EP0737252A1 - Vecteur de therapie genique pour le traitement de l'insuffisance ou des troubles de la production de globules rouges - Google Patents

Vecteur de therapie genique pour le traitement de l'insuffisance ou des troubles de la production de globules rouges

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Publication number
EP0737252A1
EP0737252A1 EP95901877A EP95901877A EP0737252A1 EP 0737252 A1 EP0737252 A1 EP 0737252A1 EP 95901877 A EP95901877 A EP 95901877A EP 95901877 A EP95901877 A EP 95901877A EP 0737252 A1 EP0737252 A1 EP 0737252A1
Authority
EP
European Patent Office
Prior art keywords
erythropoietin
control sequence
muscle
cells
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95901877A
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German (de)
English (en)
Inventor
Babru B. Samal
Yasuo Hamamori
Laurence H. Kedes
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amgen Inc
University of Southern California USC
Original Assignee
Amgen Inc
University of Southern California USC
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Publication date
Application filed by Amgen Inc, University of Southern California USC filed Critical Amgen Inc
Publication of EP0737252A1 publication Critical patent/EP0737252A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel approach to the treatment of low or defective red blood cell production.
  • the invention provides for the sustained systemic production of erythropoietin following the modification of target cells by gene transfer.
  • Erythropoiesis the production of red blood cells, occurs continuously to offset cell destruction. Erythropoiesis is a precisely controlled physiological mechanism enabling sufficient numbers of red blood cells to be available for proper tissue oxygenation, but not so many that the cells would impede circulation.
  • the formation of red blood cells occurs in the bone marrow and is under the control of the hormone, erythropoietin. Erythropoietin is normally present in very low concentrations in plasma when the body is in a healthy state wherein tissues receive sufficient oxygenation from the existing number of erythrocytes . This normal low concentration is sufficient to stimulate the replacement of red blood cells which are naturally lost through aging.
  • the amount of erythropoietin in the circulatory system is increased under conditions of hypoxia when oxygen transport by blood cells to tissue is reduced.
  • Hypoxia may be caused by loss of large amounts of blood due to hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged unconsciousness, or various forms of anemia.
  • erythropoietin increases red blood cell production by stimulating the conversion of precursor cells in the bone marrow into erythroblasts .
  • the erythroblasts subsequently mature, synthesize hemoglobin and are released into the circulatory system as red blood cells.
  • the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, erythropoietin in circulation is decreased.
  • erythropoietin is essential in the process of red blood cell formation, the hormone is useful in the treatment of blood disorders characterized by low or defective red blood cell production. While the injection of recombinantly produced human erythropoietin is a proven therapy for the treatment of blood disorders, it would be advantageous to enhance the endogenous production of erythropoietin in a patient or mammalian subject .
  • the present invention provides, for the first time, the successful development of a method for the enhancement of red blood cell production by gene therapy.
  • the invention demonstrates that expression vectors can be constructed using an expression control sequence and an erythropoietin gene, operatively linked to the control sequence and capable of expression in transfected target cells, wherein the nucleic acid construct is capable of eliciting the expression of erythropoietin sufficient to increase red blood cell production.
  • the present invention supports the development of gene therapy techniques for the treatment of low or defective red blood cell production.
  • pharmaceutical compositions involving effective amounts of the nucleic acid constructs together with a pharmaceutically acceptable delivery vehicle including suitable diluents, buffers and adjuvants.
  • the compositions can further include a carrier capable of promoting target cell uptake of the nucleic acid constructs.
  • carriers include liposomes, protein complexes and viral carriers suitable for gene transfer techniques.
  • the invention involves the development of myoblast- mediated gene therapy for the in vivo production of erythropoietin.
  • the invention further describes the use of expression vectors involving non-specific and muscle-specific promoters, and the suitability of such vectors for generating stable myogenic cell lines which, following introduction into skeletal muscle, can elicit sufficient production and secretion of erythropoietin to present a physiologically significant systemic response.
  • Figure 1 illustrates a DNA sequence for erythropoietin.
  • Gene therapy for anemia comprises the delivery of a gene for erythropoietin to cells, either in vivo or in vitro . Delivery and expression of the gene results in the production of erythropoietin in a physiologically-functional amount sufficient to increase red blood cell production.
  • the erythropoietin gene used in the present invention is a nucleic acid sequence which encodes a functional erythropoietin protein. Thus, variations in the actual sequence of the gene can be tolerated provided that functional erythropoietin is expressed.
  • An erythropoietin gene used in the practice of the present invention can be obtained through conventional methods such as DNA cloning, artificial construction or other means .
  • Gene transfer of the erythropoietin gene in accordance with the present invention can be accomplished by any suitable gene therapy technique involving a nucleic acid construct or recombinant vector containing a DNA sequence that encodes erythropoietin.
  • the nucleic acid constructs generally will be provided as an expression cassette or expression control system which will include as operatively linked components in the direction of transcription, a transcriptional initiation region, the erythropoietin nucleic acid sequence of interest and a transcriptional termination region wherein the transcriptional regulatory regions are functional in a mammalian host.
  • a recombinant vector construct may not become integrated into the host cell genome of the patient or mammalian subject, and therefore, it may be introduced into the host as part of a non-integrating nucleic acid construct.
  • a coding sequence is "operatively linked to” or “under the control of” the expression control system in a cell when DNA polymerase will bind the promoter sequence and transcribe the erythropoietin- encoding sequence into mRNA.
  • the nucleic acid construct includes a DNA sequence which encodes a polypeptide directly responsible for a therapeutic effect, as well as a sequence (s) controlling the expression of the polypeptide.
