CA2183551A1 - Gene therapy vector for the treatment of low or defective red blood cell production - Google Patents

Abstract

The present invention involves gene therapy for the enhancement of red blood cell production. The delivery and expression of the erythropoietin gene, elicits a stable increase in red blood cell production. The present invention includes recombinant delivery vectors, compositions, alternative gene therapy strategies, and transfected cells which express sufficient erythropoietin to present a physiologically significant systemic response.

Description

WO 95/13376 ;~ ~ 8 ~ 5 ~i 1 PCT/IIS94/13066 .

GENE TllERaPY VECTOR FOR TEIE 'rR~~ ~T OF ~O~ OR
Dl~ lV~: RED BI,OOD CELI- PRODUCTION
p- ~ ;r OF TEI~ INVENTION
Field of fhl~ Tnvention The present invention relates to a novel approach to the treatment of low or defective red blood cell production. In particular, the invention provides for the sustained systemic productlon of erythropoietln following the modification of target cells by gene transfer .
Desl rlption of the Baekcrround Erythropoiesis, the production of red blood cells, occurs continuously to offset cell destruction.
Erythropoiesis is a precisely controlled physiological mechanism enabling suf f icient numbers of red blood cells to be avallable for proper tissue oxygenatlon, 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 o 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.
E~ypoxia may be caused by loss of large amounts of blood due to h -rrhAge, destruction of red blood WO 9~/13376 ~ PCT/US94/13n66 cells by over-exposure to radiatlon, reduction in oxygen intake due to high altltudes or prolonged unconsciousness, or various forms of anemia. In response to tissues undergoing hypoxic stress, 5 erythropoietin increases red blood cell prorillrt ~ nn by 8t~-l1At~ng 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 10 cells. When the number of red blood cells in circulation is greater than needed for normal ~issue oxygen requirements, erythropoietin in circulation is decreased .
Because erythropoietin is essentlal in the 15 process of red ~lood cell formation, the hormone is useful in the treatment of blood disorders characteri~ed by low or defective red blood cell production. While the in~ection of re~ '~nAntly produced human erythropoietin is a proven therapy for 20 the treatment of blood disorders, it would be advantageous to enhance the endogenous production of erythropoletln in a patient or l ~ An sub~ect .
S~rMMARY OF TEIE: INVli NTION
The present invention provides, for the flrst time, the successful development of a method for the enhancement of red blood cell production by gene 30 therapy. The invention demonstrates that expresslon vectors can be constructed uslng an expresslon control sequence and an erythropoietin gene, operatlvely llnked to the control sequence and capable of expression ln transfected target cells, wherein the 35 nucleic acid construct is capable of eliciting the WO 95/13376 21~ 3 ~1 PCT/USg4/13066 S'-' expresslon of erythropoietin sufficlent to increase red blood cell production.
Having elucidated the means for the er,hancement of red blood cell production by increasing the 5 presence of erythropoietin ~n vivo, the present invention supports the development of gene therapy techniques for the treatment of low or defective red blood cell production. Also comprehended by the invention are pharmaceutical compositions involving 10 effective amounts of the nucleic acid constructs together with a pharmaceutically acceptable delivery vehicle including suitable diluents, buffers and ad~uvants. The compositions can further include a carrier capable of promoting target cell uptake of the 15 nucleic acid constructs. Such carriers include liposomes, protein complexes and viral carriers suitable for gene transfer techniques.
We have found no previous report of the use of target cells for the purpose of erythropoietin gene 20 transfer and subsequent ~n vivo production of re~ ; nAnt erythropoietin wlth a demonstrated rhA~-r~-logical response. In a speciflc embodiment, the invention involves the development of myoblast-mediated gene therapy for the in vivo production of 25 erythropoietin. ~rhe 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, 30 can elicit sufficient production and secretion of erythropoietin to present a physiologically significant systemic response.

2 ~ 8 3 ~ri 1 PCT/US94/13n66 --; 4 --. ~ .
BRIEF DESCRIPTION OF TEIE DRAW~NGS
Figure 1 illustrates a DNA sequence for erythropoietin.

DETAILED Dl!:SCRIPTION OF T~E INVENTION
Gene therapy for anemia comprises the delivery of a gene for erythropoietin to cells, either ln v~vo or 10 in vitro. Delivery and expression of the gene results in the production of erythropoietin in a physiologically-functional amount 3ufficient to increase red blood cell production. The erythropoietin gene used in the present invention, is 15 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 20 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 25 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 - --30 expression control system which will include asoperatively 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 35 transcriptional regulatory regions are fllnc~ i on~ l in a l; An host . It may be preferred that a _ _ . . . .

