Patent Grant
9284575
Current work in synthetic biology is focused on modifying protein structure and function, and regulation of gene expression circuits at the level of transcription promoters (Kwok, 2010). While promoter control of expression may be sufficiently specific in bacterial systems, heterologous promoters confer only limited control of gene expression (e.g. cell-specific expression) in eukaryotic cells, possibly because of a lack of correct chromatin assembly (Wolffe, 1999). The limited control of gene expression in eukaryotic cells by heterologous promoters alone may be insufficient for certain therapies and other biotechnological applications. Complete spatiotemporal control of gene expression is important for the use of at least a subset of biological molecules in gene therapy and other biotechnological applications.
In some embodiments, synthetic regulatory constructs are provided. In some embodiments, a synthetic regulatory construct comprises a cell-specific promoter, a heterologous gene, and a cell-specific exon. In some embodiments, the cell-specific exon is excluded in the cell in which the cell-specific promoter is most active. In some embodiments, inclusion of the exon results in the product of the heterologous gene being inactive, or no product of the heterologous gene being produced. In some embodiments, inclusion of the exon results in a frame shift in the heterologous gene or a stop codon before or within the heterologous gene, or both.
In some embodiments, the cell-specific exon is included in the cell in which the cell-specific promoter is most active. In some embodiments, exclusion of the exon results in the product of the heterologous gene being inactive, or no product of the heterologous gene being produced. In some embodiments, exclusion of the exon results in a frame shift in the heterologous gene or a stop codon before or within the heterologous gene, or both.
In some embodiments, a synthetic regulatory construct comprises a cell-specific RNA stability element. In some such embodiments, the cell-specific RNA stability element is a microRNA target sequence.
In some embodiments, a cell-specific promoter is at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold more active in a selected cell than in at least one other cell comprised in the same organism as the selected cell. In some embodiments, a cell-specific promoter is at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold more active in a selected cell than in at least two, at least three, or at least five other cells comprised in the same organism as the selected cell. In some embodiments, a cell-specific promoter is at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold more active in a selected cell than in any other cell comprised in the same organism as the selected cell.
In some embodiments, a cell is an animal cell. In some embodiments, a cell is a mammalian cell. In some embodiments, the mammal is selected from human, mouse, rat, dog, chimpanzee, and monkey. In some embodiments, a cell is a plant cell. In some embodiments, a plant is selected from a monocot and a dicot. In some embodiments, a cell is a fungal cell.
In some embodiments, cells comprising synthetic regulatory constructs are provided. In some embodiments, a cell is an animal cell. In some embodiments, a cell is a mammalian cell. In some embodiments, a cell is comprised in a mammal. In some embodiments, a cell is ex vivo or in vitro. In some embodiments, a cell is a plant cell. In some embodiments, a cell is comprised in a plant. In some embodiments, a cell is a fungal cell.
In some embodiments, methods of expressing a heterologous gene in a selected cell are provided. In some embodiments, a method comprises introducing a synthetic regulatory construct into the selected cell. In some embodiments, a cell is an animal cell. In some embodiments, a cell is a mammalian cell. In some embodiments, the mammal is selected from human, mouse, rat, dog, chimpanzee, and monkey. In some embodiments, a selected cell is selected from mesenchymal, epithelial, neuronal, heart muscle, skeletal muscle, smooth muscle, and embryonic muscle cells. In some embodiments, a cell is comprised in a mammal. In some embodiments, a cell is a plant cell. In some embodiments, a cell is comprised in a plant. In some embodiments, a cell is a fungal cell.
The present inventors have developed a synthetic gene regulation system that provides better spatiotemporal regulation than promoters used alone. In some embodiments, the synthetic gene regulation system described herein provides better cell-specificity than cell-specific promoters used alone. In some embodiments, the synthetic gene regulation system described herein provides better temporal regulation than certain promoters used alone. In some embodiments, the synthetic gene regulation system may be used for cell-specific and/or temporal regulation of expression of various agents, including, but not limited to, therapeutic agents. Accordingly, in some embodiments, artificial control of the expression of biological molecules, including, but not limited to, therapeutic biological molecules, via combinations of cell-specific promoters, cell-specific exons and cell-specific RNA stability elements, is provided. In some embodiments, artificial control of the expression of biological molecules, including, but not limited to, therapeutic biological molecules, via combinations of temporally-regulated promoters, temporally-regulated exons and temporally-regulated RNA stability elements, is provided. In some embodiments, such modular regulatory elements may come from different genes or be artificial combinations of known sub-elements.
