Patent Application
20050090006
The invention is in the field of nucleic acid sequences capable of regulating transcription, particularly sequences that may enhance expression in Schwann cells.
Schwann cells arise from a common pool of neural crest cells and differentiate into myelinating or non-myelinating glia (Mirsky and Jessen, 1996). Axonal signals control the decision to myelinate quantitative features of the sheath and are required for its subsequent maintenance (Aguayo et al., 1976a; Aguayo et al., 1976b; Berthold, 1978). These axonal instructions operate, in part, by controlling the expression of the genes encoding myelin structural proteins. In PNS myelin these include protein zero (P0), peripheral myelin protein 22 (PMP22), and myelin basic protein (MBP). High-level expression of these genes coincides with myelin elaboration, continues throughout myelinogenesis and ceases should the axon be disrupted (Gupta et al., 1988; Trapp et al., 1988; Lamperth et al., 1990; LeBlanc and Poduslo, 1990; Stahl et al., 1990; Snipes et al., 1992). Molecules implicated in the control of Schwann cell maturation and myelin formation include hormones such as progesterone, the second messenger cAMP, the zinc finger transcription factor Krox-20 and the POU domain transcription factor Tst-1/SCIP/Oct-6 (SCIP) (Lemke and Chao, 1988; Monuki et al., 1989; Monuki et al., 1990; He et al., 1991; Morgan et al., 1991; Topilko et al., 1994; Koenig et al., 1995; Weinstein et al., 1995; Bermingham et al., 1996; Jaegle et al., 1996; Zorick and Lemke, 1996). However, neither the molecules that transduce axonal instructions nor the Schwann cell signaling pathways that they control are known. Similarly, the most downstream components of such pathways, the transcription factors and DNA regulatory elements that confer expression to myelin genes, remain to be elucidated.
The presence of positive Schwann cell targeting elements has been detected from P0 or CNPase 5′ flanking sequences (Messing et al., 1992; Gravel et al. 1998). However, 5′ flanking sequences of MBP have only been shown to result in expression in oligodendrocytes, the myelin forming cell type in the central nervous system (Foran and Peterson, 1992; Gow et al., 1992; Miskimins et al., 1992; Goujet-Zalc et al., 1993; Stankoff et al., 1996). There is, therefore, a need to identify the location of other Schwann cell targeting elements.
In various aspects, the present invention provides nucleic acid molecules which modulate transcription of operably-linked sequences in Schwann cells. In some embodiments, such sequences are designated herein as SCE1 and SCE2. In some embodiments, the nucleic acid molecules enhance transcription of operably-linked sequences in Schwann cells. In other embodiments, the nucleic acid molecules repress transcription of operably-linked sequences in Schwann cells.
The invention includes recombinant nucleic acid molecules comprising at least one isolated nucleic acid molecule of the present invention that enhances transcription of operably-linked sequences in Schwann cells. Preferably, the recombinant nucleic acid molecules further comprise a heterologous promoter sequence, and a heterologous coding sequence, wherein said sequences are operably-linked.
The invention further provides a method for regulating gene expression in Schwann cells, comprising transforming a host cell with a vector comprising at least one isolated nucleic acid molecule, of the present invention, that enhances transcription of operably-linked sequences. The host cell is maintained under conditions such that an operably-linked coding sequence is expressed. The host cell may be a cell in vivo or a cell in culture. For example, the enhancer nucleic acid molecule may be used to mediate an in vitro protein production process using Schwann cells.
The invention also provides methods for gene therapy for Schwann cell-associated disease. The methods include the expression of transgenes in myelin-forming Schwann cells for the treatment of diseases of the peripheral nervous system.
In another aspect, the invention provides cells and non-human animals harbouring the recombinant nucleic acid molecules of the invention. Such animals may have a modified phenotype compared to a non-transgenic animal of the same species, such as enhanced expression of a coding sequence in Schwann cells. Methods of producing such transgenic animals are provided, for example by introducing into an animal the isolated nucleic acids of the invention.