  • the nucleic acid constructs in the invention include several forms, depending upon the intended use of the construct.
  • the transcriptional and translational initiation region (also herein referred to as a "promoter"), preferably comprises a transcriptional initiation regulatory region and a translational initiation regulatory region of untranslated 5' sequences.
  • the promoter may be modified by the addition of sequences, such as enhancers, or deletions of nonessential and/or undesired sequences.
  • the promoter will have a DNA sequence sufficiently similar to that of a native promoter to provide for the desired specificity of transcription of the erythropoietin DNA sequence.
  • the promoter may include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.
  • the expression control sequence may contain a suppresser sequence to regulate the expression of erythropoieti .
  • any region may be used with the proviso that it provides the desired level of transcription of the erythropoietin nucleic acid sequence.
  • the transcriptional initiation region may be native to or homologous to the host cell, and/or to the DNA sequence to be transcribed, or foreign or heterologous to the host cell and/or the DNA sequence to be transcribed. By foreign to the host cell is intended that the transcriptional initiation region is not found in the host into which the construct comprising the transcriptional initiation region is to be inserted.
  • Efficient promoter elements for transcription initiation include the SV40 (simian virus 40) early promoter, the RSV (Rous sarcoma virus) promoter, the Adenovirus major late promoter and the human CMV (cytomegalovirus) promoter.
  • Inducible promoters also find use with the expression control sequences where it is desired to control the timing of transcription.
  • Examples of promoters include those obtained from a ⁇ -interferon gene or those obtained from steroid hormone-responsive genes .
  • Such inducible promoters can be used to regulate transcription of the transgene by the use of external stimuli such as interferon or glucocorticoids. Because the arrangement of eukaryotic promoter elements is highly flexible, combinations of constitutive and inducible elements also can be used. Tandem arrays of two or more inducible promoter elements may increase the level of induction above baseline levels of transcription which can be achieved when compared to the level of induction above baseline achieved with a single inducible element .
  • Transcriptional enhancer elements may also be included in the expression control sequence.
  • transcriptional enhancer elements includes DNA sequences which are primary regulators of transcriptional activity and which can act to increase transcription from a promoter element.
  • the combination of promoter and enhancer element (s) used in a particular expression cassette can be selected by one skilled in the art to maximize specific effects.
  • Different enhancer elements can be used to produce a desired level of transgene expression in a wide variety of tissue and cell types.
  • the human CMV immediate early promoter-enhancer element can be used to produce high level transgene expression in vivo.
  • enhancers from SV40 and RSV-LTR examples include enhancers from SV40 and RSV-LTR.
  • the SV40 and RSV-LTR are essentially constitutive. They may be combined with other enhancers which have specific effects, or the specific enhancers may be used alone.
  • efficient enhancer elements that are active only in a tissue-, developmental-, or cell-specific fashion are of interest.
  • Tandem repeats of two or more enhancer elements or combinations of enhancer elements may significantly increase erythropoietin expression when compared to the use of a single copy of an enhancer element .
  • Enhancer elements from the same or different sources flanking or within a single promoter can in some cases produce transgene expression in each tissue in which each individual enhancer acting alone would have an effect, thereby increasing the number of tissues in which transcription is obtained. In other cases, the presence of two different enhancer elements results in silencing of the enhancer effects . Evaluation of particular combinations of enhancer elements for a particular desired effect or tissue of expression is within the level of skill in the art.
  • Gene transfer procedures are known to those skilled in the art and include cell transformation using calcium phosphate coprecipitation, lipofection of the target cells with liposome/gene or lipid/gene conjugates, plasmid-mediated transfer, DNA protein complex-mediated transfer and viral vector-mediated transfer.
  • Viral vector transfer can include suitable techniques such as transfer by recombinant retroviral vectors, adenovirus vectors and adeno-associated virus vectors.
  • the present invention includes the use of carriers to facilitate gene transfer, and different carriers may be selected as appropriate to optimize transfer to the desired cell-type which is targeted for vector delivery. It will also be appreciated that the various carriers may be selected or modified for preferential uptake by the cell-type which is targeted for vector delivery.
  • the carrier can include a selected ligand to effectively target the cells of interest.
  • the vector may contain one or more targeting sequences, generally located at both ends of the exogenous DNA sequence to be expressed. Such a construct is useful to integrate exogenous DNA into the target cell.
  • the cells targeted for gene transfer in accordance with the present invention include any cells to which delivery of the erythropoietin gene is desired. While a variety of cells may be transfected, it was determined that muscle cells are especially appropriate targets for gene transfer and the expression of physiologically active amounts of erythropoietin.
  • a physiologically active or acceptable level of erythropoietin gene function refers to a level of in vivo erythropoietin manufacture and function sufficient to cause an increase in red blood cell production. Increased red blood cell production can be readily determined by an appropriate indicator such as detection of changes in hematocrit levels.
  • the level of erythropoietin gene function sufficient to cause an increase in red blood cell production can readily be determined by a comparison of pretreatment or baseline hematocrit level to the post-treatment hematocrit level.
  • Cells or cell populations can be treated in accordance with the present invention either in vivo or in vitro .
  • recombinant erythropoietin vectors can be administered to the patient, preferably in a biologically compatible solution or pharmaceutically acceptable delivery vehicle.
  • the dosages administered can vary from patient to patient and will be determined by the level of enhancement of erythropoietin function balanced against any risk of side effects . Monitoring levels of transduction, erythropoietin expression and/or the levels of red blood cells will assist in selecting and adjusting the dosages administered.
  • In vitro transduction is also contemplated within the present invention.