As described herein, post-transcriptional regulation offers additional control of gene expression, particularly in eukaryotic cells. For example, in some embodiments, alternative cassette exons may either be included or skipped in eukaryotic mRNA in a cell-specific manner (“cell-specific exons”). In some embodiments, alternative exon inclusion may disrupt the expression of, or change the function of, a gene product. Like transcription promoters, exons are recognized by regulatory macromolecular complexes in the cell. Further, this recognition may be modular: exons may be moved into heterologous contexts and still be recognized by the cell.
As a further example, in some embodiments, post-transcriptional regulation can determine the stability of mRNAs in a cell-specific manner (“cell-specific RNA stability elements”). In some embodiments, by affecting RNA stability, such post-transcriptional regulators may determine RNA levels. This regulation may also be accomplished by an independent set of macromolecular complexes. In some embodiments, regulation of RNA stability involves small non-coding RNAs known as microRNAs (miRNAs). In some embodiments, mRNAs may be regulated at the level of translation efficiency, for example, by proteins and miRNAs, which may exert their function via signals in the 5′ and/or 3′ untranslated regions (UTRs) of the messenger.
As discussed herein, in some embodiments, the various levels of regulation (including, but not limited to, transcription, alternative splicing, and RNA stability) are orthogonal and thus provide independent modes that in combination provide multiplied specificity. Thus, in some embodiments, by combining modular control elements from different genes, novel or artificially stringent patterns of gene expression can be engineered. Currently, such modular control does not appear to be appreciated in synthetic biology.
The subject matter disclosed herein is described using several definitions, as set forth below and throughout the application.
Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided below, it is to be understood that as used in the specification, embodiments, and in the claims, “a”, “an”, and “the” can mean one or more, depending upon the context in which it is used.
As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” or “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
As used herein, the term “cell-specific promoter” refers to a promoter that is at least 3-fold more active in a selected cell than in one or more other cells. In some embodiments, a cell specific promoter is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold more active in a selected cell than in one or more other cells. In some embodiments, a cell specific promoter is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold more active in a selected cell than in other cells found in the same organism as the selected cell. In some embodiments, a selected cell is a particular cell type.
As used herein, the term “cell-specific exon” refers to an exon that is present in a transcript or absent from a transcript through alternative splicing at a rate that is at least 3-fold greater in a selected cell than in one or more other cells. In some embodiments, a cell-specific exon is present in a transcript in a selected cell at a rate that is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is present in a transcript in one or more other cells. In some embodiments, a cell-specific exon is absent from a transcript in a selected cell at a rate that is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is absent from a transcript in one or more other cells. In some embodiments, a cell-specific exon is present in a transcript in a selected cell at a rate that is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is present in a transcript in in other cells found in the same organism as the selected cell. In some embodiments, a cell-specific exon is absent from a transcript in a selected cell at a rate that is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is absent from a transcript in other cells found in the same organism as the selected cell. In some embodiments, a selected cell is a particular cell type.
In some embodiments, the rate that an exon is present is determined as the ratio of spliced transcript with the exon present to spliced transcript with the exon absent in a particular cell. In some embodiments, the rate an exon is absent is determined as the ratio of spliced transcript with the exon absent to spliced transcript with the exon present in a particular cell. As a nonlimiting example, the following table shows hypothetical rates that a hypothetical cell-specific exon A is present in a transcript in hypothetical cells x and y:
As used herein, the terms “exclusion”, “excluded”, and similar terms, when used in relation to an exon, mean that the rate that an exon is included in a transcript, as described above, is less than 1. Alternatively, the terms “exclusion”, “excluded”, and similar terms, when used in relation to an exon, mean that the rate that an exon is excluded from a transcript, determined in a similar manner as described above for inclusion of an exon (although with the numerator and denominator reversed), is greater than 1.
