell lines exhibit signs of reprogramming and appear to be B lymphocyte-derived.
- FIG. 8A DN Bright spleen cultures spontaneously produced embryoid- like bodies (top panel, brightfield, bottom panel, stained with actin-reactive phalloidin for emphasis of the three-dimensional multicellular structure) (2Ox magnification).
- FIG. 8B DN Bright spleen cultures (DNl and DN2), control spleen cultures (WTl) and ES cells were assessed for gene expression by RT-PCR.
- FIG. 8D Control spleen, brain and DNl cell line genomic DNA was amplified with multiple primers to detect J ⁇ rearrangements (Ramsden et al, 1994). DNIa was prepared 50 days prior to the DNIb sample of the DNl line. Arrows indicate expected products.
- FIG. 8D Control spleen, brain and DNl cell line genomic DNA was amplified with multiple primers to detect J ⁇ rearrangements (Ramsden et al, 1994). DNIa was prepared 50 days prior to the DNIb sample of the DNl line. Arrows indicate expected products.
- FIGS. 9A-E ARID3a knockdown results in iPS-like cell generation in a human endothelial cell line.
- FIG. 9A Western blotting indicates that ARID3a is expressed more abundantly in 293T cells as compared to human fibroblast cell lines (WL-38, BJ-h, BJ and IM-R90). Samples were developed with anti-actin to show relative loading. Lane 2 is empty.
- FIG. 9B RT-PCR of ARID3a RNA levels demonstrates efficient knockdown in 2 Bright inhibited clones compared to control 293T cells. GAPDH served as a loading control.
- FIG. 9D QRT-PCR of two clones (BriPS) versus the 293T parental cells shows fold- induction levels of KLf4, Oct4, Sox2 and c-myc transcripts relative to GAPDH.
- FIG. 10 - ARID3a knockdown iPS-like clones differentiate into cells expressing markers for all three germ line lineages.
- Conventional mouse ES cells top panels
- BriPS clone A2-P4, middle panels
- parental 293T cells bottom panels
- AFP ⁇ - fetoprotein
- SMA smooth muscle actin
- BIIIT ⁇ -3 -tubulin
- ISO isotype control
- FIG. 11 Isolation of pre-B cells from bone marrow of DN Bright transgenic mice.
- CD43TgM ⁇ B22O l0 DN Bright (left panel) and normal control pre-B cells (not shown) were sorted by flow cytometry and plated on MEFs in the presence of LIF. Post-sort analysis of the DN pre-B cells is shown (right panel).
- FIGS. 12A-C Optimization of shRNA knockdown of Bright expression in a highly expressing, mouse B cell line.
- FIG. 12A RT-PCR showed that expression of three of the shRNAs (Tables S2, shRNA 1-3) inhibited Bright mRNA production 2 and 4 days after transduction in comparison to the scrambled control shRNAs (Con), which had no effect. Transduction efficiencies were > 90%. Actin levels demonstrated equivalent sample loads.
- FIG. 12B Levels of Bright mRNA in (FIG. 12A) were corrected relative to actin levels, quantified for graphic presentation and showed >80% inhibition. Controls were arbitrarily set at 1.
- FIG. 12C Proteins from the cells in (FIG. 12A) were western blotted for Bright and actin, 2 and 4 days after viral infection.
- FIG. 13 ARID3a-deficient human iPS cells fail to express differentiation markers prior to embryoid body formation.
- the clone (BriPS A2-P4) used in FIG. 10 was subjected to nuclear (DAPI, left panels) smooth muscle actin (SMA) and ⁇ -III-tubulin ( ⁇ lll-T) staining. Scale bar shows 50 ⁇ m.
- FIG. 14 - ARID3a-deficient cells fail to form teratomas in Nod/Scid mice. Shown are representative panels of early neoplastic cells formed from BriPS cells (left) and teratoma formation from conventional ES cells (right). Magnification is 4Ox.
- FIG. 15 Adult Bright ' ' survivors express no Bright.
- Transcript expression of Bright and its ARID3b paralogue, Bdp were evaluated in wild-type (+/+), heterozygous (+/-) and homozygous null (-/-) splenic B cells (source of highest Bright expression in adult mice) by RT-PCR.
- Bright mRNA was amplified for 30 cycles using primers which span exons 3 and 4 (forward 5'-GCGGACCCCAAGAGGAAAGAGTT (SEQ ID NO:3)) and (reverse 5'- CTGGGTGAGTAGGCAAAGAGTGAGC (SEQ ID NO:4)) resulting in a 390bp product.
- RT- and GAPDH loading controls are shown.
- iPS induced pluripotent stem
- ES embryonic stem cells
- Bright B cell regulator of immunoglobulin (Ig) heavy chain transcription
- Ig immunoglobulin
- ARID3a also known as ARID3a
- Bright/ ARID3 a is the founder of the fifteen member ARID (A+T rich interaction domain) family of proteins (reviewed in (Wilsker et al, 2002; Wilsker et al, 2005)).
- ARID family members have just begun to be elucidated and include roles in cell cycle control, (Flowers et al, 2009; Ho et al, 2009a; Ho et al, 2009b) ARID domain-dependent demethylase (Tu et al, 2008), histone deacetylase (Wilsker et al, 2005; Gray et al, 2005) activities, and chromatin remodeling (Wilsker et al, 2005).
- Flowers et al, 2009; Ho et al, 2009a; Ho et al, 2009b ARID domain-dependent demethylase (Tu et al, 2008), histone deacetylase (Wilsker et al, 2005; Gray et al, 2005) activities, and chromatin remodeling (Wilsker et al, 2005).
- the transcription factor Bright (B cell regulator of IgH transcription) is a member of a growing family of proteins that interact with DNA through a highly conserved A+T-rich interaction domain, or ARID (Herrscher et al, 1995). Currently, Bright is the only member of this family for which target sequences have been identified, and which binds to DNA in a sequence-specific fashion.
- ARID family proteins include the Drosophila proteins Dead ringer and eyelid that play important roles in lineage decisions in the gut and eyelid of the fruit fly, and are required for embryonic segmentation (Gregory et al, 1996; Treisman et al, 1997); retinoblastoma binding protein (Rbpl) that interacts with retinoblastoma protein in a cell cycle-specific fashion (Fattaey et al, 1993); and BDP, a ubiquitously expressed human protein identified in a two-hybrid screen as a novel protein that also interacts with retinoblastoma protein (Rb) (Numata et al, 1999).
- the yeast protein SWI/1 has homology to Bright, and is a component of a larger protein complex that serves to modulate chromatin organization in that organism (Peterson and Herskowitz, 1992; Burns and Peterson, 1997).
- the human SWI-SNF complex contains a 270 kDa protein with non-sequence specific DNA binding activity that is also a member of the ARID family (Dallas et al., 2000).
- members of this family may participate in lineage decisions, cell cycle control, tumor suppression and modulation of chromatin.
- ARID3a Sequencing of the human genome identified fifteen members of this family, including the human Bright ortholog known as ARID3a (Wilsker et al., 2005).
- ARID family proteins have diverse functions including, chromatin remodeling, binding to retinoblastoma protein, regulating X-Y chromosome functions and participation in embryonic development (Wilsker et al, 2005). Generally, these proteins are components of large protein complexes and are tightly regulated throughout development.
- Human ARID3a can bind to E2F in cell lines of embryonic origin where its over-expression is controversial, as it has been associated with both tumor suppressor and oncogenic functions (Peeper et al, 2002; Suzuki et al, 1998; Ma et al, 2003; Fukuyo et al, 2004). Recent studies indicate that Bright activity is regulated tightly through intracellular partitioning and that it contributes to chromatin accessibility of the heavy chain enhancer (Kim and Tucker, 2006; Lin et al, 2007). It is likely that Bright/ARID3a can participate in a wide variety of regulatory functions in both embryonic and adult tissues because it functions as both a transcription factor and has a role in altering chromatin accessibility.
- ARID family proteins are expressed ubiquitously.
- murine Bright is expressed widely throughout embryonic development, but expression in the adult is largely limited to the B lymphocyte lineage where its expression is tightly regulated and is restricted at the mRNA level to the pre-B cell and peanut agglutinin-high germinal center cell populations (Herrscher et al, 1995; Webb et al, 1991; Webb et al, 1998).
