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WO1999054491A1 - Utilisation de promoteurs a chaine lourde de la myosine murine en therapie genique et dans la production d'animaux transgeniques - Google Patents

Utilisation de promoteurs a chaine lourde de la myosine murine en therapie genique et dans la production d'animaux transgeniques Download PDF

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WO1999054491A1
WO1999054491A1 PCT/US1999/008710 US9908710W WO9954491A1 WO 1999054491 A1 WO1999054491 A1 WO 1999054491A1 US 9908710 W US9908710 W US 9908710W WO 9954491 A1 WO9954491 A1 WO 9954491A1
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muscle
gene
promoter
dna
myhc
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PCT/US1999/008710
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English (en)
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Jeffrey Robbins
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Children's Hospital Medical Center
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/107—Rabbit
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0368—Animal model for inflammation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • This invention relates generally to the use of exogenous promoters for tissue specific exogenous gene expression. More specifically the present invention relates to the use of trans-species striated muscle specific promoters, such as the murine alpha and beta myosin heavy chain promoters, which can be used for tissue specific exogenous gene expression, gene therapy, gene transfer, and for the production of transgenic animals.
  • trans-species striated muscle specific promoters such as the murine alpha and beta myosin heavy chain promoters
  • a vector system is the means used to carry the exogenous genetic material into the subject for later expression.
  • retroviral vectors have tremendous potential for use as delivery or vector systems because the genetic content of the retroviral genome incorporates itself into the infected cell. By doing so, a recombinantly generated retrovirus that carries an exogenous gene of interest can be used to introduce that gene into the genome of a target cell. This incorporation means that as the target cell gives rise to progeny, each of the daughter cells will carry the exogenous gene of interest. Vectors that do not incorporate into the genome provide only transient expression of the exogenous gene.
  • retroviruses require their target cells to be dividing for incorporation to occur. Thus, the overall population of cells that are available for retroviral infection may, depending upon the target tissue, be limited.
  • Other viral vector systems have been explored as potential vector systems for gene transfer.
  • Adenoviral vectors have shown great promise in the area. These viruses can be grown in large quantities and can infect nondividing cells.
  • the adenoviral systems suffer from a limitation common to viral vectors, the adenoviral tropism limits the types cells into which the adenoviral genome can be introduced.
  • Non-viral methods include liposomal transfer.
  • a problem common to all vector systems is their cell or tissue specificity, or tropism.
  • Some vectors, such as viruses may have very specific cell types that they infect.
  • the tropism of the human immunodeficiency virus (HIV) is limited largely by the fact that the virus binds to the CD4 protein presented on T cells.
  • Other vector systems have a very low cell, tissue, or organ specificity. In view of these limitations, a gene transfer system that provided for the tissue specific expression of a gene of interest would clearly result in fewer side effects and more efficient treatment.
  • Tissue targeting can be accomplished in a number of ways. Each technique has its own advantages and disadvantages.
  • a simple technique for tissue tropism is accomplished by applying the therapeutic gene or vector only to the target tissue.
  • Another technique involves using the natural tropism of the viral vector. This tropism can be manipulated producing a viral vector that is specific for a different tissue.
  • Another technique involves making the therapeutic gene transcriptionally specific to the targeted cell or tissue. In other words, the therepeutic gene will only be expressed in the targeted tissue.
  • adenoviral vectors for the treatment of cystic fibrosis by using inhalation directly into the lungs to target the adenoviral vector to the cells which need the therapy. Because adenovirus naturally infects respiratory cells, this takes advantage of the natural tropism of the virus. However, this technique is limited to the use of vectors which naturally infect only the targeted cells. Because suitable viral vectors are limited, this severely limits the variety of tissues which can be targeted.
  • Another example involves direct injection of naked DNA into muscle tissue in the form of cDNAs, plasmids, or even viral vectors. It can be imagined that only certain diseases would be amenable to this type of localized application, thus limiting the diseases which can be targeted.
  • Muscle can be separated into two types, striated or non-striated. Striated muscle includes cardiac and skeletal muscle. This is because the two types of muscle have similar sarcomeric organization.
  • the non-striated muscle is the smooth muscle of most organs. We are interested in the study of striated muscle.
  • HSV-1 Herpes simplex virus type 1
  • a muscle-specific vector could be useful in many ways. The obvious value is in treating muscle-specific inherited and acquired diseases. However, there is also some reason to believe that a muscle specific delivery could be useful for treatment of a number of systemic diseases and more specifically, inflammatory diseases.
  • the disease prototype of muscle-specific gene therapy is Duchenne Muscular Dystropy (DMD).
