Patent 6,828,144

A method of fetal gene therapy is disclosed. In general, the method comprises the steps of identifying a fetus with a genetic defect, obtaining allantois/umbilical cord cells expressing a gene product that ameliorates the genetic defect, and exposing the fetus to the allantois/umbilical cord cells wherein a chimeric allantois is capable of supplying the gene product to the fetus is created. The present invention is also a method of examining the effect of test compounds on vasculogenesis and angiogenesis by observing the effect of the test compound on cultured allantoic explants.

Description of the Invention

BACKGROUND OF THE INVENTION

The major vascular systems of the developing fetus are formed by vasculogenesis, a developmental process in which mesoderm is transformed in situ into endothelial cells. The goal of my work is to discover how mesoderm is transformed into the endothelial cell lineage using the mouse allantois as a model in vitro system.

During its early development, the murine allantois consists of an inner core of mesoderm and an outer layer of squamous epithelium referred to as a mesothelium. The allantois undergoes two major developmental processes:

(i) maturation and fusion with the chorion to become the umbilical component of the chorioallantoic placenta, and

(ii) vascularization, forming an artery and a vein that permit within the chorionic disk the exchange of nutrients, metabolic wastes and gases with the mother during fetal gestation (K. M. Downs and R. L. Gardner, Development 121:407-416, 1995; K. M. Downs and C. Harmann, Development 124:2769-2780, 1997; K. M. Downs, et al., The Murine Allantois. In Current Topics in Developmental Biology (eds. R. Pedersen and G. Schatten). New York: Academic Press. 39:1-33, 1998; K. M. Downs, supra, 1998).

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method for evaluating the effect of test compounds, preferably potentially harmful or beneficial substances, on formation of blood vessels during vasculogenesis. In one embodiment, this method comprises direct application of a test compound to cultured allantoic explants. In another embodiment, one would evaluate test compounds by DNA uptake and expression of a test compound by the mesenchymal cells of allantoic explants.

In another embodiment, the present invention is the delivery of factors into the umbilical circulation by formation of chimeric allantoises. Preferably, this delivery ameliorates or eliminates developmental defects through delivery of therapeutic factors.

In another embodiment, the present invention is the delivery of gene expression products into the umbilical circulation by formation of a chimeric allantois. This method comprises the step of transfection of mesenchymal cells with heterologous genes expressed from appropriate endothelial cell promoters and introducing the donor mesenchymal cells into a developing allantois. Once integrated into the vasculature, the cells will express the gene product and deliver it to the bloodstream of the fetus. Preferably, the gene product is a therapeutic protein targeted to a fetus with particular developmental defects.

In another embodiment, the present invention is a method of fetal gene therapy comprising the step of creating a chimeric allantois/umbilical cord. Specifically, the method involves identifying a fetus with a genetic defect and obtaining compatible allantois/umbilical cells capable of expressing a gene product that would ameliorate this defect. The cells are introduced into the exocelomic cavity of the defective embryo or transplanted into the conceptus and assimilated into the native allantois/umbilical cord. Thus, a chimeric allantois/umbilical cord is produced. The gene product is then delivered into the fetus via the umbilical blood vessels. Preferably, the fetus is a mammalian fetus. Most preferably, the fetus is a human fetus.

In another embodiment, the present invention is a method of delivering a heterologous protein to a fetus via obtaining compatible allantois/umbilical cells capable of expressing a gene product and introducing these cells into the exocelomic cavity of an embryo or transplanting the cells onto the embryo and assimilating them into the native allantois/umbilical cord. A chimeric allantois/umbilical cord is produced and the gene product is then delivered into the fetus via the umbilical blood vessels.

In another embodiment, the present invention is a method of fetal gene therapy comprising the step of creating a chimeric allantois/umbilical cord by obtaining allantois cells compatible with a fetus with a genetic defect that express a gene product that ameliorates the genetic defect. The cells are then transplanted into the fetus wherein the transplanted cells develop into endothelial cells which line the vasculature of the umbilical cord and release the gene product into the bloodstream of the fetus.