- Activated splenic B cells in the mouse can be induced to express Bright after antigen binding, but the protein is not present in the majority of peripheral IgM + B cells (Webb et al, 1991; Webb et al, 1998).
- NF ⁇ NR nuclear factor ⁇ negative regulator
- CDP/Cut/Cux CAAAT displacement protein
- B lymphocytes While non-B cells in the mouse express NF ⁇ NR, B lymphocytes generally do not exhibit such protein complexes.
- Btk Bruton's tyrosine kinase, or Btk, associates with Bright in activated murine B lymphocytes (Webb et al, 2000).
- Btk is an X-linked gene that encodes a tyrosine kinase critical for proper development and maintenance of B lymphocytes both in humans and in mice (reviewed in (Conley et al, 1994; Satterthwaite and Witte, 1996).
- X-linked agammaglobulinemia an immunodeficiency state characterized by blocks at the pro-B cell stage of development and severely depressed serum antibody levels (Conley et al, 1994).
- Btk is clearly the defective gene product in both human and murine diseases, the molecular mechanisms by which Btk deficiencies result in blocks in B cell development are currently unknown.
- X-linked immunodeficient mice the mouse model for XLA, produce a mutated Btk protein that fails to form stable complexes with Bright (Webb et al, 2000). These data suggest that Bright may function as a component of the same signaling pathway(s) important in XLA.
- the present invention may concerns Bright/ ARID3 a protein molecules.
- a "protein” or “polypeptide” generally refers, but is not limited to, a protein of greater than about 100 amino acids or the full length endogenous sequence translated form of a gene.
- a peptide is usually from about 3 to about 100 amino acids. All the “proteinaceous” terms described above may be used interchangeably herein.
- a human ARID3a polypeptide sequence is provided in SEQ ID NO:2.
- Proteins may be produced recombinantly or purified from natural sources. Shorter peptide molecules may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, e.g., Stewart and Young (1984); Tarn et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference.
- the size of the at least one proteinaceous molecule may comprise, but is not limited to, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82,
- amino acid refers to any amino acid, amino acid derivitive or amino acid mimic as would be known to one of ordinary skill in the art.
- the residues of the proteinaceous molecule are sequential, without any non- amino molecule interrupting the sequence of amino molecule residues.
- the sequence may comprise one or more non-amino molecule moieties.
- the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.
- the proteinaceous composition comprises at least one protein, polypeptide or peptide.
- the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide.
- biocompatible refers to a substance which produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein. Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions.
- biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or peptides or synthetic proteins or peptides each essentially free from toxins, pathogens and harmful immunogens.
- Proteinaceous compositions may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials.
- the nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art.
- One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (world-wide-webe at ncbi.nlm.nih.gov).
- Genbank and GenPept databases world-wide-webe at ncbi.nlm.nih.gov.
- the coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art.
- proteins may also be fused to other proteinaceous compositions, thereby altering or supplementing their properties.
- a targeting moiety may be provided which facilitate cellular transport of the Bright derived peptide or polypeptide.
- sequences such as Tat can provide nuclear localization signals, thereby transporting peptides into the nucleus.
- a proteinaceous compound may be purified.
- purified will refer to a specific or protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as would be known to one of ordinary skill in the art for the specific or desired protein, polypeptide or peptide.
- nucleic acids derived from or encoding Bright are provided.
- the nucleic acids may comprise wild-type or a mutant version of these genes.
- the nucleic acid encodes for or comprises a transcribed nucleic acid.
- the nucleic acid comprises a nucleic acid segment of SEQ ID NO:1, or a biologically functional equivalent thereof.
- the nucleic acid encodes a protein, polypeptide, peptide.
- nucleic acid is well known in the art.
- a “nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
- a nucleobase includes, for example, a naturally-occurring purine or pyrimidine base found in DNA (e.g., an adenine "A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an "A,” a "G,” an uracil “U” or a "C”).
- nucleic acid encompass the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.”
- oligonucleotide refers to a molecule of between about 3 and about 100 nucleobases in length.
- polynucleotide refers to at least one molecule of greater than about 100 nucleobases in length.
- a nucleic acid may encompass a double-stranded molecule or a triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a molecule.
- a single-stranded nucleic acid may be denoted by the prefix "ss,” a double- stranded nucleic acid by the prefix "ds,” and a triple-stranded nucleic acid by the prefix "ts.”
- a nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production or biological production.
- a synthetic nucleic acid e.g., a synthetic oligonucleotide
- Non-limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemically synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266 032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al. (1986) and U.S. Patent 5,705,629, each incorporated herein by reference.
- one or more oligonucleotide may be used.
- Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
- a non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U.S. Patent 4,683,202 and U.S. Patent 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Patent 5,645,897, incorporated herein by reference.
- a non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et al. 2001 , incorporated herein by reference).
- a nucleic acid may be purified on polyacrylamide gels, cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al, 2001 , incorporated herein by reference).
- the present invention concerns a nucleic acid that is an isolated nucleic acid.
- isolated nucleic acid refers to a nucleic acid molecule (e.g., an RNA or DNA molecule) that has been isolated free of, or is otherwise free of, the bulk of the total genomic and transcribed nucleic acids of one or more cells.
- isolated nucleic acid refers to a nucleic acid that has been isolated free of, or is otherwise free of, bulk of cellular components or in vitro reaction components such as for example, macromolecules such as lipids or proteins, small biological molecules, and the like.
- the nucleic acid is a nucleic acid segment.
- nucleic acid segment are smaller fragments of a nucleic acid, such as for non- limiting example, those that encode only part of Bright.
- a “nucleic acid segment” may comprise any part of a gene sequence, of from about 2 nucleotides to the full length of Bright.
- the nucleic acid segment may be a probe or primer.
- a probe generally refers to a nucleic acid used in a detection method or composition.
- a “primer” generally refers to a nucleic acid used in an extension or amplification method or composition.
- the present invention also encompasses a nucleic acid that is complementary to a Bright-encoding nucleic acid.
- the invention encompasses a nucleic acid or a nucleic acid segment complementary to the sequence set forth in SEQ ID NO:1.
- a nucleic acid is a "complement(s)" or is “complementary” to another nucleic acid when it is capable of base-pairing with another nucleic acid according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules.
- another nucleic acid may refer to a separate molecule or a spatial separated sequence of the same molecule.
- complementary also refers to a nucleic acid comprising a sequence of consecutive nucleobases or semiconsecutive nucleobases (e.g., one or more nucleobase moieties are not present in the molecule) capable of hybridizing to another nucleic acid strand or duplex even if less than all the nucleobases do not base pair with a counterpart nucleobase.
- a "complementary" nucleic acid comprises a sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range derivable therein, of the nucleobase sequence is capable of base-pairing with a single or double stranded nucleic acid molecule during hybridization.
- the term "complementary" refers to a nucleic acid that may hybridize to another nucleic acid strand or duplex in stringent conditions, as would be understood by one of ordinary skill in the art.
- a "partly complementary" nucleic acid comprises a sequence that may hybridize in low stringency conditions to a single or double stranded nucleic acid, or contains a sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with a single or double stranded nucleic acid molecule during hybridization.
- Hybridization As used herein, “hybridization,” “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature.
- the term “anneal” as used herein is synonymous with “hybridize.”
- the term “hybridization,” “hybridize(s)” or “capable of hybridizing” encompasses the terms “stringent condition(s)” or “high stringency” and the terms “low stringency” or “low stringency condition(s).”
- stringent condition(s) or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but precludes hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.
- Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 5O 0 C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.
- low stringency or “low stringency conditions”
- non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20°C to about 50°C.
- wild-type refers to the naturally occurring sequence of a nucleic acid at a genetic locus in the genome of an organism, or a sequence transcribed or translated from such a nucleic acid.
- wild-type also may refer to an amino acid sequence encoded by a nucleic acid.
- wild-type encompasses all such naturally occurring allele(s).
- polymorphic means that variation exists (i.e., two or more alleles exist) at a genetic locus in the individuals of a population.
- mutant refers to a change in the sequence of a nucleic acid or its encoded protein, polypeptide or peptide that is the result of the hand of man.
- the present invention also concerns the isolation or creation of a recombinant construct or a recombinant host cell through the application of recombinant nucleic acid technology known to those of skill in the art or as described herein.
