Marcus loved his new job. Every fight he would pick up his weapon and fight hard and he would win. Nobody could stop this young man. He became so successful that he was given the opportunity to fight in one of the biggest, spectacular gladiatorial shows of all time. The fight was in a few days and word came out that Julius Caesar was going to be watching. So Marcus trained extra hard to win to impress the powerful dictator. As the fight begun Marcus drew his sword and fought for his life and many others as it was down to two people Marcus and the other gladiator, he sliced and stabbed until his opponent was dead.
Everyone cheered with joy. All comments are displayed anonymously Comments: If you think this story is one which should go in the Myths and Legends showcase, click "Yes". Initial management of acute coronary syndromes". J Am Coll Cardiol ; Accessed July 2, An Alliance for Quality". American College of Cardiology. Retrieved June 30, Archived from the original on September 28, Lifeline - a new plan to decrease deaths from major heart blockages," American Heart Association , May 31, Accessed July 3, Accessed June 16, C57BL 6 or Swiss mice or other comparable strains can be used for this purpose.
Recipient females are mated at the same time as donor females. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0. The oviducts are exposed by a single midline dorsal incision. An incision is then made through the body wall directly over the oviduct. The ovarian bursa is then torn with watchmakers forceps.
Embryos to be transferred are placed in DPBS Dulbecco's phosphate buffered saline and in the tip of a transfer pipet about 10 to 12 embryos.
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The pipet tip is inserted into the infundibulum and the embryos transferred. After the transfer, the incision is closed by two sutures. As noted above, transgenic animals and cell lines derived from such animals may find use in certain testing experiments. In this regard, transgenic animals and cell lines expressing MEF2 regulated indicator genes may be exposed to test substances.
These test substances can be screened for the ability to decrease MEF2 activity. Compounds identified by such procedures will be useful in the treatment of heart disease. The transgenic animals of the present invention include those which have a substantially increased probability of spontaneously developing cardiac hypertrophy, when compared with non-transgenic littermates. A "substantially increased" probability of spontaneously developing cardiac hypertrophy means that a statistically significant increase of measurable symptoms of cardiac hypertrophy is observed when comparing the transgenic animal with non-transgenic littermates.
In one embodiment, the transgenic animals of the present invention are produced with transgenes which comprise a coding region that encodes a gene product which modulates transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. As used herein, the term "hypertrophic signal" indicates any stimulus, mechanical or chemical, which results in measurable symptoms of cardiac hypertrophy. Hypertrophic signals include, but are not limited to. Symptoms of cardiac hypertrophy can be measured by various parameters including, but not limited to.
The transgenic mouse of the present invention has a variety of different uses. First, by creating an animal model in which MEF2 activity can be measured, the present inventors have provided a living "vessel" in which the function of MEF2 may be further dissected. In one particular scenario, the transgenic mouse may be used to elucidate the interactions of MEF2 with additional nuclear factors.
Thus, clearly, the present invention also encompasses isolation of a nuclear factors that act via an interaction with MEF2. Another use for the transgenic mouse of the present invention is in the in vivo identification of a modulator of MEF2 activity, and ultimately of cardiac hypertrophy. Treatment of a transgenic mouse with a putative MEF2 inhibitor, and comparison of the response of this treated mouse with the untreated transgenic animal, provides a means to evaluate the activity of the candidate inhibitor.
Though there have been reports that a Ca 44" mediated pathway is involved in certain heart disease, the present invention provides the first evidence of MEF2 as a central mediator of the hypertrophic response. Further it is demonstrated that the transcription activation domain of MEF2C is a nuclear target for hypertrophic signaling pathways. Thus, in a particular embodiment of the present invention, there are provided methods for the treatment of cardiac hypertrophy. These methods exploit the inventors' observation, described in detail below, that MEF2 appears to up-regulate the expression of genes involved in the hypertrophic response.
At its most basic, this embodiment will function by reducing the in vivo activity of MEF2 in individuals suspected of having undergone a hypertrophic response, currently undergoing a hypertrophic response, or in danger of cardiac hypertrophy. This may be accomplished by one of several different mechanisms. First, one may block the expression of the MEF2 protein. Second, one may directly block the function of the MEF2 protein by providing an agent that binds to or inactivates the MEF2 protein. The therapeutic compositions of the present invention may be administered in a manner similar to the administration of current treatments for heart conditions, such as aspirin, nitrates and beta blockers.