The invention also provides a method for purifying Schwann cell-specific transcription factors, the method comprises contacting cell extracts from Schwann cells with DNA comprising the isolated nucleic acids of the invention under conditions suitable for specific binding of the transcription factor(s) to the recognition site(s) on the DNA, whereupon unbound material may be removed by washing and the retained material containing the transcription factor(s) recovered.
The present invention provides isolated nucleic acid molecules which regulate transcription in Schwann cells by modulating transcriptional activity. In embodiments, such modulation comprises an enhancement of transcription or an increase in transcriptional activity. In other embodiments, such modulation comprises repression of transcription or a decrease in transcriptional activity. In a preferred embodiment, the nucleic acid is the enhancer elements SCE1 and/or SCE2, as shown in
By isolated, it is meant that the isolated substance has been substantially separated or purified away from other biological components with which it would otherwise be associated, for example in vivo. The term ‘isolated’ therefore includes substances purified by standard purification methods, as well as substances prepared by recombinant expression in a host, as well as chemically synthesized substances.
The language “regulates transcription” is intended to include a mechanism which increases or decreases the production of mRNA from a particular gene. Regulation of transcription can be measured using a promoter and regulatory region fused to a gene, preferably a reporter gene whose activity is easily measured. For example, the reporter gene may include β-galactosidase. By “transciptional enhancement”, it is meant to refer to a functional property of producing an increase in the rate of transcription of linked sequences that contain a functional promoter.
Sequences may be derived or obtainable from genomic libraries or directly from isolated DNA by deduction and synthesis based upon sequences of the present invention, such as SCE1. Derived sequences may be identified in different organisms, for example by isolation using as probes the nucleic acid sequences of the invention. Derived nucleic acids of the invention may be obtained by chemical synthesis, isolation, or cloning from genomic DNAs using techniques known in the art, such as the Polymerase Chain Reaction (PCR). Consensus sequences, such as illustrated in
Derived nucleic acids of the invention may also be identified by hybridization, such as Southern analysis. Southern analysis is a method by which the presence of DNA sequences in a target nucleic acid mixture are identified by hybridization to a labeled probe, comprising an oligonucleotide or DNA fragment of a nucleic acid of the invention. Probes for Southern analysis may for example be at least 15 nucleotides in length Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolabeled, biotinylated, or enzyme-labeled probe as described in Sambrook et al. (1989).
In alternative embodiments, a nucleic acid of the invention may be at least 70% identical when optimally aligned to the SCE1 sequence. In alternative embodiments, the degree of identity may be any integer value between 50% and 100%, such as 60%, 80%, 90%, 95% or 99%. When a position in the compared sequence is occupied by the same nucleotide, following optimal alignment of the sequences, the molecules are considered to have identity at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences. In terms of percentage, identity is the sum of identical positions, divided by the total length over which the sequences are aligned, multiplied by 100.
Various aspects of the present invention encompass nucleic acid sequences that are homologous to other sequences. As the term is used herein, a nucleic acid sequence is “homologous” to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (for example, both sequences have the ability to regulate transcription in Schwann cells; as used herein, the term ‘homologous’ does not infer evolutionary relatedness).
Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 80%, 90% or 95%. As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences.
Optimal alignment of sequences for comparisons of similarity may be automated using a variety of algorithms, such as the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence similarity may also be determined using the BLAST algorithm, described in Altschul et al. (1990), J. Mol. Biol. 215:403-10 (using the published default settings). Software and instructions for performing BLAST analysis may be available through the National Center for Biotechnology Information in the United States (including the programs BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX that may be available through the internet at http.//www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database (reference) sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919), a gap existence cost of 11, a per residue gap cost of 1, a lambda ratio of 0.85, alignments ( ) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. In the PSI-BLAST implementation of the BLAST algorithm, an expect value for inclusion in PSI-BLAST iteration may be 0.001 (Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402). Searching parameters may be varied to obtain potentially homologous sequences from database searches.
An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in O2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y.). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
In another aspect of the invention, the isolated nucleic acid may further be incorporated into a recombinant expression vector. Preferably, the vector will comprise an enhancer sequence of the present invention operably-linked to a promoter and a protein coding region such as MBP, a reporter gene or some other gene of interest. A first nucleic acid sequence is operably-linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous.
