Recombinant vectors for use in position-independent transgene expression within chromatin
The embodiments of the present invention are directed to discrete, cis-acting regulatory elements that include a barrier element, an insulating element, a silencing element, and matrix attachment regions (“MARs”). Additional embodiments of the present invention are directed to nucleic acid molecules that are useful for facilitating stable transgene expression within a chromatin environment. Additional embodiments of the present invention are directed to recombinant expression vectors including nucleic acid molecules of the present invention that can be incorporated into artificial chromosomes, eukaryotic cell-lines, non-human transgenic animals, and transgenic plants, to improve recombinant protein production in a broad range of eukaryotic hosts, and pharmaceutical compositions including nucleic acid molecules of the present invention that are also useful for gene therapy in the treatment of various genetic diseases.
This application is a continuation-in-part of application Ser. No. 10/853,581, filed May 24, 2004, which claims the benefit of provisional application No. 60/473,109, filed May 23, 2003.
SEQUENCE PROGRAM LISTING APPENDIXTwo identical CDs identified as “Copy 1 of 2” and “Copy 2 of 2,” containing the sequence listing for the present invention, is included as a sequence listing appendix.
TECHNICAL FIELDThis present invention relates to recombinant DNA technology involving compositions containing DNA elements identified within the mammalian c-myc loci that enables more efficient transgene expression. Methods for the application of the compositions to develop various expression vectors that are useful for enhancing transgene expression in eukaryotic hosts are also provided.
BACKGROUND OF THE INVENTIONEukaryotic gene expression is governed at multiple levels, involving local modes of regulation with short-range effects and global modes of regulation effective over extended distances. Within the transcriptional unit of a gene, cis-acting regulatory elements, such as promoters and enhancers, are positioned locally to efficiently recruit the transcriptional machinery to the DNA template to enable the transcription of the gene to an RNA product. Eukaryotic genes may be arranged in a linear sequence within chromosomes that are structurally and functionally subdivided into either transcriptionally active or transcriptionally inactive states. Heterochromatin comprises transcriptionally inactive domains consisting of highly condensed DNA/Histone complexes that are insensitive to DNase I treatment. Euchromatin comprises transcriptionally active domains with complexes of DNA/Histones less densely packed than in heterochromatin that are sensitive DNase I treatment.
Transcriptionally active and inactive regions of the genome are structurally distinguishable due to differential post-translational states that result from various modifications of DNA-associated histones by acetylation, methylation, phosphorylation, and ubiquitinylation. These modifications can be essential for tissue-specific regulation of gene expression, especially during differentiation processes. Euchromatin has been shown to correlate with the hyperacetylation of associated histones. In particular, the acetylation of several lysine residues and the methylation of lysine 4 in histone H3 are characteristic features of transcriptionally active genes. Transcriptionally inactive regions, by contrast, are organized into heterochromatic structures that are characterized by deacetylation of H3 and H4, and lysine 9 methylation of H3. The existence of eukaryotic genes within dynamic chromatin states contributes significantly to the phenomenon of position-effect variegation that frequently precludes stable, long-term heterologous gene expression upon random integration of exogenous DNA into a host chromosome. Because a substantial portion of the eukaryotic genome exists in the transcriptionally inactive heterochromatin state, random integration of exogenous genes into a heterochromatin environment likely results in transgene silencing. For example, gene therapy has only been partially successful, because of position-effect variegation that results from random insertion of transgenes into heterochromatin regions, leading to transgene suppression [G. H. Karpin, Curr. Opin. Genet. Dev. 4: 281-291 (1994)].
Unpredictable and low yields in recombinant protein production, which significantly reduces the rate at which therapeutic proteins are brought to the market, have also been attributed to position-effect variegation. Production yields from cultivated mammalian cells, and from transgenic plants and animals, are still not sufficient to meet growing demands. It is estimated that tons of human serum albumin are needed annually, which are currently produced by processing 50,000 liters of human plasma. Several hundred kilograms of proteins, such as collagen, thrombin, and recombinant therapeutic antibodies, are in demand on a global scale, but the demand has not been fulfilled [Florian M. Wurm, Nature Biotechnology 21: p34-35 (2003)]. There is a continuing need for improvements in recombinant technologies, in particular, improvements in vector designs for enabling more robust expression of heterologous genes within a chromatin environment, such as stably integrated exogenous genes within a host chromosome. Expression vectors that can improve the predictability, yield, and stability of protein expression are of significant value to the biotechnology industry and the biomedical community.
SUMMARY OF THE INVENTIONIn various embodiments of the present invention, compositions and methods for using DNA sequences identified from human and murine c-myc loci are provided. Nucleic acid molecules with these DNA sequences are useful for facilitating stable expression a transgene. Various embodiments of the present invention are directed to recombinant expression vectors that include the present-nucleic-acid-molecule compositions of the present invention, and that can be incorporated into eukaryotic cell-lines, artificial chromosomes, transgenic animals, and transgenic plants to improve the production of recombinant proteins in a broad range of eukaryotic hosts. Pharmaceutical compositions that include the nucleic-acid-molecule compositions of the present invention within expression vectors are also useful for gene therapy in the treatment of various genetic diseases.
In one embodiment, the nucleic-acid-molecule compositions of the present invention can function as barrier elements that preclude silencing of transgenes by stabilizing host heterochromatin, and that enhance transgene expression levels when incorporated into expression vectors that further include a transgene, a promoter, and/or an enhancer. Expression vectors including the nucleic-acid-molecule compositions of the present invention can be inserted into an artificial chromosome or a chromosome of a host cell to improve transcriptional efficiency.
In another embodiment, the nucleic-acid-molecule compositions of the present invention can function as insulating elements that facilitate transcriptional efficiency by blocking enhancer-promoter interactions, and that preclude the effect of neighboring regulatory sequences when incorporated into expression vectors that further include a transgene, a promoter, and/or an enhancer.
In another embodiment, the nucleic-acid-molecule compositions of the present invention can mediate the attachment of chromatin to a nuclear matrix, and can facilitate the regulation of transcription when incorporated into an expression vector that further includes a transgene, a promoter, and/or an enhancer. The compositions of the present invention are directed to several, discrete, cis-acting regulatory elements, that include a barrier element, an insulating element, a silencing element, and matrix-attachment regions (“MAR”), which have not been previously characterized. Each nucleic-acid-molecule compositions of the present invention can be used individually, can be combined together in various combinations, and can be used with heterologous insulators and MARs in various combinations within a broad vector-design scheme for which stable transgene expression in a chromatin environment is desired.
BRIEF DESCRIPTION OF DRAWINGS
FIGS. 8A-C show sub-mapping of multiple functional activities within MINE, particularly the activities conferred individually by the 5′boundary CTCF-binding element and the BE fragment, as described in Examples 4 and 5.
Embodiments of the present invention are directed to several, discrete nucleic acid molecules, referred to below as “the present nucleic acid molecules,” isolated from conserved regions at the 5′ and 3′ boundaries of both murine and human c-myc gene loci. By incorporating the present nucleic acid molecules into an expression vector, the present nucleic acid molecules are useful for facilitating the stable expression of any prokaryotic or eukaryotic exogenous gene, or transgene, of interest. Various embodiments are directed to recombinant expression vectors including the present nucleic acid molecules, that can be incorporated into artificial chromosomes within cultured eukaryotic cell-lines, and into chromosomes of eukaryotic cell-lines, non-human transgenic animals, and non-transgenic and transgenic plants, in order to improve recombinant protein production in a broad range of eukaryotic hosts. In other embodiments, pharmaceutical compositions including the present nucleic acid molecules within expression vectors are useful for gene therapy applications such as stem-cell therapy and tissue-engineering/tissue grafts for the treatment of various genetic diseases.
Various isolated nucleic acid molecules of the present invention confer various functions, including (1) barrier activity that inhibits the progression of heterochromatin into transcriptionally active gene loci, (2) insulating activity that blocks enhancer-promoter interactions, and (3) nuclear-matrix binding activity that regulates transcriptional efficiency. The incorporation of various nucleic acid molecules into transgene expression vectors can improve transgene expression levels, and is particularly suitable for transgenes that are randomly integrated into a host chromatin. Expression vectors comprising the present nucleic acid molecules can be inserted into an artificial chromosome or a genome of a host cell to improve transcriptional efficiency.
The present nucleic acid molecules provide many advantages. For example, because the c-myc gene is constitutively and ubiquitously expressed in most tissues of a eukaryotic organism, the activities of the present nucleic acid molecules derived from the c-myc gene are predisposed to supporting transcriptional activity in many cell types in a non-tissue-specific manner. Expression vectors including the present nucleic acid molecules, such as a c-myc Insulator Element (“MINE”) and matrix attachment regions derived from the 5′ and 3′ region of a c-myc loci (“MYC-MARs”), can be expressed in a broad range of host cells. In contrast, expression vectors comprising the chicken HS4 element, which is derived within the erythroid specific b-globin locus, is not as universally applicable, since b-globin gene expression is specific to erythroid cells. The chicken HS4 element has been shown to be functional in only a limited number of cells [Walters et al, Mol. Cell Biol. May 19 (5):3714-26 (1999). Transgene expression mediated by the chicken HS4 has not shown long-term stability in tissues that are not of the erythroid lineage. However, the present MINE variants can be utilized in divergent cells types, and expression vectors comprising MINE variants can be used for a broad range of gene therapy application which are further described in Section IIID.
