WO2001057091A1

WO2001057091A1 - Soluble, chimeric transcription factors that enter cells to modulate cell differentiation and phenotype

Info

Publication number: WO2001057091A1
Application number: PCT/US2001/003232
Authority: WO
Inventors: Mark Noble; Christoph Proschel; Margot Mayer-Proschel
Original assignee: University Of Utah Research Foundation
Priority date: 2000-02-01
Filing date: 2001-02-01
Publication date: 2001-08-09
Also published as: AU2001231268A1

Abstract

The present invention provides soluble, chimeric transcription factors that can be added to the fluids in which cells are grown and subsequently taken up by the cells and transported to the nucleus where they affect cellular differentiation and phenotype. Also provided are methods of using these soluble, chimeric transcription factors to modulate differentiation and/or phenotype of cells.

Description

Soluble, Chimeric Transcription Factors that Enter Cells to Modulate Cell Differentiation and Phenotype

Introduction

This invention was supported in part by funds from the U.S. government (NIH Grant No. R01 NS37166-01) and the U.S. government may therefore have certain rights in the invention .

Background of the Invention

One of the critical needs in the development of cell therapy is to discover means of controlling the differentiation and behavior of cells that will be implanted into a recipient host . At present there are three approaches to obtaining such control . One approach is to apply to the cells cell-extrinsic signaling molecules (sometimes called growth factors, differentiation inducing factors or morphogens) , thus causing a proportion of the cells (which may range from several percent to essentially 100%) to undergo differentiation along the desired pathway (s) . A related approach, for which few examples exist, is to utilize pharmacological agents that enter the cell and stimulate the signaling pathways regulated by the cell-extrinsic signaling molecules. Alternatively, it is possible to bypass these signaling mechanisms entirely and use various techniques of genetic engineering to cause cells to directly express transcription factors that seem to play a role in controlling the direction of cell-type specific differentiation.

The genetic modification of cells to express differentiation factors, and thus modulate their differentiation fate, was first demonstrated by experiments in which fibroblast-like cells were converted to skeletal muscle cells by expression of a cell transcription factor. In these experiments a single gene, MyoD, was found that causes 10T1/2 fibroblasts to undergo conversion to yoblasts (Weintraub et al . Science 1991 251:761-6). The same principles apply to even more complex systems, in which multipotent precursor cells can take on one of multiple, distinct cell fates, depending on the expression of certain transcription factors. An example is the requirement for the expression of the transcription factor lslet-1 for the generation of motor neurons in the vertebrate spinal cord (Pfaff et al . Cell 1996 84:309-20). Patterning of the neural tube, which gives rise to the spinal cord, converts an apparently homogenous population of neural precursor cells into a complex structure containing many different neuronal and glial cell types. This is achieved by the expression of different transcription factors, each restricted to a region of the neural tube (Tanabe, J. and Jessell, T.M. Science 1996 274:1115-23). Expression of specific transcription factors in sub-populations of neural precursors causes these sub populations to take on specific cell fates (Tsuchida et al. Cell 1994 79:957-70).

The expression of cell type specific transcription factors is often the result of integrating multiple, extracellular signals received through different pathways. Recently, a clear correlation has been made between extracellular signals, their ability to control expression of cell type-specific transcription factors in precursor cells and the resulting cell type of the differentiated offspring (Briscoe et al . Cell 2000 101:435-45; McMahon, A. P. Genes Dev. 2000 14:2261-4). Neural precursors exposed to a specific combination of growth factors, including Sonic hedgehog (Shh) and Bone morphogenetic proteins (BMPs) , integrate these signals to express Islet-1. These cells then go on to become visceral motor neurons in the ventral half of the spinal cord. Neural precursors slightly further dorsal from this location are exposed to different relative doses of Shh and BMP, express the transcription factor ChxlO and become V2 inter-neurons instead (Tsuchida et al. Cell 1994 79:957-70).

The stereotypical function of transcription factors during development and the fact that the activation of transcription factors is often the endpoint of complex signal pathways emphasizes the central role these proteins play in controlling cellular behavior. The control of differentiation by genetically modifying cells can be achieved in a variety of ways, all of which share the identical goal of introducing into a cell a DNA sequence (often referred to as a vector) that encodes a protein known to modulate differentiation fate or the specific behavior of a cell. Gene delivery can be achieved by transfection, by lipofection, by infection with a virus encoding the gene of interest (such as a retrovirus, a lentivirus, an adenovirus, a herpes virus or other comparable strategies well known to those versed in the relevant arts) , by electroporation, by direct microinjection or by other techniques known to those skilled in the arts. Furthermore, the DNA sequence is introduced in such a manner that the introduced gene will be expressed in the target cell at a desired time point. This may include conditions in which expression is not normally seen, necessitating the insertion of appropriate DNA transcription regulatory sequences into the vicinity of the gene of interest.