The recombinant expression vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by persons skilled in the art. The vectors of the present invention may also contain other sequence elements to facilitate vector propagation and selection in bacteria and host cells. In addition, the vectors of the present invention may comprise a sequence of nucleotides for one or more restriction endonuclease sites. Coding sequences for heterologous genes, sequences for selectable markers, and reporter genes are well known to persons skilled in the art. By “heterologous genes” is meant coding sequences or parts thereof which are not naturally connected to the sequences of the invention in the same manner as in the recombinant constructs of the invention.
A recombinant expression vector comprising an enhancer sequence of the present invention may be introduced into a host cell, which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector. The living cell may include both a cultured cell and a cell within a living organism. Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
Vector DNA can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals. Methods for introducing DNA into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy for diseases of the peripheral nervous system (Charcot-Marie Tooth disease) and possibly for central neurons that project axons to the periphery (ALS), for example.
A cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid, is considered “transformed”, “transfected”, or “transgenic”. A transgenic or transformed cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing a transgenic organism as a parent and exhibiting an altered phenotype resulting from the presence of a recombinant nucleic acid construct A transgenic organism is therefore an organism that has been transformed with a heterologous nucleic acid, or the progeny of such an organism that includes the transgene.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (such as resistance to antibiotics) may be introduced into the host cells along with the gene of interest Preferred selectable markers include those that-confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker may be introduced into a host cell on the same vector as that encoding the peptide compound or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid may be identified by drug selection (cells that have incorporated the selectable marker gene will survive, while the other cells die).
A nucleic acid of the invention may be delivered to cells in vivo using methods such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid based transfection, all of which may involve the use of gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a “gene gun”) for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).
Defective retroviruses are well characterized for use as gene therapy vectors (for a review see Miller, A. D. (1990) Blood 76:271). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art Examples of suitable packaging virus lines include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).
For use as a gene therapy vector, the genome of an adenovirus may be manipulated so that it encodes and expresses a peptide compound of the invention, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).
Adeno-associated virus (AAV) may be used as a gene therapy vector for delivery of DNA for gene therapy purposes. AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). AAV may be used to integrate DNA into non-dividing cells (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 may be used to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790). Lentiviral gene therapy vectors may also be adapted for use in the invention.
General methods for gene therapy are known in the art See for example, U.S. Pat. No. 5,399,346 by Anderson et al. A biocompatible capsule for delivering genetic material is described in PCT Publication WO 95/05452 by Baetge et al. Methods of gene transfer into hematopoietic cells have also previously been reported (see Clapp, D. W., et al., Blood 78: 1132-1139 (1991); Anderson, Science 288:627-9 (2000); and, Cavazzana-Calvo et al., Science 288:669-72 (2000)).
Transcription factor(s) bind to polynucleotide sequence(s) or sequence motif(s) which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding In one embodiment of the invention, the nucleic acid may be used for purifying MBP-specific transcription factors. For example, cell extracts from Schwann cells may be contacted with DNA comprising an enhancer sequence of the present invention (SCE1), under conditions suitable for specific binding of the transcription factor(s) to the recognition site(s) on the DNA, whereupon unbound material may be removed by washing and the retained material containing the transcription factor(s) recovered. Eukaryotic transcription factors known in the art include, but are not limited to: NFAT, AP1, AP-2, Spl, OCT-I OCT-2, OAP, MFKB, CREB, CTF, TFIIA, TFIIB, TFIID, Pit-I, C/EBP, SRF (Mitchell, P. J. and Tijan, R. (1989) Science 245:371), which may be assayed in accordance with the invention for binding to the sequences of the invention.