- a recombinant construct or host cell may comprise a Bright-encoding nucleic acid, and may express a Bright protein, peptide or peptide, or at least one biologically functional equivalent thereof.
- a “gene” refers to a nucleic acid that is transcribed.
- the gene includes regulatory sequences involved in transcription, or message production or composition.
- the gene comprises transcribed sequences that encode for a protein, polypeptide or peptide.
- this function term "gene” includes both genomic sequences, RNA or cDNA sequences or smaller engineered nucleic acid segments, including nucleic acid segments of a non- transcribed part of a gene, including but not limited to the non- transcribed promoter or enhancer regions of a gene.
- Smaller engineered gene nucleic acid segments may express, or may be adapted to express using nucleic acid manipulation technology, proteins, polypeptides, domains, peptides, fusion proteins, mutants and/or such like.
- isolated substantially away from other coding sequences means that the gene of interest forms the significant part of the coding region of the nucleic acid, or that the nucleic acid does not contain large portions of naturally-occurring coding nucleic acids, such as large chromosomal fragments, other functional genes, RNA or cDNA coding regions. Of course, this refers to the nucleic acid as originally isolated, and does not exclude genes or coding regions later added to the nucleic acid by the hand of man.
- nucleic acid(s) of the present invention may be combined with other nucleic acid sequences, including but not limited to, promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, and the like, to create one or more nucleic acid construct(s).
- a "nucleic acid construct" is a nucleic acid engeneered or altered by the hand of man, and generally comprises one or more nucleic acid sequences organized by the hand of man.
- one or more nucleic acid constructs may be prepared that include a contiguous stretch of nucleotides identical to or complementary to SEQ ID NO:1.
- a nucleic acid construct may be about 3, about 5, about 8, about 10 to about 14, or about 15, about 20, about 30, about 40, about 50, about 100, about 200, about 500, about 1,000, about 2,000, about 3,000, about 5,000, or about 10,000 nucleotides in length, as well as constructs of greater size, up to and including chromosomal sizes (including all intermediate lengths and intermediate ranges), given the advent of nucleic acids constructs such as a yeast artificial chromosome are known to those of ordinary skill in the art.
- intermediate lengths and “intermediate ranges,” as used herein, means any length or range including or between the quoted values (i.e., all integers including and between such values).
- Intermediate lengths include about 11, about 12, about 13, about 16, about 17, about 18, about 19, etc.; about 21, about 22, about 23, etc; about 31, about 32, etc; about 51, about 52, about 53, etc.; about 101, about 102, about 103, etc.; about 151, about 152, about 153, etc.; about 1,001, about 1002, etc.; about 10,001, about 10,002, etc
- Non-limiting examples of intermediate ranges include about 3 to about 32, about 150 to about 500, or about 5,000 to about 15,000, etc.
- biologically functional equivalent is well understood in the art. Accordingly, a sequence that has between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO:2 will be a sequence that is "essentially as set forth in SEQ ID NO:2," provided the biological activity of the protein, polypeptide or peptide is maintained.
- Table 1 provides a listing of preferred human codons.
- amino acid sequences or nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, or various combinations thereof, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein, polypeptide or peptide activity where expression of a proteinaceous composition is concerned.
- the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non- coding sequences flanking either of the 5' and/or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
- the present invention also provides for nucleic acid sequences that have between about 70% and about 79%; or more preferably, between about 80% and about 89%; or even more particularly, between about 90% and about 99%; of nucleotides that are identical to the nucleotides of SEQ ID NO:1. It will also be understood that this invention is not limited to the particular nucleic acid or amino acid sequence of SEQ ID NO:1 or 2.
- Recombinant vectors and isolated nucleic acid segments may therefore variously include these coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, and they may encode larger polypeptides or peptides that nevertheless include such coding regions or may encode biologically functional equivalent proteins, polypeptide or peptides that have variant amino acids sequences.
- nucleic acids of the present invention encompass biologically functional equivalent proteins, polypeptides, or peptides. Such sequences may arise as a consequence of codon redundancy or functional equivalency that are known to occur naturally within nucleic acid sequences or the proteins, polypeptides or peptides thus encoded.
- functionally equivalent proteins, polypeptides or peptides may be created via the application of recombinant DNA technology, in which changes in the protein, polypeptide or peptide structure may be engineered, based on considerations of the properties of the amino acids being exchanged.
- Changes designed by man may be introduced, for example, through the application of site-directed mutagenesis techniques as discussed herein below, e.g., to introduce improvements or alterations to the antigenicity of the protein, polypeptide or peptide.
- Encompassed by the invention are nucleic acid sequences encoding relatively small peptides or fusion peptides, such as, for example, peptides of from about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60
- the present invention further comprises methods for identifying inhibitors of Bright activity that are useful in the reprogramming of differentiated cells.
- These assays may comprise random screening of large libraries of candidate substances; alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to inhibit the function of Bright.
- a method generally comprises:
- a decrease in a Bright related activity as compared to Bright activity of an untreated cell, identifies the candidate substance as an inhibitor of Bright activity. Activities include stimulation of immunoglobulin production, Bright homodimerization, Bright DNA binding, Bright interaction with Btk, and Bright interaction with TFII-I. Assays also may be conducted in isolated cells, cell extracts, organs, or in living organisms.
- the term “candidate substance” refers to any molecule that may potentially inhibit the activity Bright.
- the candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to Bright/ARID3a, or a Bright/ ARID3a interacting protein, such as Btk or TFII-I.
- Using lead compounds to help develop improved compounds is known as "rational drug design” and includes not only comparisons with know inhibitors and activators, but predictions relating to the structure of target molecules.
- the goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration, or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a target molecule, or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling, or by a combination of both approaches.
- Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
- Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators.
- modulators include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for the target molecule. Such compounds are described in greater detail elsewhere in this document. For example, an antisense molecule that bound to a translational or transcriptional start site, or splice junctions, would be ideal candidate inhibitors.
- the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the structure of the modulators.
- Such compounds which may include peptidomimetics of peptide modulators, may be used in the same manner as the initial modulators.
- a quick, inexpensive and easy assay to run is an in vitro assay.
- Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time.
- a variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.
- a common form of in vitro assay is a binding assay.
- a particular format contemplated by the inventors involves the assessing of Bright/ ARID3 a binding to DNA. Both molecules are labeled with agents that can be detected individually or by virtue of fluorescence energy transfer.
- the present invention also contemplates the screening of compounds for their ability to modulate Bright/ ARID3 a activity in cells.
- Various cells and cell lines can be utilized for such screening assays, including cells specifically engineered for this purpose.
- Other cells include embryonic fibroblasts and other embryonic tissues.
- Of particular interest are cells that contain an Ig promoter linked to a selectable or screenable marker gene.
- mice are a preferred embodiment, especially for transgenics.
- other animals are suitable as well, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbons and baboons).
- Assays for inhibitors may be conducted using an animal model derived from any of these species.
- Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal, or to cells dervied from such animals. Administration will be by any route that could be utilized for clinical purposes. Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Also, measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays.
- Cells may be obtained from a wide variety of sources, including kidney, isolated B- cell subpopulations, bone marrow, and fibroblasts B. Bright Inhibitors
- the present invention contemplates the use of virtually any composition that will inhibit Bright/ ARID3 a function.
- Organopharmaceutical compounds that produce the desired effect would find great utility, and such compounds may be identified according to the screening methods described above.
- biological inhibitors, as described below, may be utilized to interfere with Bright/ ARID3 a function.
- Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
- complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G: C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
- Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
- Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
- Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
- complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions.
- sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches.
- Other sequences with lower degrees of homology also are contemplated.
- an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
- genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
- a genomic clone will need to be used.
- the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
- Ribozymes Another general class of inhibitors is ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et al, 1987; Forster and Symons, 1987).
- ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
- This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
- IGS internal guide sequence
- Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al, 1981). For example, U.S.
- Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
- sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990).
- ribozymes can elicit genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that was cleaved by a specific ribozyme.
- RNA interference also referred to as "RNA-mediated interference” or RNAi
- RNAi Double- stranded RNA
- dsRNA double- stranded RNA
- dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp et al, 2000; Tabara et al, 1999). Activation of these mechanisms targets mature, dsRNA-complementary mRNA for destruction.