Thus, the therapeutic formulations can be for oral administration in a tablet form to be swallowed such as with aspirin or to be dissolved under the tongue such as with nitrates. These medicaments also can be provided as a patch to be worn on the skin, or as a topical cream to be applied to the skin. The most direct method for blocking MEF2 expression is via antisense technology.
That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine G: C and adenine paired with either thymine A: T in the case of DNA, or adenine paired with uracil A: U in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
Targeting double-stranded ds DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject. Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene.
It is contemplated that the most effective antisense constructs for the present invention will include regions complementary to the mRNA start site. One can readily test such constructs simply by testing the constructs in vitro to determine whether levels of the target protein are affected. Similarly, detrimental non-specific inhibition of protein synthesis also can be measured by determining target cell viability in vitro. As used herein, the terms "complementary" or "antisense" mean polynucleotides that are substantially complementary over their entire length and have very few base mismatches.
For example, sequences of fifteen bases in length may be termed complementary when they have a complementary nucleotide at thirteen or fourteen nucleotides out of fifteen. Naturally, sequences which are "completely complementary" will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region e.
The polynucleotides according to the present invention may encode an MEF2 gene or a portion of those genes that is sufficient to effect antisense inhibition of protein expression. The polynucleotides may be derived from genomic DNA, i. Thus, a cDNA does not contain any interrupted coding sequences and usually contains almost exclusively the coding region s for the corresponding protein.
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In other embodiments, the antisense polynucleotide may be produced synthetically. It may be advantageous to combine portions of the genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
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It is contemplated that natural variants of exist that have different sequences than those disclosed herein. Thus, the present invention is not limited to use of the provided polynucleotide sequence for MEF2 but, rather, includes use of any naturally-occurring variants. Depending on the particular sequence of such variants, they may provide additional advantages in terms of target selectivity, i. The present invention also encompasses chemically synthesized mutants of these sequences. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of base-pairing.
For example, both binding affinity and sequence specificity of an oligonucleotide to its complementary target increase with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 base pairs will be used. While all or part of the gene sequence may be employed in the context of antisense construction, statistically, any sequence of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence.
In certain embodiments, one may wish to employ antisense constructs which include other elements, for example, those which include C-5 propyne pyrimidines. Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. As an alternative to targeted antisense delivery, targeted ribozymes may be used. Ribozymes can either be targeted directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA.
Ribozymes may be used and applied in much the same way as described for antisense polynucleotide. Ribozyme sequences also may be modified in much the same way as described for antisense polynucleotide. Alternatively, the antisense oligo- and polynucleotides according to the present invention may be provided as RNA via transcription from expression constructs that carry nucleic acids encoding the oligo- or polynucleotides.
Throughout this application, the term "expression construct" is meant to include any type of genetic construct containing a nucleic acid encoding an antisense product in which part or all of the nucleic acid sequence is capable of being transcribed. In preferred embodiments, the nucleic acid encodes an antisense oligo- or polynucleotide is placed in a replicable cloning vehicle that supports expression of the antisense molecule with cis- acting transcriptional and translational signals.
The expression constructs will comprise the gene in question and various regulatory elements as described herein below. In another embodiment, it may be desirable to block the function of an MEF2 polypeptide rather than inhibit its expression. This can be accomplished by use of organochemical compositions that interfere with the function of MEF2, by use of an antibody that blocks an active site or binding site on MEF2, or by use of a molecule that mimics an MEF2 target.
With respect to organochemical inhibitors, such compounds may be identified in standard screening assays. Once identified, such an inhibitor may be used to inhibit MEF2 function in a therapeutic context. The methods by which antibodies are generated are well known to those of skill in the art, and are detailed elsewhere in the specification.
Again, antibodies that bind MEF2 may be screened for other functional attributes. A particularly useful antibody for blocking the action of MEF2 is a single chain antibody. Methods for the production of single-chain antibodies are well known to those of skill in the art. The skilled artisan is referred to U. Patent No 5,,, incorporated herein by reference for such methods. A single chain antibody, preferred for the present invention, is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule.