In another embodiment of the invention, enhancer sequences of the present invention may be used to repress enhancer activity. For example, multiple copies of SCE1 may be introduced into a cell which exhibits SCE1 enhancer activity. These sequences may be either in the form of oligonucleotides or contained on a DNA vector separate from the vector containing the gene to be regulated. These DNA enhancer elements not in cis with the gene to be regulated compete for and reduce binding of transcription factors to the enhancer element and thereby repress enhancer activity. By “multiple copies” is meant to include a multiple of DNA sequences which is significantly high to cause competition and reduce binding of the transcription factors to the enhancer element and thereby repress enhancer activity. The number of enhancer sequences required to be provided in trans may be determined by transfecting a certain number of sequences into a cell containing a reporter gene under the control of the enhancer. The number of sequences is sufficient when the activity of the reporter gene is significantly repressed.
In another embodiment of the invention, a method of selecting agents which regulate SCE1 and/or SCE2 enhancer activity is provided. The method includes inserting the SCE1 and/or SCE2 nucleic acid sequence and a reporter gene into a vector and inserting the vector-into a cell. The cell is then exposed to the agent suspected of affecting SCE1 and/or SCE2 enhancer activity and the transcription efficiency is measured by the activity of the reporter gene. The activity can then be compared to the activity of the reporter gene in cells unexposed to the agent in question. The compounds and experimental conditions presently known to modulate myelin gene expression in Schwann cells and oligodendrocytes include progestins, that potentiate myelination both in vivo and in vitro, and electrical stimulation, that modulates the level of MBP expression in cultured Schwann cells (Koenig. et al., 1995; Stevens et al., 1998). It also is well established that elevation of cAMP levels in cultured Schwann cells results in the up-regulation of myelin gene expression (Lemke and Chao, 1988; Morgan et al., 1991). More recently, a role for myelin associated glycoprotein (MAG) in bi-directional transduction of axon-Schwann cell signals has been proposed based on the observation that homozygous MAG null animals express both axonal and myelin anomalies (Li et al., 1994; Yin et al., 1998). However, a putative downstream effector of MAG, Fyn tyrosine kinase, does not appear to be required for PNS myelination (Fujita et al., 1998; Osterhout et al., 1999).
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
Reporter Gene Constructs
Reporter gene constructs were constructed to localize cis-regulatory sequences that control MBP expression in Schwann cells. A 15 kb BamH I MBP genomic fragment, containing approximately 12.0 kb of MBP 5′ flanking sequence, was obtained by screening a lambda DASH-129 mouse genomic library (J. Rossant, Mount Sinai, Toronto). This sequence was subcloned in the BamHI site of pSK (13 clone). A Sac II (−9.0 kb)/Xba I (−3.1 kb) fragment from B clone was inserted into the reeve sites in clone pm12 (3.1 kb MBP 5′ flanking sequence in Xba I/Xma I sites of pSK- (Foran and Peterson, 1992)) to generate the −9.0 kb MBP promoter (clone 8). B clone was digested with Sac II followed by intramolecular ligation of the Sac II ends to generate the −12.0 kb (BamH I) to −9.0 kb (Sac II) MBP 5′ flanking sequence in pSK (B subclone 1A) (all vectors other than pm12 from Stratagene, La Jolla, Calif.; restriction enzymes from New England BioLabs, Mississauga, ON).
To generate reporter constructs, d10 lacZ (Foran and Peterson, 1992) was released from a pUC18 subclone with Sal I and cloned in the Sal I site of clone 8 (3′ to the −9 kb MBP promoter). This clone contained a double insert of lacZ and the second insert was released by BamH I digestion followed by intramolecular ligation (clone 5). As shown in
Two constructs were used to test the position and orientation independence of SCE1. SCE1 (0.6 kb; Sac II/Sac I) was isolated from clone 5 by Sac I digestion and cloned in the Sac I site of pSK+ (clone 6). −6.0 kb MBP-lacZ was isolated from clone S (Kpn I/BamH 1) and cloned in the same sites in clone 6 to generate −6.0 MBP-lacZ-5(SCE1)3′ (
A clone containing SCE1 and −3.1 kb MBP-lacZ also was generated. −3.1 kb MBP-lacZ was isolated from clone pm12 with Xba I and cloned in the Xba I site of clone 7, resulting in a clone containing 5(SCE1)3′- −3.1 kb MBP-lacZ). The construct was released with Sac II/Sph I.