  • Cell populations can be removed from the patient, or otherwise be provided, transfected with the erythropoietin gene in accordance with the present invention, and then administered to the patient.
  • the transfected target cells may be reintroduced by any suitable means, such as injection or implantation, and the cells will typically be delivered to target tissue of the same cell type as the target cells.
  • muscle cells may serve as the target cells.
  • Myoblasts can be isolated and manipulated in vitro, transfected with the erythropoietin vector, and the transformed cells are then reintroduced into muscle tissue.
  • the unique biology of muscle cells allows the transfected cells to form new myofibers or fuse into old ones.
  • the transplanted nuclei are sustained and active for prolonged periods of time in a normal, multinucleated environment with little or no nuclear replication for up to six months .
  • the muscle cells will sustain the production and secretion of the erythropoietin protein sufficient to result in increased red blood cell production.
  • the present invention is also amenable to the use of homologous recombination genome-modification methods. Homologous recombination is a technique originally developed for targeting genes to induce or correct mutations in transcriptionally active genes (Kucherlapati, Prog, in Nucl . Acid Res . and Mol . Biol . 36:301 (1989)) .
  • the basic technique was developed as a method for introducing specific mutations into specific regions of the mammalian genome (Thomas et al.. Cell. 44:419-428, 1986; Thomas and Capecchi, Cell . 51:503-512, 1987; Doetschman et al., Proc. Natl . Acad. Sci . 85:8583-8587, 1988) or to correct specific mutations within defective genes (Doetschman et al. , Nature . 330:576-578, 1987) .
  • a piece of DNA that one desires to insert into the genome can be directed to a specific region of the gene of interest by attaching it to "targeting DNA” .
  • Targeting DNA is DNA that is complementary (homologous) to a region of the genomic DNA.
  • the targeting DNA and the genomic DNA are in close proximity, they will hybridize to form a double stranded helix. Attached to the targeting DNA is the DNA sequence that is to be inserted into the genome.
  • Small pieces of targeting DNA that are complementary to a specific region of the genome are put in contact with the parental strand during the DNA replication process. It is a general property of DNA that has been inserted into a cell to hybridize and therefore recombine with other pieces of endogenous DNA through shared homologous regions. If this complementary strand is attached to an oligonucleotide that contains a mutation or a different sequence of DNA, it too is incorporated into the newly synthesized strand as a result of the recombination. As a result of the proofreading function, it is possible for the new sequence of DNA to serve as the template. Thus, the transfered DNA is incorporated into the genome.
  • a piece of DNA that is complementary to a selected region of the gene can be synthesized or otherwise obtained, such as by appropriate restriction of the native DNA at specific recognition sites bounding the region of interest. This piece serves as a targeting sequence upon insertion into the cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this piece of DNA, and any additional sequence attached thereto, will act as an Okazaki fragment and will be backstitched into the newly synthesized daughter strand of DNA.
  • regions of DNA will interact with the nuclear regulatory proteins present within the cell and, optionally, amplifiable and selectable DNA markers.
  • the expression of erythropoietin may be achieved not by transfection of DNA that encodes the erythropoietin gene itself, but rather by the use of targeting DNA (regions of homology with the endogenous gene of interest) coupled with DNA regulatory segments that provide the endogenous erythropoietin gene with recognizable signals for transcription.
  • targeting DNA regions of homology with the endogenous gene of interest
  • the expression of this gene is controlled by the entire genomic DNA rather than portions of the gene or the cDNA, thus improving the rate of transcription and efficiency of mRNA processing.
  • the expression characteristics of any cognate gene present within a cell type can be modified by appropriate insertion of DNA regulatory segments and without inserting entire coding portions of the gene of interest .
  • homologous recombination provides new methods for expressing a normally transcriptionally silent erythropoietin gene, or for modifying the expression of an endogenously expressing gene.
  • the erythropoietin gene will be provided with the necessary cell-specific DNA sequences (regulatory and/or amplification segments) to direct or modify expression of the gene within the muscle cell.
  • the resulting DNA will comprise the DNA sequence coding for erythropoietin directly linked in an operative way to heterologous (for the cognate DNA sequence) regulatory and/or amplification segments.
  • a positive selectable marker is optionally included within the construction to facilitate the screening of resultant cells.
  • neomycin resistance gene is preferred, although any selectable marker may be employed. Negative selectable markers may, optionally, also be employed.
  • HSVtk Herpes Simplex Virus thymidine kinase
  • the fused DNAs, or existing expressing DNAs, can be amplified if the targeting DNA is linked to an amplifiable marker.
  • a myoblast cell line which stably expressed the human erythropoietin gene.
  • the cell line was established by transfecting the cells with a plasmid containing the erythropoietin gene driven by a CMV promoter.
  • the plasmid was derived from pCD vector 1 (Okayama et al., A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol . Cell . Bio . 3:280-289, 1993) as described in the following examples.
  • erythropoietin Genes encoding erythropoietin are described in United States Patent Number 4,703,008 issued October 27, 1987, and entitled DNA Sequences Encoding Erythropoietin, and Figure la-d.
  • the plasmid included a gene for neomycin resistance such that transformed cells could be selected by antibiotic resistance . After the expansion of 23 randomly selected clones, the clones were screened for the secretion of erythropoietin into the culture media by Western blot and radioimmunoassay.
  • EPO Erythropoietin
  • the present invention demonstrates the efficacy of gene transfer to obtain sustained in vivo production of a therapeutic polypeptide, such as erythropoietin, at levels sufficient to enhance red blood cell production.
  • a therapeutic polypeptide such as erythropoietin
  • refinements in the selection of promoter and enhancer genes will serve to optimize the expression of erythropoietin in the transfected target cells.