- RNAi offers major experimental advantages for study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp, 1999; Sharp et al, 2000; Tabara et al, 1999). Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C. elegans, Trypanasoma, Drosophila, and mammals (Grishok et al, 2000; Sharp, 1999; Sharp et al, 2000; Elbashir et al, 2001).
- RNAi acts post- transcriptionally, targeting RNA transcripts for degradation, and possibly by inhibiting translation. It appears that both nuclear and cytoplasmic RNA can be targeted (Bosher et al , 2000).
- siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e. those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siRNA's guide function while including sequences that are specific to the gene or genes.
- siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above (Montgomery et al, 1998). Of particular interest are those siRNAs that span an exon-intron junction.
- siRNAs has been mainly through direct chemical synthesis; through processing of longer, double stranded RNAs through exposure to Drosophila embryo lysates; or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive.
- Chemical synthesis proceeds by making two single stranded RNA-oligomers followed by the annealing of the two single stranded oligomers into a double stranded RNA. Methods of chemical synthesis are diverse. Non-limiting examples are provided in U.S. Patents 5,889,136, 4,415,732, and 4,458,066, expressly incorporated herein by reference, and in Wincott et al. (1995).
- siRNAs are found to work optimally when they are in cell culture at concentrations of 25-100 nM. This had been demonstrated by Elbashir et al. (2001) wherein concentrations of about 100 nM achieved effective suppression of expression in mammalian cells. siRNAs have been most effective in mammalian cell culture at about 100 nM. In several instances, however, lower concentrations of chemically synthesized siRNA have been used (Caplen et al, 2000; Elbashir et al., 2001).
- RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference.
- the enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6) via the use and production of an expression construct as is known in the art. See U.S. Patent 5,795,715.
- the contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene.
- the length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length.
- RNA single strands of RNA can be produced enzymatically or by partial/total organic synthesis.
- single stranded RNA is enzymatically synthesized from the PCRTM products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides.
- WO 01/36646 places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures.
- the templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence.
- the templates can be attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences.
- the inventors propose to inhibit ARID3a expression in adult tissues in vitro using siRNA or shRNA in a lentiviral vector.
- a GFP marker can be utilized to determine that cells took up the vector, and thus permit checking for appropriate inhibition of ARID3a production.
- the B cell line BCg3R-ld and/or over-expressing transgenic mouse spleen cells can be utilized. After the inventors confirm that inhibition of ARID3a occurs in these cells, they will inhibit ARID3a expression in mouse embryo fibroblasts and culture the GFP+ cells to confirm that pluripotent stem cells develop.
- the use of an inducible promoter discussed below that allow induction of the siRNA or shRNA only under specific growth conditions permit reversible inhibition of ARID3a.
- cells can be induced to dedifferentiate into a pluripotent and self-renewing state in vitro, and can then be induced to differentiate into mature lineage cells under different growth conditions without inhibition of ARID3a.
- These methods offer considerable advantages over current methodologies, which involve introduction of multiple viral copies and genes, some of which are known to be oncogenic. Self-deleting vectors may also be used.
- antibodies may find use as inhibitors of Bright.
- the term "antibody” is intended to refer broadly to any appropriate immunologic binding agent such as IgG, IgM, IgA, IgD and IgE.
- IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
- antibody also refers to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
- DABs single domain antibodies
- Fv single chain Fv
- scFv single chain Fv
- Monoclonal antibodies are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred.
- the invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.
- Single-chain Mabs are described in U.S. Patents 4,946,778 and 5,888,773, each of which are hereby incorporated by reference. The present invention would most likely utilize single-chain antibodies expressed from expression vectors, as described below.
- Humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. Methods for the development of antibodies that are "custom-tailored” to the patient's dental disease are likewise known and such custom-tailored antibodies are also contemplated.
- Peptides may prove to be useful inhibitors of Bright/ ARID3 a function by competiting with or mimicking Bright domains that bind or interact with DNA, Btk, TFII-I or other molecules, or compete with Bright dimerization sequences. Also contemplated are regions of Bright that comprise nuclear shuttling sequences. Bright-derived peptides are therefore a particular type of compound that may prove useful in inhibiting Bright function.
- the peptides may be designed around an existing structure, i.e., portions of Bright, or they may be selected for function from a randomized library. Of particular interest is a region of SEQ ID NO:1 from about residue 444 to residue
- the peptides will be less than 50 residues, and comprising at least about 10 consecutive residues of Bright/ARID3a.
- the number of consecutive Bright/ ARID3 a residues may be 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, with additional non-Bright sequences attached thereto.
- Ranges of peptide length of 10-50 residues, 10-40 residues, 15-50 residues 15-40, residues, 15-35 residues, 15-30 residues, 15-25 residues, 15- 20 residues and 20-25 residues are contemplated.
- the number of additional non- Bright/ARID3a residues may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues.
- the overall size of the peptides may therefore range from 8 reisudes to 75 or more residues, with 10-70 residues, 10-60 residues, 10-50 residues, 10-40 residues, 10-30 residues and 15-70 residues, 15-60 residues, 15-50 residues, 15-40 residues, 15-30 residues, 20-70 residues, 20-60 residues, 20-50 residues, 20-40 residues, and 20-30 residues being specifically contemplated ranges.
- Peptides may be produced by cleavage of polypeptides, such as Bright, with proteolytic enzymes (trypsin, chymotrypsin, etc.), or chemicals. They may also be produced recombinantly using vectors and techniques described supra. However, it may be be most advantageous to produce peptides using the solid-phase synthetic techniques (Merrifield, 1963). Other peptide synthesis techniques are well known to those of skill in the art (Bodanszky et al, 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984). Appropriate protective groups for use in such syntheses will be found in the above texts, as well as in Protective Groups in Organic Chemistry, 1973.
- the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
- the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted with the residue already attached to the solid support.
- the protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide.
- the peptides of the invention are preferably devoid of benzylated or methylbenzylated amino acids.
- Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.
- non-standard amino acids Aside from the twenty standard amino acids can be used, there are a vast number of "non-standard” amino acids. Two of these can be specified by the genetic code, but are rather rare in proteins. Selenocysteine is incorporated into some proteins at a UGA codon, which is normally a stop codon. Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG. Examples of non-standard amino acids that are not found in proteins include lanthionine, 2- aminoisobutyric acid, dehydroalanine and the neurotransmitter gamma-aminobutyric acid.
- Non-standard amino acids often occur as intermediates in the metabolic pathways for standard amino acids - for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism.
- Non-standard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while hydroxyproline is made by a posttranslational modification of proline.
- the present invention may utilize L-conf ⁇ guration amino acids, D-configuration amino acids, or a mixture thereof.
- L-amino acids represent the vast majority of amino acids found in proteins
- D-amino acids are found in some proteins produced by exotic sea-dwelling organisms, such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria. D-serine may act as a neurotransmitter in the brain.
- the L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can theoretically be synthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde is levorotary).
- Retro-inverso modification of naturally occurring polypeptides involves the synthetic assemblage of amino acids with ⁇ - carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D-amino acids in reverse order with respect to the native peptide sequence.
- a retro-inverso analogue thus has reversed termini and reversed direction of peptide bonds (NH-CO rather than CO- NH) while approximately maintaining the topology of the side chains as in the native peptide sequence. See U.S. Patent 6,261,569, incorporated herein by reference.
- Peptides may advantageously be attached or fused to certain additional peptide segments for beneficial properties associated therewith.
- such domains are cell delivery domains (also called a cell delivery vector, or cell transduction domain).
- cell delivery domains also called a cell delivery vector, or cell transduction domain.
- These types of domains are well known in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides (Fischer, 2007).
- poly-D-Arg and poly-D-Lys sequences ⁇ e.g., dextrorotary residues, eight residues in length). Others are listed below in Table 2.
- Linkers or cross-linking agents may be used to fuse peptides to other proteinaceous sequences.
- Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies.
- Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites.
- Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially.
- the bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied.
- a majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group.
- heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described in U.S. Patent 5,889,155, specifically incorporated herein by reference in its entirety.
- the cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols.