The present invention is also a population of transgenic allantois cells, wherein the transgene may be a therapeutic gene and/or a marker or reporter gene.

It is an object of the present invention to provide a method of human fetal gene therapy.

It is another object of the present invention to provide a method of human fetal gene therapy that would benefit continued therapy after birth because the supplemented gene product would be recognized as "self" by the adult immune system.

DETAILED DESCRIPTION OF THE INVENTION

1. Method of Delivering Therapeutic Molecules to a Fetus

The present invention is a method of delivering molecules, preferably therapeutic molecules, to a fetus. In one embodiment, this method begins with identifying a fetus with a genetic defect. Once one has identified this defect, one then must obtain compatible allantois cells capable of expressing a gene that would ameliorate this defect. (These cells could be recombinant cells expressing a foreign gene product or non-recombinant cells expressing native gene products.) The Examples below describe a proposed method of ameliorating hemophilia A in transgenic mice defective in production of Factor VIII with genetically-engineered cells which can assimilate into the allantois and/or umbilical cord to form a chimeric umbilical cord. Of course, other genetic defects and diseases are known to one of skill in the art of molecular biology.

We propose below to obtain compatible allantois or umbilical cord cell lines that will then be manipulated to express factor VIII. By "compatible," we mean cells that may be assimilated into the native allantois to make a chimeric allantois or umbilical cord. As described in the Examples, whole allantoises from appropriate conceptuses are removed with glass capillary pipettes and placed in tissue culture dishes. When the explants are fairly large, they will be disaggregated and passaged into cells to reach confluence. These cells will be subcloned, transfected and tested for the appropriate gene activity. Alternatively, one might infect explants rather than cell lines and subclone after infection.

The present invention is also a transgenic allantois cell. The transgene may be a therapeutic gene or a marker gene.

The Examples below demonstrate that cells taken from different areas of the allantois seem to have different developmental potentials.

In the allantois, the transplanted cells appeared as various cell types, including endothelial cells which would be expected to line the vasculature of the umbilical cord. Thus, these transplanted cells could potentially be useful for releasing the products of transfected genes into the bloodstream of a developing fetus. Genetic engineering of these allantoic cells could also be useful for studying the function of various genes in normal vasculogenesis and in malfunctions of vasculogenesis.

It may therefore be advantageous in some applications to obtain cells from only one portion of the allantois and manipulate and transplant these specially selected cells. For example, the examples below describe the appearance of transgenic endothelial cells after transplantation of transgenic allantois cells onto a non-transgenic fetus. Other experiments suggest to us that cells of the mid-region, as described below in the Examples, might be the most effective. Alternatively, the cells of the base of the allantois seem to be more pluripotent and these cells might be selected for other applications.

Additionally, the Examples below suggest that the location of the cell transplant can determine allantoic cell development. Other applications might require that the allantois cells be transplanted specifically to a particular location to achieve redirected allantoic development, according to the new host site. For example, allantoic cells from the appropriate allantoic region may be placed into the liver where the liver redirects allantoic cells to differentiate into the endothelium characteristic of the vasculature of liver cells.

In a particularly advantageous form of the present invention, one obtains allantois cells compatible with the fetus and transplants the allantois cells to the fetus wherein the transplanted cells develop into endothelial cells which line the vasculature of the umbilical cord and release the desired gene product into the bloodstream of the fetus.

It is useful in the Examples below to work with cells containing a marker gene, such as the LacZ gene. The presence of a marker gene enables one to easily monitor whether the foreign cells have integrated with the native allantois.

Once appropriate allantois cells have been produced, the cells will be injected into the exocelomic cavity of the defective embryo or transplanted directly onto a particular region of the fetus. Assimilation of the cells into the native allantois should commence and thus, delivery of factor VIII to the fetus. Preferably, the cells will be transplanted directly into the umbilical cord or into a particular region of the fetus.

For human gene therapy, allantois or fetal umbilical cord cells may be obtained from an aborted or miscarried fetus.