Genetic modification of cells has been sufficiently effective as to be widely utilized, but it is not without its problems. For example, many viral vectors (such as retroviruses) insert DNA molecules randomly into the genome of the infected cell, causing the problem of insertional mutagenesis in which the function of a normal gene can be altered as a sequel of the infection (Gray, D.A. Cancer Invest. 1991 9:295-304). Alternatively, as another example, viral coat proteins may be expressed in the membrane of the infected cell, thus making the cell a target of immune attack by the host organism. Other problems include, but are not limited to, the low efficiency of transfection, lipofection, electroporation and other means of moving desired DNA molecules into cells by physical means and difficulties in controlling the quantity and timing of gene expression.

The need to discover better means of controlling differentiation has prompted the development of efforts to use reporter gene technologies to discover small molecules that can enter cells and mimic the effects of transcription factors to control differentiation. This approach, in its general form, involves genetically modifying a cell to express a protein that can be visualized (such as Green Fluorescent Protein) under the control of the transcriptional regulatory regions of a gene that is an indicator of a desired state of differentiation (Zhu, M. and Fahl, W.E. Anal. Biochem. 2000 287:210-7). For example, the promoter/enhancer region controlling the expression of the gene encoding a desired transcription factor can be used to search for pharmacological compounds that stimulate the transcription of that transcription factor. Alternatively, the promoter/enhancer region of a gene that is only expressed by a desired differentiated cell type can be used to search for compounds that turn on the expression of the indicator gene.

Summary of the Invention

In the present invention, compositions and methods are provided for building soluble chimeric transcription factors which can be delivered to cells (whether in vivo or in vi tro) and subsequently taken up by cells so that the transcriptional regulatory protein can function to affect the cellular differentiation and/or phenotype of the cells. In in vi tro or ex vivo applications, soluble, chimeric transcription factors of this invention can be added to cells in similar fashion to addition of growth factors, thus greatly enhancing control of the differentiation process. In in vivo applications the chimeric transcription factors can be delivered locally to selected cells via standard methods. The chimeric transcription factors of the present invention can also be genetically modified for tailoring of target specificity (such as DNA binding activity) and biological activity of these molecules to further enhance the desired effect and reduce unwanted side-effects.

Brief Description of the Drawings

Figure 1 provides a schematic example of a chimeric transcription factor of the present invention. This schematic representation of GCM2 protein constructs shows the DNA binding domain at the amino-terminus (N, dark gray box) and the transcription activating domain at the carboxy-terminus (C, light gray box) . The dark box at amino- terminus of pTAT fusion proteins represents TAT- polypeptide. Asterisks indicate point mutations. Figure 2 is a bar graph from experiments demonstrating that addition of GCM2-pTAT protein to neuroepithelial stem cell (NEP) cultures alters the differentiation potential of neuroepithelial stem cells (NEPs) . Different purified GCM2-pTAT fusion proteins were added to NEP cultures as indicated. After allowing NEP cultures to differentiate, the proportion of beta-III- tubulin expressing neuronal cells (hatched bars) and the proportion of GFAP expressing glial cells (open bars) was determined by immunofluorescent labeling. Detailed Description of the Invention

In the present invention, compositions and methods are provided wherein transcription factors are used as soluble agents to be added to cells, and be taken up by said cells, in order to modulate their differentiation and/or phenotype. It has now been found that attachment of a transport promoting peptide to a transcriptional regulatory protein results in transportation of the transcriptional regulatory protein and the peptide across the cell membrane into the cell. These compositions which comprise a transcriptional regulatory protein and a transport promoting peptide are referred to herein as soluble, chimeric transcription factors.

For purposes of the present invention by "transport promoting peptide" , it is meant a peptide or system of peptides which facilitates movement of a protein across a cell membrane into the cytoplasm of the cell. Examples of transport promoting peptides useful in the present invention include, but are not limited to, the penetratins such as that derived from the Drosophila Antennapedia protein (Joliot et al . Proc . Natl Acad. Sci . 1991 88:1864- 8) , the herpes simplex virus-1 VP22 peptide (Elliott, G. and O'Hare, P. Cell 1997 88:223-33) and the pTAT peptide derived from the similarly named HIV TAT protein (Frankel, A.D. and Pabo, CO. Cell 1988 55:1189-93; Green, M. and

Loewenstein, P.M. Cell 1988 55:1179-88). The peptides have been used to introduce proteins up to 120 kD in size into the cytoplasm of cells (Anderson et al . Bioconjug. Chem.