Single-chain antibody variable fragments Fvs in which the C-terminus of one variable domain is tethered to the N-terminus of the other via a 15 to 25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or specificity of the binding Bedzyk et al, ; Chaudhary et al. These Fvs lack the constant regions Fc present in the heavy and light chains of the native antibody. With respect to inhibitors that mimic MEF2 targets, the use of mimetics provides one example of custom designed molecules. Such molecules may be small molecule inhibitors that specifically inhibit MEF2 protein activity.
Such molecules may be sterically similar to the actual target compounds, at least in key portions of the target's structure and or organochemical in structure. Alternatively these inhibitors may be peptidyl compounds, these are called peptidomimetics. Peptide mimetics are peptide-containing molecules which mimic elements of protein secondary structure. See, for example, Johnson et al.
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The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of ligand and receptor. An exemplary peptide mimetic of the present invention would, when administered to a subject, bind to MEF2. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains, as discussed in Johnson et al, In order to prevent MEF2 from interacting with these targets, one may take a variety of different approaches.
For example, one may generate antibodies against the target and then provide the antibodies to the subject in question, thereby blocking access of MEF2 to the target molecule. In yet another embodiment, antisense methodologies may be employed in order to inhibit the interaction of MEF2 with its target, seeing as the MEF2 binding partner is a DNA molecule. Alternatively, one may design a polypeptide or peptide mimetic that is capable of interacting with the MEF2 target in the same fashion as MEF2, but without any MEF2-like effect on the target.
In a preferred embodiment, the present invention will provide an agent that binds competitively to GATA4. Affinity for the GATA4 can be determined in vitro by performing kinetic studies on binding rates. Other compounds may be developed based on computer modeling and predicted higher order structure, both of the MEF2 molecule and of the identified target molecules.
This approach has proved successful in developing inhibitors for a number of receptor-ligand interactions. Genetic Constructs and Gene Transfer. In particular aspects of the present invention, it may be desirable to place a variety of cardiac genes into expression constructs and monitor their expression. For example, a cardiac hypertrophy gene such as BNP, MHC and the like may be tested by introducing into cultured cardiomyocytes an expression construct comprising a promoter operably linked to a hypertrophy- sensitive gene or genes and monitoring the expression of the hypertrophy-sensitive gene or genes.
Expression constructs are also used in generating transgenic animals include a promoter for expression of the construct in an animal cell and a region encoding a gene product which modulates transcription of at least one gene that is expressed in cardiomyocytes in response to a hypertrophic signal. In other embodiments, the expression construct encodes an antisense oligo- or polynucleotide is placed in a replicable cloning vehicle that supports expression of the antisense molecule for the therapeutic purposes discussed above.
Genetic Constructs Throughout this application, the term "expression construct" is meant to include any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding genes of interest. Transcriptional regulatory elements which are suitable for use in the present invention include which direct the transcription of a coding region to which they are operably linked preferentially in cardiomyocytes.
By "preferentially" is meant that the expression of the transgene in cardiomyocytes is at least about fold, more preferably at least about fold to about fold, even more preferably at least about fold to fold, even more preferably more than fold greater than that in non-cardiomyocytes. Preferably, expression of the transgene is below detectable limits in cells other than cardiomyocytes, as indicated by reporter gene assays well known to those of skill in the art.
The nucleic acid encoding a gene product is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase U.
Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase tk and SV40 early transcription units.
These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a.
TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product. For example in the case where expression of a transgene, or transgenes when a multicistronic vector is utilized, is toxic to the cells in which the vector is produced in, it may be desirable to prohibit or reduce expression of one or more of the transgenes.
Examples of transgenes that may be toxic to the producer cell line are pro-apoptotic and cytokine genes. Several inducible promoter systems are available for production of viral vectors where the transgene product may be toxic. The ecdysone system Invitrogen, Carlsbad, CA is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells.
It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over fold inducibility. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mRNA transcripts are attained.