  • muscle-specific expression control sequences for high level recombinant protein expression in transformed muscle cells.
  • High activity promoter and enhancer cassettes can be used to intensify recombinant gene expression.
  • Such promoters will increase the levels of therapeutic recombinant proteins synthesized and secreted by both newly formed myofibers as well as muscle fibers that contain a mixture of donor and recipient myonuclei.
  • the major contractile proteins of thin and thick filaments e.g., alpha-actins, troponin C, myosin heavy chains, as well as several muscle enriched enzymes, such as creatine kinase and carbonic anhydrase III
  • Muscle-specific gene expression is usually associated with muscle-specific transcription factors including members of the MyoD family (Weintraub, et al., The myoO gene family: nodal point during specification of the muscle cell lineage.
  • muscle-specific genes have been cloned, and the promoters analyzed: these include skeletal actin, cardiac actin, Troponin C fast, Troponin C slow and Troponin I slow, as well as beta and gamma cytoskeletal actins .
  • Other muscle specific promoters that have been the subject of detailed analysis include creatine kinase, myosin light chains and various myosin heavy chain genes as well as Troponins I, T and C. Detailed analyses of such enhancer and promoter regions that provide muscle specificity are available, as illustrated by the following brief summary.
  • Skeletal ⁇ -actin The tissue specific distal promoter of the human skeletal ⁇ -actin gene (-1282 to -708) induces transcription in myogenic cells approximately 10-fold and, with the most proximal promoter domain (-153 to -87), it synergistically increases transcription 100-fold (Muscat, et al. , Multiple 5' flanking regions of the human skeletal actin gene synergistically modulate muscle specific gene expression. ⁇ fol. Cell Biol . 7:4089-4099, 1987) .
  • the human skeletal ⁇ -actin gene is regulated by a muscle-specific enhancer that binds three nuclear factors .
  • Cardiac ⁇ -actin The cardiac ⁇ -actin gene is the fetal isoform of ⁇ -actin in rodents and human muscle, but it does not express after birth in rodents. The down regulation appears to be dependent on nucleotide sequences far downstream of the transcribed gene. In adult human muscle, cardiac ⁇ -actin represents about 5% of the ⁇ -actin mRNA. The cardiac ⁇ -actin promoter and endogenous gene are highly expressed in cell lines derived from skeletal muscle. Thus, the cardiac actin gene promoter and upstream elements are candidate elements as positive regulators of muscle specific gene expression in skeletal muscle cells.
  • Skeletal Fast-twitch Troponin C gene The expression of the human fast-twitch skeletal muscle troponin C (TnC or TnCfast) gene is muscle-specific and confined to the class of fast-twitch myofibers in adult skeletal muscle. There is a strong classical enhancer element within the 5'-flanking sequence of this gene which is required for the transcriptional activity. A MEF-2 site alone in this enhancer is sufficient to support high level transcription. Interestingly, and unlike enhancers of other muscle genes, the human fast TnC enhancer is muscle cell specific, but only if linked to its own basal promoter which is itself not muscle cell restricted. This suggests that interactions between the enhancer and the basal promoter of the human fast TnC gene are responsible for its muscle restricted expression.
  • TnC or TnCfast The expression of the human fast-twitch skeletal muscle troponin C (TnC or TnCfast) gene is muscle-specific and confined to the class of fast-twitch myofibers in adult skeletal muscle
  • Slow-Twitch/cardiac Troponin C gene At least four separate elements cooperate to confer muscle specific expression on the human slow twitch skeletal/cardiac troponin C (HcTnC or TnCslow) gene: a basal promoter (from -61 to -13) augments transcription 9-fold, upstream major regulatory sequences (from -64 to -1318 and from -1318 to -4500) augment transcription 18-fold and 39-fold, respectively, and a position and orientation independent enhancer in the first intron (from +58 to +1519) augments transcription 5-fold.
  • This enhancer increases muscle specific CAT activity when linked to its own promoter elements or to a heterologous SV40 promoter, and the effects appear to be multiplicative rather than additive.
  • constructs carrying either the TnC promoter or the first intron of the gene are >500-fold induced.
  • MyoD myogenic determination factor
  • Slow-twitch Troponin I ⁇ ene At least three separate elements spaced over 1 kb of the 5' upstream regions of the human slow twitch troponin I gene (HsTnl) combine to synergistically regulate muscle specific gene expression.
  • a basal promoter lies within 300 base pairs of the transcription start site and two independent muscle specific enhancers 800 and 1000 base pairs upstream. All three appear to be required for expression.
  • cDNAs encoding the erythropoietin gene may be cloned into a variety of in-frame expression vectors.
  • a non-muscle-specific beta-actin promoter constructed as a high level expression vector with neomycin selection capacity, contains the promoter and first intron of the human ⁇ -actin gene, a neomycin resistance gene, a bacterial origin and the SV40 late region polyadenylation signal (Gunning, et al. , A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc. Natl . Acad. Sci . USA . 84:4831-5; 1987) . The use of such a construct fosters high level transcription of inserted sequences in mammalian cells.
  • the erythropoietin sequence may also be cloned into an internally deleted human skeletal ⁇ -actin gene promoter that carries high level muscle specific expression.
  • This construct carries an upstream element (from -1282 to -1177) linked to its own promoter from -153.
  • This enhancer/promoter combination may be inserted in the place of beta-actin sequences to create a new muscle specific expression vector with neomycin selectability (pHaSKApr-1-neo) .
  • the plasmid vectors are sequenced after construction to insure in-frame accuracy.
  • the plasmids may be co-transfected into C2 myogenic cells along with a ⁇ -galactosidase expression vector.
