Cardiac-cell specific enhancer elements and uses thereof

Details Cardiac-cell specific enhancer elements and uses thereof Cardiac-cell specific enhancer elements and uses thereof

2001-01-16

2004-11-16

Faulk, Anne-Marie (Department: 1636)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C435S320100

Reexamination Certificate

active

06818757

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to reagents and methods for expressing gene products in cardiac cells or precursors to cardiac cells in vitro and in vivo.
Adult mammalian cardiomyocytes do not de-differentiate or re-enter the cell cycle. After being placed into cell culture, neonatal cardiomyocytes soon lose their ability to proliferate. Several cell lines, including P19 teratocarcinoma cells, embryonic stem (ES) cells, AT-1, H9c2, QCE-6, or 10T1/2 cells, have some molecular characteristics of cardiomyocytes. These cells are very difficult to manipulate, however, or are lacking important characteristics of cardiomyocytes. Because of these reasons, cultured neonatal cardiomyocytes from rats or mice are often used in in vitro systems, even though these cells are difficult to transfect (usually less than 0.1% transfection rate) and require long preparation procedures.
Recently, it has been demonstrated that immortalized cardiac myogenic (CMG) cells can be differentiated from mouse bone marrow stromal cells. This is evidence of the generation of cardiomyocytes from a tissue of extra-cardiac origin.
The possibility of bone marrow being an in vivo source of circulating cardiomyocyte progenitors has been previously suggested. A distribution of transplanted bone marrow-derived cells in a dystrophic mouse heart has been observed. Although the molecular characteristics of these cells were not identified, their location in the heart tissue indicated these cells were cardiomyocytes. Taken together, it appears that bone marrow stromal cells are an extra-cardiac source of cardiomyocytes in vivo, and in vitro induction of beating cardiomyocytes from a heterogeneous population of bone marrow cells is possible by the introduction of inductive agents such as 5-azacytidine.
The molecular mechanisms which guide development of cardiac cells (and the heart in general) in vertebrates have been the subject of intense investigation (Fishman and Chien, Cell 91: 153-156, 1997; Olson and Srivastava, Science 272: 671-676, 1996). In most vertebrates, the heart tissue initially develops as a crescent shaped mesodermal structure located anteriorly and laterally. This precardiac mesoderm is brought ventrally and caudally, by folding of the embryo, to a form a single midline heart tube with the inflow region located most rostrally. This heart tube undergoes looping, bringing the inflow, ventricular, and outflow regions of the heart into the alignment seen in the mature heart. Later, chamber septation occurs, valves develop in the atrioventricular (AV) junction as well as in the outflow tract, and the outflow tract itself is divided into two great vessels. Additional refinements occur with the development of the coronary arteries and the cardiac conduction system.
Heart development is governed by complex signals including inductive and positional signals from adjacent structures, as well as signals from a number of transcription factors (Fishman and Chien, Cell 91: 153-156, 1997; Lyons, Curr. Opin. Genet. Dev. 6: 454-460, 1996; Mohun and Sparrow, Curr. Opin. Genet. Dev. 7: 628-633, 1997; Olson and Srivastava, Science 272: 671-676, 1996). Since transcriptional factors have the ability to activate multiple genes, they are generally considered important regulators of organ development. A number of cardiac transcription factors have been identified that have important influences on the early stages of specification and differentiation of the cardiac mesoderm (Tanaka et al., Dev. Genet. 22: 239-249, 1998). Csx/Nkx2.5 (Komuro and Izumo, Proc. Natl. Acad. Sci. USA 90: 8145-8149, 1993; Lints et al., Development 119: 419-431, 1993), MEF-2C (Edmondson et al., Development 120: 1251-1263, 1994), GATA4 (Heikinheimo et al., Dev. Biol. 164: 361-373, 1994; Kelley et al., Development 118: 817-827, 1993) and dHAND and eHAND are members of four different classes of transcriptional factors all expressed in the heart at early stages of development. Targeted disruption of any one of these genes yields severe cardiac and extracardiac phenotypes, and results in death of the embryo between E9.5 and E10.5 of development.
The mouse Csx/Nkx2.5 gene is first expressed in the cardiac progenitor cells at embryonic day 7.5 (E7.5), and during this stage is detected principally in the heart and tongue (and, to a lesser extent, in spleen, stomach, liver, and larynx). The extra-cardiac expression of Csx/Nkx2.5 is markedly reduced after birth, however, and in the adult, the expression is confined to the heart. Mice in which the Csx/Nkx.2-5 gene has been deleted have no functional heart, causing an embryonic lethality by E9.5-11.5.
Most tissue-specific gene expression is controlled by enhancer and repressor sequences at the transcriptional level. Generally, to confer tightly-regulated expression, enhancers adopt complex regulatory mechanisms that require the collaboration of multiple transcription factors. The binding sites for these transcription factors may be many kilobases (kb) from the gene promoter and dispersed relative to each other.
It is desirable to be able to express genes in a cardiac cell-specific manner. This would be useful, for example, for the targeted expression of genes encoding therapeutic proteins for the treatment of damaged heart tissue. Moreover, to maximize the utility of stem cell-derived cardiomyocytes, for example, in the treatment of damaged heart tissue in humans and other animals, it is desirable to be able to rapidly purify cardiac cells from a potentially heterogenous cell population.
Accordingly, there is a need for the development of reagents and methods for achieving cardiac cell-specific gene expression. The present invention provides these reagents and methods.
SUMMARY OF THE INVENTION
In a first aspect, the invention features a substantially purified nucleic acid molecule comprising an enhancer element having: (a) 100% identity to 40 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1 or SEQ ID NO.: 3; (b) at least 91% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 2; (c) at least 97% identity to 60 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1 or SEQ ID NO.: 3; or (d) at least 95% identity to 70 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1 or SEQ ID NO.: 3.
In a second related aspect, the invention features a substantially purified nucleic acid molecule comprising a cardiac-specific enhancer element derived from a human, wherein the enhancer element has at least 60% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1, SEQ ID NO.: 2, or SEQ ID NO.: 3. Preferably, the element has at least 70% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1, SEQ ID NO.: 2, or SEQ ID NO.: 3. More preferably, the identity is at least 80%, and most preferably, the identity is at least 90%, when compared to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1, SEQ ID NO.: 2, or SEQ ID NO.: 3.
Preferably, when expressed in vivo, the enhancer element is active in all four cardiac chambers. The enhancer element of the first or second aspect may be naturally occurring, or it may be non-naturally occurring.
Preferably, the enhancer element of the first or second aspect includes a binding site selected from the group consisting of Mef2, dHAND, GATA, TGF-&bgr;, CarG, E-box, and Csx/Nkx2.5 binding sites. More preferably, the enhancer element includes at least two binding sites selected from this group. The enhancer element preferably also includes an Sp-1 binding site.
In a third aspect, the invention also features a substantially purified non-naturally occurring nucleic acid molecule that includes at least three transcription factor binding sites selected from Mef2, dHAND, GATA, TGF-&bgr;, CarG, E-box, and Csx/Nkx2.5 binding sites. More preferably, the nucleic acid molecule includes four transcription factor binding sites, and most preferably includes five transcription factor binding sites selec

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