WO 95/13376 '2 1 ~ 3 ~ 1 ; PC'r/US94113066 ", -- 5 --' !
re~ ~ nAnt vector construct not become lntegrated into the host cell genome of the patient or 7 ~ An sub~ect, 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. Thus, the nucleic acld construct includes a DNA sequence which encodes a polypeptide directly responsible for a therapeutic effect, as well as a sequence ~s) controlling the expresxion 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 . In alternate embodlments, the promoter may be modified by the addition of sequences, such as ~nh~n~rs~ or deletions of nonessential and/or undesired sequences. The promoter will have a DNA sequence suEficiently 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. It will also be appreciated by those skilled in the art that the expression control sequence may contain a suppresser sequence to regulate the expression of erythropo iet in .
For the transcriptional initiation region, or promoter element, any region may be used with the 2~3551 ~ .

proviso that it provides the desired level of tr2nscription of the erythropoietin nucleic acid sequence. The transcrlptional initiation region may be native to or homologous to the host cell, and/or to 5 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 lO comprising the transcriptional initiatlon region is to be inserted . By f oreign to the DNA sequence is intended a transcriptional initiation region that is not normally assoclated with the DNA sequence of interest. Efficient promoter elements for 15 transcrlption initiation include the SV40 (simian virus 40) early promoter, the RSV ~Rous sarcoma virus) promoter, the Adenovirus ma~or late promoter and the human CMV (cytomegalovirus) promoter.
Inducible promoters also find use with the 20 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 25 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 30 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 35 inducible element.

WO 95/13376 2 1 8 3 ~ 1 PCrlUS94113066 Transcriptional enhancer ~ q may also be included in the expresæion control seguence. The term "transcriptional enhancer elements" includes DNA
seguences which are primary regulators of 5 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.
lO Different enhancer elements can be used to produce a desired level of transgene expression in a wide variety of tissue and cell types. For example, the human CMV immediate early promoter-enhancer element can be used to produce high level transgene expression 15 in vivo.
Examples of other en_ancer elements which confer a high level of transcription on linked genes in a number of different cell types from many species include enhancers from SV40 and RSV-LTR. The SV40 and 20 RSV-~TR are essentially constitutive. They may be combined with other enhancers which have specific effects, or the specific ~nh;ln~-Pr~ may be used alone.
Thus, where specific control of transcription is desired, efficient ~nh~nC~r elements that are active 25 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 signif icantly increase erythropoietin expression when compared to 30 the use of a single copy of an enhancer element.
Fnh~ncl~r 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 35 effect, thereby increasing the number of tissues in which transcription is obtained. In other cases, the W0 95/13376 2 1 8 ~ 5 5 1 PCrlUS94/13066 ~