  • the cells may be split 1:20 into 60 mm dishes with DMEM containing 20% fetal calf serum (FCS) and 0.4 mg/ml of neomycin (Geneticin® G418; Gibco Laboratories, Grand Island, NY) . After 14 days of selection, individual clones may be isolated and expanded in DMEM containing 20% FCS and 0.2 mg/ml of G418. Since the ability of transferred cells to differentiate into yotubes in vivo is a likely requirement for their stability and longevity in situ, the clones are tested for their ability both to differentiate into myotubes in DMEM supplemented with 2% horse serum and to express beta- galactosidase.
  • FCS fetal calf serum
  • G418 Gibco Laboratories, Grand Island, NY
  • the ⁇ -galactosidase expression serves as a histological marker to monitor survival, as well as both macroscopic and microscopic location, of injected myoblasts at the conclusion of the studies .
  • Individual clones are expanded and the culture media tested for polypeptide production by Western blot and radioimmunoassay. High level expressing clones are selected for further analysis.
  • Novel combinations of muscle-specific enhancer and promoter elements may be constructed and tested for increased polypeptide expression in vitro .
  • the creation of myogenic cells expressing exceptionally high levels of recombinant polypeptides provides a means of reducing both the numbers of primary cells required for ex vivo manipulation and the numbers of cells required for gene therapy muscle cell transplant.
  • Plasmid expression vectors may be constructed from several of these components linked together.
  • a construct may include the parent skeletal actin chloramphenicol acetyltransferase (CAT) expression vector containing the upstream enhancer and the muscle specific promoter.
  • CAT chloramphenicol acetyltransferase
  • To this may be added single copies of the TnCfast upstream enhancer, the TnCslow first intron enhancer element, and the MCK enhancer (Johnson, et al. , Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice. Mol . Cell . Biol . 9:3393-3399;
  • One or more of these elements may be added as 3-5 multimers. After checking the validity of the constructs by DNA sequencing, they may be used in calcium phosphate mediated gene transfer for transient transfection CAT assays by standard techniques.
  • the plasmids may be cotransfected into C2 cells with RSV- luciferase as a positive control standard in the search for promoters with heightened transcriptional activity.
  • the plasmids may also be transfected into non-muscle (Hela or CV1) cells to evaluate their degree of muscle specificity. Beta-actin and RSV CAT constructs may be transfected into the same cells in parallel to serve as comparisons.
  • the regulatory region is transferred to replace the promoter in the neomycin vector (pHaSKApr-1-neo) described above along with a beta-galactosidase expression vector.
  • a polylinker was inserted in the unique PstI site of a pCD vector 1 (Okayama et al., A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol . Cell . Bio . 3:280-289, 1993) to generate the VI9 vector.
  • the VI9.1 vector was derived from the VI9 vector by switching direction of Eco R I and Hind III sites in relation to the SV40 promoter (see Table 2) . This vector was then digested with
  • EPO cDNA was isolated from the vector VI9.1 as an Eco R I-Hind III fragment. The sticky ends were filled in using T4 DNA polymerase in the presence of deoxyribonucleotides.
  • a human cytomegalovirus vector (CMV/RC; Invitrogen Corporation, San Diego, CA) , was digested with the restriction enzyme Hind III and was blunted using T4 DNA polymerase.
  • An erythropoietin cDNA fragment was ligated to the CMV/RC vector to form a pRC/CMV-huEPO expression construct.
  • E. coli DH5 alpha competent cells were transformed with the pRC/CMV-huEPO plasmid. Plasmid DNA was isolated, sequenced and used for transfection of mammalian cells.
  • Mouse myogenic C2 cells (Yaffe and Saxel, A myogenic cell line with altered serum requirements for differentiation, Differen . 7:159-166; 1977; and Serial passaging and differentiation of myogenic cells isolated form dystrophic mouse muscle. Nature . 270:725-727; 1977) were cultured in growth medium consisting of Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum, 0.5% chick embryo extract (Gibco Laboratories) and 100 ⁇ g/ml of kanamycin (Gibco Laboratories) in 10% CO 2 . Subconfluent C2 myoblast cells, in 100 mm dishes, were split 1:4 the day before transfection.
  • DMEM Dulbecco's modified Eagle medium
  • Subconfluent C2 myoblast cells in 100 mm dishes, were split 1:4 the day before transfection.
  • C2 cells were transfected by the calcium phosphate precipitation method using the pRC/CMV-huEPO plasmid of Example 1.
  • Transfection mixtures were prepared as follows: a solution of 250 mM CaCl2 (0.5 ml) was added dropwise to 8 ⁇ g of DNA in 0.5 ml of 2 x N-2-hydroxyethylpiperazine-N 1 -2-ethanesulfonic acid (HEPES)-buffered saline (42 mM HEPES [pH 7.05], 270 mM ⁇ aCl, 10 mM KC1, 1.4 mM ⁇ a 2 HP0 4 , 11 mM dextrose) . This was done with constant mixing.
  • HEPES N-2-hydroxyethylpiperazine-N 1 -2-ethanesulfonic acid
  • the calcium phosphate-DNA precipitate was left for 20 minutes at room temperature after which it was added to the cells.
  • the cells were incubated for 16 hours, washed with phosphate-buffered saline (PBS) , and incubated in growth medium (10 ml of 10% fetal calf serum in DMEM) for 48 hours.