As used herein, the terms “inclusion”, “included”, and similar terms, when used in relation to an exon, mean that the rate that an exon is included in a transcript, as described above, is greater than 1. Alternatively, the terms “inclusion”, “included”, and similar terms, when used in relation to an exon, mean that the rate that an exon is excluded from a transcript, determined in a similar manner as described above for inclusion of an exon (although with the numerator and denominator reversed), is less than 1.
As used herein, the term “heterologous gene” refers to any gene or coding sequence that is not controlled in its natural state (e.g., within a non-genetically modified cell) by the cell-specific promoter to which it is operably linked in a particular construct, and whose gene, in its natural state, does not contain the cell-specific exon included in the particular construct. In some embodiments, the gene or coding sequence is described as being heterologous to the cell-specific promoter and/or heterologous to the cell-specific exon.
As used herein, the terms “cell-specific RNA stability element” and “cell-specific RNA stability element” refer to a regulatory element, which may be in the 3′-untranslated region or 5′-untranslated region of a transcript, that increases the stability or translation of a transcript in a selected cell and/or decreases the stability or translation of the transcript in one or more cells other than the selected cell, such that the stability or translation of the transcript in the selected cell is at least 2-fold greater than the stability or translation of the transcript in one or more other cells. In some embodiments, the stability or translation of the transcript in the selected cell is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than in one or more other cells. In some embodiments, the stability or translation of the transcript in the selected cell is at least 2-fold, 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than in other cells found in the same organism as the selected cell. In some embodiments, a selected cell is a particular cell type. In some embodiments, an RNA stability element is an element that decreases the stability or translation of the transcript in one or more cells other than the selected cell. In some embodiments, a cell-specific RNA stability element is a microRNA target sequence. In some embodiments, a cell-specific RNA stability element is a binding site for an hnRNP protein. Nonlimiting exemplary hnRNPs include AUF-1, AUF-2, and HuR.
The term “microRNA target sequence” refers to a RNA stability element that comprises a seed match sequence for a particular microRNA. A seed match sequence is a sequence that is complementary to at least nucleotides 2 to 7 of the microRNA. In some embodiments, a microRNA target sequence comprises a sequence that is complementary to more of the microRNA than just nucleotides 2 to 7. In some embodiments, a microRNA target sequence decreases the stability or translation of a transcript in cells that express the particular microRNA.
Nonlimiting Exemplary Cell-Specific Promoters
Many cell-specific promoters are known in the art. Nonlimiting exemplary mammalian cell-specific promoters have been characterized and used in mice expressing Cre recombinase in a cell-specific manner. See, e.g., The Jackson Laboratory Mouse strain database which can be found at the website jaxmice.jax.org/list/xprs_creRT.html#xprs1801. Certain nonlimiting exemplary mammalian cell-specific promoters are listed in Table 1.
In some embodiments, a cell-specific promoter is a promoter that is active in plants. Many exemplary cell-specific plant promoters are known in the art. See, e.g., U.S. Pat. Nos. 5,097,025; 5,783,393; 5,880,330; 5,981,727; 7,557,264; 6,291,666; 7,132,526; and 7,323,622; and U.S. Publication Nos. 2010/0269226; 2007/0180580; 2005/0034192; and 2005/0086712, which are incorporated by reference herein in their entireties for any purpose.
Nonlimiting Exemplary Cell-Specific Exons
Many cell-specific exons are known in the art. Certain nonlimiting exemplary cell-specific exons are described in Table 2 and in the examples provided herein. The literature references provided in Table 2 are each incorporated by reference herein in their entireties for any purpose.