1993 4:10-18; Ezhevsky et al . Proc. Natl Acad. Sci. USA 1997 94:10699-704; Fawell et al . Proc. Natl Acad. Sci. USA

1994 91:664-8; Nagahara et al . Nat. Med. 1998 4:1449-52; and Schwarze et al . Trends Pharmacol. Sci. 1999 21:45-8). However, the extent of function preserved in these proteins following transport was not clear. Further, there are indications that proteins must be denatured before they can traverse the cell membranes. Accordingly, prior to the present invention preservation of function was unpredictable, particularly for proteins with actions as complex as transcriptional regulatory proteins, as the denatured proteins must refold inside of the cell.

Moreover, it is believed that further translocation into the nucleus of the cells may be required for the transcriptional regulatory proteins to function. Chimeric transcription factors that can be transported into cells after being added to the medium in which cells are growing have now been constructed, thus creating for the first time transcription factors that can be used similarly to growth factors. In these experiments, the transcription factor known as GCM2 (where GCM stands for glial cell missing) , a mammalian homologue of the Drosophila gem gene, was used as the transcriptional regulatory protein. The gem protein was first identified as being important for the formation of glial cells in the Drosophila nervous system. In the absence of this protein, the precursor cells that normally make glial cells generate neurons instead.

The cell system used in these experiments was the neuroepithelial stem cell (NEP) , a precursor cell from which all of the cell types of the brain and spinal cord are derived. NEPs were utilized in these studies to demonstrate that the modified transcription factors of the instant invention can modulate the differentiation of a precursor cell, such modulation being one of the primary intents of this invention. The GCM2 protein was fused with a transport promoting peptide comprising 11 amino acids of the pTAT expression construct (Ezhevsky et al . Proc. Natl Acad. Sci. USA 1997 94:10699) to produce the chimeric transcription factor. Recombinant GCM-pTAT protein was purified by standard procedures known to those skilled in the arts. The purified GCM-pTAT protein was then added directly to cultures of NEPs and the cultures were induced to differentiate. The effect of GCM-pTAT protein on the differentiation potential of these NEPs was then determined by scoring the number of neuronal and glial cells generated in these cultures .

Specifically, NEPs were isolated from embryonic day 10.5 (Post coitum) , rat embryonic spinal cords. These cells were maintained in culture in an undifferentiated state by growth on a fibronectin substrate in the presence of 20 ng/ml basic fibroblast growth factor (bFGF) and 10% (v/v) chick embryo extract (Mayer-Proschel et al . Neuron 1997 19:773-85; Rao, M. and Mayer-Proschel, M. Dev . Biol . 1997 188:48-63). During this time NEPs were fed every 24 hours for two days with GCM-pTAT protein at a final concentration of 300 μM . After completing the GCM-pTAT treatment, cells were transferred into differentiation promoting conditions (fibronectin/laminin substrate, 20 ng/ml bFGF and 20 ng/ml neurotrophin-3 , NT-3), which also support the growth of both neuronal and glial cells derived from the NEPs. After five days, cells were immunofluorescently stained for the presence of beta-III- tubulin, a neuronal marker, and glial fibrillary acidic protein (GFAP) , a marker of glial cells. To evaluate the specific activity of the GCM2-pTAT protein, three different variants of GCM2-pTAT (Figure 1) were tested along with buffer only and untreated control samples. Based on previous observations (Akiyama et al . Proc. Natl. Acad. Sci. USA 1996 93:14912-14916; Schreiber et al . Nucleic Acids Res. 1998 26:2337-43; Schreiber et al . Proc. Natl Acad. Sci. USA 1997 29:4739-4744), mutations were introduced into GCM2 to generate what are considered to be an activated form (GCM2-C) , an inactive form (GCM2- KC) and a dominant negative form (GCM2-N) of GCM2. Mutating cysteine¹⁰¹ to alanine results in a form of GCM2 (GCM2-C) which can bind to DNA in both oxidizing and reducing conditions. This mutant represents a potentially activated form of GCM2 which can no longer be down regulated by changes in the redox state of the cell. Conversely, point mutations in the DNA binding domain of GCM2 create a form of GCM2 which has become unable to bind to DNA target sequences (GCM2-KC) and is thus unable to act as a transcription factor. The third GCM2-pTAT construct is a carboxy-terminal truncation of GCM2 (GCM2-N) . This truncation removes the GCM2 transactivation domain, which is required for the transcriptional activation of GCM2 target genes. Since GCM2-N still contains an intact DNA- binding domain but is unable to activate transcription, this mutant is thought to act as a dominant negative form of GCM2 by displacing endogenous, active GCM in target Cultures treated with GCM2-C-pTAT consistently exhibited a highly significant decrease in the number of neurons and an increase in the number of glial cells present, as compared to cultures treated with either untreated, buffer treated or the inactive GCM2-KC-pTAT form of GCM2 (see Figure 2) . In addition, the putative dominant negative form of GCM2 (GCM2-N-pTAT) actually had the opposite effect by increasing the number of glial cells while reducing the number of neuronal cells formed. Labeling of mitotic cells and relative cell numbers indicated that the effects of GCM-pTAT proteins was not due to differences in either cell survival or cell proliferation. These results are consistent with the proposed role for GCM2 in the formation of glial cells during NEP differentiation. The specific effects of the various GCM2 mutants employed here, also suggest that the GCM2-pTAT proteins are mediating this effect through their ability to act as transcriptional modulators.