  • PBS phosphate-buffered saline
  • the highest producing clone was cultured in 150 mm dishes in growth medium containing 200 ⁇ g/ml of G418. When the cells reached 80% confluence, they were trypsinized and collected in ice-cold PBS. Total cell number was determined by hemocytometer. Cells were rinsed once again with PBS to remove residual trypsin, pelleted, and resuspended in a small volume of PBS to a concentration of 1 x 10 s cells/ml. We routinely collected 2-3 x 10 8 cells for each experiment . Cells were kept on ice until use to prevent aggregation.
  • C3H mice syngeneic to the C2 cell line and nude mice (both 6-8 week-old) were used for transplantation.
  • Animals were anesthetized by intraperitoneal administration of a mixture of ketamine (30 mg/kg) and xylane (4 mg/kg) .
  • a total of 4 x 10 7 cells per mouse were injected percutaneously through a 27 gauge needle at 40 different sites (1 x 106 cells/10 ⁇ l/site) into skeletal muscle tissue of both hind limbs.
  • the same number of parental C2 cells were transplanted in the same manner.
  • Hematocrit was measured by microhematocrit method. Under general anesthesia, a total amount of approximately 200 ⁇ l of blood was collected by a retroorbital approach into three heparinized capillary tubes. Blood collection was performed one week prior to transplantation, three days and one week after transplantation, and weekly thereafter. Control animals showed that this amount of blood collection did not significantly affect basal hematocrit levels. On several occasions, the hematocrit was also measured using a Coulter counter which showed parallel results with micro-hematocrit method. After hematocrit measurement, the plasma was collected and stored at -20°C for huEPO concentration measurement.
  • mice were sacrificed by cervical dislocation for the histochemical detection of ⁇ -galactosidase expression.
  • Skeletal muscle tissue was excised and frozen immediately on dry ice.
  • the excised muscles were then sectioned with a freezing microtome.
  • the 10 ⁇ m thick sections were attached to microscope slides, fixed in 0.25% glutaraldehyde for 10 minutes, washed in PBS for 10 minutes, and stained in PBS containing 1 mg/ml of 5-bromo-4-chloro-3-indolyl- ⁇ —D-galactoside, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl 2 • Sections were incubated at 37°C overnight, rinsed in PBS, mounted and studied under microscope.
  • RT-PCR Reverse transcription-poly erase chain reaction
  • RNAse-free DNAsel Boehringer Mannheim, Indianapolis, IN
  • placental RNAse inhibitor Promega, Madison, WI
  • 20 mM MgCl 2 20 mM MgCl 2
  • 2 mM dithiothreitol 20 mM MgCl 2
  • DNAse stopping mixture containing 50 mM EDTA, 1.5 M sodium acetate (pH 4.8) and 1% sodium dodecyl sulfate.
  • the RNA was treated with phenol/chloroform, chloroform, and ethanol- precipitated.
  • RNA and 4 pmol primer complimentary to the 3 ' untranslated region of huEPO RNA were incubated at 70°C, rapidly cooled on ice, and treated with 100 U of reverse transcriptase (Superscript; Gibco Laboratories) .
  • the obtained cDNA was amplified by known PCR methods using primers including the initiation and stop codons.
  • Table 3 shows the time course of mean hematocrit change after the transplantation of C2 cells expressing huEPO gene.
  • Hematocrit started to increase three days after the transplantation of 4 x 10 7 cells into C3H syngeneic mice (A in Table 3) .
  • the peak hematocrit was achieved two weeks after the transplantation.
  • Hematocrit declined gradually thereafter, becoming lower than the basal level after week 4.
  • RT-PCR showed persistent huEPO mRNA expression for at least one month in the injected muscle, but not in the muscle from uninjected left hind limb. Mice transplanted with parental C2 cells did not show significant hematocrit increase.
  • nude mice (B) showed significantly higher and more sustained hematocrit increase for at least two months.
  • the net hematocrit increase was also approximately half of that observed with 4 x 10 7 cells, thereby indicating that huEPO production can be regulated by cell number.
  • mice transplanted with the C2 cells expressing ⁇ -galactosidase showed positive myofibers over the entire injected sites three months later.
  • the injected C2 cells appeared to fuse among themselves as well as with preexisting myofibers. No ⁇ -galactosidase positive myoblasts were observed.
  • Serum huEPO concentration as determined at several points after transplantation by either radioimmunoassay or bioassay using an erythropoietin- responsive human leukemic cell line (UT-7/EPO) showed significantly elevated erythropoietin concentrations ranging from 90 to 3500 U/ml. Erythropoietin concentrations before transplantation in these mice were ⁇ 25 mU/ml. Table 3
  • the current major indication for recombinant human EPO administration is anemia associated with end-stage renal failure (Faulds et al. , Drugs. 38:863- 899 (1989)) .
  • the efficacy of a myoblast gene therapy approach is demonstrated using an animal model of renal failure in nude mice.
  • the experiment was designed to determine whether myoblasts can be transplanted and then secrete functional human EPO in an amount sufficient to correct anemia, for., a long-term in these uremic subjects.
  • Transplantation of EPO- producing C2 cells generated marked erythropoiesis as efficiently as in non-uremic mice, indicating that a myoblast gene transfer approach can be applied in renal failure subjects as effectively as in normal subjects.
  • myoblast gene transfer is means to correct anemia associated with renal failure as well as other types of EPO-responsive anemia.
  • Human EPO-secreting C2 myoblast clones were prepared as described above.
  • the clones carry the 1.34 kb human EPO cDNA (starting at + 190 nucleotide from the major transcription initiation site to the end of poly A tail) cloned into the plasmid pRC/CMV (Invitrogen, San Diego, CA.) .
  • This plasmid bears the cytomegalovirus enhancer/promoter to drive the EPO gene, and a neomycin resistance gene.