- the cross-linking reagent can be modified to cross-link various functional groups and is thus useful for cross-linking polypeptides. In instances where a particular peptide does not contain a residue amenable for a given cross-linking reagent in its native sequence, conservative genetic or synthetic amino acid changes in the primary sequence can be utilized.
- Peptides modified for in vivo use by the addition, at the amino- and/or carboxyl- terminal ends, of a blocking agent to facilitate survival of the peptide in vivo are contemplated. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake.
- blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues.
- the inventors also contemplate that certain non-natural amino acids that satisfy the structural constraints of the inhibitory peptides of the present invention without a loss, and perhaps with an improvement in, biological function.
- the present inventors also contemplate that structurally similar compounds may be formulated to mimic the key portions of Bright/ ARID3 a.
- Such compounds which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and, hence, also are functional equivalents.
- peptide mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993).
- the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and/or antigen.
- a peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
- Methods for generating specific structures have been disclosed in the art. For example, ⁇ -helix mimetics are disclosed in U.S. Patents 5,446,128; 5,710,245; 5,840,833; and 5,859,184.
- molecular modeling is meant quantitative and/or qualitative analysis of the structure and function of protein- protein physical interaction based on three-dimensional structural information and protein-protein interaction models. This includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models. Molecular modeling typically is performed using a computer and may be further optimized using known methods. Computer programs that use X-ray crystallography data are particularly useful for designing such compounds. Programs such as RasMol, for example, can be used to generate three dimensional models.
- INSIGHT Error Light
- GRASP Alignment-Proty Nicholls
- Dock Molecular Design Institute, University of California at San Francisco
- Auto-Dock Unmanned Equipment Deformation
- the methods can involve the additional step of outputting to an output device a model of the 3-D structure of the compound.
- the 3-D data of candidate compounds can be compared to a computer database of, for example, 3-D structures.
- Compounds of the invention also may be interactively designed from structural information of the compounds described herein using other structure-based design/modeling techniques (see, e.g., Jackson, 1997; Jones et al, 1996). Candidate compounds can then be tested in standard assays familiar to those skilled in the art. Exemplary assays are described herein.
- the 3-D structure of biological macromolecules can be determined from data obtained by a variety of methodologies. These methodologies, which have been applied most effectively to the assessment of the 3-D structure of proteins, include: (a) x-ray crystallography; (b) nuclear magnetic resonance (NMR) spectroscopy; (c) analysis of physical distance constraints formed between defined sites on a macromolecule, e.g., intramolecular chemical crosslinks between residues on a protein (e.g., PCT/USOO/14667, the disclosure of which is incorporated herein by reference in its entirety), and (d) molecular modeling methods based on a knowledge of the primary structure of a protein of interest, e.g., homology modeling techniques, threading algorithms, or ab initio structure modeling using computer programs such as MONSSTER (Modeling Of New Structures from Secondary and Tertiary Restraints) (see, e.g., International Application No.
- MONSSTER Modeling Of New Structures from Secondary and Tertiary Restraints
- Dominant-negative proteins are defective proteins with can negate the effects of normal, functional proteins when both are present in the same environment. In many cases, dominant-negative proteins homo-multimerize and are thus able to "poison" a complex that contains one or more functional proteins. Dominant-negative forms of Bright have been produced which act in just this manner. In designing dominant-negative Bright molecules, several regions present useful points for mutation. First, changes in the DNA binding domain (ARID) that block DNA binding produce dominant-negative effects. Second, alterations in the nuclear localization sequence which block nuclear translocation result in a dominant- negative form of Bright/ARID3a. Third, manipulation of the interaction and dimerization domains cause a dominant-negative function. Other dominant-negative proteins may be produced by interfering with the amino-terminal domain. Dominant-negative forms of Bright are described in Nixon et al (2004).
- the present invention addresses culturing of de-differentiated cells for expansion.
- the culturing of multipotent cells is performed for the purpose of re-differentiation.
- the present invention also contemplates the use of naturally- occuring or engineered cells that are Bright/ ARID3a-defective to enhance the growth of natural ESCs. Such cells have proven to provide signals/factors that stimulate the growth/differentiation of stem cells in culture.
- A. Cell Identification and Separation Techniques Methods of separating ESCs from mixed cell populations are well known in the art and may be applied to the cell populations of the present invention. Cells purified in this fashion may then be used for genetic engineering/gene replacement therapy. Sources of such cells include, but are not limited to, bone marrow and chord blood. In addition, they can be used for tissue regeneration purposes. The following description sets forth exemplary methods for separation of stem cells based upon the surface expression of various markers.
- FACS facilitates the quantitation and/or separation of subpopulations of cells based upon surface markers.
- Cells to be sorted are first tagged with a fluorescently labeled antibody or other marker specific ligand.
- labeled antibodies and ligands are specific for the expression of a phenotype specific cell surface molecule.
- cells may be labeled internally.
- the labeled cells are then passed through a laser beam and the fluorescence intensity of each cell determined.
- the sorter distributes the positive and negative cells into label -plus and label-minus wells at a flow rate of approximately 3000-10,000 cells per second.
- fluorescent labels exciting at different wavelengths allows for sorting based upon multiple or alternate criteria.
- fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
- a single PBMC sample may be analyzed with alternatively labeled anti-Ig antibody, anti-CD3 antibody, anti-CD8 antibody and anti-CD4 antibody to screen for the presence of B cells and T cells within the sample, as well as distinguishing specific T cell subsets.
- a flow cytometer generally consists of a light source, normally a laser, collection optics, electronics and a computer to translate signals to data. Scattered and emitted fluorescent light is collected by two lenses (one positioned in front of the light source and one set at right angles) and by a series of optics, beam splitters and filters, which allow for specific bands of fluorescence to be measured.
- Flow cytometer apparatus permit quantitative multiparameter analysis of cellular properties at rates of several thousand cells per second. These instruments provide the ability to differentiate among cell types. Data are often displayed in one-dimensional (histogram) or two-dimensional (contour plot, scatter plot) frequency distributions of measured variables.
- the partitioning of multiparameter data files involves consecutive use of the interactive one- or two-dimensional graphics programs.
- Quantitative analysis of multiparameter flow cytometric data for rapid cell detection consists of two stages: cell class characterization and sample processing.
- cell class characterization partitions the cell feature into cells of interest and not of interest.
- sample processing each cell is classified in one of the two categories according to the region in which it falls.
- Analysis of the class of cells is very important, as high detection performance may be expected only if an appropriate characteristic of the cells is obtained. Also, one can use forward side scatter (granularity and size) for separative purposes.
- Certain particles in the stream may be selectively charged and then separated by deflecting them into designated receptacles.
- a classic form of this separation is via fluorescent tagged antibodies, which are used to mark one or more cell types for separation.
- Micro-bead separations involve contacting a cell suspension with a slurry of microbeads labeled with a cell surface specific antibody or ligand. Cells labeled with the micro-beads are then separated using an affinity capture method specific for some property of the beads. This format facilitates both positive and negative selection.
- Magnetic beads are uniform, superparamagnetic beads generally coated with an affinity tag such as recombinant streptavidin that will bind biotinylated immunoglobulins, or other biotinylated molecules such as, for example, peptides/proteins or lectins.
- Magnetic beads are generally uniform micro- or nanoparticles of Fe 3 O 4 . These particles are superparamagnetic, meaning that they are attracted to a magnetic field but retain no residual magnetism after the field is removed. Suspended superparamagnetic particles tagged to a cell of interest can be removed from a matrix using a magnetic field, but they do not agglomerate (i.e., they stay suspended) after removal of the field.
- a common format for separations involving superparamagnetic nanoparticles is to disperse the beads within the pores of larger microparticles. These microparticles are coated with a monoclonal antibody for a cell-surface antigen. The antibody-tagged, superparamagnetic microparticles are then introduced into a cellular suspension. The particles bind to cells expressing the surface antigen of interest and may be separated out with the application of a magnetic field. This may be facilitated by running the suspension over a high gradient magnetic separation column placed in a strong magnetic field. The magnetically labeled cells are retained in the column while non-labeled cells pass through. When the column is removed from the magnetic field, the magnetically retained cells are eluted. Both, labeled and non-labeled fractions can be completely recovered. iii. Affinity Chromatography
- Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
- the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
- the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
- the ligand should be coupled in such a way as to not affect its binding properties.