Thus, these experiments demonstrate that the soluble chimeric transcription factors of the present invention can be added to cells as growth factors would be added, that such chimeric translocatable transcription factors can be taken up by the cells to which they are added and that such chimeric transcription factors can modulate the differentiation and phenotype of these cells. The soluble chimeric transcription factors of the present invention provide a new means of modulating cellular differentiation and phenotype as determined by behavior of the cell. As would be understood by those of skill in the art upon reading this disclosure, the soluble chimeric transcription factors can modulate the phenotype or behavior of a cell without modulating differentiation. For example, the soluble transcription factors of the present invention can be used to increase protein expression levels of a selected cell by exposure to a transcriptional regulatory protein that causes increased expression of the desired protein. Other functions may be similarly regulated.

Thus, also provided in the present invention are methods for modulating the differentiation of cells and methods for modulating the phenotype of cells using these soluble chimeric transcription factors. The methods of the present invention can be applied to in vi tro and ex vivo cell cultures by adding the soluble chimeric transcription factor to media in which the cells are grown so that the soluble chimeric transcription factor is taken up by the cells and the transcriptional regulatory protein functions in the cells. The methods can also be used in in vivo applications wherein the soluble chimeric transcription factor is delivered to the cells preferably via local administration of the soluble chimeric transcription factor to a tissue comprising the cells to modulate differentiation and/or phenotype of the cells. Methods for local administration of an agent to a selected tissue are well known in the art and can be adapted routinely by those of skill in the art to the soluble chimeric transcription factors of the present invention.

The present invention is intended to encompass all transcriptional regulatory proteins that modulate cellular differentiation and behavior, and all transport promoting peptides and/or systems that can be used to transport such transcriptional regulatory proteins across the cell membrane in a manner that would be directly analogous to the chimeric molecule exemplified in this application. Examples of transport promoting peptides or systems which can be used in this invention include, but are not limited to, pTAT, penetratins, retro-inverso peptides and other equivalent agents. The invention may be applied to the modulation of the differentiation and/or phenotype of any cell type in which manipulation is a desired goal.

Claims

What is Claimed is:

1. A soluble chimeric transcription factor comprising a transcriptional regulatory protein and a transport promoting peptide attached thereto, said peptide facilitating transport of the transcriptional regulatory protein across a cell membrane into a cell wherein the transcriptional regulatory protein functions.

2. A method for facilitating transportation of a functioning transcriptional regulatory protein across a cell membrane into a cell comprising:

(a) attaching a transport promoting peptide to the transcriptional regulatory protein to produce a soluble chimeric transcription factor; and

(b) adding the soluble chimeric transcription factor to media in which the cells are grown so that the soluble chimeric transcription factor is taken up by the cells and the functioning transcriptional regulatory protein is transported into the cells.

3. A method for facilitating transportation of a functioning transcriptional regulatory protein across a cell membrane into a cell comprising:

(a) attaching a transport promoting peptide to the transcriptional regulatory protein to produce a soluble chimeric transcription factor; and (b) delivering to the cells a soluble chimeric transcription factor so that the soluble chimeric transcription factor is taken up by the cells and the functioning transcriptional regulatory protein is transported into the cells.

4. A method for modulating differentiation of cells comprising adding to a media in which the cells are growing a soluble chimeric transcription factor of claim 1 so that the soluble chimeric transcription factor is taken up by the cells and the transcriptional regulatory protein of the soluble chimeric transcription factor functions to modulate differentiation of the cells.

5. A method for modulating differentiation of cells comprising delivering to cells a soluble chimeric transcription factor of claim 1 so that the soluble chimeric transcription factor is taken up by the cells and the transcriptional regulatory protein of the soluble chimeric transcription factor functions to modulate differentiation of the cells.

6. A method for modulating phenotype of cells comprising delivering to cells a soluble chimeric transcription factor of claim 1 so that the soluble chimeric transcription factor is taken up by the cells and the transcriptional regulatory protein of the soluble chimeric transcription factor functions to modulate phenotype of the cells.

7. A method for modulating phenotype of cells comprising delivering to cells a soluble chimeric transcription factor of claim 1 so that the soluble chimeric transcription factor is taken up by the cells and the transcriptional regulatory protein of the soluble chimeric transcription factor functions to modulate phenotype of the cells.