In some embodiments, a cell-specific exon is included in a selected cell. In some embodiments, the inclusion of the cell-specific exon allows for expression of an active product from a heterologous gene into which the exon is incorporated. In some embodiments, the inclusion of a cell-specific exon results in expression of an inactive product from a heterologous gene. In some embodiments, the inclusion of a cell-specific exon results in a decrease or elimination of expression of the heterologous gene product, for example, by inserting a stop codon upstream of the start codon of the heterologous gene and/or by frame-shifting the heterologous protein coding region.
In some embodiments, a cell-specific exon is excluded in a selected cell. In some embodiments, the exclusion of the cell-specific exon allows for expression of an active product from a heterologous gene into which the exon is incorporated. In some embodiments, the exclusion of a cell-specific exon results in expression of an inactive product from a heterologous gene. In some embodiments, the exclusion of a cell-specific exon results in a decrease or elimination of expression of the heterologous gene product, for example, by frame-shifting the heterologous gene coding region.
Nonlimiting Exemplary Cell-Specific RNA Stability Elements
Various cell-specific RNA stability elements are known in the art. In some embodiments, a cell-specific RNA stability element is a microRNA target site. Many cell-specific microRNAs are known in the art. Nonlimiting exemplary mammalian cell-specific microRNAs are shown in Table 3.
In some embodiments, a cell-specific microRNA is a plant microRNA.
Nonlimiting Exemplary Constructs
In some embodiments, a construct for synthetic regulation of gene expression is provided. In some such embodiments, the construct comprises a cell-specific promoter and a cell-specific exon. In some embodiments, the construct further comprises a cell-specific RNA stability element.
In some embodiments, a construct may further contain one or more additional elements that facilitate the propagation, use, and/or functioning of the construct, such as, without limitation, one or more coding sequences for selectable markers, one or more origins of replication, localization domains, etc. Elements for use in constructs for in vitro and in vivo gene expression are known in the art, and one skilled in the art can select suitable elements to include in a construct for synthetic regulation of gene expression described herein. In some embodiments, the selected elements facilitate the propagation, use, and/or functioning of a construct in a mammal. In some embodiments, the selected elements facilitate propagation, use, and/or functioning of a construct in a plant. In some embodiments, an element facilitates propagation of a construct in vitro, although the construct is intended for use in vivo.
Nonlimiting Exemplary Methods
In some embodiments, methods of synthetic regulation of gene expression in a cell, mammal, or plant are provided. In some embodiments, methods of cell-specific expression of a gene in a cell, mammal, or plant are provided. In some embodiments, a method comprises introducing into a cell, mammal, plant, or introducing into a selected cell in a mammal or plant, a construct comprising a cell-specific promoter, a heterologous gene, and a cell-specific exon, under conditions allowing expression of the heterologous gene in the selected cell. In some embodiments, a construct further comprises a cell-specific RNA stability element.
In some embodiments, the heterologous gene is expressed in the selected cell at levels at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is expressed in one or more other cells of the same organism. In some embodiments, the heterologous gene is expressed in the selected cell at levels at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is expressed in other cells of the organism. In some embodiments, the heterologous gene is expressed in a set of selected cells at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is expressed in other cells of the organism. A set of selected cells may be any combination of cells in which a particular construct will express a heterologous gene. Nonlimiting exemplary sets of cells include, but are not limited to, cells of the cortex, hippocampus, and cerebellum; epithelial cells located in various tissues (such as kidneys and mammary glands); and muscle cells located throughout the body (such as skeletal muscle).
In some embodiments, a method comprises gene therapy in a mammal. In some such embodiments, the method allows expression of a heterologous gene in a selected cell type, with little or no expression of the heterologous gene in one or more other cell types. In some embodiments, a method comprises creating a transgenic animal. In some embodiments, a method comprises creating a transgenic plant. In some such embodiments, the method allows expression of a heterologous gene in a selected cell in the plant, with little or no expression of the heterologous gene in one or more other cells in the plant. In some embodiments, a method comprises creating transgenic fungi, for example, for temporal control of gene expression.
In some embodiments, the heterologous gene is expressed at a higher level in a selected cell and/or is expressed at a lower level in one or more other cells, than it would be expressed if it were only under the control of a cell-specific promoter.
The following examples are illustrative and are not intended to limit the claimed and/or disclosed subject matter.