To generate SCE1-hsp-lacZ constructs (
Transgenic Mice
Transgenic mice were derived by injection of DNA into the pronuclei of B6C3F2 zygotes as previously described (Foran and Peterson, 1992). Injected zygotes were transplanted into the oviducts of B6C3F1 females rendered pseudopregnant by mating with vasectomized males. Litters were delivered either spontaneously or by cesarean section 18 days later. Primary transgenic mice and mice derived from established lines were investigated for transgene expression (Table 1).
Histochemical Detection of β-Galactosidase Activity
Mice were anesthetized with avertin intraperitoneally and perfused transcardially with 0.5% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) all at 4° C. Tissues to be analyzed were recovered and postfixed for an additional hour. Samples were then rinsed in 0.1 M phosphate buffer and incubated as whole mounts at 37° C. for various times ranging from less than one hour to overnight in stain consisting of 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 and 0.4 mg/ml Bluo-gal (Gibco/BRL, Burlington, Ontario). In some preparations, detergents sodium deoxycholate and NP-40 were added to the stain at 0.01% and 0.03% respectively to permeabilize the tissue thus assisting the penetration of β-galactosidase substrate. Following the whole mount histochemical reaction, some samples were processed for plastic embedding. Typically, tissue was osmicated prior to dehydration and embedding in Epon. Following polymerization, the blocks were sectioned at 1 μm, and the sections were mounted on slides and viewed either directly or after staining with toluidine blue. Additional tissue was cryoprotected by immersion in 30% sucrose prior to freezing and 12 μm cryostat sections were made and subsequently incubated in stain containing 0.8 mg/ml X-gal (Gibco/BRL, Burlington, Ontario). To investigate prenatal expression, we applied a histochemical technique capable of detecting low-level β-galactosidase activity in sections. Fetuses were recovered and immersion fixed for 1 hour in the same aldehyde mix at 4° C. To cryoprotect, they were then incubated at 4° C. in 30% sucrose overnight prior to freezing in isopentane pre-cooled in liquid nitrogen. Cryostat sections, 12 μm thick, were picked up on slides and dried for 30 min at room temperature and 20 min at 37° C. Sections were then post-fixed by immersion in 4% formalin and 7.5% sucrose in 0.1 M phosphate buffer for 20 min at 4° C., washed for 5 min in buffer alone 3 times, then transferred to β-galactosidase stain (X-gal at 0.8 mg/ml) and incubated at 37° C. overnight. Slides were then cover-slipped using 10% glycerol as the mounting medium.
Identification of Mouse SCE1
Using the reporter gene constructs described above, the expression conferred to MBP was analyzed in transgenic mice. In the initial constructs evaluated, lacZ was promoted by approximately 6, 7, 8.5 or 9 kb of MBP 5′ flanking sequence (
To assess whether this distal sequence had functions characteristic of an enhancer, constructs were generated in which the slightly longer −9 to −8.4 kb sequence was ligated, in both orientations, to the 3′ end of the lacZ reporter promoted by 6 kb of MBP 5′ flanking sequence (
Identification of Mouse SCE2
To search for further sequences capable of conferring Schwann cell expression, the 3 kb sequence immediately upstream of SCE1 (−12.0 to −9.0) was ligated to the 3′ end of the lac Z reporter gene, driven by 6 kb of MBP 5′ flanking sequence (
Heterologous Promoter Constructs
As shown in
Identification of Homologous Human SCE1 Sequence
Using sequence comparison algorithms with human and mouse MBP 5′ flanking sequence, a homologous and potentially enhancer-bearing human sequence was identified at approximately −16 kb in the human locus (
SCE1 Directs Endogenous MBP Expression Program
To determine how closely the expression conferred by SCE1 tracks the expression phenotype of the endogenous MBP gene, prenatal and postnatal transgenic mice bearing SCE1-containing constructs were analyzed. The endogenous MBP locus is expressed at low but detectable levels in the developing PNS of prenatal mice (Bachnou et al., ISN Satellite and UConn-Kroc Symposium, Mystic, 1997). Transgenic mice bearing SCE1-hsp68-lacZ constructs similarly express low but detectable levels of β-galactosidase activity in fetal peripheral nerves from E14 through birth. Also reflecting the endogenous expression program, high-level expression of β-galactosidase appeared in Schwann cells coincident with myelin formation when accumulation of endogenous MBP mRNA markedly increases. As predicted by the temporally discordant myelination programs in dorsal and ventral spinal roots of mice (Baron et al., 1994), ventral roots began to label intensely one to two days prior to dorsal roots. During the first postnatal week of mouse development, the number of myelinating Schwann cells greatly increases (Bray et al., 1977) and the apparent level of β-galactosidase accumulation, assessed by the rapidity and intensity of histochemical labeling, increased in parallel. As similar expression programs were observed for SCE1 containing constructs promoted either by hsp68 or proximal MBP sequence, it appears that this short enhancer sequence is capable of directing the major features of the endogenous MBP expression program during Schwann cell maturation.