The present invention is also a method of fetal gene therapy comprising identifying a fetus with a genetic defect, and obtaining allantois or umbilical cord cells compatible with the fetus, wherein the cells express a gene product that ameliorates the genetic defect. These cells are also capable of colonizing a fetal organ. One then exposes the fetus to the cells. A chimeric fetal organ capable of supplying the gene product to the fetus is created. By "organ" we mean to include the blood circulatory system. Preferably, the fetal organ is a liver or aorta. Preferably, the method of exposure is microinjection of the cells into the fetal organ.

2. In Vitro System of Vasculogenesis

We have discovered, as demonstrated below in Section II, that the allantois vascularizes by vasculogenesis that is not accompanied by erythropoiesis. Transformation of core allantoic mesoderm into the endothelial cells of blood vessels initially occurs along a spatial gradient, with the cells more distal to the embryo farther along in their developmental program than those more proximal (K. M. Downs and C. Harmann, supra, 1997; K. M. Downs, et al., supra, 1998). How core allantoic mesoderm differentiates is not known, though results of our studies strongly implicate allantoic mesothelium as a key player in this transformation, because mesothelial cell formation coincides with, or possibly precedes vascularization in core mesoderm.

Moreover, Flk-1, an early marker of endothelial cells, is expressed only in core mesoderm, whereas Flk-1's ligand, vascular endothelial growth factor (VEGF), is expressed in the overlying mesothelium, invoking a paracrine system of differentiation between mesothelium and core mesoderm.

Therefore, in one embodiment, the present invention is a method for evaluating the effect of test compounds, preferably potentially harmful or beneficial substances, on formation of blood vessels during vasculogenesis. This method may comprise direct application of a test compound to cultured allantoic explants, as described in Section II and U.S. Ser. No. 08/838,384. In another embodiment, one would evaluate test compounds by DNA uptake and expression of a test compound by the mesenchymal cells of allantoic explants.

Inhibitors of vasculogenesis are known to affect angiogenesis (see M. S. Pepper, et al., Cytokine and Growth Factor Reviews 8:21-43, 1997). Pepper, et al. show that transforming Growth Factor beta (TGFbeta) has different functions on vessel formation at different stages in the process and regulates formation of blood vessels (vasculogenesis) by potentiating or inhibiting the activity of positive regulators such as basic Fibroblast Growth Factor (bFGF) and Vascular Endothelial Growth Factor (VEGF) in a concentration-dependent manner. On the other hand, once a vessel has formed, TGFbetal promotes maintenance of endothelial cell quiescence and induces vessel maturation (both vasculogenesis and angiogenesis). Therefore, we expect that the test compounds found to modulate vasculogenesis will modulate angiogenesis as well.

Relevance of the Mouse Conceptus as a Model System for Development of the Chorioallantoic Placenta

Many eutherian mammals, including humans and rodents, survive and develop within the uterine environment through the formation of a chorio-allantoic placenta. Although fine details may vary, all placentae contain an umbilical circulation that consists of at least one major artery and vein. Without exception, these major blood vessels transport fetal blood to and from the chorionic disc for the exchange of nutrients, metabolic wastes and gases with the mother (H. W. Mossman, Vertebrate Fetal Membranes. MacMillan Press Ltd: Basingstoke, UK, 1987).

The mouse is an ideal model system for the study of umbilical development for several reasons. First, formation of the placenta occurs on schedule in whole embryo culture of living mouse conceptuses (K. M. Downs and R. L. Gardner, supra, 1995; K. M. Downs, et al., supra, 1998). Second, the allantois, precursor of the umbilical cord, is particularly amenable to manipulation in vitro and can be isolated free of contamination from the conceptus (K. M. Downs and R. L. Gardner, supra, 1995; K. M. Downs and C. Harmann, supra, 1997; K. M. Downs, et al., supra, 1998; reviewed in K. M. Downs, supra, 1998) Third, transgenic mouse technology has enabled the identification of genes involved in formation of the placenta, either because its two major components, the allantois and the chorion, do not unite in the mutant mice (G. C. Gurtner, et al., Genes and Dev. 9:1-14, 1995; L. Kwee, et al., Development 121:489-503, 1995; J. T. Yang, et al., Development 121:549-560, 1995) or because vasculogenesis has not occurred in the umbilicus (R. J. Akhurst, et al., Development 108:645-656, 1990; M. C. Dickson, et al., Development 121:1845-1854, 1995; F. Shalaby, et al., Nature 376:62-66, 1995). Thus, the mouse is an ideal system in which to elucidate the genetic control of major developmental processes. There exists no other mammalian model at this time that exhibits all of these significant strengths.