To demonstrate synthetic regulation of gene expression, a set of plasmids was designed that would provide differential expression of firefly luciferase in mesenchymal and epithelial cells. As shown in
Mesenchymal-like rat prostate cancer cells, AT3 cells, and epithelial-like rat prostate cancer cells, DT cells, were grown (separately) in 6-well plates overnight in low glucose DMEM. DT and AT3 cells are described, for example, in Tennant et al. (2000). Each type of cell was transfected with 50 ng of each of the plasmids shown in
The results of that experiment are shown in
A system was designed to provide synthetic regulation of Cre expression in mesenchymal and epithelial cells, which would lead to a color change from red to green predominantly in epithelial cells.
AT3 cells were transfected with 250 ng RG plasmid, 250 ng EcadCreIIIc plasmid, and 1.5 μg pUC19 as carrier DNA using lipofectamine (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. Dt cells were transfected with 100 ng RG plasmid, 100 ng EcadCreIIIc plasmid, and 1.8 μg pUC19 as carrier DNA also using lipofectamine according to the manufacturer's instructions. The cells were incubated at 37° C. overnight.
The fraction of EGFP-positive cells was then tracked over time during selection for stable transfectants using hygromycin (selecting for the RG plasmid) and blastocidin (selecting for the EcadCreIIIc plasmid). Those results are shown in
To determine whether the synthetic regulation systems discussed herein can be used to limit toxin expression to particular cell types, a plasmid was designed to express diphtheria toxin only in epithelial cells.
AT3 cells were transfected with 200 or 2,000 ng CMV-DipIIIc plasmid and 1.8 or 0 μg pUC19 as carrier DNA using lipofectamine (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. DT cells were transfected with 20 or 100 ng CMV-DipIIIc plasmid and 1.95 or 1.9 μg pUC19 as carrier DNA also using lipofectamine according to the manufacturer's instructions. The cells were incubated at 37° C. overnight with blasticidin selection, which selects for the CMV-DipIIIc plasmid. The presence or absence of the FGFR2 IIIc exon flanked by diphtheria toxin coding sequences was detected in mRNA isolated from the transfected cells using RT-PCR.
The results of that experiment are shown in
A mesenchymal-specific expression vector was created using a vimentin promoter and by including FGFR2 exons IIIb and IIIc. Exon IIIb is skipped in mesenchymal cells, while exon IIIc is retained. A diagram of the vector is shown in
AT3 cells grown for one day in 6-well plates were transfected with 250 ng VimIIIbIIIc plasmid and 1.75 μg pUC19 as carrier DNA using lipofectamine (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. Dt cells were transfected with 250 ng VimIIIbIIIc plasmid and 1.75 μg pUC19 as carrier DNA also using lipofectamine according to the manufacturer's instructions. The cells were incubated at 37° C. overnight in low glucose DMEM. RNA was extracted from the cells using RNeasy (Qiagen) and the various splice products amplified by RT-PCR as previously described. See, e.g., Baraniak et al., Mol. Cell Biol. 2006 February; 26(4):1209-22. The amplified products were then cleaved using AvaI or HincII, which cleave the four splice products in such a way that products of various sizes can be used to identify each of the four splice products. Uncut RT-PCT products separate into a longer band, which corresponds to inclusion of both exons IIIb and IIIc (“double inclusion”), and a shorter band, which corresponds to inclusion of either exon IIIb or IIIc (“single inclusion”). AvaI cleaves once in the IIIb exon. HincII cleaves twice in the IIIc exon. See, e.g., Carstens et al., Mol. Cell Biol., 20(19): 7388-7400 (2000), which is incorporated by reference herein in its entirety for any purpose.
The results of that experiment are shown in
This application claims the benefit of U.S. Provisional Application No. 61/607,312, filed Mar. 6, 2012, which is incorporated herein by reference in its entirety for any purpose.
This invention was made with government support under Federal Grant Nos. IF32CA142095 and R01CA127727, both awarded by the National Institutes of Health: National Cancer Institute. The government has certain rights in the invention.
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