  • the highest EPO-producing clone, hereafter called C2-EP09 produced approximately 33 U/10 * ⁇ cells/day of human EPO as determined by radioimmunoassay.
  • the functional activity of EPO produced by this clone was confirmed by an in vitro bioassay.
  • Myoblasts from C2-EP09 were cultured and harvested as previously described. Under general anesthesia, a total of 4 x 10 7 cells were injected through a 27-gauge needle at 40 different sites (1 x 10*- * cells/lOml/site) of skeletal muscle of both hind limbs in nude mice.
  • Anesthetic agents included 20 mg/kg of ketamine hydrochloride and 3 mg/kg of xylazine hydrochloride (Sigma, St. Louis, MO) .
  • Hematocrit was measured by the microhematocrit method (Koepke, J.A., ed. Practical Laboratory Hematology, . 1991, Churchill Livingstone: New York.
  • a renal failure model was created by a two-step nephrectomy (Chanutin et al . , Arch . Intern Med. 49:767-787 (1932)) using 7-8 week old male nude mice (Charles River Labs., Wilmington, MA) . Under general anesthesia using sterile techniques, the right kidney was exposed through a flank incision and decapsulated, and the upper and lower poles (2/3 of the right kidney) were resected. The remnant right kidney was allowed to recover from swelling for a week, and then the total left kidney was resected.
  • the animals were fed standard chow (Harlan Tekland #8656; Harlan Tekland, Madison, WI) containing 24.0% protein and 1.0% phosphorus, and water ad libitum. Renal failure was confirmed by the development of both anemia and uremia.
  • blood urea nitrogen (BUN) was determined weekly with a BUN kit (Sigma 535-A; Sigma, St. Louis, MO) using four milliliters of serum.
  • Serum concentrations of human EPO were determined by an enzyme linked immunosorbent assay (ELISA) system using a mouse monoclonal antibody according to the manufacturer's protocol (Quantikine IVD; R & D Systems, Minneapolis, MN) . This method has a linear range between 2.5 and 200 mU/ml of human EPO with a detection threshold of 0.25 mU/ml .
  • ELISA enzyme linked immunosorbent assay
  • skeletal muscle tissue was excised and frozen immediately on dry ice.
  • the excised muscles were then sectioned with a freezing microtome.
  • the sections were attached to microscope slides, fixed in 0.25% glutaraldehyde for 10 minutes, washed in PBS for 10 minutes, and stained in PBS containing 1 mg/ml of X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, and 2 mM o
  • mice Ten of twenty-seven nephrectomized mice died within four days after surgery (two after the first, and eight after the second surgery) , a mortality rate comparable to a previous report (Gibb et al. , Clinical Immunology and Immunopathology. 35:276-284 (1985)) .
  • Group II included 13 mice that showed persistent anemia with a hematocrit decrease of more than 15% from the preoperative level in three consecutive measurements after the second nephrectomy and were used for transplantation experiments.
  • the mean hematocrit decreased from a preoperative level of 45.2 ⁇ 2.7 to 33.9 ⁇ 3.7 (%0) three weeks after the second nephrectomy.
  • the Group I mice did not develop further anemia, and the degree of BUN increase was much lower than that of the Group II mice (52.6 ⁇ 13.2 vs. 95.4 ⁇ 16.5 three weeks after the second nephrectomy) ; presumably due to insufficient nephrectomy.
  • mice Eight mice were transplanted with C2-EP09 cells, and three mice were followed without transplantation as a non-transplantation control (one mouse died just before transplantation, presumably due to severe uremia) . All of the transplanted mice of Group II had a marked hematocrit increase, despite the presence of severe uremia as indicated by the high BUN levels. The rise in hematocrit was comparable to that observed in normal nude mice (Table 4) . A mean hematocrit of 68.6 ⁇ 4.2 was achieved two weeks after the transplantation, and this hematocrit increase persisted thereafter. Those without transplantation showed persistent or even deteriorating anemia.
  • mice All of the Group II mice, except one, died between six and eleven weeks (8.2 ⁇ 1.8 weeks) after the second nephrectomy, while in Group I only one mouse died during the experimental period.
  • the observed survival rate is consistent with previous observations (Kumano et al., Kidney International . 30:433-436 (1986)) .
  • the one long term survivor in Group I also had the lowest levels of BUN in that group.
  • serum human EPO concentration was measured using an ELISA. As previously determined, this method did not detect a significant level of mouse EPO ( ⁇ 2.5 mU/ml) in sera of nude mice phlebotomized (150 ml) weekly over three months (unpublished observation) . This observation was confirmed in the non-transplanted renal failure mice in Group II (not shown) . Serum EPO measured by this method, therefore, represents just the human EPO produced by the transplanted muscle cells and not endogenous EPO levels. A week after transplantation with C2-EP09 cells, the serum EPO level was 87.3 ⁇ 22.lmU/ml in group II uremic mice.
  • transplanted C2-EP09 cells persistently produced human EPO at a steady rate for at least two months after transplantation into mice with severe renal failure.
  • C2-EP09 cells were transduced with BAG retrovirus (Price et al. , Proc . Natl . Acad. Sci . USA . 84:156-160 (1987)) bearing ⁇ -galactosidase and neomycin resistance genes . Since the C2-EP09 clone had already been maintained in the presence of G418, BAG- transduced clones were selected by positive X-gal staining.
  • End-stage renal failure patients as well as patients with hypoproliferative anemia secondary to 3'-azido-3 'deoxythymidine (AZT) administration are currently treated with 100-150 U of recombinant EPO per kg of body weight per week to maintain a target hematocrit level between 30 and 33, which is equal to 857-1286 U/day for a 60 kg patient. Since C2-EP09 secreted 32.8 U of EPO/10 6 cells/day, 2.6-3.9 x 10 7 cells would, in theory, be sufficient to provide 1286 U/day.