Description of the Technique of in vitro Vasculogenesis, Applications, and Preliminary Results

We have recently demonstrated that the murine allantois vascularizes via vasculogenesis, an intrinsic process in which mesoderm is transformed into the endothelial cell lineage in situ, rather than by angiogenesis, which is the extension of blood vessels formed elsewhere (K. M. Downs, et al., supra, 1998). Further, unlike all other vasculogenic systems heretofore described (reviewed in W. Risau and I. Flamme, Ann. Rev. Cell Dev. Biol. 11:73-91, 1995), allantoic vasculogenesis is not accompanied by erythropoiesis, i.e., formation of red blood cells.

In the course of our studies, we demonstrated that when allantoises are removed from headfold-stage conceptuses (approximately 8.0 days postcoitum) and cultured under relatively simple conditions in isolation, they rapidly undergo reproducible and stereotypic vasculogenesis (K. M. Downs, et al., supra, 1998). With feeding, the allantoic vasculature is maintained and remodeled for up to 3 days. The cultured explants consist of at least three cell lineages, endothelial, mesothelial, and mesenchymal, all of which are normally found in intact allantoises. Further, correct topographical relations between at least two of these lineages, the endothelial and mesothelial cells, are maintained in the explants. Moreover, cells from explanted cultured allantoises can be returned to developmentally-equivalent host allantoises where they correctly colonize appropriate cell types. Lastly, one of the explanted cell populations, the mesenchyrnal cells, can take up and express exogenous DNA. On the basis of our findings, we propose that the murine allantois will be a powerful and extremely valuable model system for at least two novel applications (Method 1 and Method 2), described below:

Method 1. Evaluation of Potentially Harmful/beneficial Substances on Formation of Blood Vessels During Vasculogenesis

In one embodiment of the present invention, allantoic explants will be used to evaluate the effect of potentially toxic environmental compounds and specific gene products on either abrogation or enhancement of blood vessel formation.

Two preferred methods are envisioned to achieve this goal: (a) direct application of a test compound to the cultured allantoic explants or (b) DNA uptake and expression of a test compound by the mesenchymal cells of allantoic explants. We envision that the allantoic explants will, preferably, be created as described below.

In general, allantoises are mouth-aspirated into a hand-pulled glass microcapillary (K. M. Downs and R. L. Gardner, supra; K. M. Downs, et al., supra, 1998) and either cultured in suspension (K. M. Downs, et al., supra, 1998) or placed individually either into wells of 24-well tissue-culture plats (Falcon 304), or onto glass cover slips (12 mm, Fisher) inserted into wells of 24-well tissue-culture plates and coated for 30 minutes in filtered (0.45 .mu.m, cellulose acetate) poly-D-lysine (1 mg/ml double processed tissue culture water; Sigma) and rinsed 3-4 times with sterile water.

Allantoises are then cultured in 0.5 ml of culture medium (Dulbecco's modified Eagle's medium (DMEM) containing either (i) equal parts of immediately-centrifuged and heat-inactivated rat serum as previously described (K. M. Downs and R. L. Gardner, supra, 1995), or (ii) varying concentrations of heat-inactivated rat or fetal calf serum (the latter obtained from Gibco-BRL; frozen and thawed twice before using). For culture longer than one day, allantoises are given completely fresh medium at 24-hour intervals.

At the onset of culture, or at varying times thereafter, known test compounds, such as Vascular Endothelial Growth Factor (VEGF, R & D Systems, Minneapolis, Minn.), are prepared according to the manufacturer's instructions and added to the cultures in concentrations recommended by the manufacturer (e.g., 1-20 ng VEGF/ml culture medium) or, in the case of unknown test compounds, in varying concentrations to determine the one that either inhibits or enhances vasculogenesis.