- the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
- affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed elsewhere in this document.
- cell surface markers are often used to identify.
- Table 3 provides a list of markers and the ESC type with which they are most commonly associated, as well as markers for differentiated cells.
- Endothelial endothelial cell progenitor marker of cell- 1 (FIkI) cell contacts
- Osteoblast phosphatase indicates bone formation (BAP)
- Osteocalcin Osteoblast synthesized by osteoblast; marker of bone formation
- BMPR bone committed mesenchymal cell types from morphogenetic Mesenchymal stem mesenchymal stem and progenitor cells; protein receptor and progenitor cells BMPR identifies early mesenchymal (BMPR) lineages (stem and progenitor cells)
- CD4 and CD8 mature T lymphocyte (WBC subtype)
- HSC bone marrow cell, cell
- CD34 endothelial progenitor CD34 also identifies muscle progenitor satellite, a muscle stem cell
- CD38 Present on WBC lineages. Selection of CD34 + /CD38 " cells lineages allows for purification of HSC populations
- CD44 Mesenchymal identify specific types of mesenchymal cells
- CFU assay detects the ability of a single stem
- Bone marrow forming unit fibroblastic cells such identified cells are fibroblast (CFU-F) precursors of differentiated mesenchymal lineages
- HSC Hoechst dye Absent on HSC extrudes the dye and stains lightly compared with other cell types
- HSC HSC
- MSC markers of mature blood cell
- Lin-negative cells antigen (Lin) and WBC lineages assists in the purification of HSC and hematopoietic progenitor populations
- Mac-1 WBC granulocyte and macrophage WBC subtypes
- Bone marrow superfamily found on bone marrow
- Muc-18 (CD146) fibroblasts fibroblasts, which may be important in endothelial hematopoiesis; a subpopulation of Muc-18+ cells are mesenchymal precursors
- HSC HSC
- MSC cell indicative of HSC and MSC Bone (Sca-1) Marrow and Blood cont.
- Stro-1 antigen (mesenchymal) marrow stromal (mesenchymal) cells marrow stromal (mesenchymal) cells; precursor cells, selection of Stro-1 + cells assists in isolating hematopoietic cells mesenchymal precursor cells, which are multipotent cells that give rise to adipocytes, osteocytes, smooth myocytes, fibroblasts, chondrocytes, and blood cells
- Fatty acid Transport molecule located specifically in
- CD133 stem cells which give rise to neurons and HSC glial cells
- Glial fibrillary acidic protein Astrocyte Protein specifically produced by astrocyte
- Dendrite-specific MAP protein found associated protein- Neuron specifically in dendritic branching of neuron
- Oligodendrocyte oligodendrocytes located in the myelin protein (MPB) sheath surrounding neuronal structures
- Neural tubulin Neuron identifies differentiated neuron
- Neuron NF identifies differentiated neuron
- Neurosphere differentiating ES cells indicates presence of (EB), ES early neurons and glia
- CKl 9 identifies specific pancreatic epithelial
- Cytokeratin 19 Pancreatic cells that are progenitors for islet cells and (CKl 9) epithelium ductal cells
- pancreatic progenitor progenitor cell lines including pancreatic progenitor cell lines
- pancreatic Pancreatic islet Expressed by gamma-islet cell of pancreas polypeptide
- ES Alkaline
- EC plu ⁇ potent phosphatase carcinoma
- PSC stem cell
- Endoderm primitive endoderm reflects endodermal (AFP) differentiation
- TDGF-I Cripto (TDGF-I) ES, cardiomyocyte cells, primitive ectoderm, and developing cardiomyocyte
- HNF-4 Endoderm endoderm formation
- Neuronal cell- Cell-surface molecule that promotes cell-cell adhesion molecule Ectoderm interaction indicates primitive (N-CAM) neuroectoderm formation
- SCF or c-Kit ES Stem cell factor proliferation of ES and EC cells, (SCF or c-Kit ES, EC 5 HSC 5 MSC hematopoietic stem cell (HSCs) 5 and ligand) mesenchymal stem cells (MSCs); binds the receptor c-Kit
- TRA-I -60 ES 5 EC molecule is synthesized by undifferentiated PSCs
- MyoD and Pax7 Myoblast myocyte differentiation of myoblasts into mature myocytes
- Stem cells are generally defined as having both the capacity to self-renew (make more stem cells by cell division) as well as being able to differentiate into mature, specialized cells.
- a progenitor cell is an early descendant of a stem cell that can only differentiate, but it cannot renew itself anymore. In contrast, a stem cell can renew itself (make more stem cells by cell division) or it can differentiate (divide and with each cell division evolve more and more into different types of cells).
- a progenitor cell is often more limited in the kinds of cells it can become than a stem cell. In scientific terms, it is said that progenitor cells are more differentiated than stem cells.
- Cell culture facilitates the maintenance and propagation of cells in vitro under controlled conditions.
- Cells may be cultured in a variety of types of vessels constructed of, for example, glass or plastic.
- the surfaces of culture vessels may be pre-treated or coated with, for example, gelatin, collagen, polylysine, or components of the extracellular matrix, to facilitate the cellular adherence.
- Some sophisticated techniques utilize entire layers of adherent cells, feeder cells, which are used to support the growth of cells with more demanding growth requirements.
- Cells are normally cultured under conditions designed to closely mimic those observed in vivo. In order to mimic the normal physiological environment cells are generally incubated in a CO 2 atmosphere with semi-synthetic growth media. Culture media is buffered and contains, among other things, amino acids, nucleotides, salts, vitamins, and also a supplement of serum such as fetal calf serum (FCS) horse serum or even human serum. Culture media may be further supplemented with growth factors and inhibitors such as hormones, transferrin, insulin, selenium, and attachment factors. As a rule, cells grown in vitro do not organize themselves into tissues. Instead, cultured cells grow as monolayers (or in some instances as multilayers) on the surface of tissue culture dishes. The cells usually multiply until they come into contact with each other to form a monolayer and stop growing when they come into contact with each other due to contact inhibition.
- FCS fetal calf serum
- Anchorage-dependent cells show the phenomenon of adherence, i.e., they grow and multiply only if attached to the inert surface of a culture dish or another suitable support. Such cells cannot normally be grown without a solid support. Many cells do not require this solid surface and show a phenomenon known as Anchorage-independent growth. Accordingly, one variant of growing these cells in culture is the use of Spinner cultures or suspension cultures in which single cells float freely in the medium and are maintained in suspension by constant stirring or agitation. This technique is particularly useful for growing large amounts of cells in batch cultures.
- Anchorage-independent cells are usually capable of forming colonies in semisolid media ⁇ e.g., matrigel). Some techniques have been developed that can be used also to grow anchorage-dependent cells in spinner cultures. They make use of microscopically small positively-charged dextran beads to which these cells can attach.
- the starting material for the establishment of a cell culture may be tissue from a suitable donor obtained under sterile conditions.
- the tissues may be minced and treated with proteolytic enzymes such as trypsin, collagenase of dispase to obtain a single cell suspension that can be used to inoculate a culture dish.
- proteolytic enzymes such as trypsin, collagenase of dispase to obtain a single cell suspension that can be used to inoculate a culture dish.
- dispersion of tissue is also effectively achieved by treatment with buffers containing EDTA.
- a particular form of initiating a cell culture is the use of tiny pieces of tissues from which cells may grow out in vitro.
- cells are cultured with differentiating agents.
- Cells will be cultured under specified conditions to achieve particular types of differentiation, and provided various factors necessary to facilitate the desired differentiation.
- Cell growth and differentiation factors are molecules that stimulate cells to proliferate, first while being maintained in a multipotent state, and second, to induce (re)differentiation.
- Leukemia inhibitory factor (LIF) may be utilized to impede spontaneous differentiation.
- culturing with factors to promote growth and differentiation of multipotent cells into functionally mature forms can be undertaken.
- Administration of the growth and/or differentiation factors may be repeated as needed.
- a growth and/or differentiation factor may constitute a hormone, cytokine, chemokine, hematapoietin, colony stimulating factor, interleukin, interferon, growth factor, other endocrine factor or combination thereof that act as intercellular mediators.
- intercellular mediators are lymphokines, monokines, growth factors and traditional polypeptide hormones.