  • DMD Duchenne Muscular Dystropy
  • DMD Duchenne Muscular Dystropy
  • dystrophin cDNAs into skeletal muscle fibers of dystrophin-deficient mice (mdx) through direct DNA injection into plasmid expression vectors, and by replication-defective recombinant retrovirus or adenovirus vectors. Less than 10% of adult mdx fibers of the plasmid and retrovirus injected muscle expressed dystrophin. This very low efficiency provides some hope for such treatment, however, it is widely believed that specific tropism or gene transcriptional activity is vital for treatment of DMD. Other Uses for a Muscle-Specific Vector System
  • muscle-specific vector system there are a number of other uses for a muscle-specific vector system particularly in research-related activities.
  • One use is for producing transgenics which express various muscle-specific genes or turn off others. These transgenics would be useful in
  • muscle-specific vector systems could be used in vitro to more efficiently transfer genes into muscle-related cell types.
  • the invention is a selectively modified myosin promoter which drives high levels of protein expression very efficiently in muscle tissue such that they offer the ability to direct in a specific manner to striated muscle, very high and efficient delivery of transgene expression.
  • the ⁇ and ⁇ myosin heavy chain promoter (MyHC) will drive expression of gene therapeutics at high levels in striated muscle.
  • MyHC myosin heavy chain promoter
  • the promoter is inactive in non-muscle tissue or in smooth muscle, lending the desired degree of specificity to the biological delivery systems. This has previously been unobtainable for a promoter that is able to drive very high levels of transgene expression in striated muscle types of large mammalian species. Therefore, there has long been a need for a tissue specific method of gene transfer. The promoter of the invention fills this need.
  • One object of the invention is to provide a vector for expressing an exogenous DNA in a muscle specific manner which is made up of a murine myosin heavy chain promoter or variants thereof which are capable of expressing in a muscle-specific manner which is expressing an exogenous DNA.
  • the vector preferably uses the ⁇ or ⁇ murine myosin heavy chain promoter. It is prefered that the muscle specificity is to striated muscle. More preferably, the promoter and exogenous DNA are contained in a delivery system, preferably, a virus, plasmid, or liposomes.
  • the exogenous DNA is preferably a muscle-specific gene, heart-specific gene, anti-inflammatory gene, antisense DNA, ribozyme, or systemic disease gene.
  • the muscle-specific gene is preferably, the Dystrophin gene, the Dystrophin mini- gene, the Utrophin gene, or variants thereof, dystroglycans, emerin, and tropomyosin.
  • the systemic disease genes are preferably Factor IX or decorin.
  • DNA in a muscle-specific cell, organism, or tissue having the steps of a) selecting an exogenous gene b) genetically attaching it to the murine myosin heavy chain promoter or variants thereof producing a promoter construct, such that the promoter controls expression of the exogenous DNA, and c) delivering the promoter construct to a cell, organism or tissue
  • the promoter is the ⁇ or ⁇ murine myosin heavy chain promoter, preferably confering muscle specificity to striated muscle.
  • the exogenous DNA is a muscle-specific gene, heart-specific gene, anti-inflammatory gene, antisense DNA, ribozyme, or systemic disease gene. More preferably, the muscle-specific gene is the Dystrophin gene, the Dystrophin mini-gene, the Utrophin gene, or variants thereof, dystroglycans, emerin, and tropomyosin. More preferably, the systemic disease genes are Factor IX or decorin.
  • the delivery system is a viral vector, a plasmid, a liposome, or Naked DNA.
  • the exogenous DNA can be delivered to said cell, organism, or tissue in vitro or in vivo.
  • a further object of the invention is a method for producing a transgenic with muscle- specific expression of an exogenous DNA having the steps of a) selecting an exogenous gene or DNA, b) producing a functional promoter by attaching it to the murine myosin heavy chain promoter or variants thereof producing a promoter construct, and c) delivering the promoter construct to an egg, blastocyst or zygote.
  • the promoter is preferably the ⁇ or ⁇ murine myosin heavy chain promoter. It is prefered that the muscle specificity is to striated muscle.
  • the exogenous gene or DNA is a muscle-specific gene, heart- specific gene, antisense DNA, and variants thereof.
  • FIGS 2A-2B CAT expression is homogeneous throughout muscle.
  • FIGS 3A-3B CAT expression in skeletal muscle.
  • Figure 4 Endogenous expression of ⁇ -MyHC and ⁇ -MyHC in transgenic rabbits in Alpha CAT line 290, the highest expressing line.
  • ⁇ -MyHC alpha myosin heavy chain
  • ⁇ -MyHC beta myosin heavy chain
  • GAPDH glyceraldehyde phosphatase dehydrogenase
  • RA right atrium
  • LA left atrium
  • V ventricle
  • TG transgenic
  • NTG nontransgenic.
  • Figures 5A-5B CAT expression driven by the mouse ⁇ -MyHC promoter in transgenic rabbits.
  • B Beta CAT line 492 muscle expression.
  • the present invention relates to the use of modified and unmodified myosin promoters from an exogenous species that drive high levels of protein expression in striated muscle tissue.