The advantage of technique (a) is that a controlled amount of substance can be delivered to the allantois at specific and different times in vascular development and its effect on vascularization assessed. Moreover, that compound can be removed at particular times in order to assess the long-term affect of the compound on the formation of blood vessels.

For technique (b), one would preferentially begin by culturing individually-plated headfold-stage allantoises for 12 or 18 hours in 24-well dishes before transfection, at which time 0.5 ml of heat- and gas-equilibrated DMEM containing 5% fetal calf serum (Gibco-BRL) replaces the culture medium. Allantoises are transfected using a standard CaHPO₄ method (F. L. Graham and A. J. van der Eb, Virology 54:536-539, 1973) for 6 hours in 5.0% CO₂ at 37^oC. by addition of 50 .mu. of precipitate containing, for example, 1 .mu.g of plasmid containing the test gene of interest whose expression is driven by the immediate early promoter of human cytomegalovirus (Clontech, Palo Alto, Calif.). Controls are explants cultured in either DMEM containing 50% rat serum or 5% FCS for the 6 hour transfection period. Following incubation, allantoises are washed once with warm PBS and returned to incubate for varying periods at 37^oC. in 6.2% CO₂ in DMEM containing 50% rat serum to assess the affect of the test compound on vascularization. Expression of the gene of interest is monitored by immunohistochemistry.

The advantage of technique (b) is that, for compounds with relatively short half-lives, the mesenchymal cells of allantoic explants can take up the gene whose product is of interest and express that product continuously in the culture to assess its affect on growth, development and/or maintenance of the vasculature.

Vascular Endothelial Growth Factor (VEGF) is expressed in the allantoic mesothelium (D. J. Dumont, et al., Dev. Dyn. 203:80-92, 1995) before spreading into the core (K. M. Downs, unpublished data). We have demonstrated that culture of allantoic explants in high rat serum (20-50% rat serum) is optimal for the formation of blood vessels. Culture of explants in low serum (fetal calf serum, FCS, 5-10%) favors formation of angioblasts, as revealed by expression of Flk-1 and Flt-1, early markers of angioblasts, but not their conversion into nascent blood vessels. Moreover, despite feeding, allantoises cultured in 5% FCS are typically devoid of vascular channels by 48 hours. By 72 hours, explants cultured in and fed 5% FCS at 24 hour intervals consist predominantly of mesenchymal cells. Increasing the concentration of FCS to 10-20% FCS results in partial maintenance of vascular channels for up to 72 hours, though significant breakdown of the channels is observed in about 87.5% of explants. Thus, a high concentration of some factor(s) must be required for both formation and maintenance of endothelial cells in allantoic explants. To test that possibility, recombinant VEGE (1-10 ng/ml culture medium) was added to explants at the start of culture in 5% FOS. Feeding at 24 hour intervals in the presence of Vascular Endothelial Growth Factor (2-10 ng/ml) resulted in formation of many vascular channels containing Flk-1 and Flt-1, and cell survival (78% cell retention compared with 36% in untreated explants) whereas untreated explants or those treated with 1 ng/ml of VEGF were devoid of such channels.

Together these findings suggest that varying the culture conditions of allantoic explants through serum starvation or enrichment varies the state of the endothelial cells, with low serum favoring formation of non-epithelialized angioblasts, and high serum favoring formation of endothelial channels. At least one of the key growth factors required in formation and maintenance of vascularity appears to be VEGF.

Method 2. Amelioration/elimination of Developmental Defects Through Delivery of Blood-borne Therapeutic Factors into the Umbilical Circulation by Formation of Chimeric Allantoises

In another embodiment, the present invention is the delivery of factors into the umbilical circulation by formation of a chimeric allantois. Preferably, this delivery results in a therapeutic effect or the amelioration or elimination of developmental defects.