Differential Expression of SCE1
As mice bearing the 9.0 kb promoted construct matured, there was a modest decline in β-galactosidase labeling intensity throughout their mixed nerves (lines 17 and 32) and in the mosaic line 24, expression ceased. Surprisingly and consistently, in mice over 3 months of age from line 17 and 32, dorsal roots continued to label but a further precipitous decline in transgene expression was observed in ventral roots. In line 17 mice, expression appeared to be shut off while in line 32, ventral root down regulation was not absolute but labeling intensity was clearly weaker than that observed in dorsal roots. A similarly dramatic difference in dorsal and ventral root labeling intensity was observed in one line of SCE1-hsp68-lacZ mice (also designated 17). These combined observations suggest a novel level of heterogeneity amongst Schwann cells that coincides with the modality (sensory versus motor) of the innervating axon. Although a striking finding in the multiple lines where it was observed, this phenomenon was not encountered in all SCE1 bearing lines. Notably, both dorsal and ventral roots in mice from the SCE1-hsp68-lac Z lines 49 and 54 continued to label at an apparently similar level throughout maturity.
Analysis of SCE1 and SCE2 Sub-Regions and their Effects on SCE1 and SCE2 Enhancer Activity
A 510 bp SCE1 sub-sequence defined by 5′ NaeI and 3′ NaeI restriction sites (
A 488 bp SCE1 sub-sequence defined by 5′ SacII and 3′ Sbf1 restriction sites (
A shorter, 213 bp SCE1 sub-sequence defined by SacII and AvrII restriction sites (
A construct bearing the 213 bp sequence noted above, plus 203 bp of contiguous 5′ mouse sequence from SCE2 (a total of 416 bps;
The above results suggest that different regions of SCE1 confer different types and levels of modulation of transcriptional activity. For example, the higher level of enhancer activity observed with the above noted 213 bp sequence relative to the larger 510 bp and 486 bp sequences, demonstrates the presence of regions in the 510 bp and 486 bp sequences which may confer some repression of transcriptional activity when present in a certain context. These results thus further demonstrate that the nucleic acids of the invention can provide a variable and fine control for the modulation of transcriptional activity, both in the type of modulation (enhancement or repression) and the degree, which can thus be tailored to meet the requirements of various specific applications.
transgenic mice used for the functional analysis of the MBP promoter. The designations + (indicates reporter gene expression) and − (no expression) were made based on the β-galactosidase histochemical assay as described. Designations were made based on comprehensive analysis
*1° indicates that a primary transgenic mouse, rather than mice from a transgenic line, was analyzed for a given construct.
**Lines known to show unique patterns of expression outside the PNS attributed to enhancer trapping at the site of transgene insertion.
All publications, including patent applications referred to herein and including the following, are hereby incorporated by reference (no admission is hereby made with respect to whether such references constitute prior art in any jurisdiction):
| Number | Date | Country | Kind |
|---|---|---|---|
| 2,347,583 | May 2001 | CA | national |
| 2,343,953 | May 2001 | CA | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA02/00657 | 5/2/2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 60290501 | May 2001 | US |