  • the promoters of the present invention are substantially inactive in non- muscle tissue or in smooth muscle. This tissue specific activity provides the desired degree of specificity to the various gene delivery systems. This specificity has previously been unobtainable for a promoter that is able to drive very high levels of transgene expression in striated muscle types of large mammalian species. Therefore, this technology will fill a long felt need for a strong, striated-muscle specific promoter suitable for biologic delivery.
  • the exogenous promoters described by the present invention are highly useful for efficacious and selective gene transfer protocols.
  • the promoters of the present invention are used as part of a therapeutic modality for the treatment of inherited skeletal muscle disorders.
  • these promoters can be used in any gene delivery system where a striated muscle limited expression of the gene of interest is desired. Additionally, since striated muscle is a secretory tissue, the general approach holds open the possibility of systemic delivery if desired.
  • exogenous promoters taught by the present invention can be accomplished using a number of vector or delivery systems.
  • vector systems include but are not limited to viral vectors, plasmid DNA, cDNA constructs, liposomes, naked DNA constructs, and other delivery systems known to those of skill in the art.
  • viral delivery systems are retroviral or lentiviral. These are very efficient but have the disadvantage that current vectors only incorporate into proliferating cells. Current work on modifying the vectors so that they incorporate into nonproliferating cells is showing promise.
  • Another example of a suitable viral vector system is the adenoviral system. Adenoviral vectors will incorporate into nonproliferating cells. However, adenoviral DNA does not integrate into host nuclei, but nonetheless it persists in postmitotic myofibers for up to 6 months. Herpesviral and other viral vectors are also being devoloped.
  • This type of therapy does not possess the biohazard that many of the current viral vectors have.
  • the present invention contemplates the use of myosin heavy chain promoters from one species to drive striated muscle specific gene expression in a different species.
  • a murine myosin heavy chain promoter is a promoter which is operably linked to murine myosin heavy chain.
  • myosin heavy chain promoters of murine origin are used to drive striated muscle specific expression in a rabbit model. However, use of these promoters in any non-murine host is contemplated.
  • myosin heavy chain In both lower organisms and in mammals, an important component of the contractile apparatus is the myosin heavy chain (MyHC).
  • the myosin heavy chain protein is encoded by a large gene family. The members of this multigene family are differentially expressed in a developmental stage- and muscle type-specific manner. In mammalian cardiac muscle, two of the gene family's members, termed ⁇ -MyHC and ⁇ -MyHC and are thought to play a critical role in determining the speed of contraction. Other myosin heavy chain promoters are specifically expressed in skeletal muscle and even more specifically in fast skeletal muscle (fast skeletal myosin heavy chain promoter). In adult murine atrium, ⁇ -MyHC is expressed constitutively.
  • the ⁇ -gene is predominantly expressed.
  • At or around birth there is an antithetic switch of ⁇ to ⁇ in the ventricle such that the V3 isoform is gradually replaced by the VI protein.
  • >95% of the MyHC transcripts in the mature ventricle are transcribed from ⁇ -MyHC with only trace amounts of the ⁇ -gene-encoded RNA being present.
  • the murine myosin heavy chain promoter contains thyroid response elements (TREs) identified in the proximal promoter region. Expression from the TREs is controlled by thyroid hormone (TH).
  • TREs thyroid response elements
  • TH thyroid hormone
  • Direct injections of DNA into the myocardium have shown that 612 bp of the gene's upstream region is sufficient to confer TH modulation to a reporter gene construct in vivo (Kitsis et al., 1991P.N.A.S. Vol 88, pp. 4138-42)
  • Site- directed mutagenesis of the ⁇ -MyHC promoter in a transgenic analysis has been used to define those elements responsible for high levels of transcription in vivo. Because of the similarity between the promoters, these studies can be applied to other myosin heavy chain
  • TRE, and TRE 2 are located at -129 to -149 and -102 to -120, respectively, on the ⁇ -MyHC promoter. Although the elements' ablation had differential effects on transgene expression, neither single mutation abolished transgene expression completely, however, each TRE alone only had about 10% of normal activity. Mutating both elements resulted in a complete inactivation of the transgene in both ventricles and atria under conditions with no thyroid hormone. In hyper thyroid conditions, expression can still be detected. Therefore, although TRE, and TRE 2 elements are critical elements for high levels of ⁇ -MyHC transcription in vivo, other promoter sites can mediate at least some degree of transcriptional activation. Both elements are needed for the high level of gene expression as well as developmental regulation. This suggests that other parts of the promoter would not be necessary for this high level of expression. Exogenous DNA or The Gene of Interest
  • the gene of interest is any gene which is capable of being expressed in the system.
  • the gene may be of interest for experimental reasons or for treatment of a disease.
  • Preferably expression of the gene product would alleviate a disease.
  • diseases due to loss of a functional gene product such as Duchenne Muscular Dystrophy (DMD) which has lost the gene dystrophin, limb-girdle dystrophy which has lost dystroglycans, Emery-Dreifuss disease which has lost emerin, and nemaline rod myopathy which has lost tropomyosin.