I have recently proposed that cultured allantoic cells may be a valuable source of genetically-manipulable cells that could be re-introduced into developing allantoises where they would express a therapeutic gene and deliver it to the fetal bloodstream for the amelioration/cure of certain developmental defects (K. M. Downs, supra, 1998). We have recently demonstrated that cells of cultured explanted allantoises can be returned to the nascent umbilical cord where they integrate into all three allantoic cell lineages, endothelium, mesothelium and mesenchyme. At least 34% of injected cells colonize the host allantois. In addition, 5.5-6.0% of mesenchymal cells in allantoic explants are able to take up and express exogenously introduced DNA. Mesenchymal cells are thought to be relatively undifferentiated. Then, following transfection of mesenchymal cells with therapeutic genes expressed from appropriate endothelial cell promoters, we propose that donor mesenchymal cells will be introduced into the developing allantois of a developmentally-compromised fetus and, once integrated into the vasculature, they will express the therapeutic compound and deliver it directly into the bloodstream of the affected fetus.

The following is a preferred method of the present invention: Individually-plated headfold-stage allantoises are cultured for 12-18 hours in 24-well dishes before transfection at which time, 0.5 ml of heat- and gas-equilibrated DMEM containing 5% fetal calf serum (Gibco-BRL) replaces the culture medium. Allantoises are transfected using a standard CaHPO4 method (F. L. Graham and A. J. van der Eb, supra, 1973) for 6 hours in 5.0% CO₂ at 37^oC. by addition of 50 .mu.l of precipitate containing the plasmid of interest driven either by the immediate early promoter of human cytomegalovirus or the TIE1 endothelial cell specific promoter (Korhonen, et al., Blood 86:1828-1835, 1995; T. M. Schlaeger, et al., Development 11:1089-1098, 1995). Following incubation, allantoises are washed once with warm PBS and returned to incubate for 20-24 hours at 37^oC. in 6.2% CO₂ in DMEM containing 50% rat serum. Antibodies to the gene product of interest are applied to some of the cultures to ensure that transfection has taken place and that the gene of interest is being expressed.

Transfection is the process of macromolecule transfer to cells by physical or chemical means. One may decide to use various methods of transfection in the method of the present invention. For nearly twenty years, physical, chemical and viral-based methods have been widely available for introducing DNA into mammalian cells in culture. Physical methods may employ high-voltage electric pulses to create pores in membranes ("electroporation"; E. Neumann, et al., EMBO J. 1:841-845, 1982) or a gun to "shoot" genes into individual cells. Most commonly, chemical methods such as calcium phosphate or DEAE-dextran (or its analogues) are used as carrier to deliver DNA into cells (F. L. Graham and A. J. van der Eb, supra, 1973); alternatively, cationic liposomes containing DNA within them fuse with cell membranes to deliver DNA (J. P. Behr, et al., Proc. Natl. Acad. Sci. USA 86:6982, 1989; J. P. Loeffler, et al., J. Neurochem. 54:1812-1815, 1990; F. Barthel, et al., DNA Cell Biol. 12:553, 1993; J. S. Remy, et al., Bioconjugate Chem. 5:647-654, 1994). Virus-mediated transfer involves host-specific viruses that either replicate and express DNA in the cytoplasm as episomes (e.g., adenoviruses) or that integrate into the host's genome (e.g., retroviruses).

In the present invention, transfection by calcium phosphate and cationic liposomes (lipofection) will be the two methods of choice used to determine uptake and efficiency of DNA transfer to cultured allantoic cells. These are the most reliable, cost-efficient, and safe means by which to introduce DNA into mammalian cells. However, virus-based transfer is also suitable.

Calcium phosphate precipitation (F. L. Graham and A. J. van der Eb, supra, 1973; M. Wigler, et al., Cell 16:777-785, 1977) and lipofection (J. P. Behr, et al., supra, 1989; J. P. Loeffler, et al., supra, 1990; F. Barthel, et al., supra, 1993; J. S. Remy, et al., supra, 1994; Delaplace, 1991) have both been described and reliable and practical kits for both of these methods are available (e.g., ProfectionR Mammalian Transfection Systems and TfxTM products, Promega, Fitchburg, Wis.). Our overall strategy for transfection will involve a timecourse, i.e., application of the reporter gene between the time of removal of the allantois from the conceptus and up to 24 hours in culture.

Calcium phosphate-mediated transfection involves mixing DNA directly with CaCl₂ and a phosphate buffer to form a precipitate that is added to the cultured cells. This method achieves both transient and stable expression of DNA, the latter following integration of the transfected DNA into the host cell genome (M. Wigler, et al., Cell 16:777-785, 1979; M. Botchan, et al., Cell 20:143-152, 1980; S. Kato, et al., Mol. Cell. Biol. 6:1787-1795, 1986) or by autonomous replication in mini-chromosomal structures (D.H. Hamer, et al., Cell 17:725-735, 1979; D. DiMaio, et al., Proc. Natl. Acad. Sci. USA 79:4030-4034, 1982; R. Reeves, et al., Nucl. Acids Res. 13:3599-3615, 1985). As described above, allantoises will be removed and plated in individual wells of 24-well tissue culture dishes. One or more allantoises will be plated per well. Because CaP-mediated transfection requires that cells be 30-60% confluent, allantoises will be cultured for 12 hours, which is ample time for them to flatten out and spread somewhat on the bottom of the dish. Prior to transfection, the culture medium will be changed to medium containing 5% fetal calf serum, and the CaP/GFP complex added to the wells. The cells will be returned to the incubator and exposed to the precipitate for 6 hours, after which they will be washed with phosphate buffered saline (PBS, Sigma) and exposed to fresh media. They will then be re-fed every 24 hours up to the time of analysis, which will take place 36-54 hours after transfection (54-72 hours total time in culture) To increase the efficiency of transformation, some of the available "shock" methods, such as application of DMSO, will be applied to the cell cultures 14-16 hours after transfection and immediately removed and replaced with fresh medium.

Delivery of DNA into the nucleus of allantoic cells via lipofection involves close association of the cationic liposome-DNA complex with the cell membrane, followed by internalization of the complex into the cell, perhaps by fusion with the cell membrane and endocytosis (X. Gao and L. Huang, Gene Ther. 2:710-722, 1995; P. Hug and R. G. Sleight, Biochim. Biophys. Acta 1097:1-17, 1991). This method results in both transient and stable transfection. GFP-plasmid (as a control) and a plasmid of interest will be combined with lipofection reagents (e.g., TfxTM reagent, Promega, Fitchburg, Wis.) according to the manufacturer's instructions. The complexes will then be added to allantoic cell cultures at different timepoints following removal and plating of the allantois. We will do this in both the presence (10% FCS) and absence of serum. Lipofection in the absence of serum is more efficient, but this may of course be, cell line dependent. The advantage of this method of delivery is that two hours, instead of six, is all the time needed to expose cells to the exogenous DNA.

It is possible that chemical methods will not achieve the highest frequency of transfection and expression in whole allantoises. In that case, alternative strategies may be used, which employ viral-mediated DNA transfer. Infection with viruses would be preferred over microinjection of DNA into the nucleus because the latter method is labor intensive and can target only a small number of cells at a time. The general methodology would be similar to that described above for transfection. Allantoises would be plated and soon thereafter infected according to standard protocols with a high titer virus genetically engineered with a reporter gene, for example, GFP whose genetic size is suitable for most viral vectors. Scoring would be as described above, and infected cultures trypsinized and introduced into living embryos to assay for appropriate integration into the vasculature and gene expression.

Most viral vectors have common limitations. Among these are the size of the foreign gene that they may accommodate (maximal insert sizes for SV40 and retroviruses are 2.5 kb and 6 kb, respectively), and/or the fact that they may be subjected to rearrangement upon propagation of the viral stock, a serious consideration in the case of adenoviruses (H. Lochmuller, et al., Hum. Gene Ther. 5:1485-1491, 1994) and requiring therefore constant monitoring of viral stocks. Another limitation is the cytopathic effect of some viruses, particularly adenoviruses, on the host cell, which limits expression to a relatively short period of time. Finally, the variability in gene expression depends upon many parameters which are not completely clear. Among these are proper translation, processing, and modification of the resulting protein.