Various embodiments of the present invention are directed to a c-myc insulator element (“MINE”) (114), located at the 5′-boundary (116), which is positioned within a transitional region between euchromatin and heterochromatin at the 5′ end of the c-myc gene locus. The MINE comprises four sub-elements, including two main sub-fragments referred to as “BE” (118) and “CTCF” (120). The BE sub-fragment (118) represents the region of MINE that confers barrier activity. The CTCF sub-fragment (120) contains the 5′CTCF-binding site located within the 5′-boundary region.
Other elements, embodied in the compositions, relate to the identification and isolation of MYC-5′MAR (122) and MYC-3′MAR (124), which are distinct, nuclear matrix-associated regions located within two discrete regions of the heterochromatin domains flanking the transcribed sequences, i.e., c-myc gene (100). The MYC-5′MAR (122) is located approximately 76 kb upstream, and the MYC-3′MAR (124) is located approximately 32 kb downstream, from the c-myc locus. The MYC-5′MAR is positioned between the euchromatin domain of c-myc and the nearest neighboring gene (NCBI, accession number XM—299806) located 85 kb upstream of c-myc. The MYC-3′MAR is positioned between the euchromatin domain of c-myc and the PVT-1 gene (126), located 60 kb downstream of c-myc gene.
The embodiments of the present disclosure are discussed in the following order. Section I provides definitions. Section II provides functional characterizations for the cis-regulatory elements that includes a MINE, a BE, a 5′CTCF-binding site, and 5′ and 3′-MYC-MARs. Section III provides advantages and various embodiments of the present invention that include variants of MINE and MYC-MARs, various expression vector constructs, and practical applications for these variants. The references listed are incorporated herein by reference.
I. DefinitionsThe term “heterochromatin” refers to transcriptionally inactive regions of a chromosome consisting of highly condensed DNA/Histone complexes that are insensitive to DNAse I treatment. Heterochromatin can be characterized by detecting the deacetylation states of Histone 3 and Histone 4 and the methylation state of Histone 3 at lysine 9.
The term “position-effect variegation” refers to a phenomenon that occurs when a transgene becomes transcriptionally silenced, or transcriptionally inactivated, upon random integration within the heterochromatin of a host.
The term “operably linked” refers to the joining of distinct DNA molecules, or DNA sequences, to produce a functional transcriptional unit.
The terms “gene,” “gene of interest,” and “transgene” are used interchangeably in the present disclosure. Generally, the terms refer to a deoxyribonucleotide (DNA) molecule, or a DNA sequence, that contains a coding sequence for a particular polypeptide. For example, a “transgene” may refer to the coding sequence for a recombinant protein of interest such as a therapeutic antibody. A transgene may contain heterologous sequences to encode a fusion protein. A “gene,” “gene of interest,” and “transgene” are not intended to include polyadenylation sites, promoters, enhancers, insulators, and MARs.
The term “upstream” refers to a relative direction or position with respect to a reference point along a linear nucleic acid molecule, or a nucleic acid sequence, towards the 5′ end of a nucleotide sequence. “Upstream” may be used interchangeably with “5′”.
The term “downstream” refers to a relative direction or position with respect to a reference point along a linear nucleic acid molecule, or a nucleic acid sequence, towards the 3′ end of a nucleotide sequence. “Downstream” may be used interchangeably with “3′”.
The term “promoter” refers to a nucleic acid molecule, or a nucleic acid sequence, that is typically positioned upstream of a gene and that recruits transcriptional machinery, such as the RNA polymerase and associated factors, that, in turn, initiates transcription of the gene.
The term “enhancer” refers to a nucleotide molecule, or a nucleic acid sequence, that can recruit transcriptional regulatory proteins, such as transcriptional activators, to enhance the transcriptional activation of a gene. Enhancer elements from heterologous sources can be used as a component within expression vectors that represent embodiments of the present invention. Some enhancers can operate in any orientation with respect to a particular heterologous transgene, and enhancers may be positioned upstream or downstream of a gene.
The term “orientation” refers to a particular order in the placement of a nucleic acid molecule, or a nucleic acid sequence, in the context of a DNA sequence. A linear DNA sequence can have, at a given point in time, one of two possible orientations: a 5′-to-3′ orientation or a 3′-to-5′ orientation. Generally, relative to a horizontal reference molecule/sequence, the orientation of a second horizontal nucleic acid molecule/sequence may be the same, or may be reverse or inverted.
The terms “expression vector,” or “vector,” refer to a nucleic acid vehicle that contains a combination of recombinant DNA sequence components for enabling transgene expression. An expression vector enables transport of a combined DNA sequence as an entity. Expression vectors embodied by the present invention include: episomes that are capable of extra-chromosomal replication, such as a circular, double-stranded DNA plasmid; a linearized double-stranded DNA plasmid; and other expression vectors that can serve equivalent functions.
The term “flanking” refers a relative position of one nucleic acid sequence with respect to another nucleic acid sequence. A flanking sequence proceeds or follows a flanked sequence but need not be contiguous with, or immediately adjacent to the flanked sequence. For example, in the context of a transcription unit comprising a promoter and a transgene, a MINE may be placed within an upstream flanking region of the transcription unit. A MYC-3′MAR may be placed within a downstream flanking region of the transcription unit.
The terms “homology,” “identity,” and “similarity” refer to the degree of sequence similarity between two polypeptides or between two nucleic acid molecules being compared by sequence alignment. The degree of homology between two discrete nucleic acid sequences being compared is a function of the number of identical, or matching, nucleotides at comparable positions. The degree of homology between two discrete amino acid sequences being compared is a function of the number of identical, or matching, amino acids at comparable positions.
II. Identification of Cis-Regulatory Elements at the c-Myc Gene Loci The present inventors have isolated cis-regulatory elements, discussed below at the human and mouse c-myc loci, and have characterized the respective functions of each element [Wendy M Gombert, Stephen D. Farris, Eric D. Rubio, Kristin M. Morey-Rosler, William H. Schubach, and Anton Krumm, “The c-myc-Insulator Element and Matrix Attachment Regions Define the c-myc Chromosomal Domain,” Molecular and Cellular Biology, Vol. 23, No. 24, p. 9338-9348 (2003), which is incorporated by reference]. Methods used to establish boundaries between euchromatic and heterochromatic domains at the human and mouse c-myc locus are provided in Example 1.
A. Isolation and Characterization of c-Myc Insulator Elements (“MINE”)
DNA sequences, referred to as “insulators,” have evolved as a complementary mechanism for structurally and functionally distinguishing regions of euchromatin from heterochromatin. In their native context, insulator elements are peripherally positioned with respect to a given transcriptional unit. Insulators function by establishing boundaries between neighboring transcriptional units to prevent encroachment by neighboring regions of heterochromatin. Insulators also serve as efficient gatekeepers in permitting or preventing access to a transcription unit by transcriptional regulatory proteins. Insulating elements are thought to serve at least two functions: (1) to provide a protective shield against deleterious effects of neighboring enhancer regions on the transcriptional activity of a gene, and (2) to facilitate or to amplify the activity of distantly positioned, multi-element enhancer complexes within a transcriptional unit, such as the Locus Control Regions of the β-globin gene.
Insulators, originally identified in Drosophila and subsequently found in yeast and vertebrate cells, protect transcriptionally active regions from the silencing effects of inert chromatin. In fission yeast, the deletion of boundary elements that are positioned at the borders of silenced domains and that separates euchromatin from heterochromatin results in the spreading of K9-methylated H3, a characteristic of heterochromatin, into neighboring euchromatin sequences. When insulator elements isolated from flies, such the scs/scs′ and gypsy elements, are placed between an enhancer and a promoter, these insulator elements can associate with DNaseI-hypersensitive sites, block enhancer-promoter interactions, and provide position-independent expression of exogenous genes, or transgenes. Insulator elements, identified within yeast telomeres and within rDNA and HMR loci, also confer protective properties against negative influences from surrounding heterochromatin. Within the chicken β-globin locus, a barrier function is conferred by the insulator HS4 element (“cHS4”), which includes a binding site for CTCF, a 11-zinc-finger protein. The barrier activity of cHS4 associates with a peak in histone acetylation over the insulator element, which is independent of the expression status of the transcribed gene [Recillas-Targa et al., Proc. Natl. Acad. Sci. U.S.A. 99: 6883-88 (2002)].
1. Characterization of Enhancer-Blocking and Barrier Activities of c-Myc INsulator Element (MINE)
To determine the functional activity of the 1.6 kb MINE (SEQ ID NO:2) isolated from the 5′ boundary of human c-myc gene, the MINE was placed at variable positions relative to an exogenous enhancer and promoter that are contained within several transcription-specific, reporter expression cassettes with a selectable marker as an exemplary transgene (see Example 3). Random integrations of an expression vector comprising a 1.6 kb region of MINE into the chromatin of an eukaryotic host demonstrated that the MINE confers at least three functional activities: (1) an enhancer-blocking activity; (2) a heterochromatin barrier activity; and (3) a transcriptional silencing activity.