  • the exogenous DNA is a gene product which would alleviate diseases due to mutation or aberrent expression of a gene product or virus. These could be treated with antisense DNA or ribozymes.
  • the promoter is used to produce a gene of interest which acts as a vaccine.
  • the method of the present invention can also be used to generate transgenic animals.
  • a gene transfer vector containing a gene of interest and exogenous promoters is introduced into a target cell line. Those cells are then used to generate an entire subject animal in which the gene of interest has been incorporated.
  • ⁇ -MyHC promoter is capable of driving high levels of transgene expression in a developmental stage- and cardiac compartment- specific fashion, with promoter-driven expression corresponding to the endogenous expression pattern of ⁇ -MyHC. Additionally, the expression level is generally
  • the mouse ⁇ -MyHC promoter also displays developmental stage- and compartment specific activity and in the adult mouse expresses in the cardiac ventricle and the slow soleus muscle.
  • proximal rabbit ⁇ -MyHC and ⁇ -MyHC promoters share approximately 85% homology with the mouse promoters in the most proximal 600 base pairs. Since the proximal promoter is responsible for cardiac specificity and this region is essentially conserved between mouse and rabbit, we hypothesized that, as in murine transgenics, the mouse promoters might be useful in remodeling the protein complement of the rabbit heart. Additionally, heterologous promoters have been used successfully to create transgenic animals (including transgenic rabbits). It was found that heterologous use of the murine myosin heavy chain promoters does result in the efficient transcription of a target transgene in the heart of the rabbit. However, surprisingly, it also resulted in efficient transcription in the striated muscles.
  • muscle-specific promoters in Gene transfer
  • a specific promoter which is capable of a very high level of expression in striated muscle has a clear use in in vitro and in vivo studies.
  • Vectors for expressing exogenous genes in tissue culture which can express at high levels and only in specific tissues are needed for experimental systems.
  • the promoter of the present invention is very useful for expressing exogenous genes in muscle-related cell lines such as, myoblasts, myotubes, myogenic cell lines, transformed cell lines and possibly muscle-related cancers such a rhabdomyosarcoma, etc.
  • muscle-related cell lines such as, myoblasts, myotubes, myogenic cell lines, transformed cell lines and possibly muscle-related cancers such a rhabdomyosarcoma, etc.
  • muscle-related cell lines such as, myoblasts, myotubes, myogenic cell lines, transformed cell lines and possibly muscle-related cancers such a rhabdomyosarcoma, etc.
  • genes are expressed in undifferentiated cell lines to determine if they are involved in differentiation of the cells toward the muscle phenotype, antisense DNA is expressed in cell lines to determine the effect of a newly discovered gene product, developmental genes are expressed to determine the effect on a differentiated muscle cell, etc.
  • Utrophin (Dystrophin) expression vector For example genes are expressed in undifferentiated cell lines to determine if they are involved in differentiation of the cells toward the muscle phenotype, antisense DNA is expressed in cell lines to determine the effect of a newly discovered gene product, developmental genes are expressed to determine the effect on a differentiated muscle cell, etc.
  • DMD Duchenne muscular dystrophy
  • dystrophin or a related protein
  • the main goal of gene therapy for Duchenne muscular dystrophy (DMD) is to restore dystrophin (or a related protein) into as many muscle cells as necessary to be therapeutic.
  • DMD Duchenne muscular dystrophy
  • Experiments outlined in the Background have supported the concept of treating DMD in this way by demonstrating that regional expression of recombinant dystrophin in dystrophic muscle leads to regional restoration of normal muscle morphology.
  • dystrophic mini-genes driven by muscle specific regulatory elements are more effective than the full-length dystrophin gene.
  • the related gene utrophin has been used to prevent
  • ⁇ -MyHC and ⁇ -MyHC promoters are prime examples of such muscle-specific regulatory elements.
  • the muscles serve as an excellent site for the production of genetically engineered proteins that may be therapeutic for conditions other than primary myopathies.
  • vaccines can be produced or antisense and ribozymes.
  • cardiovascular system has benefited tremendously from the use of genetically altered animals, specifically gene-targeted and transgenic mice.
  • Virtually all facets of the cardiovascular system, including cardiac development, the conduction system, the coronary vasculature, the adrenergic system, and the components of the sarcomere have been explored using these technologies.
  • Augmentation of in vitro preparations with in vivo models has been invaluable in providing integrative data regarding physiological and pathological states in the heart, such as cardiac hypertrophy and dilation.
  • These animals provide the potential reagents to explore complex signaling pathways mediating the transitions from normal cardiac function through compensated cardiac dysfunction to heart failure.
  • Cardiovascular disease remains the leading cause of death in developed countries. There is an urgent need for valid experimental systems to study the pathological progression of cardiovascular disease at all levels (molecular to whole animal) in order to dissect the pathological basis of disease and facilitate the discovery of novel therapeutic agents.
  • mice Because of the ease with which the genome may be manipulated and the relatively low cost of maintaining large colonies, most molecular investigations of the cardiovascular system to date have used mice, although in some cases, transgenic rats have been studied. However, the mouse and rat do not accurately reflect potentially crucial facets of human cardiovascular physiology. Indeed, a number of experimental models aimed at duplicating human pathological states by expressing correlative genetic mutations of human genes in small mammals have failed to accurately reproduce the human phenotype. This should not be surprising since the murine heart differs from the human in several very significant features. From a functional standpoint, the mouse heart beats 600 - 700 times per minute and supplies cardiac output for a body mass of 20-40 grams. In contrast, the adult human heart at rest beats 50 - 100 times per minute, supplying cardiac output to a body mass of 50-
  • myosin heavy chains are present as two isoforms: the "fast” alpha MyHC isoform, ( ⁇ -MyHC) and the “slow” beta MyHC ( ⁇ -MyHC) designated “fast” and “slow” in reference to the relative rates of ATPase activity inherent to these enzymatically active proteins.
  • the normal adult mouse ventricle expresses only the “fast” ( ⁇ -MyHC) isoform, while the normal human ventricle expresses a mixture of the "slow” ⁇ -MyHC and fast” ⁇ -MyHC, with the ⁇ -MyHC isoform predominating in the healthy adult state.
  • Transthoracic echocardiography has been widely used as a method to repeatedly assess cardiac function in mice but the quality of data obtained is highly user dependent and complex, load- independent measurements cannot be reliably obtained. All told, despite a great deal of effort over the last eight years, only a limited number of laboratories are capable of performing these assays, leaving the bulk of the research community with serious accessibility issues. Thus, reproducible data remain limited.
  • the rabbit was a good choice to start with because the gestation period is relatively short (30 days) and sexual maturity occurs relatively quickly (20 - 24 weeks).
  • the rabbit is a very useful model for studying aspects of human heart disease and transgenics can be made in a relatively straightforward manner.
  • rabbit atria express the ⁇ -MyHC isoform at all stages of development while the ventricles express both the ⁇ - and ⁇ -MyHC isoforms, with the ⁇ -MyHC isoform dominating in adulthood.
  • This MyHC expression pattern is essentially identical to that of the human heart. With a mature weight of 4 - 6 kg, the rabbit is intermediate in size
  • the rabbit has a significantly slower heart rate than the mouse and approaches that of a human neonate.
  • the promoter of the present invention will be used to produce non-murine transgenics for the purpose of understanding the role a protein plays in muscle development and disease. Much can be learned by over-expressing the protein product or a mutated version, or by producing an antisense DNA. The effect will be limited to striated muscle.
  • Example 1 describes the construction of a murine myosin heavy chain promoter drive gene expression cassette.
  • the full-length mouse ⁇ -MyHC and ⁇ -MyHC promoters has been extensively characterized using chloramphenicol acetyl transferase (CAT) as the reporter gene (Rindt, H. et al. 1995, Transgenic Research 4, 397-405). Briefly, all critical transcriptional components are conserved upstream of the cDNA insertion site. This includes exon-intron splicing junctions and a strong translational start signal. Downstream are three stop codons in all possible frames and a polyadenylation site.
  • CAT chloramphenicol acetyl transferase
  • ⁇ -MyHC/CATand ⁇ -MyHC/CAT constructs ( ⁇ /CAT and ⁇ /CAT, respectively) are free of cloning artifacts and thus were used in the generation of transgenic rabbits without modification.
  • the promoter sequence and CAT reporter gene were excised from the plasmid by Not I digest and the desired fragment isolated by gel purification and subsequent dialysis against TE (10 mM Tris, pH 7.0, O.lmM EDTA).
  • the promoter itself is excised and subcloned into the vector, virus, plasmid, cDNA, or other delivery mechanism of choice. The promoter will then be used to express exogenous DNA.
  • Example 2 The murine promoter described in Example 1 was used to produce a rabbit transgenic as shown in Examples 2-9
  • the full-length mouse ⁇ -MyHC and ⁇ -MyHC promoters were used in the generation of transgenic rabbits without modification.
  • the promoter sequence and CAT reporter gene were excised from the plasmid by Not I digest and the desired fragment isolated by gel purification and subsequent dialysis against TE (10 mM Tris, pH 7.0, O.lmM
  • the standard injection protocol for transgenic mice was modified to a four-day procedure in the rabbit to account for timing differences in ovulation and fertilization. All experiments were performed with New Zealand White rabbits under a protocol approved by the Animal Care Committee.
  • the oocyte donor doe was super-ovulated on day one of the protocol with 150 units pregnant mare serum gonadotropin (PMSG) delivered subcutaneously under the scruff of the neck.
  • PMSG pregnant mare serum gonadotropin
  • the donor doe was mated with a non-transgenic buck.
  • both the donor and recipient does received 150 units of human choriogonadotropin (HCG) administered in an ear vein.
  • HCG human choriogonadotropin
  • the eggs were harvested from the donor doe and the pronucleus of viable eggs injected with purified DNA.
  • the injected eggs were then transplanted into the fallopian tube of the pseudopregnant donor.
  • the recipient doe was moved to a nesting cage two to three days prior to the expected delivery date.
  • Transgenic offspring were identified by PCR (using CAT specific primers) and genomic Southern (with 32P-labelled CAT cDNA as the probe).
  • the founder rabbits were aged to five months (females) or six months (males) before attempting to breed for FI offspring.
  • FI and/or F2 offspring were used for all subsequent analyses. Table 1 summarizes our experience in founder generation.
  • Numbers include our experience with both the mouse ⁇ -MyHC/CAT( ⁇ /CAT) and the mouse ⁇ - MyHC/CAT( ⁇ /CAT) constructs.
  • Diploid copy number was determined with DNA dot blots using a 32 P-labelled CAT cDNA probe. The blots were placed on a phosphor screen, the image scanned with a STORM 760 machine and the results analyzed using Image Quant Mac 1.2 (Molecular Dynamics, Sunnyvale, CA). The overall success rate is shown in Table 1. From 1000 reimplanted embryos, 87 liveborn rabbits were obtained, of which 11 were transgenic. These results gave an overall efficiency of approximately 1%, (approximately 13% for live born rabbits). The success rate for the generation of transgenic mice was approximately 25%. Thus, the success rate for rabbits was less than the success with mice, but similar to what has been reported by others.
  • RNA dot blots were performed on nitrocellulose with atrial and ventricular total RNA using one microgram of total RNA per
  • Hybridization proceeded for five hours. After three ten minute washes with 0.7X SSC/1% SDS, the blots were placed on a phosphor screen overnight then scanned and analyzed as described above. Expression of the transgene was analyzed by CAT enzyme-linked immunoabsorption assay (ELISA). Transgenic rabbits were sacrificed at 3-5 days, 8-12 days, 4-6 weeks, 8-12 weeks, and > 16 weeks as described above. Tissue samples were dissected from multiple regions in the heart (right atrium, left atrium, ventricular apex, aorta, and pulmonary artery) for use in CAT ELISA.
  • Proteins for CAT ELISA were obtained by homogenizing the tissues in a small volume (200 -400 mL) of 0.25M Tris (pH 7.8) using a Tekmar homogenizer (Tekmar Company, Cincinnati, OH). The homogenate was incubated at 65°C for ten minutes then centrifuged for ten minutes at 12,000 rpm in a tabletop microfuge. The supernatant was transferred to a new tube and the protein concentration determined.
  • CAT ELISAs were performed with a microtiter kit according to the manufacturer's instructions (Boehringer-Mannheim, Indianapolis, IN). A standard curve was performed with each analysis so that test results could be compared between different experiments and production lots. The initial experiments in each line used 50 mg protein samples; this was decreased as needed depending upon the expression level of a given line to ensure that the test results remained within the linear range of the standard curve.
  • RNA dot blots were performed using total RNA extracted from the biceps, vastus lateralis, tibialis anterior, gastrocnemius, soleus, masseter, tongue, and diaphragm of 10 day old, 6 week old, and 16- week-old rabbits. They were hybridized with the ⁇ -MyHC, ⁇ -MyHC, and GAPDH probes as described in Example 3. Alpha MyHC is strongly expressed in the masseter at 6 weeks and 16 weeks, but not at 10 days.
  • ⁇ -MyHC and ⁇ -MyHC are expressed in the diaphragm, with ⁇ -MyHC present at low levels at all three time points and ⁇ -MyHC expression increasing with age.
  • the soleus muscle had ⁇ -MyHC expression at all time points, with a very low level of ⁇ -MyHC detectable at 10 days but not at 6 weeks or 16 weeks.
  • Beta MyHC expression was demonstrated at very low levels in the biceps and gastrocnemius at all three timpoints assayed.
  • Figure 3 shows the level of CAT expression in the masseter, diaphragm, and soleus as determined by CAT ELISA in lines 286 and 290 (with 2 and 14 diploid copies of the
  • CAT expression in smooth muscle and non-muscle tissue A critical point for the specificity and usefulness of these promoters is that expression be restricted to the desired tissue types, that is, striated muscle.
  • CAT ELISA's were performed on protein extracts from a number of smooth muscle (stomach, small intestine, urinary bladder, and uterus) and non-muscle sites (liver, lung, kidney, spleen, brain, and ovary). These results are summarized in Table 2 and show that the mouse ⁇ -MyHC promoter is striated muscle specific in the rabbit. Table 2.
  • Nontransgenic papillary muscle was distributed homogeneously throughout the muscle.
  • the staining protocol has been described in detail elsewhere (Knotts S, Sanchez A,
  • cryosections rather than paraffin embedded tissue.
  • Papillary muscle tissue was embedded in Tissue-Tek O.C.T. compound (Miles, Inc., Elkhart, IN). Twelve micrometer cryosections were placed on positively-charged slides and the sections allowed to air dry for one hour before fixing with ice-cold acetone for twenty minutes. Excess acetone was blotted away and the slide allowed to air dry. Dehydration and bleaching of the tissue and all subsequent steps were then performed basically as described by Knotts et al, (1996, Dev Dyn, Vol. 206, ppg. 182-192) with a primary antibody concentration of 1 :1000, secondary antibody concentration of 1 :500, and exposure time of 24 hours.
  • EXAMPLE 7 Developmental expression in the rabbit transgenic To analyze mouse promoter activity in transgenic rabbits, the amount of the reporter protein, CAT, was examined by CAT enzyme linked immunoabsorption assay (ELISA). CAT ELISA was chosen over CAT transcript analysis (which may not reflect protein accumulation) or CAT activity assay as a standardized and reproducible method to quantitate the amount of CAT protein.
  • CAT enzyme linked immunoabsorption assay CAT enzyme linked immunoabsorption assay
  • CAT chloramphenicol acetyl transferase
  • LA left atrium
  • RA right atrium
  • APEX ventricular apex
  • ⁇ -MyHC expression is initially high in the ventricle but gradually decreases as the rabbit matures, being replaced by the ⁇ -MyHC isoform. None of our three ⁇ /CAT lines exactly mimicked the endogenous pattern. Line 222, with 8 diploid copies of the transgene, showed a progressive increase in the amount of CAT present in the atria with age to approximately 300 pg CAT/mg protein seen at the oldest age assayed. There was low and relatively constant expression of CAT in the ventricular apex (Fig.lA). Line 286, with 2 copies of the transgene had very low levels of CAT in the atria at all time points tested and modest and essentially unvarying expression in the ventricular apex (Fig. IB).
  • Line 290 with 14 copies of the transgene, initially had high levels of CAT in the atria (approximately 800-1000 pg CAT/mg protein) with attenuation of expression over time to almost undetectable levels at 16 weeks (Fig. 1C).
  • Ventricular expression was extremely high earlier in development, in the order of 3000-7000 pg CAT/mg protein decreasing to 300 - 500 pg CAT/mg protein at 16 weeks.
  • the levels of CAT expression seen in these three lines compare favorably with the levels seen in transgenic mice when the mouse ⁇ - and ⁇ -MyHC promoters were first characterized and are sufficient to drive transgene expression at a level in which abundant proteins in the heart or other striated muscle tissues can be replaced by transgenically- encoded sequences.
  • FIG. 4 shows an RNA dot blot experiment comparing the expression of ⁇ -MyHC and ⁇ -MyHC in the right atrium (RA), left atrium (LA), and ventricle (V) in a line 290 heart at 12 weeks.
  • Transgenic (TG) expression is compared to an age-matched nontransgenic rabbit (NTG). No significant difference was found between the TG and NTG animals in endogenous rabbit ⁇ -MyHC and ⁇ -MyHC expression despite the very high levels of transgene expression in line 290, suggesting that even extremely high levels of transgene expression do not lead to inhibition of endogenous RNA expression.
  • EXAMPLE 9 Activity of the mouse ⁇ -MvHC promoter in the rabbit As noted above, unlike the mouse, in the rabbit ventricle it is the ⁇ -MyHC promoter that is most active. This is also the case in the human ventricle. To determine if the mouse ⁇ -MyHC promoter was capable of driving significant levels of transgene expression in the rabbit, the corresponding ⁇ /CAT construct was used to generate transgenic rabbits. A similar strategy as for the generation of ⁇ -CAT founders was used with the ⁇ -MyHC construct. Four founders were obtained and we have analyzed CAT expression in one line (Fig.
  • the generation of technique involves genetically engineering the ⁇ -MyHC and ⁇ - MyHC promoters to express the gene of interest producing a construct. Any type of vector could be used, viral, plasmid, or naked DNA.
  • the construct is transfected into the cell line using a variety of techniques known by those of skill in the art. If the cell line contains the correct transcription factors, or is related to a striated muscle cell, the gene of interest will be expressed. Analysis of the outcome of expression of the gene is specific to the experimental system.
  • This example addresses the use of a gene transfer vector to express the dystrophin, utrophin, dystrophin mini gene, or related genes in a target muscle cell line.
  • a vector is constructed using recombinant techniques to express the gene of interest under control of the murine MyHC promoters.
  • a recombinant construct is then transferred to the animal or human in an appropriate manner.
  • viral vectors can be injected intraveneously, intramuscularly, or subcutaneously. Naked DNA and liposomes will be injected intramuscularly.
  • Vectors or DNA are mixed with an appropriate buffer and solutions supportive to the virus, liposomes, or DNA.
  • Muscle-related disease expression vector A muscle-related disease gene such as dystroglycan (for use in limb-girdle dystrophy), emerin (for use in Erery-Dreifuss disease), and tropomyosin (for use in nemaline rod myopathy), is genetically engineered to be expressed by the murine MyHC promoters.
  • a delivery system is chosen, then it is transferred to the animal or human in an appropriate manner.
  • viral vectors can be injected intraveneously, intramuscularly, or subcutaneously. Naked DNA and liposomes will be injected intramuscularly. Vectors or DNA will be mixed with an appropriate buffer and solutions supportive to the virus, liposomes, or DNA.
  • a non-muscle-related disease gene such as a gene encoding an antigen or an antisense gene, or Factor IX or decorin, or any other gene of interest, is genetically engineered to be expressed systemically by the murine MyHC promoters.
  • a delivery system is chosen and then used to transferred the gene of interest under MyHC promoter control to the animal or human in an appropriate manner.
  • viral vectors can be injected intraveneously, intramuscularly, or subcutaneously. Naked DNA and liposomes will be injected intramuscularly. Vectors or DNA will be mixed with an appropriate buffer
  • the exogenous DNA will be expressed in the muscle and secreted into blood or lymph where it can travel to the therapeutic site.
  • trans-species transgenics are produced using the murine ⁇ -MyHC and ⁇ -MyHC promoters.
  • a construct is engineered containing a cardiac-related or therapeutic gene under the control of one of these promoters.
  • the transgenic will be produced following the steps outlined in Example 2. Following introduction of the exogenous gene, cardiac function in these transgenic animals is monitored to determine the effect of the exogenous gene. Monitoring of cardiac function is performed using standard methods known to those of ordinary skill in the art.
  • Trans-species transgenics containing genes controlled and regulated by the murine ⁇ -MyHC and ⁇ -MyHC promoters are constructed using the methods described above.
  • a construct is produced containing a muscle-related gene or antisense under the control of one of these promoters.
  • the promoter construct will be injected into the fertilized egg, zygote, or blastocyst. Following the introduction of the exogenous gene of interest, muscle function in the transgenic animal is observed and compared to wild type muscle function using standard techniques well known to those of skill in the art.

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Abstract

Selon l'invention, on utilise un promoteur à chaîne lourde de la myosine murine α et β, dans le transfert de gènes, la thérapie génique et la production d'animaux transgéniques. Chez les grands animaux, ce promoteur s'exprime seulement dans les muscles striés. Ainsi, on peut l'utiliser pour obtenir des niveaux d'expression efficaces et élevés d'un gène d'intérêt, notamment dans un tissu musculaire strié, ce qui rend ce promoteur parfait pour une utilisation en thérapie génique des maladies associées aux muscles, telles que la dystrophie musculaire progressive de Duchenne, et même des maladies systémiques, notamment des maladies inflammatoires.
PCT/US1999/008710 1998-04-20 1999-04-20 Utilisation de promoteurs a chaine lourde de la myosine murine en therapie genique et dans la production d'animaux transgeniques WO1999054491A1 (fr)

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WO2002012297A1 (fr) * 2000-06-21 2002-02-14 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine 9 de liaison a la tropomoduline, et polynucleotide codant ce polypeptide
EP1282433A4 (fr) * 2000-05-05 2004-08-11 Gtc Biotherapeutics Inc Decorine transgenique
WO2010106295A1 (fr) * 2009-03-18 2010-09-23 Genethon Utilisation de la décorine pour augmenter la masse musculaire
CN115992178A (zh) * 2022-08-08 2023-04-21 首都医科大学附属北京天坛医院 核心蛋白聚糖转基因小鼠的制备方法及应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1282433A4 (fr) * 2000-05-05 2004-08-11 Gtc Biotherapeutics Inc Decorine transgenique
AU2001259465B2 (en) * 2000-05-05 2006-12-07 Gtc Biotherapeutics, Inc. Transgenically produced decorin
WO2002012297A1 (fr) * 2000-06-21 2002-02-14 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine 9 de liaison a la tropomoduline, et polynucleotide codant ce polypeptide
WO2010106295A1 (fr) * 2009-03-18 2010-09-23 Genethon Utilisation de la décorine pour augmenter la masse musculaire
FR2943249A1 (fr) * 2009-03-18 2010-09-24 Genethon Utilisation de la decorine pour augmenter la masse musculaire
US9474782B2 (en) 2009-03-18 2016-10-25 Association Francaise Contre Les Myopathies Use of decorin for increasing muscle mass
CN115992178A (zh) * 2022-08-08 2023-04-21 首都医科大学附属北京天坛医院 核心蛋白聚糖转基因小鼠的制备方法及应用

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