  • the delivery of this number of muscle cells appears to be feasible, since in a phase I clinical trial of myoblast transfer in Duchenne muscular dystrophy patients, as many as 10*-- myoblasts could be prepared from small muscle biopsy (0.5-1. Og) of first degree relatives and transplanted into patients (Gussoni et al. , Nature . 356:435-438 (1992)) .
  • the present myoblast gene transfer system could further be optimized for clinical applications .
  • the technique may be modified to include: (1) the use of primary myoblasts and/or (2) the use of an implantable immunoisolation device.
  • myoblast gene transfer technology could correct a disease condition (correction of anemia) as a systemic response to EPO transgene expression, and (2) that myoblast gene transfer is feasible for the delivery of genes of interest (not restricted to EPO) in the setting of severe uremia, a disease condition previously untested for this approach.
  • GTCTCCGCCC AAGACCGGGA TGCCCCCCAG GGGAGGTGTC CGGGAGCCCA GCCTTTCCCA 120
  • AACAGCCCCG ACCCCCGGCC AGAGCCGCAG AGTCCCTGGG CCACCCCGGC CGCTCGCTGC 420
  • Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg 125 130 135 GCT CTG GGA GCC CAG AAG GAA GCC ATC TCC CCT CCA GAT GCG GCC TCA 1083 Ala Leu Gly Ala Gin Lys Glu Ala lie Ser Pro Pro Asp Ala Ala Ser 140 145 150
  • GCT GCT CCA CTC CGA ACA ATC ACT
  • GCT GAC ACT TTC CGC AAA CTC TTC 1131 Ala Ala Pro Leu Arg Thr lie Thr Ala Asp Thr Phe Arg Lys Leu Phe 155 160 165 CGA
  • GTC TAC TCC AAT TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG 1179 Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly 170 175 180 185

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Abstract

La présente invention concerne la thérapie génique pour améliorer la production de globules rouges. L'apport et l'expression du gène de l'érythropoïétine déclenchent une augmentation stable de la production de globules rouges. La présente invention traite également de vecteurs d'apport de recombinaison, de compositions, d'autres stratégies de thérapie génique, et de cellules transfectées qui expriment assez d'érythropoïétine pour présenter une réponse systémique physiologiquement significative.
EP95901877A 1993-11-10 1994-11-09 Vecteur de therapie genique pour le traitement de l'insuffisance ou des troubles de la production de globules rouges Withdrawn EP0737252A1 (fr)

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PCT/US1994/013066 WO1995013376A1 (fr) 1993-11-10 1994-11-09 Vecteur de therapie genique pour le traitement de l'insuffisance ou des troubles de la production de globules rouges

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GB9402857D0 (en) 1994-02-15 1994-04-06 Isis Innovation Targeting gene therapy
US6506379B1 (en) 1995-06-07 2003-01-14 Ariad Gene Therapeutics, Inc. Intramuscular delivery of recombinant AAV
US5962313A (en) * 1996-01-18 1999-10-05 Avigen, Inc. Adeno-associated virus vectors comprising a gene encoding a lyosomal enzyme
US5858351A (en) 1996-01-18 1999-01-12 Avigen, Inc. Methods for delivering DNA to muscle cells using recombinant adeno-associated virus vectors
US5846528A (en) 1996-01-18 1998-12-08 Avigen, Inc. Treating anemia using recombinant adeno-associated virus virions comprising an EPO DNA sequence
JP4301347B2 (ja) 1996-03-14 2009-07-22 ジェネンテク， インコーポレイテッド Ｇｄｎｆおよびｇｄｎｆ受容体の用途
AU737129B2 (en) * 1996-08-23 2001-08-09 Arch Development Corporation Long-term expression of gene products by transforming muscle cells
US5952226A (en) * 1996-11-05 1999-09-14 Modex Therapeutiques Hypoxia responsive EPO producing cells
JP2002533124A (ja) 1998-12-31 2002-10-08 カイロン コーポレイション Ｈｉｖポリペプチドの改善された発現およびウイルス様粒子の生成
EP1177285A1 (fr) * 1999-05-07 2002-02-06 Genentech, Inc. Nouveaux polypeptides d'erythropoietine du chimpanze (chepo) et acides nucleiques codant pour ces memes polypeptides
US6831060B2 (en) 1999-05-07 2004-12-14 Genentech, Inc. Chimpanzee erythropoietin (CHEPO) polypeptides and nucleic acids encoding the same
US6555343B1 (en) 1999-05-07 2003-04-29 Genentech Inc. Chimpanzee erythropoietin (CHEPO) polypeptides and nucleic acids encoding the same
KR20030070702A (ko) * 2002-02-26 2003-09-02 사회복지법인삼성생명공익재단(삼성서울병원) 전기적 자극을 이용한 ｄｎａ 전달방법 및 이를 이용한 에리스로포이에틴 발현 방법

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DK173067B1 (da) * 1986-06-27 1999-12-13 Univ Washington Humant erythropoietin-gen, fremgangsmåde til ekspression deraf i transficerede cellelinier, de transficerede cellelinier sa
US5538722A (en) * 1989-06-13 1996-07-23 Stanford University Isolation, growth, differentiation and genetic engineering of human muscle cells
WO1994001129A1 (fr) * 1992-07-02 1994-01-20 The Salk Institute For Biological Studies Utilisation de myoblastes en vue de l'administration prolongee de produits geniques

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