The orientation specificity of MINE was tested by inserting the 1.6 kb fragment between the enhancer and promoter elements in s reverse orientation to reporter constructs. Unexpectedly, MINE blocked enhancer-promoter interaction more efficiently in the 3′to-5′ orientation compared to the alternative, 5′-to-3′ orientation. Thus, these experiments show that MINE provides at least two functional activities, an enhancer-blocking activity and a barrier activity, both of which are integral to the proper functioning of the 5′boundary of c-myc (see
Thus, when an expression vector comprising a MINE sequence is integrated into an eukaryotic host chromatin, the barrier activity of MINE can be used to protect against position-effect variegation that often results in transgene silencing, or that precludes robust transgene expression. The enhancer-blocking activity of MINE enables MINE-containing expression vectors to shield against the deleterious activity of neighboring enhancers that may have long-range, over-reaching effects on neighboring transcriptional units. Various embodiments are directed to compositions comprising a MINE (human, SEQ ID NO:1; mouse, SEQ ID NO: 9), which are further described in Section III.
2. Characterization of Activities of MINE Sub-Fragments Comprising a Barrier Element (BE), 5′CTCF-Binding Site, and Silencing Elements S1 and S2
In
(a) Barrier Activity of BE
The BE is defined within a 1.0 kb fragment flanked by SpeI and HindIII restriction sites, and is designated as SEQ ID NO:3, which is a sub-fragment of SEQ ID NO:1 (1-1256). To determine BE function, the 1 kb BE fragment (FIG. 8A, Construct 7) was inserted into a parent reporter construct E-P-neo-scs′ (described in Example 3). Expression cassettes comprising a BE (SEQ ID NO:3) placed upstream of an enhancer can enhance the expression level of a transgene, when randomly integrated into host chromatin. However, expression cassettes comprising a BE placed between an enhancer and a promoter do not affect the transgene expression level, when randomly integrated into host chromatin. Thus, the barrier activity observed in MINE is attributed to BE. Various embodiments are directed to compositions comprising a BE (human SEQ ID NO: 3; mouse SEQ ID NO:10), which are further described in Section III.
(b) In Vivo CTCF-Binding Activity at the 5′Boundary of c-myc
The CTCF fragment is a 640 bp fragment ((214),
(c) Silencing Activity of “S1” and “S2”
Also within the 640 bp CTCF fragment ((214),
S1 and S2 confer the silencing properties of MINE when positioned upstream of enhancers, as described in Example 4 and
B. Nuclear-Matrix-Attachment Activity of 3′ and 5′ MYC-MARs
Matrix attachment regions (“MARs”), or scaffold attachment regions (“SARs”), affect gene regulation by mediating the anchoring of chromatin fibers to the nuclear matrix through AT-rich DNA sequences. Proteins bound to these elements recognize structural features such as narrow minor grooves, DNA bends and kinks, and topoisomerase II cleavage sites. The association of the MARs with some enhancers [Scheuermann and Garrard., Crit. Rev. Eukaryot. Gene Expr. 9: 295-310 (1999)] suggests that they participate in long-range control of gene expression by facilitating the activity of enhancers. However, MARs of the chicken lysozyme gene and the human apolipoprotein B (“apo B”) gene are not associated with enhancer sequences [Kalos and Fournier., Mol. Cell. Biol. 15: 198-207 (1995); Wang et al., J. Lipid Res. 37: 2117-14 (1996)].
The human MYC-5′MAR (SEQ ID NO:6) was identified within a 3.4 kb region flanked by XbaI and EcoRI restriction sites. The MYC-5′MAR is positioned 76 kb upstream of the 5′-end of c-myc gene. The nuclear-matrix binding activity of the mouse MYC-5′MAR (SEQ ID NO:11) was independently identified by the present inventors within a comparable region flanked by XbaI and EcoRI restriction sites.
The human MYC-3′MAR is designated as SEQ ID NO:7, and comprises a sub-region (SEQ ID NO:8) that appears to be unique to the 3′ boundary of human c-myc. The MYC-3′MAR is located 32 kb downstream of the 3′end of the c-myc. Various embodiments are directed to compositions comprising mouse MYC-5′MAR (SEQ ID NO:11), and mouse MYC-3′MAR (SEQ ID NO:12), which are further described in the following Section III, below.
A functionally comparable region also exists within the 5′ boundary of murine c-myc loci (mouse MYC-5′MAR designated as SEQ ID NO:11), and a conserved region (80% identity relative to human) within the 3′ boundary of murine c-myc loci (mouse MYC-3′MAR designated as SEQ ID NO:12). Various embodiments of the present invention, are directed to comparable mouse MYC-5′MAR, and mouse MYC-3′MAR identified at the boundary of mouse c-myc loci, which are further described in the following Section III, below.
III. Advantages of the Present Invention and Related EmbodimentsThe cis-regulatory elements that have been isolated and characterized by the present invention are positioned endogenously within the 5′ and 3′ regions of mammalian c-myc loci. The present inventors have shown that these nucleic acid elements may be removed from their native context and incorporated into various expression vectors. The present nucleic acid sequences that include a barrier element, an insulating element, a silencing element, and MYC-MARs, may be incorporated into a broad vector-design scheme in order to enhance the predictability and stability of recombinant protein production. Examples of various transgenes, promoters, and enhancers that may be incorporated within expression vectors that represent embodiments of the present invention are first provided. Examples of MINE and MYC-MAR variants that may be interchanged are provided. Examples of various heterologous insulators, barrier elements, and MARs that may be interchanged with MINE and MYC-MAR variants within expression vectors are provided. Examples of host cells that may be transfected or infected with various mechanisms of gene delivery are provided. Examples of various uses for expression vectors are provided.
A. Operable Transgenes, Promoters, and Enhancers
1. Trangenes
In general, an expression vector embodied by the present invention comprises a transcription unit that includes the coding region of a transgene of interest operably linked to a transcriptional regulatory element. A transgene of interest is any exogenous gene that encodes a polypeptide, the recombinant production of which is desired by a practitioner. A transgene may be a prokaryotic gene that encodes a prokaryotic polypeptide, or a eukaryotic gene that encodes a eukaryotic polypeptide. A transgene can be a chimeric molecule, comprising two or more distinct polynucleotide sequences that encode a fusion protein. The coding region of any gene may be used, for example, the cDNA of a transgene or the cDNA fragment of a transgene. Examples of suitable transgenes include structural proteins, enzymes, antibodies, membrane receptors, neurotransmitters, hormones, regulatory proteins, cytokines, antigens, and clotting factors. Stable and reproducible production of such therapeutic proteins over an extended period of time is highly desirable.
More than one transgene may be operably positioned under the transcriptional control of a promoter and/or an enhancer within a transcriptional unit by a person skilled in the art of molecular biology. More than one transcriptional unit may be included within an expression vector. The techniques for combining any DNA sequence of the present invention can be found in various sources, such as a standard laboratory molecular biology treatises [Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., 2001, Cold Spring Harbor, which is incorporated in its entirety]. Conventional techniques that are practiced in the art for recombinant engineering include the use of restriction enzyme digestion, nucleic acid ligation, and polymerase chain reaction (PCR).
2. Promoter and Enhancers
A transcriptional regulatory element of a transcriptional unit may include one or more promoters and enhancers. Generally, a promoter is positioned upstream of a transgene. In contrast, an enhancer can be placed upstream, downstream, or both upstream and down stream of a transgene. With respect to the transgene of interest, promoter and enhancer elements may be derived from a homologous source, for example, derived from the native gene locus of the transgene (i.e., the endogenous promoter of gene A may be operably linked to gene A within the vector). Alternatively, promoter and enhancer elements may be derived from a heterologous source such as from the same gene locus of a different species of an organism, from a different gene locus of the same species, or from a different gene locus of a different species of organism. A suitable promoter can be constitutive (i.e., transcriptionally activated at steady-state) or can be inducible (i.e., transcriptionally activated upon proper stimulus). Constitutive and inducible promoters are generally known by persons skilled in the art. Examples of constitutive promoters include various promoters derived from infectious viruses, such as retroviruses, herpes virus, lentivirus, adenovirus and adenovirus variants, mumps and poliovirus. Promoter and enhancer selection is generally independent of the particular transgene of interest, but is dependent on the presence or absence of host-specific factors, such as tissue-specific regulatory factors that are present within the nuclear compartment of a host cell. Promoter and enhancer selection can vary depending on the type of eukaryotic host cell in which the present expression vectors need to be operable, so that a given transgene can be expressed pursuant to host-chromatin integration. Examples of suitable host cells are provided below, and include eukaryotic animal and plant cells. Thus, operable promoters of the present invention include a broad range of constitutive, inducible, tissue-specific, non-tissue-specific, and developmentally-specific promoters that can be derived from animals, plants, and viruses, which can be activated, or recognized, by nuclear regulatory factors present within a eukaryotic host. Various promoters and methods for operably linking promoters to transgenes are known by persons skilled in the art of recombinant technology.
3. Additional Regulatory Elements
An expression vector can include one or more additional sequences necessary for transgene expression. For example, the expression vectors that represent embodiments of the present invention (“present expression vectors”) can include a 3′ untranslated sequence (“UTS”), such as a polyadenylation site that can be derived from the 3′ end of most eukaryotic genes. Various polyadenylation sites can be positioned downstream of the transgene, including a 3′ UTS derived from the native gene locus of the transgene of interest (i.e., homologous polyadenylation site), and heterologous sites. In addition, a 5′ UTS element can be positioned upstream of a transgene of interest to enhance long-term transgene expression. With respect to the transgene, homologous 5′ UTS derived from the endogenous transgene locus, or various heterologous 5′ UTS can be used including, for example, a gamma-actin 5′ UTS, an aldolase 5′ UTS, an EFl-alpha 5′ UTS, or a beta-actin 5′ UTS. The length of a suitable 5′ UTS sequence can extend up to 500 base pairs, but preferably between 25 to 150 base pairs. Alternatively, a heterologous U1 intron sequence can be positioned adjacently to the 5′ UTS sequence, or fused to a 5′ UTS region [Korb & Johnson, Nucleic Acids Res. 21(25):5901-8, (1993)] Examples of U1 introns include a EF1-alpha U1 intron, a GAPDH U1 intron, and a beta-actin U1 intron. Suitable 5′ UTS sequences for the various genes are known by persons skilled in the art and can be readily identified by searching a reputable database, such as the GenBank database.
4. Selectable Marker Gene
An expression construct can also include the coding sequence for a selectable marker used to confer a selectable phenotype upon introduction into various mammalian cells. The selectable phenotype is used to identify and to isolate recipient primary or secondary cells. For example, a selectable marker which confers a selectable phenotype, such as drug resistance, nutritional auxotrophy, resistance to a cytotoxic agent, or expression of a surface protein, can be used. Examples of suitable selectable markers include genes that confer resistance to neomycin, puromycin, hygromycin, and histidine D. A selectable marker can be introduced into primary or secondary cells by incorporating the selectable marker gene within expression constructs comprising a transgene and the present nucleic acid sequences, or by incorporating the marker gene into a distinct vector than can be co-transfected for example, with the present expression constructs.
B. Use of MINE, MYC-MARs, and Variants of MINE and MYC-MARs within Expression Vectors
1. Expression Vectors Comprising c-Myc Insulator Element
(a) Variants of MINE
The various expression vectors described in the previous sub-section A can be embellished, or further incorporated, with the present nucleic acid sequences comprising MINE, sub-fragments of MINE, and homologous variants of MINE and sub-fragments. In one embodiment, the present expression vector comprises a MINE sequence, or a functionally equivalent sequence that includes sub-fragments of MINE, and are derived from the c-myc gene of any mammal. Examples of mammals include human, mouse, rat, monkey, horse, cat, dog, pig, cow, sheep, goat, and varying marsupials. MINE sequence variants exhibit activities that include: an enhancer-blocking activity, an insulating activity, a silencing activity, and a barrier activity. The homology between human MINE and murine MINE is approximately 90%, for example. The present embodiments are directed to variant sequences of MINE that have one or more activities associated with MINE, and that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% identities.
In the described embodiments, a MINE, sub-fragments of MINE, and MINE sequence variants are positioned in a 5′-to-3′ direction in the following order: a MINE, a enhancer, a promoter, and a transgene of interest. In addition, a MINE, sub-fragments of MINE, and MINE sequence variants can be positioned with respect to the enhancer, the promoter, and the transgene, in an orientation that is either 5′-to-3′ direction, or 3′-to-5′ direction. Furthermore, one or more molecules of MINE, sub-fragments of MINE, and MINE sequence variants may be used within an expression vector molecule.
In another embodiment, the present expression vectors comprise a human MINE sequence, including SEQ ID NO:1, and sequences that have at least 50% identity with respect to SEQ ID NO:1. In another embodiment, the present expression vector comprises a murine MINE sequence, including SEQ ID NO:9, and sequences that have at least 50% identity with respect to SEQ ID NO:9.
(b) Variants of BE
In another embodiment, the present expression vectors comprise a barrier element (BE), which is a sub-fragment of MINE, derived from the c-myc gene of any mammal. In a related embodiment, the present expression vectors comprise a human BE sequence, including SEQ ID NO:3, and sequences that have at least 50% identity with respect to SEQ ID NO:3. In a related embodiment, the present expression vectors comprise a murine BE sequence, including SEQ ID NO:10, and sequences that have at least 50% identity with respect to SEQ ID NO: 10.
(c) Variants of 5′CTCF-Binding Site
In another embodiment, the present expression vectors comprise a CTCF-binding site, which is a sub-fragment of MINE, derived from the c-myc gene of any mammal. In a related embodiment, the present expression vectors comprise a human CTCF-binding site, including SEQ ID NO:15, and sequences that have at least 50% identity with respect to SEQ ID NO:15. In a related embodiment, the present expression vectors comprise a murine CTCF-binding site, including SEQ ID NO:16, and sequences that have at least 50% identity with respect to SEQ ID NO:16.
(d) Variants of S1 and S2
In another embodiment, the present expression vectors comprise two silencing elements, which are sub-fragments of MINE, derived from the c-myc gene of any mammal. In a related embodiment, the present expression vectors comprise a first human silencing element (SI), including SEQ ID NO:4, and sequences that have at least 50% identity with respect to SEQ ID NO:4. In a related embodiment, the present expression vectors comprise a second human silencing element (S1), including SEQ ID NO:5, and sequences that have at least 50% identity with respect to SEQ ID NO:5. In a related embodiment, the present expression vectors comprise a first mouse silencing element (S2), including SEQ ID NO:207, and sequences that have at least 50% identity with respect to SEQ ID NO:207. In a related embodiment, the present expression vectors comprise a second mouse silencing element (S2), including SEQ ID NO:208, and sequences that have at least 50% identity with respect to SEQ ID NO:208.
(d) Combination Variants of MINE
In one embodiment, the present expression vectors comprise any combination of mammalian MINE sub-fragments. In another embodiment, the present expression vectors comprise any combination of human and murine MINE sub-fragments. In another embodiment, the present expression vectors comprise one or more of any combination of the following sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:9; SEQ ID NO:10, and sequences that have at least 50% identity with respect to SEQ ID NOS:1-5, 9, and 10.
2. Expression Vectors Comprising MYC-MARs (“MYC-5′MAR and MYC-3′MAR”)
The various expression vectors described in the previous sub-sections A and B(1) can be embellished, or further incorporated, with the present nucleic acid sequences comprising MYC-5′MAR, MYC-3′MAR, sub-fragments of MYC-5′MAR and MYC-3′MAR, and homologous variants of MYC-5′MAR and MYC-3′MAR. In one embodiment, the present expression vector comprises a MAR sequence, or a functionally equivalent sequence that includes sub-fragments of MYC-5′MAR and MYC-3′MAR, that is derived from a c-myc locus of a mammal, and functions as a nuclear-matrix attachment region (“MAR”) when inserted into a host chromosome. Exemplary mammalian sources for MINE also apply to MYC-MARs (Sub-section B (1)). In the described embodiments, MYC-5′MAR variants can be positioned 5′ (upstream) from a transgene, and the MYC-3′MAR variants can be positioned 3′ (downstream) from a transgene. Alternatively, MYC-5′MAR variants can be positioned 3′ (downstream) from a transgene, and the MYC-3′MAR variants can be positioned 5′ (upstream) from a transgene.
By incorporating MYC-MAR sequences into a vector design, a practitioner may regulate the nuclear organization of host cells by permitting more efficient interaction among trans-acting regulatory proteins present within the nuclear compartment of a host cell and cis-regulatory DNA elements, such as promoters and enhancers. Structural re-organization of the nuclear sub-compartments may be achieved by sub-regions of MYC-5′MAR and MYC-3′MAR sequences.
(a) Variants of Human MYC-MARs
In one embodiment, the present expression vectors comprise a human MYC-5′MAR sequence that includes SEQ ID NO:6, sequences that have at least 50% identity with respect to SEQ ID NO:6 within any given 1000 base pairs of alignment, and preferably sequences that have at least 70% identity with respect to SEQ ID NO:6 within any given 500 base pairs of alignment.
In another embodiment, the present expression vectors comprise a human MYC-3′MAR sequence that include SEQ ID NO:7, sequences that have at least 50% identity with respect to SEQ ID NO:7 within any given 1000 base pairs of alignment, and preferably sequences that have at least 70% identity with respect to SEQ ID NO:7 within any given 500 base pairs of alignment. In another embodiment, the present expression vectors comprise a sub-region of MYC-3′MAR that contain the sequence of SEQ ID NO:8, or that has at least 50% identity to SEQ ID NO:8. Various embodiments are directed to variant sequences of SEQ ID NO:8, which appears to be unique to human MYC-3′MAR, and have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% identities.
In another embodiment, the present expression vectors comprise both a human MYC-5′MAR and a human MYC-3′MAR. Such human MYC-5′MAR includes SEQ ID NO:6, sequences that have at least 50% identity with respect to SEQ ID NO:6 within any given 1000 base pairs of alignment, and preferably sequences that have at least 70% identity with respect to SEQ ID NO:6 within any given 500 base pairs of alignment. Such human MYC-3′MAR sequence includes SEQ ID NO:7, sequences that have at least 50% identity with respect to SEQ ID NO:7 within any given 1000 base pairs of alignment, and preferably sequences that have at least 70% identity with respect to SEQ ID NO:7 within any given 500 base pairs of alignment.
(b) Variants of Murine MYC-MARs
In another embodiment, the present expression vectors comprise a murine MYC-5′MAR sequence that includes SEQ ID NO:11, sequences that have at least 50% identity with respect to SEQ ID NO:11 within any given 1000 base pairs of alignment, and preferably sequences that have at least 70% identity with respect to SEQ ID NO:11 within any given 500 base pairs of alignment.
In another embodiment, the present expression vectors comprise a murine MYC-3′MAR sequence that includes SEQ ID NO:12 and variants of SEQ ID NO:12. With respect to the human MYC-3′MAR, a comparable sequence is reasonably identifiable within the 3′ region of murine c-myc locus. A sequence containing a sub-fragment of the murine MYC-3′MAR (SEQ ID NO:12) was identified within an approximately 10 kb, 3′ region of c-myc. The murine MYC-3′MAR (SEQ ID NO:12) shares approximately 80% sequence identity with a sub-region of human MYC-3′MAR (SEQ ID NO:7). The present embodiments are directed to variant sequences of MYC-3′MAR (SEQ ID NO:12) that can attach to the nuclear matrix, and that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% identities with respect to MYC-3′MAR (SEQ ID NO:12).
(c) Variants of Human/Murine MYC-MARs
Various embodiments are directed to expression vectors comprising any combination of the following: a human MYC-5′MAR, a human MYC-3′MAR, a murine MYC-5′MAR, and a murine MYC-3′MAR. Such embodiments include the combination of any of the following sequences: SEQ ID NO:6, sequences that have at least 50% identity with respect to SEQ ID NO:6 within any given 1000 base pairs of alignment, sequences that have at least 70% identity with respect to SEQ ID NO:6 within any given 500 base pairs of alignment; SEQ ID NO:7, sequences that have at least 50% identity with respect to SEQ ID NO:7 within any given 1000 base pairs of alignment, and preferably sequences that have at least 70% identity with respect to SEQ ID NO:7 within any given 500 base pairs of alignment; SEQ ID NO:11, sequences that have at least 50% identity with respect to SEQ ID NO:11 within any given 1000 base pairs of alignment, and preferably sequences that have at least 70% identity with respect to SEQ ID NO:11 within any given 500 base pairs of alignment; and SEQ ID NO:12 and sequences that can attach to the nuclear matrix, and that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% identities with respect to MYC-3′MAR (SEQ ID NO:12).
(d) The Combined Effect of MINE and MYC-MARs on Transgene Expression
In one embodiment, an expression vectors comprise a MINE that is operably linked to a MYC-5′MAR in an arrangement which is functionally equivalent to the endogenous, intact 5′boundary region of c-myc. The MINE and the MYC-5′MAR are operably linked to a transcriptional unit within an expression vector so that the insulating (i.e., enhancer-blocking) and barrier activities of MINE, and the nuclear-matrix attachment activity of MYC-5′MAR can operate synergistically to enhance trangene expression level. An example of an optimal arrangement includes positioning a MYC-5′MAR upstream of a MINE, which is positioned upstream of a enhancer/promoter element, which is positioned upstream of a transgene. In these embodiments, a MINE can be positioned in any orientation.
In another embodiment, expression vectors comprise a MINE that is operably linked to a transcriptional unit of a transgene, which is operably linked to a MYC-3′MAR. In these embodiments, a MINE can be positioned in any orientation. In another embodiment, an expression vector comprises a MINE, a MYC-5′MAR, and a MYC-3′MAR, that are operably linked to a transcriptional unit of a transgene. An example of an optimal arrangement includes positioning a MYC-5′MAR upstream of a MINE, which is positioned upstream of the enhancer/promoter element, which is positioned upstream of a transgene, which is positioned upstream of a MYC-3′MAR.
C. Use of MINE with Heterologous MARs and the Use of MYC-MARs with Heterologous Insulators within Expression Vectors
1. Protective Effect of Insulators on Transgene Expression
The incorporation of insulators into an expression vector including a transgene of interest can provide: (1) an effective insulating shield against negative influences from external enhancers, i.e., preventing background promoter-enhancer interactions; and (2) an effective barrier against the encroachment by host heterochromatin after random integration. Expression vectors comprising insulator elements with barrier activity, such as MINE, can inhibit heterochromatin progression at sites of integration, and therefore, substantially improve transcriptional efficiency by overcoming the problem of heterochromatin-induced silencing of transgenes.
It has been shown by the present inventors that the barrier activity of MINE is separable from the nuclear-matrix anchoring activity of MYC-5′MAR. The separability of insulators from endogenous nuclear-matrix-attachment regions appears to be a general phenomenon. In various embodiments, a MINE, sub-fragments of MINE, and MINE sequence variants may be operably linked with a heterologous MAR sequence, derived from a gene locus other than c-myc. Examples of heterologous MARs include: Human IFN-α MAR, Human interferon-beta 1, Apo-B MAR, Serpin MARs, Human β-globin MARS, and Human IgH MARs.
In various embodiments, variants of MYC-MARs, including variants of human MYC-5′MAR, human MYC-3′MAR, murine MYC-5′MAR, and murine MYC-3′MAR, can be operably linked with heterologous insulators, derived from a gene locus other than c-myc. Examples of heterologous insulators include: Chicken β-globin HS4, Human β-globin 3′ HS1/5′ HS5, Human Bead-1 (TCR α/β locus), Human Dad-1 (TCR α/β locus), Drosophila scs and scs′ (Hsp70), Drosophila Gypsy (retrotransposon), Drosophila Fab-7 (Bithorax complex), Drosophila Mcp (bithorax complex), Drosophila eve-promoter (even-skipped gene), and Xenopus Repeat Organizer (RO) (Ribosomal RNA genes).
D. Gene Delivery Methods and Host Cells
1. Gene Delivery Vectors and Related Methods
The present expression vectors may be introduced into a host cell by receptor-mediated gene delivery, by microinjection, and by conventional methods of transfection such as liposome fusion, electroporation, and calcium-phosphate precipitation. To produce recombinant proteins using various mammalian cell lines, transfection and receptor-mediated gene delivery (i.e., virus infection) methods may be preferred. For protein production using insect cell-lines such as Hi-fives and Sf-9 cells, viral infection is a preferred method for gene delivery. Vectors designed to target insect tissues for making therapeutic and biopharmaceutical proteins are contemplated [Florian W. Wurm, Nature Biotechnology 21: 34-35 (2003)]. Primary or secondary cells can also be transfected by any transfection method listed.
Various gene delivery vectors that are practiced by persons skilled in the art can be used to introduce the present expression vectors. Examples of viral vectors that may be used to infect host cells include: improved adenoviral vectors that can target pulmonary tissues [Reynold et al., Nature Biotechnology 19: 838-842 (2001)]; gene-deleted adenovirus-transposon vectors that can stably maintain virus-encoded transgenes in vivo through integration into host chromosomes [Yant et al., Nature Biotechnology 20: 999-1005 (2002)]; recombinant adenoviruses that can target rat brain neurons [Bilang-Bleuel et al., Proc. Natl. Acad. Sci. U.S.A. 94: 8818-8823 (1997)]; Moloney-murine-leukemia-virus (“Mo-MuLV”) based retroviral vectors that can target CD4+ and CD+8 Tcells and monocyte-macrophages [Auten et al., Human Gene Therapy 10: 1389-99 (2003)]; and poliovirus-replicon-based vectors that can target tissues of the central nervous tissue [Bledsoe et al., Nature Biotechnology 18: 964-969 (2000)]. Examples of other suitable viral vectors include: herpes virus, mumps virus, Sindbis virus, and vaccinia virus, such as the canary pox virus. The usage of viral vectors is well known by persons skilled in the art, and for gene therapy uses, viral infection is preferred generally [Robbins and Ghizzani, Mol. Med. Today 1:410-417, (1995)].
2. Host Cells
(a) Eukaryotic Cell-Lines for Recombinant Protein Production
In various embodiments, suitable host cells for recombinant protein production include cell-lines that can be maintained and propagated in culture. Examples of transformed or immortalized cells include: a Bowes Melanoma cell (ATCC Accession No. CRL 9607); a Daudi cell (ATCC Accession No. CCL 213); a HeLa cell and a derivative of a HeLa cell (ATCC Accession Nos. CCL 2, CCL2.1, and CCL 2.2); a HL-60 cell (ATCC Accession No. CCL 240); a HT-1080 cell (ATCC Accession No. CCL 121); a Jurkat cell (ATCC Accession No. TIB 152); a KB carcinoma cell (ATCC Accession No. CCL 17); a K-562 leukemia cell (ATCC Accession No. CCL 243); a MCF-7 breast cancer cell (ATCC Accession No. BTH 22); a MOLT-4 cell (ATCC Accession No. 1582); a Namalwa cell (ATCC Accession No. CRL 1432); a Raji cell (ATCC Accession No. CCL 86); a RPMI 8226 cell (ATCC Accession No. CCL 155); a U-937 cell (ATCC Accession No. CRL 1593); WI-38VA13 sub line 2R4 cells (ATCC Accession No. CLL 75.1); a CCRF-CEM cell (ATCC Accession No. CCL 119); a 2780AD ovarian carcinoma cell (Van Der Blick et al., Cancer Res. 48: 5927-5932, 1988); and heterohybridoma cells produced by the fusion of human cells and cells of another species. In another embodiment, the immortalized cell line can be a non-human cell-line, such as a CHO cell-line or a COS cell-line.
(b) Primary and Secondary Cells for Ex Vivo Gene Therapy
In one embodiment, primary and secondary cells may be transfected or infected with expression constructs, that represent embodiments of the present invention (“present expression constructs”), to enable stable, long term transgene expression within the tissues of a diseased patient. Primary and secondary cells may be removed from a diseased animal or a cancerous animal host prior to ex vivo transfection or infection. Examples of suitable host cells for use in gene therapy include various primary and secondary cells derived from mammalian tissues. Primary and secondary cells that can be transfected or infected ex vivo include fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, lymphocytes, bone marrow cells, muscle cells, and precursors of these somatic cell types. The use of MINE, MYC-MARs, and any combination of MINE and MYC-MARs with other insulators and other MARs within the expression vectors may substantially enable the stable, long term expression of transgenes. Preparation of mammalian tissues for ex vivo adaptation are known to persons skilled in the art.
In another related embodiment, the present expression vectors may be used to genetically engineer tissues under ex vivo culture systems. For example, healthy stem cells may be removed from a donor so that the cells can be induced to differentiate into a desired tissue type. Stem cells and other embryonic tissues can be induced by known differentiation factors in a controlled microenvironment established within a tissue-growing laboratory. Such cells can be further manipulated by introducing the present expression vectors comprising the transgene of interest. The use of MINE, MYC-MARs, and any combination of MINE and MYC-MARs with other insulators and other MARs within the expression vectors may substantially enable the stable, long term expression of transgenes.
(c) Delivery of Pharmaceutical Compositions for In Vivo Gene Therapy
In one embodiment, the present expression vectors can be supplemented with sequences derived from recombinant viral vectors to enable stable, long term transgene expression within specific tissues of a diseased patient. Specific examples of viral vectors are described above in Subsection IIID(1). A pharmaceutical composition comprising the present expression vectors can be administered to a diseased mammal. The use of MINE, MYC-MARs, and any combination of MINE and MYC-MARs with other insulators and other MARs within the expression vectors may substantially enable the stable, long term expression of transgenes. These expression vectors may be used to produce proteins with substantial therapeutic value.
Because c-myc is widely expressed in mammalian systems, many diseases have been attributed to aberrant regulation of c-myc. Many types of cancers have been associated with the overexpression of a c-myc gene, including 80% of breast cancers, 70% of colon cancer, 90% of gynecological cancers, 50% of hepatocellular carcinomas, and hematological tumors. N-myc amplifications associated with neuroblastoma and chromosomal translocations affecting c-myc in Burkitt's lymphoma are noted examples. Furthermore, cancers that are associated with Epstein-Bar Virus (EBV) have been associated with c-myc overexpression [Cutrona et al, “Transfection of the c-myc oncogene into normal Epstein-Barr virus-harboring B cells results in new phenotypic and functional features resembling those of Burkitt lymphoma cells and normal centroblasts,” J. of Experimental Medicine, Vol. 181, p. 699-711 (1995)]. Such diseases may be targeted by pharmaceutical agents or drugs that are able to interact with MYC-MAR sequences in vivo, either directly or indirectly.
E. Artificial Chromosomes Comprising MINE and MYC-MARs
As another embodiment, the present expression vectors may be used to genetically engineer artificial chromosomes for the production of recombinant protein.
F. Transgenic Plants and Animals Comprising MINE and MYC-MARs
In another embodiment, the present expression vectors may be used to engineer non-human transgenic animals for the production of recombinant protein products. Various vectors systems that can target animal tissues of vertebrates, which are known and practiced by persons skilled in the art, are contemplated. Examples of suitable vectors and suitable cells were provided in Subsection III.D(1) and (2). Methods for making transgenic animals are known by persons skilled in the art [Taube et al., Transgenic Res. 11(4): p397-410].
In another embodiment, the present expression vectors may be used to engineer transgenic plants for the production of recombinant protein. Examples of suitable host that may be transformed by standard transformation techniques include: Nicotiana bethamiana, Arabidopsis thaliana, tomato, banana, turnip, black-eyed bean, oilseed rape, Ethiopian mustard, potato, rice, wheat, and maize [Giddings et al., Nature Biotechnology 18: 1151-1155 (2000) is hereby incorporated by reference]. In addition, non-transgenic plants may be infected with recombinant viruses, such as the tobacco mosaic virus (TMV) and the cowpea mosaic virus (CPMV), that can be engineered to contain the present compositions to express transgenes during their replication cycles. The present compositions may be operably linked to plant virus or plant promoters such as the CaMV 35S promoter, glutelin Gt3 promoter, and the legume B4 promoter [Giddings et al., Nature Biotechnology 18: 1151-1155 (2000)]. Higher production levels of vaccines, antibodies, and biopharmaceuticals may be achievable using the present compositions within vectors that are known and practiced by persons skilled in the art.
EXAMPLESThe following examples are offered as illustrations of certain embodiments of the present invention. In Example 1, regions of the c-myc locus that associate with either acetylated or methylated histones were defined in order to identify the boundaries between euchromatin and heterochromatin. In Example 2, at least two CTCF-binding elements that are functionally occupied with CTCF in vivo were defined within the 5boundary of c-myc. In Example 3, the enhancer-blocking activity and barrier activity of MINE were defined. In Example 4, enhancer-blocking activity and silencing activity within the subregions of MINE were functionally distinguished. In Example 5, the barrier activity within a subregion of MINE was defined. In Example 6, the 5′ and 3′ matrix attachment regions (MARs) at the c-myc locus were identified.
Example 1 Boundary Definitions of Euchromatic and Heterochromatic Domains at the c-myc Locus Transitions in the histone modification patterns mark the boundaries between euchromatic and heterochromatic domains at a gene locus [Litt et al., Embo J. 20: 2224-35 (2001)]. A high resolution map of histone acetylation and methylation patterns was generated within a 25 kb region of c-myc loci in both human and mouse (
The enrichment of c-myc specific histone modifications was measured by duplex PCR, by isolating the DNA from immunoprecipitated chromatin from the mouse (
RESULTS: Patterns of histone acetylation and methylation detected over a 25 kb region of human and mouse c-myc loci are highly conserved.
The distribution of K9-methylated histones along the 25 kb region of the human and murine (
Data indicate that the mammalian c-myc gene resides within a large hyperacetylated domain flanked by chromatin with heterochromatin characteristics. The patterns of methylation and acetylation in mouse and human are basically similar by the fact that in both systems, hyperacetylated domain extends approximately 2 kb upstream of the c-myc exon 1 sequences, and is flanked by transcriptionally inert chromatin containing K9-methylated, hypo-acetylated histone H3. The degree of hypermethylation is similar to that of the transcriptionally inactive murine β-globin gene in CTLL2 cells (see bars to the right of
CELL CULTURE: The human HL60 and Jurkat cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics penicillin (50 U/ml) and streptomycin. HL60 cells were induced to differentiate with 1.5% DMSO for 2 h. The IL2-dependent murine CD8+T-cell line CTLL2 was maintained in RPMI 1640 supplemented with 1 mM sodium pyruvate, 10% fetal bovine serum, 2 mM L-glutamine media, antibiotics and 50 u/ml rIL2 (Chiron, Emeryville, Calif.). To inhibit proliferation and to generate a resting T-cell population, CTLL2 cells were placed into media lacking IL2 for 8 h. For mitogenic induction, 100 U/ml rIL2 was added.
CHROMATIN IMMUNOPRECIPITATION ASSAY: ChIP assays were performed according to Shang et al. [Cell 103: 843-52 (2000)]. Chromatin was cross-linked in the presence of 1% formaldehyde for 5 min. at room temperature. After the addition of glycine to a 0.125 M concentration, the cells were washed in ice-cold PBS containing protease inhibitors (Roche Molecular Biochemicals). Cells were pelleted, resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1× protease inhibitor cocktail and 10 mM butyrate), and sonicated 6 times 20 seconds each at setting 6.5 in a Branson sonicator with microtip. The length of the DNA fragments ranged from 200 bp to 800 bp. After centrifugation to remove cell debris, the whole-cell extract was diluted 10-fold with ChIP dilution buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl, 1× protease inhibitor cocktail and 10 mM butyrate). Before immunoprecipitation, the chromatin solution was pre-cleared with protein A agarose slurry and normal rabbit serum to reduce non-specific background. Anti-acetylated histone H3, anti-methylated K9 or anti-CTCF antibody (Upstate Biotechnology) was added and incubated overnight at 4° C. with agitation. A no-antibody control was also performed for each CHIP assay. Immune complexes were collected by addition of 25 μl of protein A agarose slurry that had been preincubated with bovine serum albumin and salmon sperm DNA. After incubation at 4° C. for 2 h the beads were washed several times according to Orlando et al. [Methods 11: 205-14 (1997)]. The DNA contained within the immune complexes was recovered by the addition of proteinase K and SDS (0.1%) for 1 hour at 55° C. followed by incubation at 65° C. for at least 4 h to reverse the cross-links. The DNA was then purified by phenol/chloroform extraction and precipitated with ethanol precipitation.
DUPLEX PCR: DNA recovered from immunoprecipitations was analyzed by duplex PCR using c-myc specific primers and reference primers (β-globin, MyoD), the sequences of which are provided below. The reactions were performed with the Failsafe™ Taq polymerase in 1× buffer F (Epicenter), 10 pmol of each c-myc primer in the presence of a 32P-dCTP. After electrophoresis on a native 6% acrylamide gel, the signals were quantified with the Cyclone™ phosphoimager. The fold enrichment in each immunoprecipitation was determined by calculating the ratio of the signal obtained with c-myc primers to that of the reference primers.
C-MYC SPECIFIC PRIMERS: The primer sets for each sub-region of the c-myc loci from A to Z (human primer sequences) and a to w (mouse primer sequences) are provided below in Table 1, also represented in
To determine the existence of functional binding sites for CTCF at the 5′boundary of the human and murine c-myc gene loci, in vivo associations between CTCF and regions of the 5′boundary were assayed by ChIP using a CTCF-specific antibody. Several potential CTCF-binding sites within the c-myc locus are listed in Table 2. The consensus sequence 5′-CCRNNAGRGG-3′ (SEQ ID NO:18) was previously characterized by Bell et al. [Cell 98: 387-96 (1999)]. Sites A (“P2”) (human SEQ ID NO: 20; mouse SEQ ID NO: 21) and B (“P1”) (SEQ ID NO: 19) were identified by in vitro binding studies [Fillipova et al., Nat. Genet. 28: 335-43 (2001)]. Alignment of CTCF sites positioned near the 5′boundary (human SEQ ID NO: 15; mouse SEQ ID NO: 16) located upstream of exon 1, and at the P2 promoter (human SEQ ID NO: 20; mouse SEQ ID NO: 21) located downstream of the beginning of exon 1, suggested a high level of conservation between the human and mouse c-myc genes, which coincidentally occur near DNase I hypersensitivity sites [Siebenlist et al., Cell 37: 381-91 (1984)].
In
RESULTS: Of the potential CTCF-binding sites tested, only two highly conserved CTCF sites within human and mouse c-myc gene exhibited CTCF-binding activity.
To determine the functional activity of the 1.6 kb MINE (SEQ ID NO:2) constituting the 5′boundary sequences of the human c-myc gene, it was placed at variable positions relative to enhancer and promoter elements within expression cassettes.
The following constructs were tested. All constructs contained the element scs′ to inhibit transcriptional interference originating from the integration site at the 3′end. Construct 1 representing E-P-neo-scs′ consists of: the TCRδ-enhancer, the Vδ-promoter, the neomycin resistance gene, and the Drosophila hsp70 scs′ boundary element, respectively. Construct 2 represents the P-neo-scs′ lacking the Eδ enhancer. Constructs 4, 7, and 8 have the 1.6 kb MINE fragment (SEQ ID NO:2) or the 1.2 kb chicken β-globin insulator cHS4 (SEQ ID NO:13) positioned upstream of the enhancer. Constructs 3, 5, and 6 have the 1.6 kb MINE fragment or the chicken β-globin insulator cHS4 positioned between the enhancer and promoter. The MINE fragment consists of the 1.0 kb BE-fragment and the 640 bp CTCF-fragment (method of construction described below).
RESULTS: At least two functional activities, the enhancer-blocking activity and the barrier activity, are conferred by MINE.
When the 1.6 kb MINE fragment derived from the c-myc 5′boundary (−1.7 kb to −3.3 kb relative to exon 1) was tested under parallel conditions, its ability to modulate the transcription of the reporter gene was analogous to the activity of the HS4 insulator element. When placed between Eδ and Vδ (comparing Constructs 5 and 1), MINE inhibited the enhancer activity by two-fold. When positioned upstream from Eδ (Constructs 7 and 1), no effect was observed.
The orientation specificity of MINE was tested by inserting it between the enhancer and promoter elements in the reverse orientation, into the E-P-neo-scs′ construct (Construct 6). Unexpectedly, MINE blocked enhancer-promoter interaction more efficiently in the 3′ to 5′ orientation (comparing Constructs 6 and 5) than it does in the alternative, 5′ to 3′ orientation, resulting in seven-fold reduction of G418 resistant colonies, equivalent to the number of colonies obtained after the deletion of the enhancer (comparing Constructs 6 and 2). In contrast, MINE increased the number of G418 resistant colonies by three-fold when positioned in the reversed orientation upstream of the enhancer, suggesting that it provides protection against position-effect variegation (comparing Constructs 8 and 7). Thus, these set of experiments show that MINE provides at least two functional activities such as the enhancer-blocking activity and the barrier activity, both of which are integral to the proper functioning of the 5boundary of c-myc.
DNA CONSTRUCTS: E-P-neo-scs′ was supplied by M. Krangel [Zhong and Krangel, Proc. Natl. Acad. Sci. USA 94: 5219-24 (1997)]. The “E”, “P”, “neo”, and “scs′” elements represent the TCRδ-enhancer, the Vδ-promoter, the neomycin resistance gene, and the Drosophila hsp70 scs′ boundary element, respectively. The P-neo-scs′ construct lacking the Eδ enhancer was generated by Xba1/Cla1digestion, treatment with T4 polymerase, and re-ligation. All boundary and control fragments were introduced into the NotI and/or XbaI sites upstream of Eδ, or the ClaI and/or SalI sites between Eδ and the Vδ promoter. The derivative construct E-MINE-P-neo-scs′ was generated by triple ligation of PCR-generated 1.0 kb ClaI/HindIII BE-fragment (as provided below) and the 640 bp HindIII/SalI CTCF into ClaI-SalI sites between Eδ and the Vδ promoter. The 1.0 kb BE fragment from the c-myc locus was generated by PCR amplification of cosmid DNA using forward primer (5′-3′; CTATGAGATCGATGTGGACC)(SEQ ID NO:113) and reverse primer (5′-3′; GCTAATGAAAGTCGAACTATGG) (SEQ ID NO:114). The 640 bp CTCF was generated by PCR amplification of cosmid DNA using forward primer (5′-GAGTTTCATCGATGTGGGGG-3′) (SEQ ID NO:115) and reverse primer (5′-GGGGTGCGTCGACAGCATGT-3′) (SEQ ID NO:116). All constructs were verified by sequencing.
THE SOFT AGAR COLONY-FORMING ASSAY: Jurkat cells (107 cells) were electroporated with 1.5 pmol (˜5.0-10.0 μg) of linearized plasmid using a BTX-Electroporator (Hercules, Calif.) at 250 V, 1700 μF, RF 72 (time constant ˜40 msec). After the electroporation, cells were placed on ice for 20-30 min. The cell suspension was then transferred into 10 ml of RPMI 1640/10% fetal bovine serum and cultured at 37° C. After 48 h, 5×105 cells were resuspended in a 7 ml RPMI plating medium (11% FBS, 31% Jurkat-conditioned medium, 50 μl Penicillin/Streptomycin (78 μg/ml), 100 μl L-glutamine (78 U/ml) and 750 μg/ml active G418 (Life Technologies, Gaithersburg, Md.) in RPMI. After addition of 3 ml of soft agar plating medium [1:1 volumes of 0.2% agar (Difco) and 2×RPMI 1640 medium], cells were plated and incubated for three to four weeks. Transfections of Jurkat cells with each construct were performed in triplicate.
Example 4 Identification of Enhancer-Blocking Activity and Silencing Activity within MINE To functionally define the components of MINE within the 1.6 kb fragment, subfragments of MINE were individually tested using the colony assay (Example 3). In
RESULTS: The enhancer-blocking activity of MINE (demonstrated by Example 3) is attributed to a 40 bp fragment containing the constitutive 5′CTCF-binding site (SEQ ID NO: 15) derived from sequences 2 kb upstream of the c-myc gene.
Sequences within the 1.6 kb MINE flanking the 40 bp CTCF element confer barrier and silencing activities. The fact that the 120 bp (
To confirm the loss of CTCF-binding in the mutated 640 bp fragment, chromatin derived from Jurkat cells that were transfected with constructs containing the 640 bp fragment or the 640 bp ACTCF fragment were immunoprecipitated using anti-CTCF antibody. Primers specific for the DM1 locus [Filippova et al., Nat. Genet. 28: 33543 (2001)] were used as a positive control. Primers specific for 5′CTCF-binding site within the stably integrated transgene do not detect sequences of the endogenous c-myc gene.