The viral vectors of choice will be adenovirus (Karpati, et al., 1996; Yeh and Perricaudet, 1997) and helper-virus-free retrovirus vectors (K. Shimotohno and H. M. Temin, Cell 26:66-77, 1981).

Significance and Summary

Use of the murine allantois as a model in vitro system of vasculogenesis will have enormous impact on the study of formation of blood vessels for several reasons. First, abnormalities during the development of two major vascular systems, the heart and circulation, are the leading cause of birth defects (March of Dimes Web Page, 1999). A valuable approach for elucidating the cellular and molecular mechanisms of endothelial cell formation would be the discovery of practical and reproducible in vitro systems of vasculogenesis. Embryonic stem cells from mice (W. Risau, et al., Development 102:471-478, 1988; R. Wang, et al., Development 114:303-316, 1992; R. L. Gendron, et al., Dev. Biol. 177:332-346, 1996; D. Vittet, et al., Blood 88:3424-3431, 1996) and avian epiblast (I. Flamme and W. Risau, supra, 1992; I. Flamme, et al., Anat. Rec. 237:49-57, 1993; K. Krah, et al., Dev. Biol. 164:123-132, 1994) have been used to this end. A major drawback to these is that differentiation of embryonic stem cells into the endothelial cell lineage is always accompanied by erythropoiesis. The presence of blood cells makes it difficult to determine which factors are essential for vasculogenesis alone. In addition, the types of cells present and their topographical relationships to each other vary from culture to culture, differentiation occurs over relatively long time periods, and the frequency of vasculogenesis/erythropoiesis is typically much lower than 100 percent. Pure cultures of endothelial cells have been isolated but, because most of them have been derived from adult organs (e.g., J. D. Rone and A. L. Goodman, Proc. Soc. Exp. Biol. Med. 184:495-503, 1987; L. C. Masek and J. W. Sweetenham, Brit. J. Haem. 88:855-865, 1994; K. Uchida, et al., Am. J. Physiol. 266:F81-88, 1994; G. Haraldsen, et al., Gut 37:225-234, 1995; T. Sakamoto, et al., Curr. Eye Res. 14:621-627, 1995; L. K. Christenson and R. L. Stouffer, Biol. Reprod. 55:1397-1404, 1996; Q. Yan, et al., Invest. Ophthal. Vis. Sci. 37:2185-2194, 1996), they offer little in the way of recapitulating the early steps of vasculogenesis. Second, although vasculogenesis and angiogenesis are typically described as distinct developmental process, it is not known in what way, if any, these processes involve different factors. Angiogenesis, which is the extension of blood vessels from elsewhere, is critical for the growth and metastatic spread of tumors. In the absence of a blood supply, tumor size remains fixed; without access to the vasculature, metastatic tumor cells are denied access to travel about the body. Vascularization of tumors involves the microvasculature, composed of endothelial cells. Comparison of the effect of particular proteins on the formation of endothelial cells via vasculogenesis in the allantoic explants with current models of endothelial cell formation via angiogenesis may lead to the identification of tumor-specific angiogenic proteins (reviewed in R. Auerbach, Int. J. Radiat. Biol. 60:1-10, 1991).

Lastly, the ability of allantoic cells to be genetically-manipulated and to colonize the developing allantois may prove therapeutically valuable for in utero gene therapy in cases where a blood-borne circulating factor might ameliorate or cure certain fetal defects

Claim 1 of 2 Claims

I claim:

1. A method of determining whether a compound alters the development of allantoic mesoderm into blood vessels in vitro comprising:

a) isolating a first and second allantoic tissue;

b) culturing the first and second allantoic tissues in vitro;

c) treating the first allantoic tissue with a compound, but not treating the second allantoic tissue with said compound; and

d) observing the development of allantoic mesoderm into blood vessels in the first and second allantoic tissues, wherein an alteration in the development of allantoic mesoderm into blood vessels in the first allantoic tissue as compared to the second allantoic tissue indicates the compound alters the development of allantoic mesoderm into blood vessels.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]