- growth factors include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factors- ⁇ and - ⁇ .; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor-I and -II; erythropoietin (E
- growth and/or differentiation factors include proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence, including synthetic molecules and mimetics.
- a support is BD Biosciences MatrigelTM Basement Membrane Mix. This material can be used for (re)differentiation of hepatocytes, epithelial cells, endothelial, smooth muscle cells and neurons (Biederer & Scheiffele, 2007; Li et al, 2005; Hadley et al, 1985; Ireland, 1997; McGuire & Orkin, 1987; Bissel, et al, 1987; Page et al., 2007; Li et al., 1987; Barcellof et al., 1989; Roskelley et al, 1994; Xu et al, 2007; Debnath et al, 2003; Muthuswamy et al, 2001; Madison et al, 1985; Xu et al, 1994; Fouad et al, 2005).
- Other (re)differentiation methods would seek to induce pancreatic cell and
- the vitamin A derivative, retinoic acid can be used to differentiate B cells (Chen et al, 2008) and neuronal cells (Guan et al, 2001).
- Fico et al (2008) provide methods and compositions useful for single step differentiation of neurons. The relevant teachings of these papers are incorporated by reference herein.
- expression vectors are employed to express various products including Bright, peptides, dominant negative Bright proteins, antibodies or fragments thereof, antisense molecules, ribozymes or interfering RNAs.
- Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
- Elements designed to optimize messenger RNA stability and translatability in host cells also are defined.
- the conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
- expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
- the transcript may be translated into a protein, but it need not be.
- expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
- the nucleic acid encoding a gene product is under transcriptional control of a promoter.
- a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
- under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
- promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
- Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
- At least one module in each promoter functions to position the start site for RNA synthesis.
- the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
- promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
- the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
- the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
- CMV cytomegalovirus
- the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
- promoter By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
- Tables 2 and 3 list several regulatory elements that may be employed, in the context of the present invention, to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof.
- Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
- enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Below is a list of viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct (Table 4 and Table 5).
- Eukaryotic Promoter Data Base EPDB any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene.
- Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
- promoters/regulatory regions that are inducible.
- the present invention contemplates the use of vectors that can be delivered to cells to express an inhibitor of Bright/ ARID3 a when induced, but upon removal of the inducing agent, the Bright/ ARID3 a inhibition is relieved and the cell may differentiate and lose immortality, optionally for the purpose of reimplantation in an individual.
- the present inventors contemplate the use of retroviral and lentiviral vectors for delivery of nucleic acids, and thus they envision use of the endogenous promoters in these vectors.
- a cDNA insert where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
- the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
- a terminator Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
- the cells contain nucleic acid constructs of the present invention
- a cell may be identified in vitro or in vivo by including a marker in the expression construct.
- markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
- a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, histidinol, GFP, and lacZ are useful selectable markers.
- enzymes such as herpes simplex virus thymidine kinase (tk) chloramphenicol acetyltransferase (CAT), or HAT selection may be employed.
- Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
- IRES elements are used to create multigene, or polycistronic, messages.
- IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
- IRES elements from two members of the picanovirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
- IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages.
- each open reading frame is accessible to ribosomes for efficient translation.
- Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
- Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
- the expression construct comprises a virus or engineered construct derived from a viral genome.
- the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
- Adenoviral Vectors One particular mode of in vivo delivery involves the use of an adenovirus expression vector.
- Adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.
- the expression vector comprises a genetically engineered form of adenovirus.
- adenovirus a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992).
- retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
- adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
- Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
- ITRs inverted repeats
- the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
- the El region (ElA and ElB) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
- the expression of the E2 region results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990).
- the products of the late genes are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).
- MLP located at 16.8 m.u.
- TPL 5'- tripartite leader
- recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
- adenovirus generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh- Choudhury et al, 1987), providing capacity for about 2 extra kb of DNA.
- Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.
- Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
- natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
- Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows.
- the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
- Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
- the typical vector according to the present invention is replication defective and will not have an adenovirus El region.
- the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
- the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, as described by Karlsson et al. (1986), or in the E4 region where a helper cell line or helper virus complements the E4 defect.
- Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo.
- This group of viruses can be obtained in high titers, e.g., 10 -10 plaque-forming units per ml, and they are highly infective.
- the life cycle of adenovirus does not require integration into the host cell genome.
- the foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
- Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford- Perricaudet et al, 1990; Rich et al, 1993).
- the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription (Coffin, 1990).
- the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
- the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
- the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
- a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
- Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
- a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
- a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983).
- Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
- retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al, 1981).
- Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
- new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988; Hersdorffer et al, 1990).
- Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al, 1996; Zufferey et al, 1997; Blomer et al, 1997; U.S. Patent 6,013,516 and 5,994,136). Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-I, HIV-2 and the Simian Immunodeficiency Virus: SIV.
- Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
- Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.
- recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Patent 5,994,136, incorporated herein by reference.
- a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
- viral vectors may be employed as expression constructs in the present invention.
- Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
- Epstein-Barr virus is a member of the herpesvirus family and one of the most common human viruses. The virus occurs worldwide, and most people become infected with EBV sometime during their lives. In the United States, as many as 95% of adults between 35 and 40 years of age have been infected. When infection with EBV occurs during adolescence or young adulthood, it causes infectious mononucleosis 35% to 50% of the time. EBV vectors have been used to efficiently deliver DNA sequences to cells, in particular, to B lymphocytes. Robertson et al (1986) provides a review of EBV as a gene therapy vector.
- Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al , 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al , 1986; Potter et al , 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
- the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
- the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
- the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
- the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
- Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
- a naked DNA expression construct into cells may involve particle bombardment.
- This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987).
- Several devices for accelerating small particles have been developed.
- One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990).
- the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
- Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990; Zelenin et al, 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.
- the expression construct may be entrapped in a liposome.
- Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
- Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
- Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
- Nicolau et al (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.
- the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
- HVJ hemagglutinating virus
- the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-I) (Kato et al, 1991).
- HMG-I nuclear non-histone chromosomal proteins
- the liposome may be complexed or employed in conjunction with both HVJ and HMG-I. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
- bacterial promoter is employed in the DNA construct
- Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
- Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer.
- the delivery vehicle may comprise a ligand and a liposome.
- a ligand and a liposome For example, Nicolau et al, (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
- a nucleic acid encoding a particular gene also may be specifically delivered into a cell type by any number of receptor-ligand systems with or without liposomes.
- epidermal growth factor (EGF) may be used as the receptor for mediated delivery of a nucleic acid into cells that exhibit upregulation of EGF receptor.
- Mannose can be used to target the mannose receptor on liver cells.
- antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.
- gene transfer may more easily be performed under ex vivo conditions.
- Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
- kits are also within the scope of the invention.
- kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, in particular, a Bright inhibitor.
- the kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial end user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
- a label can be provided on the container to indicate that the composition is used for a specific therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.
- the inventors have generated self-renewing pluripotent cells from seven ARID3a- deficient spleens, four ARID3a-deficient bone marrow cultures and one kidney culture. Therefore, ARID3a-deficiency appears to result in cells with self-renewing potential from multiple tissue sources.
- One of the bone marrow cultures has been tested in matrigel cultures for the ability to produce endothelial tubes, but instead gave rise to neural spheres and differentiated neuron-like cells over a two-three week culture period (FIGS. 2C-D). Bone marrow cultures also readily formed adipocyte and stromal cells (not shown). Kidney cultures contain multiple morphologically distinct cell types of currently unknown identity.
- FIGS. 3A-D indicate that the inventors have observed Sox-2, nanog, myc, and Klf4 expression in their ARID-3a- deficient cultures, suggesting that early pluripotent cells are present.
- Surface stains for stem cell markers also demonstrate early stem cell lineage markers such as c-kit, sca-1 and CD9 (not shown). These data further indicate that pluripotent cells are present within these self- renewing cultures.
- Supernatants from the pluripotent spleen cells were shown to be effective in inducing growth and differentiation of standard ES cell lines (FIG. 4).
- FIGS. 5A-F show pluripotent stem cell production from B cells. Bone marrow pro-B cells from dominant-negative Bright transgenic mice were isolated by flow cytometry as CD43- IgM- B220+ cells (FIG. 5A) to >95% purity as shown by the post sort in (FIG. 5B). After 4 weeks of growth on irradiated murine embryonic fibroblasts with added LIF, the control C57B1/6 background pre-B cells still resembled pre-B cells as shown in (FIG. 5C).