presence of two different enhan'cer elements results in ~;1 f.n.; ng of the enhancer e~fects . 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 con 5ugates, 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 . Thus, the present invention; n~ rl.o.c 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. For example, the carrier can include a selected ligand to effectively target the cells of interest. In addition, 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 WO 9S113376 2 1 ~ 3 ~ ~ 1 PCTll~S94113066 _ 9 _ erythropoletin. For the purposes of the present invention, a physiologically active or acceptable level of erythropoletin gene function refers to a level of ln vivo erythropoietin manufacture and 5 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 hematpcrit levels.
The level of erythropoietin gene function sufficient lO 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 15 accordance with the present invention either ~n v~ vo or ln vltro. For example, in in vivo treatments, recombinant erythropoietin vectors can be administered to the patient, preierably in a biologically compatible solution or pharmaceutically acceptable 20 delivery vehicle. ~rhe dosages administered can vary from patient to patient and will be determined by the level of ~nhAnc~ of erythropoietin function h~l~n~ ed against any risk of side effects . Monitoring levels of transduction, erythropoietin expression 25 and/or the levels of red blood cells will assist in selectlng and ad~usting 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, 30 transfected with the erythropoietin gene in accordance wlth the present invention, and then administered to the patient. ~he transfected target cells may be reintroduced by any suitable means, such as in~ection or implantation, and the cells will typically be 35 delivered to target tissue of the same cell type as the target cells. For example, muscle cells may serve ~1~3~51 as the target cells. Myoblasts can be isolated and ~-nlr~ ted in vltro, transfected with the erythropoietin vector, and the transformed cells are then relntroduced into muscle tissue. The unique 5 biology of muscle cells allows the transfected cells to form new myofibers or fuse into old ones. It was discovered that the transplanted nuclei are sustained and active for prolonged periods of time in a normal, mult;nl~r1e~te~ environment with little or no nuclear 10 replication for up to six months. Moreover, it was discovered that 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. E~omologous recombination is a technique originally developed for targeting genes to induce or correct mutations in transcriptionally active genes (Kucherlapati, Prog. in Nucl_ Ac~d Res. and ~ol. Biol.
36:301 (1989) ) . The basic technique was developed as a method for introducing specific mutations into specific regions of the l; ~n genome (Thomas et al., Cell. 44:419-428, 1986; Thomas and Capecchi, Cell. 51:503-512, 1987; Doetschman et al., Proc. Natl.
Acad. SCl. 85:8583-8587, 1988) or to correct specific mutations within defective genes (Doetschman et al., Nature. 330:576-578, 1987).
Through homologous recombination, 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. When two homologous pieces of single stranded DNA (i.e., the targeting DNA and the genomic DNA) are in close proximity, they will WO 95/13376 ~18 3 5 51 PCT/USg4ll3066 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 5 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 10 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 15 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.
If the sequence of a particular :gene is known, a piece of DNA that is complementary to a selected 20 region of the gene can be synthesized or otherwlse 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 25 hybridize to lts 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 30 daughter strand of DNA.
In the present invention, attached to these pieces of targeting DNA are regions of DNA will - interact with the nuclear regulatory proteins present within the cell and, optionally, amplifiable and 35 selectable DNA markers. Thus, the expression of erythropoietin may be achieYed not by transfection of WO 95/13376 ~ l 8 3 55 1 PCr/lTS94113066 DNA that encodes the erythropoietin gene itself, but rather by the use of targeting~ ~NA (regions of homology with the endogenou$ gene of interest) coupled with DNA regulatory segments that provide the 5 endogenous erythropoletin gene with recognizable signals for transcription. With this technology, it is possible to express and to amplify any cognate gene pre~ent within a cell type without actually transfecting that gene. In addition, the expression lO 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. Furthermore, the expression characteristics of any cognate gene present within a 15 cell type can be modified by appropriate insertion`of DNA regulatory segments and without inserting entire coding portions of the gene of interest.
In accordance with the present invention, homologous recombination provides new methods for 20 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 25 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) 30 regulatory and/or amplification segments. A positive selectable marker is optionally included within the construction to facilitate the screening of resultant cells. The use of the neomycin resistance gene is preferred, although any selectable marker may be 35 employed. Negative selectable markers may, optionally, also be employed. For instance, the .. . , . ... .. . .. _ _ _ _ _ _ _ _ _ _ _ WO 95113376 2 1 8 3 ~5 1 PCTII~S94113066 He~pes Simplex Virus thymidine kinase (HSVtk) gene may be used as a marker to select against randomly integrated vector DNA. The fused DNAs, or existing expressing DNAs, can be amplified if the targeting DNA
5 is linked to an amplifiable marker.
In the specific examples which follow, a myoblast cell line was est~hl; shf~d which stably expressed the human erythropoietin gene. The cell line was l0 established by transfecting the cells with a plasmid containing the erythropoieti~L gene driven by a CMV
promoter. The plasmid was derived from pCD vector l (Okayama et al., A cDNA cloning vector that permits expression of cDNA inserts in ~ n cells. Mol.
Cell. Bio. 3:280-289, 1993) as described in the following examples. 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 20 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 radio~ Rsay.

2183~5i Table 1~L
Erythropoietln (EPO) Ex~prèssion in Transformed Myoblasts as Determined by RIA
C2 cell clone Erythropoietin (units/ml) 60k, EPO2, #6, 1/14 4. 9 60k, EPO1, #5, 1/11 28.7 30k, 7, 1/11 0.16 60k, EPO1--2, #1, 1/11 11.8 60k, EPO1, #4, 1/19 2.37 60k, 3a, #6, 1/11 ~ 1.27 120k, 4, 1/11 0.38 60k, EPO1-1, #1, 1/11 2. 6 60k, EPO1, #10, 1/11 2.17 60k--2, EPO1, #10, 1/11 22.8 60k--2, EPO1, #8, 1/11 2.3 60k, EPO1, #8, 1/11 0.18 As illustrated in Table 1, twelve clones had measurable erythropoietin production. The assay involved a typical RIA procedure and was performed substantially in accordance with the method described 10 by Egrie et al., .Journal of rznrnunolo~ical Nethods, 99:235-241 (1987). Values for erythropoietin levels in the media of the positive clones ranged from 0.18 to 28 l~nits per ml. This represented a production/secretion range of approximately 2 x 1o-8 15 to as much as 5 x 10-7 Units per cell per hour (assuming linear synthesis and secretion and no significant decay of activity) .
The present invention demonstrates the efficacy of gene transfer to obtain sustained in v~vo 20 production of a therapeutic polypeptide, such as 21~3~1 WO 95113376 - PCr/US94113066 erythropoietin, at levels sufficient to enhance red blood cell production. It will be appreciated by those skilled in the art that refinements in the selection of promoter and enhancer genes will Yerve to optimize the e~pression of erythropoietin in the transfected target cells. For example, it is also within the present invention to use muscle-specific expre3sion control sequences for high level recombinant protein expression ln transformed muscle cells. High activity promoter and erhancer cassettes can be used to intensify recombinant gene expression.
Such promoters will increase the levels of therapeutic re, ~ n~nt proteins synthesized and secreted by both newly formed myofibers as well as muscle fibers that contain a mixture of donor and recipient myonuclei.
The ma ~or contractile proteins of thin and thick :Eilaments (e.g., alpha-actins, troponln C, myosin heavy chains, as well as several muscle enriched enzymes, such as creatine kinase and carbonic anhydrase III) all have genes that are expressed at high levels in muscle. Promoters and enhancers of most of these genes have been the sub ~ects of intense investigation and analysis (reviewed in Bishopric, et al., The molecular biology of cardiac myocyte hypertrophy p. 399-412. In L.EI. Kedes and F.E.
Stockdale [ed. ] . Cellular and Molecular E!iology of Muscle DevelopmeDt. vol. 93, Alan R. Liss, Inc. New York 1986; and Wade, et al., Developmental regulation of contractile protein genes p. 179-188 . [ed. ], Annu.
Rev. Phys~ol. vol. 51, Annual Reviews, Inc. Palo Alto, 1989). Muscle-specific gene e~pression is usually associated with muscle-specific transcription factors including members of the MyoD family (Weintraub, et al., The myoD gene family: nodal point during ~pe~-~ f i ~tion of the muscle cell lineage .
Science. 251:761-766, 1991) and the MEF-2 site binding 2183~1 factors (Cser~esi, P. and E.N. Olson Myogenin lnduces the myocyte-speclflc enhanceriblnding factor MEF-2 in~el~elldently of other muscl~e--specific gene products.
Molecular And Cellular ~loiogy. 11:4854-62, 1991; and 5 Olson, et al., Molecular control of myogenesis:
antagonism between growth and differentiation. Mol.
Cell . Biochem. 104 :1-2, 1991. ) . The approprlate selectlon and combination of vector elements will provlde for optlmal regulation of muscle cell gene 10 expression and sustained high levels of expresslon of recomblnant genes lntroduced lnto muscle cells.
A number of muscle-speclfic genes have been cloned, and the promoters analyzed: these lnclude skeletal actln, cardiac actin, Troponln C fast, 15 Troponin C slow and Troponin I slow, as well as beta and gamma cytoskeletal actins. Other muscle specific promoters that have been the sub~ect of detailed analysis include creatine kinase, myosin light chains and various myosin heavy chain genes as well as 20 Troponins I, T and C. Detailed analyses of such enhancer and promoter regions that provide muscle speclflcity are available, as illustrated by the following brief summary.
Skeletal C~-acti n, The tissue speclfic distal 25 promoter of the human skeletal a-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 lncreases transcrlptlon 100-fold (Muscat, et al., 30 Multiple 5 ' flanking regions of the human skeletal actin gene synergistically modulate muscle specific gene expression. Nol. Cell Biol. 7:4089-4099, 1987) A short fragment of the distal promoter, the distal regulatory element (DRE) from -1232 to -lI77,--35 functions as a muscle-specif ic composite enhancer (Muscat, et al., The human skeletal c~-actin gene is . . . _ _ _ _ _ _ . . . _ _ _ _ _ _ _ . _ _ . _ . .

WO 95/13376 2 1 % 3 ~ 1 PCT/US94113066 regulated by a muscle-specific enhancer that binds three nuclear factors.
~ ;.r~i~s,c a-actln: The cardiac a-actln gene is the fetal isoform of a-actin in rodents and human muscle, 5 ~ut 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 a-actin represents about 5~6 of the a-actin mRNA. The cardiac a-actin promoter lO 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.
SkeletA 1 F~ Ct-twitch Tro~on; n C ~ene: 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 20 PnhAnr~Pr 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 o~her muscle 25 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 30 responsible for its muscle restricted expression.
Slow-Twitch/c~r~ c Trop~nln C crene: At least four separate elements cooperate to confer muscle specific expression on the human slow twitch skeletal/cardiac troponin C ~HcTnC or TnCslow) gene:
35 a basal promoter ~from -61 to -13) aug~ents wo gs1l3376 2 ~ 8 3 ~ 5 ~ PCTIUS94/13066 transcription 9-fold, upstream ma~or regulatory ser~uences ~from -64 to -1318 and from -1318 to -4500) augment transcription 18-fold ànd 39-fold, respectively, and a positipr~`and orlentation 5 ~n~l.or~n~iont enhancer in the first intron (from +58 to +1519) augments transcription 5-fold. This enhancer increa~es muscle specific CAT activity when linked to its own promoter elements or to a heterologous SV40 promoter, and the effects appear to be multiplicative 10 rather than additive. When the various promoter/CAT
chimeric plasmids are cotransfected with a MyoD
expression vector into 10 T 1/2 cells, constructs carrying either the TnC promoter or the f irst intron of the gene are >500-fold induced. Thus, each of 15 these regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD. These observations suggest that skeletal muscle expression of the HcTnC gene is cooperatively regulated by the complex interactions of 20 multiple regulatory elements.
Slow-twitrh Tro~rn ~ n I ~ene: At least three separate elements spaced over 1 kb of the 5 ' upstream regions of the human slow twitch troponin I gene (HsTnI) combine to synergistically regulate muscle 25 specific gene expression. A basal promoter lies within 300 base pairs of the transcription start site and two in~l~r~n~ipnt muscle specific enhancers 800 and lO00 base pairs upstream. All three appear to be re~uired for expression. These observations suggest 30 that muscle expression of the HsTnI gene is cooperatively regulated by the complex interactions of multiple regulatory elements.
cDNAs encoding the erythropoietin gene may be 35 cloned into a variety of in-frame expression vectors.
A non-muscle-specific beta-actin promoter, constructed _ _ _ _ _ _ . _ _ _ _ = = .. . . .

2~ 83'S~1 WO 95113376 PCrlUS94113066 as a high level expresslon 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 ~-CIlml-lAtion of antisense transcripts.
Proc. Natl. Acad. Scl. USA. 84:4831-5; 1987). The use of such a construct fosters high level transcription of inserted sequences in 1; iln cells .
The erythropoietin sequence may also be cloned into an internally deleted human skeletal ~c-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-l-neo) .
Typically, 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.
Twenty four hours after infection, the cells may be split 1:20 into 60 mm dishes with D~EM containing 20%
fetal calf serum (FCS) and 0 . 4 mg/ml of neomycin (Geneticin~lD G418; Gibco Laboratories, Grand Island, NY). After 14 days of selection, individual clones may be isolated and .o~r7~ntl~1 in DME~ containing 20%
FCS and 0 . 2 mg/ml of G418 . Since the ability of - transferred cells to differentiate into myotubes ln vivo is a likely requirement for their stability and - longevity ln situ, the clones are tested for their ability both to differentiate into myotubes in D~IEM
supplemented with 2% horse serum and to express beta-galactosidase. The ,13-galactosidase expression serves ,, Wo 95/13376 PCT/US94/13066 as a histological marker to monltor survival, as well a3 both macroscoplc and microycopic location, of =~
in~ected myoblasts at the conclusion of the studies.
Individual clones ar~ PYr~n~ and the culture media tested for polype`ptide 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 ~n vltro. 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 v~vo manipulation and the numbers of cells required for gene therapy muscle cell transplant .
As described above, there are a number of strong muscle specific promoter and enhancer elements in the genes for contractile proteins Plasmid expression vectors may be constructed from several of these components linked together. For example, a construct may include the parent skeletal actin chl~ hPnl col acetyltransferase (CAT) expression vector containing the upstream enhancer and the muscle specific promoter. 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. ~ol. Cell. Biol. 9:3393-3399;
1989. ) . 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-WO 95/13376 2 1~ 3 S 5 ~ PCT/US94/13066 luciferase as a positive control standard in the search for promoters with heightened transcriptional activity. The plasmids may also be transfected into non .t~r~lr~ (Hela or CVl) cells to evaluate their 5 degree of muscle specificity. Beta-actin and RSV CAT
constructs may be transfected into the same cells in parallel to serve as comparisons. Once a ~ nAt~r n of enhancer/promoter elements that significantly augment transcription is identified, the regulatory l0 region is transferred to replace the promoter in the neomycin vector (pHaSEApr-l-neo) described above along with a beta-galactosidase expression vector.
The present invention is further described by the following specific examples, which are illustrative 15 but non-limiting.
EXA~IP I.E S
Example l Construction of Erythropoietin cDNA for expression A polylinker was inserted in the unique PstI site of a pCD vector l (Okayama et al., A cDNA cloning vector that permits expression of cDNA inserts in l~An cells. ~ol. Cell. Bio. 3:280-289, 1993) to generate the Vl9 vector. The Vl9 . l vector was derived from the Vl9 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 - Eco R I and Hind III to which the Bst E II to Hind III
fragment of EPO cDNA and a Bst E II-Eco R I linker - (see Table 2) were ligated to form Vl9 . l EPO.

2183~51 WO 95113376 pcTlus94ll3n66 ; ~ -- 2 2 Table 2 Linker and Adapter Constructs the ~fl~ter to c~nctruct V19 from Drr~
5' agctgaattctctagaaaagctt 3' 3' cttaagagatcttttcgaattaa 5' 10 thf~ Bst E II-Eco R I 1 inker:
5 ' aattcccccccgtgtg 3 ' 3' yyyyyyy~:~caccagtg 5' EPO cDNA was isolated from the vector V19.1 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 20 (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 25 alpha competent cells were transformed with the pRC/CMV-huEPO plasmid. Plasmid DNA was isolated, sequenced and used for transfection of 1 ~;~n cells .
3 0 Example 2 Transfection and screening of clones ~ouse 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 WO 95113376 2 18 ~ S ~ ~ PCTIUS94113066 1 .
~ i - 23 --consisting of Dulbecco's modified Eagle medium ~DMEM) supplemented with 20% fetal bovine serum, 0 . 5% chick embryo extract ~Gibco Laboratories) and 100 llg/ml of kanamycin ~Gibco ~aboratories) in 10% Co2.
5 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
pla3mid of Example 1. Transfection mixtures were prepared as follows: a 501ution of 250 mM CaC12 ~ 5 ml) was added dropwise to 8 llg of DNA in 0 . 5 ml of 2 x N-2-hydroxyethylpiperazine-N' -2-ethanesul f onic acid ~HEPES) -buffered saline (42 m~ E~EPES [pH 7 . 05], 270 mM
NaCl, 10 mM KCl, 1.4 mM Na2HPO4, 11 mM dextrose).
This was done with constant mixing. 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 ~8 hours.
Three days a~iter transfection, the cells were split at 1:10 and incubated for 12 hours. For neomycin resistance selection, G418 was added to the medium at a final concentration of 400 llg/ml. The cells were supplemented with fresh growth medium c~nt~n~ng 400 llg/ml of G418 every three days. After two weeks of incubation, 23 colonies were selected, and expanded.
For detection of erythropoietin producing clones, each clone was cultured at 1 x 106/100 mm dish in growth medium overnight, and incubated in serum-free - DMEM (3 ml) for three days. The culture medium from each clone was collected, and erythropoietin concentration was det~rm~n~l by radi~ qc~ay. The clones were aliquoted and stored in liquid nitrogen.

2 7 83~51 Wo 95/13376 PCTNS94113066 Example 3 Myoblast Transplantation The highest produclng clone was cultured in 150 mm dishes in growth medlum contalnlng 200 ~Lg/ml of G418. When the cells reached 80% cnnfl~lonce~ they were trypslnized and collected ln lce-cold PBS. Total cell number was determined by hemocytometer. Cells 10 were rinsed once again with PBS to remove resldual trypsln, pelleted, and resuspended ln a small volume of PBS to a concentratlon of 1 x 108 cells/ml. We routlnely collected 2-3 x 108 cells for each experiment. Cells were kept on lce until use to 15 prevent aggregation.
C3H mice syngeneic to the C2 cell llne and nude mlce (both 6-8 week-old) were used for transplantation. Animals were anesthetized by intraperltoneal admlnlstratlon of a mlxture of 20 ketamlne ~30 mg/kg) and xylane (4 mg/kg). A total of 4 x 107 cells per mouse were ln~ected percutaneously through a 27 gauge needle at~40 dlfferent sites (1 x 106 cells/10 ~Ll/site) into skeletal muscle tissue of both hlnd limbs. As a control, the same number of 25 parental C2 cells were transplanted ln the same manner .
Example 4 Hematocrit Measurement Hematocrlt was measured by mlcrohematocrlt method. Under general anesthesia, a total amount of approxlmately 200 111 of blood was collected by a retroorbltal approach lnto three heparlnized capillary 35 tubes. Blood collection waS performed one week prior to transplantation, three days and one week after _ .

WO 9S/13376 2 1 8 ~ 5 5 ~ PCTIUS9~113D66 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 5 using a Coulter counter which showed parallel results with micro-hematocrit method. After hematocrit measurement, the plasma was collected and stored at -20C for huEPO concentration measurement.
Example 5 Cell monitoring a) Tr~nq~l~ntation of C2 cel 1~: oYnre88~ n~ B-ctosi~lA~e To monitor the fate of in~ected cells, another C2 cell line which stabily expressed ,l~-galactosidase was established. The cells were selected by neomycin resistance, and several clones expressing p-galactosida8e were collected. One clone was used for transplantation into right hind limb (2 x 107 cells/mouse) .
b) B ~AlActo8i~1ARe Assays The mice were sacrificed by cervical dislocation for the histochemical detection of ~-galactosidase expression. Skeletal muscle tissue was excised and frozen i ~ Ately 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 PP,S containing 1 mg/ml of 5-bromo-4-chloro-3-indolyl-~D-galactoside, 5 - mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgC12. Sections were incubated at 37C overnight, rinsed in PBS, mounted and studied under microscope.

; s' c) Reveræe tran~cription-pol -ra~e ehA;n reaction (RT--PCR ) To confirm ~n vivo huEPO gene expression, muscle 5 tissue was excised from the mlce ln~ected with the huEPO expresslng C2 cells. The excised muscles were frozen by li~uid nitrogen and homogenized by Polytron homogenizer (Brinkman Instruments, Company, NY) .
Total RNA was isolated using Tri Reagent (Molecular 10 Research Center, ~-ln~lnnptl, OH) accordlng to the manufacturer's protocol. The RNA (7011g) was resuspended in 50 ~Ll of Tris/EDTA (pH 7 . 4) . To this was added 50 111 of Tris/EDTA (pH 7.4) containing 8 U
of RNAse-free DNAseI (30ehringer MAnnh~;m, 15 Indianapolis, IN), 4 U of placental RNAse inhibitor (Promega, Madison, WI), 20 mM MgC12, and 2 mM
dithiothreitol. The reaction was stopped by the addition of DNAse stopping mixture l -~ntAln;ng 50 mM
EDTA, 1.5 M sodium acetate ~pH 4.8) and 196 sodium 20 dodecyl sulfate. The RNA was treated with phenol/chloroform, chloroform, and ethanol-precipitated. For reverse transcription reaction, approximately 5 llg of RNA and 4 pmol primer complimentary to the 3 ' untranslated region of huEPO
25 RNA were incubated at 70C, rapidly cooled on ice, and treated with 100 U of reverse transcriptase (Superscript; Gibco Labor~tories). The obtained cDNA
was amplified by known PCR methods using primers including the initiation and stop codons.
d) Results Table 3 shows the time course of mean hematocrit change after the transplantation of C2 cells expressing huEPO gene. Hematocrit started to increase 35 three days after the transplantation of 4 x 107 cells into C3H syngeneic mice (A in Table 3). The pea3c WO95/13376 218 3~ ~ PCT/us94ll3n66 hematocrit was achieved two weeks after the transplantation. Hematocrit declined gradually thereafter, becoming lower than the basal level after wee3c 4.
RT-PCR showed persistent huEPO mRNA expression for at least one month in the injected muscle, but not in the muscle from unin~ected left hind limb. Mice transplanted with parental C2 cells did not show significant hematocrit increase. In contrast to C3H
mice, nude mice (B) showed significantly higher and more sustained hematocrit increase for at least tw~
months. When half the number of cells (2 x 107) were in~ected, the net hematocrit increase was also approximately half of that observed with 4 x 107 cells, thereby indicating that huEPO production can be regulated by cell number.
The mice transplanted with the C2 cells expressing ~-galactosidase showed positive myofibers over the entire in ~ected sites three months later.
The in~ected C2 cells appeared to fuse among themselves as well as with preexisting myofibers. No ¦3-galactosidase positive myoblasts were observed.
Serum huEPO c~nc~on~rAtion as determined at several points after transplantation by either 25 r~ say or bioassay using an erythropoietin-responsive human leukemic cell line (UT-7/EPO) showed significantly elevated erythropoietin concentrations ranging from 90 to 3500 mU/ml. Erythropoietin concentrations before transplantation in these mice 30 were <25 m~/ml.