To determine the function of BE (SEQ ID NO:3) located upstream from the CTCF fragment (640 bp) positioned at the 5′ boundary of the c-myc locus, the 1 kb BE fragment (
In summary, the data in
To identify regions of the c-myc locus that associate in vivo with the nuclear matrix, sub-regions of DNA spanning a stretch of approximately 200 kb across c-myc were assayed by duplex PCR as described below. Jurkat cell nuclei were subjected to high-salt extraction procedures to remove non-scaffolding proteins (i.e., proteins not associated with the nuclear matrix), and were subjected to digestions with specific restriction enzymes. The DNA contained in the supernatant fraction representing fragments that did not associate with the matrix were separated from the non-soluble DNA (matrix-bound) in the pellet (P) by centrifugation, and their distribution was quantitatively determined by duplex PCR as described below. As positive control, a primer set specific for a known matrix attached region previously characterized at the apo B gene was used. Specific primer sets (provided below) were used to perform this analysis on a total of 68 genomic DNA fragments generated by the digestion with restriction enzymes HindIII/XbaI/EcoR1/ClaI (
RESULTS:
IN VIVO MAR AND DUPLEX PCR ASSAYS: Nuclei from Jurkat and HL60 cells was isolated according to Izaurralde et al., J. Mol. Biol. 200: 111-25 (1988) with some modification, and were kept at −20° C. in storage buffer (5 mM Tris.Cl pH 7.5, 20 mM KCl, 125 μM Spermidine, 50 μM Spermine, 500 μM EDTA, 1% v/v Thioglycol, 50% v/v glycerol) at a density of ˜108 nuclei/ml. Matrices were stably stored in 50% glycerol at −20° C. for at least six months. To isolate matrix-bound (P, pellet) or soluble DNA (S, supernatant), nuclei from Jurkat T cells were subjected to a lithium 3,5-diiodosalicylate (LIS) extraction protocol. Nuclei of ˜107 matrices (1 OD260 unit) were used per assay. Non-scaffold proteins were extracted by the addition of 1.0 ml 10 mM LIS (Sigma D-3635) extraction buffer, the salt-extracted nuclei were collected and subjected to restriction enzyme digestion with EcoRI, HindIII, Xba I and Cla I (˜500-1000 U/sample) and/or with the addition of Eco1CR1 (Promega). After proteinase K treatment, DNA samples were then subjected to phenol/chloroform extraction and ethanol precipitation.
To quantify the relative distribution of specific DNA sequences in the P and S fractions, duplex PCR assay was performed. Two nanograms of each P and S fractions were used as templates for amplification with primer pairs corresponding to each restriction fragment (generated by Hind III, Xba I, Cla I, Eco R1 and Eco1CR1) across ˜200 kb of the c-myc locus. The PCR reactions contained a primer set specific for the MAR at the apo B gene as a positive control (forward primer: 5′-GGGTGAATGAATGCCCTATG-3′ (SEQ ID NO:117); reverse primer: 5′-TACTCCATGCGAGGTCCACT-3′(SEQ ID NO:118). Results were expressed as a percentage of the signal from the matrix bound portion (T) or the supernatant portion (S), divided by the signal from the supernatant and pellet together, IP/IT(100) and IS/IT(100).
PRIMERS FOR APO B: Forward primer was 5′-GGGTGAATGATGCCCTATG-3′ (SEQ ID NO:119) and reverse primer was 5′-TACTCCATGCGAGGTCCACT-3′ (SEQ ID NO:120). PRIMERS USED TO DEFINE MYC-5′MAR: Primers sets are provided in Table 3.
PRIMERS USED TO DEFINE MYC-3′MAR: Primers sets are provided in Table 4.
It will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
Claims
1. An isolated nucleic acid molecule having one or more activities including an enhancer-blocking activity, an insulating activity, a silencing activity, and a barrier activity, the isolated nucleic acid derived from a c-myc locus of a mammal, and having at least 50% nucleotide identity with a corresponding subsequence of the c-myc locus, the nucleic acid molecule comprising one of:
- a MINE (c-myc Insulator Element) sequence;
- a MINE sequence fragment;
- a variant sequence; and
- a variant sequence fragment.
2. The nucleic acid molecule of claim 1 wherein the mammal is a human or a rodent.
3. The nucleic acid molecule of claim 1 wherein the MINE sequence is SEQ ID NO:1, a variant of SEQ ID NO:1, SEQ ID NO:9, or a variant of SEQ ID NO:9.
4. The nucleic acid molecule of claim 1 wherein the fragment of the MINE comprises a BE (barrier element).
5. The nucleic acid molecule of claim 4 wherein the BE has the sequence of: SEQ ID NO:3; a variant of SEQ ID NO:3; SEQ ID NO:10; or a variant of SEQ ID NO:10.
6. The nucleic acid molecule of claim 1 wherein the fragment of the MINE comprises a 5′CTCF-binding site.
7. The nucleic acid molecule of claim 6 wherein the 5′CTCF-binding site has the sequence of: SEQ ID NO: 15; a variant of SEQ ID NO: 15; SEQ ID NO:16; or a variant of SEQ ID NO: 16.
8. The nucleic acid molecule of claim 1 wherein the fragment comprises a first silencing element that suppresses an expression of an exogenous gene.
9. The nucleic acid molecule of claim 1 wherein the fragment comprises a second silencing element that suppresses an expression of an exogenous gene.
10. The nucleic acid molecule of claim 8 wherein the first silencing element has the sequence of: SEQ ID NO:4; a variant of SEQ ID NO:4; SEQ ID NO:207; or a variant of SEQ ID NO:207.
11. The nucleic acid molecule of claim 9 wherein the second silencing element has the sequence of: SEQ ID NO:5; a variant of SEQ ID NO:5; SEQ ID NO:208; or a variant of SEQ ID NO:208.
12. An isolated nucleic acid molecule that functions as a nuclear-matrix attachment region (“MAR”) when inserted into a host chromosome, the isolated nucleic acid derived from a c-myc locus of a mammal, and having at least 50% nucleotide identity with a corresponding subsequence of the c-myc locus, the nucleic acid molecule comprising one of:
- a MAR sequence;
- a MAR fragment;
- a variant sequence of MAR; and
- a variant fragment.
13. The nucleic acid molecule of claim 12 wherein the MAR is derived from a region 5′ of a human c-myc gene (MYC-5′MAR), and has the sequence of SEQ ID NO:6.
14. The nucleic acid molecule of claim 12 wherein the MAR is derived from a region 3′ of a human c-myc gene (MYC-3′MAR), and has the sequence of SEQ ID NO:7.
15. The nucleic acid molecule of claim 14 that comprises a sub-region of the MYC-3′MAR that has the sequence of SEQ ID NO:8, or that has at least 50% identity to SEQ ID NO:8.
16. The nucleic acid molecule of claim 12 wherein the MAR is derived from a region 5′ of a mouse c-myc gene (MYC-5′MAR), and has the sequence of SEQ ID NO:11.
17. The nucleic acid molecule of claim 12 wherein the MAR is derived from a region 3′ of a mouse c-myc gene (MYC-3′MAR), and has the sequence of SEQ ID NO:12.
18. The nucleic acid molecule of claim 17 that has at least 50% identity to SEQ ID NO: 12.
19. An expression vector comprising:
- a transgene that is operably linked to a promoter and an enhancer; and
- a MINE (c-Myc Insulator Element), or a MINE fragment, that is operably linked to the gene, the promoter, and the enhancer.
20. The expression vector of claim 19 wherein the components are positioned in a 5′-to-3′ direction in the following order: the MINE sequence or the MINE fragment, the enhancer, the promoter, and the gene of interest.
21. The expression vector of claim 19 wherein the MINE, or the MINE fragment, is positioned relative to the enhancer, the promoter, and the gene of interest, in an orientation that is either 5′-to-3′ direction, or 3′-to-5′ direction.
22. The expression vector of claim 19 wherein the MINE fragment comprises a BE.
23. The expression vector of claim 19 wherein the MINE fragment comprises a 5′CTCF-binding site.
24. The expression vector of claim 19 wherein vector includes a selectable marker.
25. The expression vector of claim 19 that further comprises a matrix attachment region (MAR) derived from any gene locus of any eukaryotic organism, and wherein the MAR is positioned 5′ (upstream) of a MINE or positioned 3′ (downstream) of the gene of interest.
26. The expression vector of claim 25 wherein the MAR is derived from the 5′ region of a c-myc gene (MYC-5′MAR), or the 3′ region of a c-myc gene (MYC-3′MAR).
27. An artificial chromosome comprising the vector of claim 19.
28. An eukaryotic host cell comprising the vector of claim 19.
29. An eukaryotic host cell comprising the artificial chromosome of claim 27.
30. A non-human transgenic animal comprising the vector of claim 19.
31. A transgenic plant comprising the vector of claim 19.
32. A pharmaceutical composition comprising:
- the vector of claim 19; and
- a pharmaceutical carrier.
33. A method for decreasing a position-effect variegation effect on an expression level of an exogenous gene, the method comprising:
- introducing the vector of claim 19 into a eukaryotic host cell; and
- permitting the vector to randomly integrate into heterochromatin of the host cell, wherein a MINE, a MINE fragment, or a MINE variant within the vector inhibits the progression of the heterochromatin at an integration site.
34. The method of 33 wherein the sequence comprises the one or more of the following sequences, or variants of the sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; and SEQ ID NO:12.
35. A method for decreasing a position-effect variegation effect on an expression level of an exogenous gene, which is expressed to treat a diseased eukaryotic host animal, the method comprising:
- administering a pharmaceutical composition comprising the vector of claim 19 into the diseased host;
- permitting the vector to enter a eukaryotic cell of the diseased host;
- permitting the vector to randomly integrate into an heterochromatin genome of the host cell; and
- enabling a sequence within the vector to inhibit the progression of the heterochromatin at an integration site.
36. The method of claim 35 wherein the eukaryotic host contains a mutant allele the expression of which causes a diseased phenotype that is amenable to treatment by expression of a transgene comprising a wildtype allele.
Type: Application
Filed: Dec 10, 2004
Publication Date: Dec 22, 2005
Inventors: Anton Krumm (Seattle, WA), Wendy Gombert (Bellevue, WA)
Application Number: 11/009,389