In this system, both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone- responsive promoter which drives expression of the gene of interest is on another plasmid. Engineering of this type of system into the gene transfer vector of interest would therefore be useful. Cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would then allow for the production of the gene transfer vector without expression of a potentially toxic transgene.
At the appropriate time, expression of the transgene could be activated with ecdysone or muristeron A. This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline. These systems are based on two regulatory elements derived from the tetracycline resistance operon of E. The tetracycline operator sequence to which the tetracycline repressor binds, and the tetracycline repressor protein.
The gene of interest is cloned into a plasmid behind a promoter that has tetracycline- responsive elements present in it. Thus in the absence of doxycycline, transcription is constitutively on. In some circumstances, it may be desirable to regulate expression of a transgene in a gene transfer vector. For example, different viral promoters with varying strengths of activity may be utilized depending on the level of expression desired. In mammalian cells, the CMV immediate early promoter if often used to provide strong transcriptional activation.
Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired. Similarly tissue specific promoters may be used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues.
For example, promoters such as the PSA, probasin, prostatic acid phosphatase or prostate-specific glandular kallikrein hK2 may be used to target gene expression in the prostate. Similarly, the following promoters may be used to target gene expression in other tissues. It is envisioned that any of the above promoters alone or in combination with another may be useful according to the present invention depending on the action desired. In addition, this list of promoters is should not be construed to be exhaustive or limiting, those of skill in the art will know of other promoters that may be used in conjunction with the promoters and methods disclosed herein.
Enhancers Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements.
On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. In preferred embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome.
The ability of certain viruses to enter cells via receptor-mediated endocytosis and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells Ridgeway, ; Nicolas and Rubenstein, ; Baichwal and Sugden, ; Temin, The first viruses used as gene vectors were DNA viruses including the papovaviruses simian virus 40, bovine papilloma virus, and polyoma Ridgeway, ; Baichwal and Sugden, and adenoviruses Ridgeway, ; Baichwal and Sugden, These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum.
Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals Nicolas and Rubenstein, ; Temin, Polyadenylation Signals Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human or bovine growth hormone and SV40 polyadenylation signals.
Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
There are a number of ways in which expression vectors may introduced into cells. In certain embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. In other embodiments, non-viral delivery is contemplated. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells Ridgeway, ; Nicolas and Rubenstein, ; Baichwal and Sugden, ; Temin, Delivery mechanisms are discussed in further detail herein below.
The present section provides a discussion of methods and compositions of non-viral gene transfer. DNA constructs of the present invention are generally delivered to a cell, and in certain situations, the nucleic acid or the protein to be transferred may be transferred using non-viral methods. Several non-viral methods for the transfer of expression constructs into cultured mammalian cells are contemplated by the present invention.
Once the construct has been delivered into the cell the nucleic acid encoding the particular gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination gene replacement or it may be integrated in a random, non-specific location gene augmentation. In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.
Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. In another particular embodiment of the invention, the expression construct may be entrapped in a liposome.
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Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers Ghosh and Bachhawat, The addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules Radler et al, These DNA-lipid complexes are potential non-viral vectors for use in gene delivery.
Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Also included are various commercial approaches involving "lipofection" technology. In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus HVJ. This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA Kaneda et al, In other embodiments, the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins HMG-1 Kato et al, In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
Other vector delivery systems which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific Wu and Wu, Receptor-mediated gene targeting vehicles generally consist of two components: Several ligands have been used for receptor- mediated gene transfer.
Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle Ferkol et al, ; Perales et al, and epidermal growth factor EGF has also been used to deliver genes to squamous carcinoma cells Myers, EPO In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane.
This is applicable particularly for transfer in vitro, however, it may be applied for in vivo use as well. Benvenisty and Neshif also demonstrated that direct intraperitoneal injection of CaPO 4 precipitated plasmids results in expression of the transfected genes. Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them Klein et al, Several devices for accelerating small particles have been developed.
One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force " Yang et al, The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene application refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal.
One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. In this context, expression does not require that the gene product be synthesized. The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb Grunhaus and Horwitz, In contrast to retroviruses, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
As used herein, the term "genotoxicity" refers to permanent inheritable host cell genetic alteration. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification of normal derivatives. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage.