STEM CELL GENE TARGETING
The invention provides a method for generating a transgenic eukaryotic cell population having a modified human Rosa26 locus, which method includes introducing a functional DNA sequence into the human Rosa26 locus of starting eukaryotic cells. Also provided are targeting vectors useful in the method, as well as a cell population and a transgenic non-human animal comprising a modified human Rosa26 locus. Finally, the invention provides an isolated DNA sequence corresponding to the human Rosa26 locus.
This application claims priority of U.S. Application Ser. No. 60/881,226 filed Jan. 19, 2007, the disclosure of which is incorporated herein by reference.
GOVERNMENT SUPPORTThis invention was made with government support under grant numbers HL80627 and P20GM075019 awarded by the National Institutes of Health. The United States government has certain rights in this invention.
BACKGROUND OF THE INVENTIONThe derivation of human embryonic stem cells (hESCs) has opened new avenues for studies on human development and it also provides a potential source of cells for replacement therapy. For example, the ability to genetically alter hESCs offers unique opportunities to study the mechanisms regulating lineage commitment during human development, establish new approaches to identify and screen drugs, and develop in vitro models of human disease. The feasibility of this approach is dependent on the identification of a locus in the genome that is easy to access through targeting and one that will be permissive to expression of the introduced genetic material in the undifferentiated ES cells as well as in a broad range of differentiated cell types generated from these cells. A general review of this approach is provided in Yates et al., Gene Therapy (2006) vol. 13: 1431-1439.
Previous studies aimed at expressing genes in hESCs and derivative lineages have used either lentiviral vectors or transgenes that integrate randomly into the genome. These approaches are problematic for a number of reasons, e.g., a randomly integrated vector can activate or suppress expression of endogenous genes through insertional mutagenesis, the vectors are often present in multiple copies, and their expression is subject to silencing.
Homologous recombination in mouse embryonic stem cells has been used to produce mice carrying a single copy of the transgene integrated into a predetermined site of the genome (see e.g., Shaw-White et al., Transgenic Res.; (1):1-13 (1993); Bronson et al., Proc. Natl. Acad. Sci. USA, 93(17:9067-72 (1996); Hatada et al., J. Biol., Chem., 274(2):948-55 (1999); Tang et al., Genesis, 32(3):199-202 (2002)). In these studies, the ubiquitous Hprt locus was used with limited and unpredictable success. It would be desirable to define an autosomal locus that allows strong and predictable expression of transgenes inserted through homologous recombination, but is difficult to identify chromosomal loci that fulfill these criteria. Exogenous transgenes may not harbor all of the sequences necessary and sufficient for proper regulation of transcription and may therefore be influenced by cis-regulatory elements near the site of insertion.
In the mouse, a locus known as Rosa26 locus meets these criteria because it is expressed in ES cells and many derivative tissues both in vitro and in vivo and new genetic material can be easily introduced into it through homologous recombination. WO 99/53017 describes a process for making transgenic animals that ubiquitously express a heterologous gene, wherein the heterologous gene is under the control of a ubiquitously expressed endogenous promoter, e.g., that of the mouse Rosa26 locus. R. Dacquin et al., Dev. Dynamics 224:245-251 (2002) and K. A. Moses et al., Genesis 31:176-180 (2001) utilize the transgenic mouse strain R26R obtained according to WO 99/53017 for the expression of heterologous genes. WO 02/098217 describes a method of targeting promoter-less selection cassettes into transcriptionally active loci, such as the Rosa26 locus. WO 03/020743 describes the expression of transgenes in vivo by targeting protected transgene cassettes into predetermined loci (e.g. the Rosa26 locus), such that the introduced tissue specific exogenous promoter has at least some tissue specific activity.
US 2006/0205077 describes a method for targeted transgenesis using the mRosa26 locus. U.S. Pat. No. 6,461,864 also describes the use of the mRosa26 locus in the production of genetically engineered non-human animals that express a heterologous DNA segment.
SUMMARY OF THE INVENTIONThe present invention is based on the identification of the human Rosa26 (hRosa26) locus, which is capable of preserving the activity of heterologous promoters inserted through homologous recombination at the locus. Human Rosa26 is therefore useful for the efficient generation of transgenic animals, tissues and cell populations with a predictable transgene expression pattern.
Therefore, the present invention provides a method for generating a transgenic eukaryotic cell population having a modified human Rosa26 locus, which method comprises introducing a functional DNA sequence into the human Rosa26 locus of starting eukaryotic cells. In one embodiment, the functional DNA sequence is a gene expression cassette comprising a gene of interest operatively linked to a heterologous promoter; alternatively, the functional DNA sequence is a gene expression cassette comprising a gene of interest, wherein the DNA sequence becomes integrated into the locus by homologous recombination, thereby inserting the DNA sequence into the locus such that expression of the DNA sequence is under the control of the endogenous hRosa26 promoter.
In the method of the present invention, the functional DNA sequence is introduced into the eukaryotic cells by homologous recombination with a targeting vector comprising the functional DNA sequence flanked by DNA sequences homologous to the human Rosa26 locus. The eukaryotic cells are selected from the group consisting of primary cells and immortalized cells, and in a particular embodiment the eukaryotic cells are human embryonic stem (ES) cells.
The gene of interest may be any DNA sequence. A non-limiting list of genes that may be used in the method of the present invention includes recombinases, reporter genes, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, disease causing gene products and toxins, and mutations and combinations thereof.
In one embodiment, the functional DNA sequence is a gene expression cassette comprising a gene of interest operatively linked to a heterologous promoter, wherein the promoter is selected from the group consisting of a constitutive ubiquitous promoter, a constitutive tissue specific promoter, an inducible ubiquitous promoter and an inducible tissue specific promoter. A non-limiting list of suitable promoters includes CAGGS, hCMV, PGK, FABP, Lck, CamKII, CD19, Keratin, Albumin, aP2, Insulin, MCK, MyHC, WAP, Col2A, Mx, tet and Trex promoter.
The functional DNA sequence or gene expression cassette used in the method of the present invention further comprises one or more additional functional sequences selected from the group consisting of marker genes, one or more recombinase recognition sites which may be the same or different, poly A signal, introns, and combinations thereof. For example, the expression cassette comprises one or more functional sequences selected from the group consisting of a viral splice acceptor, a loxP-flanked promoterless neomycin resistance gene, an inverted RFP variant, mutant loxP2272 sites, and combinations thereof. In a particular embodiment, the expression cassette comprises the following elements in sequential order: (a) a viral splice acceptor, (b) a loxP-flanked promoterless neomycin resistance gene, and (c) an inverted RFP variant (tdRFP), wherein said inverted RFP variant is flanked by mutant loxP2272 sites. Specifically, the expression cassette comprises a DNA sequence coding for a Cre recombinase and said loxP and mutant loxP2272 sites are positioned such that following expression of Cre recombinase, the neomycin resistance cassette is removed and the tdRFP inverted, placing it under control of the endogenous hROSA26 promoter.
Still further, the targeting vector used in the invention further comprises functional sequences selected from the group consisting of tags for protein detection, enhancers, selection markers, and combinations thereof.
The transgenic eukaryotic cells are derived from human and the DNA sequences homologous to the human Rosa26 locus are derived from the 5’ and 3′ flanking arm of the human Rosa26 locus. In one embodiment, the targeting vector comprises a functional DNA sequence flanked by DNA sequence homologous with a human Rosa26 locus.
The invention also comprises a eukaryotic cell population comprising a modified human Rosa26 locus.
The invention additionally provides an isolated DNA sequence substantially homologous to a nucleotide sequence located between nucleotide positions 9′415′082 and 9′414′043 on chromosome 3. Alternatively, the invention provides an isolated DNA sequence substantially homologous to SEQ ID NO:2. Still further, the invention provides an isolated DNA sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:2.
Also provided is a transgenic non-human animal comprising a modified human Rosa26 locus.
human. Putative exons 1 and 2 were mapped according to the positions of electronically identified ESTs/transcripts.
The present invention provides the human homologue of the mouse Rosa26 locus and methods for site-specific integration of a transgene into the human Rosa26 locus. Therefore, the invention provides an isolated DNA sequence substantially homologous to a nucleotide sequence located between nucleotide positions 9′415′082 and 9′414′043 on chromosome 3. The invention further provides an isolated DNA sequence substantially homologous to SEQ ID NO: 2. And also provided is an isolated DNA sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:2.
The terms “substantially homologous”, “substantially corresponds to”, and “substantial identity” as used herein denotes a characteristic of a nucleic acid sequence such that a nucleic acid sequence has at least about 90% sequence identity, and most preferably at least about 95% sequence identity as compared to a reference sequence. The percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, the reference sequence is at least 18 nucleotides long, typically at least about 30 nucleotides long, and preferably at least about 50 to 100 nucleotides long.
“Substantially complementary” as used herein refers to a sequence that is complementary to a sequence that substantially corresponds to a reference sequence. In general, targeting efficiency increases with the length of the targeting transgene portion (i.e., homology region) that is substantially complementary to a reference sequence present in the target DNA (i.e., crossover target sequence). In general, targeting efficiency is optimized with the use of isogenic DNA homology clamps, although it is recognized that the presence of various recombinases may reduce the degree of sequence identity required for efficient recombination.
“Stringent conditions” refer to conditions under which a specific hybrid is formed. Generally, stringent conditions include conditions under which nucleic acid molecules having high homology, for example, preferably 90% or most preferably 95%, hybridize with each other. Alternatively, stringent conditions generally include conditions whereby nucleic acid molecules hybridize with each other at a salt concentration corresponding to a typical washing condition of Southern hybridization, i.e., approximately 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, at 60° C.
Any method known in the art may be used for the site-specific integration of a transgene using the human Rosa26 locus. Suitable, but non-limiting examples of various methods that may be used in connection with the hRosa26 locus are described in Yates et al., Gene Therapy (2006) vol. 13: 1431-1439; Shaw-White et al., Transgenic Res.; (1):1-13 (1993); Bronson et al., Proc. Natl. Acad. Sci. USA, 93(17:9067-72 (1996); Hatada et al., J. Biol., Chem., 274(2):948-55 (1999); Tang et al., Genesis, 32(3):199-202 (2002); WO 99/53017; R. Dacquin et al., Dev. Dynamics 224:245-251 (2002); K. A. Moses et al., Genesis 31:176-180 (2001); WO 03/020743; US 2006/0205077; and U.S. Pat. No. 6,461,864; the disclosures of which are incorporated herein by reference in their entirety.
For example, a targeting vector may be engineered for the site-specific integration of a transgene using the hRosa26 locus by methods known in the art. A targeting vector generally comprises a first sequence homologous to a portion or a region of a target gene sequence, i.e., the hRosa26 locus, and a second sequence homologous to a second portion or region of a target gene sequence, i.e., a second portion of the hRosa26 locus. The targeting vector may also include a selectable marker cassette that comprises a selectable marker gene. Preferably, the selectable marker cassette is positioned in between the first and the second sequence homologous to a region or portion of the target gene sequence. The selectable marker cassette may further comprise a sequence that initiates, directs, or mediates transcription of the selectable marker and the targeting vector also comprises a regulator that has the ability to control or regulate the expression of the selectable marker.
In one embodiment of the invention, the functional DNA sequence introduced into the hRosa26 locus is a gene expression cassette comprising a gene of interest operatively linked to a heterologous promoter. As used herein, the term “promoter”, generally refers to a regulatory region of DNA capable of initiating, directing and mediating the transcription of a nucleic acid sequence. Promoters may additionally comprise recognition sequences, such as upstream or downstream promoter or enhancer elements, which may influence the transcription rate.
In one embodiment of the present invention, a promoter may be used in the gene expression cassette (which is a heterologous promoter relative to the hRosa26 locus) that is a ubiquitous or tissue specific promoter, either constitutive or inducible. This ubiquitous promoter is selected from polymerases I, II and III dependent promoters, preferably is a polymerase II or III dependent promoter including, but not limited to, a CMV promoter, a CAGGS promoter, a snRNA promoter such as U6, a RNAse P RNA promoter such as H1, a tRNA promoter, a 7SL RNA promoter, a 5 S rRNA promoter, etc. Suitable examples of ubiquitous promoters are CAGGS, hCMV, PGK, and examples of tissue specific promoters are FABP (Saam & Gordon, J. Biol. Chem., 274:38071-38082 (1999)), Lck (Orban et al., Proc. Natl. Acad. Sci. USA, 89:6861-5 (1992)), CamKII (Tsien et al., Cell 87: 1317-1326 (1996)), CD19 (Rickert et al., Nucleic Acids Res. 25:1317-1318 (1997)); Keratin (Li et al., Development, 128:675-88 (201)), Albumin (Postic & Magnuson, Genesis, 26:149-150 (2000)), aP2(Barlow et al., Nucleic Acids Res., 25 (1997)), Insulin (Ray et al., Int. J. Pancreatol. 25:157-63 (1999)), MCK (Bruning et al., Molecular Cell 2:559-569 (1998)), MyHC (Agak et al., J. Clin. Invest., 100:169-179 (1997), WAP (Utomo et al., Nat. Biotechnol. 17:1091-1096 (1999)), Col2A (Ovchinnikov et al., Genesis, 26:145-146 (2000)); examples of inducible promoter sites are Mx (Kuhn et al. Scinence, 269: 1427-1429 (1995)), tet (Urlinger et al., Proc. Natl. Acad. Sci. USA, 97:7963-8 (2000)), Trex (Feng and Erikson, Human Gene Therapy, 10:419-27). Suitable inducible promoters are the above-mentioned promoters containing an operator sequence including, but not limited to, tet, Gal4, lac, etc.
Alternatively, as described hereinabove, the expression cassette may comprise a gene of interest and other integration elements such that when the DNA sequence is integrated into the locus by homologous recombination, expression of the DNA sequence is under control of the endogenous hRosa26 promoter. For example, the expression cassette may include a sequence flanked by a recombinase recognition site, e.g., loxP, and the cassette is engineered such that following expression of the recombinase, the functional DNA sequence is placed under the control of the endogenous hRosa26 promoter. In particular, the expression cassette may include a viral splice acceptor, followed by a loxP-flanked promoterless neomycin resistance gene, which is followed by an inverted RFP variant (a tandem-dimer RFP or tdRFP), flanked by mutant loxP2272 sites. As described above, the loxP and loxP2272 sites are positioned such that after Cre expression, the neomycin resistance cassette is removed and the tdRFP is inverted, thereby placing the cassette under the control of the endogenous hRosa26 promoter.
The targeting vector, functional DNA sequence or gene expression cassette may further comprise one or more additional sequences including but not limited to (selectable) marker genes (such as the neomycin phosphotransferase gene of E. coli transposon, etc.), recombinase recognition sites (which include loxP, FRT, variants thereof, etc.), poly A signals (such as synthetic polyadenylation sites, or the polyadenylation site of human growth hormones, etc.), splice acceptor sequences (such as a splice acceptor of adenovirus, etc.), introns, tags for protein detection, enhancers, selection markers, etc.
In a preferred embodiment, the targeting vector comprises a functional DNA sequence flanked by DNA sequences homologous to the human Rosa26 locus. Although the size of each flanking region is not critical and can range from as few as 100 base pairs to as many as 100 kb, preferably each flanking fragment is greater than about 1 kb in length, more preferably between about 1 and about 10 kb, and even more preferably between about 1 and about 5 kb. Although larger fragments may increase the number of homologous recombination events in ES cells, larger fragments will also be more difficult to clone.
In another embodiment, the method of the invention includes homologous recombination and the expression cassette is free of a transcriptional stop signal 5′ to the (heterologous) promoter of the cassette (i.e. is a non-protected cassette); and/or the exogenous promoter is a ubiquitous (constitutive or inducible) promoter.
The hRosa26 locus may be used for the site-specific integration of a transgene, wherein the transgene includes a gene of interest. As used herein, a transgene includes a gene or any DNA sequence that has been introduced into a targeting vector and ultimately into a different cell population or organism. This non-native segment of DNA may retain its original biological properties and functions, e.g., to produce RNA or protein, once transferred or introduced into the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code.
The gene of interest includes any gene or DNA sequence of natural or synthetic origin. A non-limiting list of genes that may be used in the method of the present invention is selected from the group of genes consisting of recombinases, reporter genes, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, disease causing gene products and toxins, and mutations and combinations thereof. The term “mutation” is understood to mean any changes introduced into the DNA sequence of a reference gene.
In one embodiment, the ES cell is a human ES cell. ES cells may be obtained commercially or isolated from blastocysts by methods known in the art, as described for example by U.S. Pat. No. 5,843,780; Thompson et al. (1998) Science 282:1145-1147; U.S. Pat. No. 6,492,575; Evans et al. (1981) Nature 292:154-156; and Reubinoff et al. (2000) Nature Biotech. 18:399. The method described herein may also be used to deliver a transgene to an adult, i.e. somatic, stem cell. Adult stem cells include, for example, hematopoietic stem cells, bone marrow stromal stem cells, adipose derived adult stem cells, olfactory adult stem cells, neuronal stem cells, skin stem cells, and so on. Adult stem cells have a similar ability as ES cells to give rise to many different cell types, but have the advantage that they can be harvested from an adult.
The undifferentiated ES cells are preferably maintained under conditions that allow maintenance of healthy colonies in an undifferentiated state. For example, human ES cells may be maintained on a feeder layer such as irradiated mouse embryonic fibroblasts in the presence of serum, or with serum replacement in the presence of bFGF, or in medium conditioned by mouse embryonic fibroblasts, or under serum free conditions using human feeder layers derived from, for example, human embryonic fibroblasts, fallopian tube epithelial cells or foreskin.
The method of the present invention results in site-specific integration of the transgene at the hRosa26 locus of the ES cell genome. The ES cells having the integrated transgene undergo normal embryoid body (EB) development and retain the capacity to differentiate into multiple cell types. Expression of the transgene is maintained throughout differentiation. Further, the ES cells having the integrated transgene maintain the capacity to generate cells of multiple lineages.
Stem cells having a transgene integrated therein as made by the method of the present invention are useful, inter alia, for generating transgenic non-human animals, for generating differentiated cells and tissues having a transgene integrated therein, for studying differentiation of stem cells, for evaluating strategies for safe and effective gene targeting in stem cells, and for targeted therapeutic gene transfer. Methods for generating differentiated cells from stem cells are known in the art. The model system for ES cell in vitro differentiation is based on the formation of three dimensional structures known as embryoid bodies (EBs) that contain developing cell populations presenting derivatives of all three germ layers and is disclosed in the art, for example by Keller (1995) Curr. Qpin. Cell Biol. 7:862-869.
For example in one embodiment, prior to differentiation, ES cells are removed from feeder cells prior to differentiation by subcloning the ES cells directly onto a gelatinized culture vessel. Twenty-four to 48 hours prior to the initiation of EB generation, ES cells are passaged into IMDM-ES. Following 1-2 days culture in this medium, cells are harvested and transferred into liquid medium (IMDM, 15% FBS, glutamine, transferrin, ascorbic acid, monothioglycerol and protein free hybridoma medium II) in Petri-grade dishes. Under these conditions, ES cells are unable to adhere to the surface of the culture dish, and will generate EBs.
Culture conditions are known in the art for the differentiation to cell types found in blood (Wiles et al. (1991) Development 111: 259-67), heart (Maltsev et al. (1993) Mech. Dev. 44:41-50), muscle (Rohwedel et al. (1994) Dev. Biol. 164:87-101), blood vessels (Yamashita et al. (2000) Nature 408:92-96), brain (Bain et al. (1995) Dev. Biol. 168:342), bone (Buttery et al. (2001) Tissue Eng. 7:89-99) and reproductive system (Toyooka et al. (2003) Proc. Natl. Acad. Sci. USA 100:11457-11462).
The differentiated cells and/or tissue generated therefrom may be introduced in an animal for therapeutic purposes. Accordingly, in another embodiment the present invention provides an animal comprising differentiated cells having a transgene integrated into the hRosa26 locus thereof, or comprising a tissue generated from such cells. In one embodiment the differentiated cell is a hemotopoietic cell, endothelial cell, cardiomyocyte, skeletal muscle cell or neuronal cell. The cells or tissues may be transplanted into the animal by methods known in the art.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated herein by reference in their entireties.
Claims
1-14. (canceled)
15. A targeting vector comprising an expression cassette comprising a nucleic acid encoding a protein, said expression cassette flanked by DNA sequences homologous to the human Rosa26 gene.
16. An ex vivo human cell population comprising an exogenous nucleic acid in the human Rosa26 gene.
17.-20. (canceled)
21. The targeting vector of claim 15 wherein the expression cassette further comprises a promoter operably linked to the nucleic acid encoding the protein.
22. The targeting vector of claim 21 wherein the promoter is selected from the group consisting of a constitutive ubiquitous promoter, a constitutive tissue specific promoter, an inducible ubiquitous promoter and an inducible tissue specific promoter.
23. The targeting vector of claim 21 wherein the promoter is heterologous to the human Rosa26 gene.
24. The targeting vector of claim 21 wherein the promoter is the endogenous human Rosa26 promoter.
25. The targeting vector of claim 15 wherein the sequences homologous to the human Rosa26 gene are derived from the 5′ and 3′ flanking arms of the human Rosa26 gene.
26. The targeting vector of claim 15 further comprising tags for protein detection, enhancers, selection markers, and combinations thereof.
27. The targeting vector of claim 15 wherein the nucleic acid encodes a recombinase or a reporter.
28. The targeting vector of claim 15 wherein the expression cassette further comprises a marker gene, one or more recombinase recognition sites, a poly A signal, an intron, or combinations thereof.
29. The targeting vector of claim 15 wherein the expression cassette further comprises a viral splice acceptor, a loxP-flanked promoterless neomycin resistance gene, an inverted RFP variant, loxP2272 sites, or combinations thereof.
30. The targeting vector of claim 15 wherein the expression cassette comprises the following elements in sequential order: (a) a viral splice acceptor, (b) a loxP site, (c) a promoterless neomycin resistance gene, (d) a loxP2272 site, (e) an inverted nucleic acid sequence encoding the protein, (f) a loxP site, and (g) a loxP2272 site.
Type: Application
Filed: Oct 21, 2016
Publication Date: Oct 19, 2017
Inventors: Gordon M. Keller (Toronto), Stefan Irion (Toronto), Herve Luche (Ulm), Paul Gadue (New York, NY), Hans J. Fehling (Ulm)
Application Number: 15/331,299