- Multicellular stem cell-like colonies were observed in the dominant-negative cultures even in the absence of LIF (FIGS. 5D and 5E).
- Amplification of genomic DNA from bulk splenic cultures from dominant-negative mice (SCDND*36 and 50) showed increased D-J rearrangement relative to control (SC57#1) cultures suggesting increased numbers of cells were derived from B cell progenitors.
- Nod.CB17-Prkdc sc ⁇ d /J mice were obtained from Jackson Laboratories. Animals were used with institutional approval and within review board-specified guidelines. Tissue culture and iPS induction. Whole spleen, kidney, lymph node or bone marrow from Bright ' ' ' or DN transgenic mice were teased into single cell suspension and fed 2-3 times per week with RPMI 1640 containing 5% FBS and standard supplements (Webb et al, 1989). Mouse ES cells and iPS-like cells were grown with 10 ng/ml LIF on MEFs, and were passaged using trypsin as described (Meissner et al, 2009).
- Embryoid body formation and differentiation assays were performed using standard protocols (Meissner et al., 2009). MEFs were prepared from 129sv mice as described (Meissner et al, 2009). Teratoma formation was induced by intramuscular injection of Nod/Scid mice with 2 x 10 6 cells. Surgically dissected tumors were paraffin-embedded, stained with heamatoxylin and eosin and evaluated by a licensed pathologist (S. Kosanke, OUHSC, OK). Immunofluorescence staining and microscopy.
- Mouse Sox2 and Lin28 primers were 5'-GAAAGGAGAGAAGTTTGGAGCCC-S' (SEQ ID NO:49) and 5'-GCTGTTCTTCTGGTTGCCGC-S ' (SEQ ID NO:50), and 5'- CCCTGGTGGTGTGTTCTGTATTGG-3' (SEQ ID NO:51) and 5'-
- TGGC AAGGGAAATATC ACACAGC-3' SEQ ID NO:52
- Other human primers Yama et al, 2007; Nixon et al, 2004; Park et al, 2008
- mouse primers Teanaka et al, 2007; Liu et al, 2007; Kinoshita et al, 2007; Shaffer et al, 2002
- Western blotting was performed with polyclonal anti-Bright and anti-actin as described (Nixon et al, 2008).
- shRNAs (Table 7) were subcloned into the pSIF-Hl-copGFP lentiviral vector (System Biosciences, Mountain View, CA) and packaged by co-transfection with ⁇ FIV-34N and pVSV-G plasmids with the LipoD293TM DNA transfection reagent (SignaGen Laboratories, Gaithersburg, MD) according to the manufacturer's protocol. Virus was harvested, filtered using a 0.45 ⁇ m sterile filter and concentrated by ultracentrifugation 48-72 hours later. Titers were determined by numbers of GFP-positive cells by flow cytometry.
- BCg3R-ld or 293T cells were treated with virus and 6 ⁇ g/ml polybrene (Sigma) for 24 hours.
- iPS-like cells were fed with ES medium (DMEM containing 20% FBS, 0.1 mM non-essential amino acid, and 0.1 mM ⁇ -mercaptoethanol) with 10 ng/ml LIF, and seeded onto irradiated MEF feeder cells in gelatin-coated plates. Medium was changed every other day.
- ES medium DMEM containing 20% FBS, 0.1 mM non-essential amino acid, and 0.1 mM ⁇ -mercaptoethanol
- the inventors plated Bright ' ⁇ cells onto mouse embryonic fibroblast feeders (MEFs) in the presence of the differentiation inhibiting cytokine, leukemia inhibitory factor (LIF), as is done routinely for maintenance of normal ES and iPS cells. After a period of 4-6 weeks, the inventors were able to isolate clones with iPS-like morphology that expressed the ES cell markers SSEA-I (FIG. 7B) and Nanog (not shown). These cells exhibited a stable iPS-like phenotype and indicate that iPS-like cells can be isolated from multiple tissues of Bright '1 ' ' mice.
- MEFs mouse embryonic fibroblast feeders
- spleen and bone marrow from DN Bright transgenic mice exhibited the ability to spontaneously form embryoid-like bodies (FIG. 8A) and to spontaneously convert into cells with variable lineage surface marker expression ⁇ e.g., CD3, Mac-1, and GR-I; data not shown) and upregulation of Nanog (FIG. 8B).
- the inventors' transgenic mice express DN Bright from the B cell-specific CDl 9 promoter (Nixon et al, 2008). DN transgenic mice did not generate CD19 + mature, DN Bright-expressing B cells (Nixon et al., 2008). Thus, instead of following normal B cell differentiation pathways, loss of Bright function may halt and reprogram these B lymphocytes.
- DN-derived, non-B cell containing tissues e.g., kidney and liver
- long term cell lines established from DN transgenic bone marrow and spleen exhibited D-J H rearrangements of their IgH loci (FIG. 8C, and not shown).
- PCR products demonstrating unrearranged germline bands were also present, and may result from unrearranged alleles or they may indicate the presence of non-B cell-derived stromal cells within the cultures.
- the inventors also detected evidence of K light chain rearrangement (FIG.
- ARID3a mouse and human
- the human embryonic epithelial cell line, 293T met both of these criteria.
- 293T epithelia constitutively express high levels of ARID3a relative to the human fibroblast lines more commonly used for iPS production (FIG. 9A), as well as large T antigen.
- the inventors found that shRNA knockdown efficiently silenced ARID3a expression in these cells (FIG. 9B).
- FIG. 9C After only six days of infection, cells underwent morphological changes and resembled tight iPS-like colonies after multiple passages rather than the typical 293T adherent monolayer.
- the ARID3a-inhibited clones maintained the original 293T transformed karyotype (data not shown).
- ARID3a inhibition may lead to reprogramming of cells without teratoma production in vivo as has been observed for other human iPS-like cell lines (Shih et al., 2007).
- ARID3a-deficient cells resemble iPS cells in their ability to express early markers of mesoderm, endoderm, and ectoderm, but that they differ from true iPS cells in that they do not generate teratomas derived from terminally- differentiated tissues of multiple types.
- ShRNA primer sequences for Bright are listed and their point of origin within the coding sequence is indicated.
- the DNA binding domains of mouse Bright and human ARID3a are sufficiently homologous that these shRNAs effectively inhibited ARID3a.
- the inventors have provided three independent lines of evidence indicating that inhibition of a single transcription factor, Bright/ ARID3 a, promotes reprogramming of somatic cells into an iPS-like state.
- Bright ' ' ' cells from multiple tissues exhibit self- renewing growth properties, form embryoid-like bodies, express stem cell markers and exhibit the potential to differentiate into cells of multiple lineages.
- bone marrow and spleen cells from DN Bright transgenic mice exhibit similar properties. These iPS-like cells retain evidence of Ig recombination, indicating that they originated from B lymphocyte lineage cells in which the DN Bright transgenic protein was specifically expressed.
- Bodanszky et al J. Antibiot, 29(5):549-53, 1976. Bodine and Ley, EMBO J. , 6:2997, 1987.
- Nicolas and Rubenstein In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (Eds.), Stoneham: Butterworth, 494-513, 1988.
- Racher et al Biotechnology Techniques, 9:169-174, 1995. Ragot et al, Nature, 361 :647-650, 1993.
- Robertson et al Nature 322:445-448, 1986.
- Rooney et al Rheumatol. Int., 10:217-219, 1990.
- Vasseur et al Proc Natl. Acad. ScL U.S.A., 77:1068, 1980.
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EP1385937A4 (en) * | 2001-04-20 | 2005-11-09 | Chiron Corp | Delivery of polynucleotide agents to the central nervous sysstem |
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Also Published As
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EP2313494A2 (en) | 2011-04-27 |
CA2730546A1 (en) | 2010-01-21 |
JP2011527905A (en) | 2011-11-10 |
WO2010009015A3 (en) | 2010-03-11 |
IL210638A0 (en) | 2011-03-31 |
AU2009271149A1 (en) | 2010-01-21 |
US9453204B2 (en) | 2016-09-27 |
US20100008891A1 (en) | 2010-01-14 |
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