2183~1 Wo 95/13376 - PCTIUS94113066 ~ ':
Tab~1e ~3 Mean Hematocrits in C3H Mice (A, n=7 ) and Nude Mice (B, n=5) pre-injection 3d lw 2w 3w 4w 5w 6w 7w (A) 42.9 45.1 53.5 58.6 50.2 41.8 36.5 33.1 34.4 ~B) 43.0 55.8 60.0 67.0 59.1 67.4 57.3 64.0 66.3 Example 6 Myoblast Gene Transfer to Correct Anemia Associated with Renal Failure The current ma~or indication for recombinant human EPO administration is anemia associated with end-stage renal failure (Faulds et al ., Drugs. 38: 863-899 (1989) ) . Here, 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 20 a~L~a~o~ sllff~ri~nt ~o c4rrec:t anemia for a long ter~in these uremic sub~ects. 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 25 renal failure subjects as effectively as in normal sub~ects. Thus, myoblast gene transfer is means to correct anemia associated with renal failure as well as other types of EPO-responsive anemia.

WO 95/13376 2 1 ~ ~ ~ 5 1 PCrlUS94113066 ~Q~
a) TrAnRfeoti~n and soreen~ng of ~10neR
Human EPO-secreting C2 myoblast clones were prepared as described above. The clone3 carry the 1.34 kb human EPO cDNA (starting at + 190 nucleotide from the major transcription inltiatlon 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-EPO9, produced approximately 33 IJ/106 cells/day of human EPO
as determined by radioi OARqay. The fllnt-t;onAl activity of EPO produced by this clone was confirmed by an ln vitro bioassay.
b) ~yohlARt tranq~pl AntAtion Myoblasts from C2-EPO9 were cultured and harvested as previously described. Under general anesthesia, a total of 4 x 107 cells were in~ected through a 27-gauge needle at 40 different sites (1 x 106 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).
c) il :oerit - Rll Hematocrit was measured by the microhematocrit method (Koepke, J.A., ed. Practical Laboratory Hematolo~y,. 1991, Churchill Livingstone: New York.
112-114). Each week, under general anesthesia, 150 ml of blood was collected by a retroorbital approach into two heparinized capillary tubes. On several occasions, the hematocrit was also measured using a 35 Coulter Counter ~which showed results parallel to those obtained by the microhematocrit method (not shown).

~83~51 After hematocrit measurement, serum was recovered from the capillary tubes and stored at -20 C degree for the measurement of EPO concentration,~ànd BUN.
d) rr~AtiOn of r~nAl fA~ lure 1 del u~in~ nude mlce A renal failure model was created by a two-step nephrectomy (Chanutin et al., Arch. ~ntern ~ed.
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 kldney 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, Madlson, WI) ~r~ntA~n~n~ 24.0% protein and 1.0% phosphorus, and water ad libitum. Renal failure was ronf~ ~ by the development of both anemia and uremia. For uremia, blood urea nitrogen (BUN) was deternLined weekly with a BUN kit (Sigma 535--A; Sigma, St. Louis, MO) using four milliliters of serum.
e) ~oA,'Ill -nt of serum EPO . r,ncentrat~on Serum concentrations of human EPO were determined by an en2yme linked immunosorbent assay ~ELISA) system using a mouse monoclonal antibody accordlng 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.
f) B--~A 1 ACtosi(:Ase assavs After euthanasia, skeletal muscle tissue was excised and frozen immediately on dry ice. The exclsed muscles were then sectloned with a freezinc _ _ _ _ _ _ . _ . . . , . _ WO 9~113376 ~18 3 5 ~ i PCT/IJS94113066 mlcrotome. The sections were attached to microscope 31ides, fixed in 0.25~ glut~rAl~7~hyde for 10 minutes, washed in PBS for 10 minutes, and stained in PBS
c~nts~n;ng 1 mg/ml o~ X-gal, 5 mM potassium 5 ferrocyanide, 5 mM potassium ferrocyanide, and 2 mM
MgCl2. Sections were incubated at 37 C overnight, rinsed in PBS, mounted, studied under microscope, and photographed .
g) Stat ~ Rti- A 1 ~nA 1 ysis Statistical signi~icance was assessed by Student ' s t-test . p<0 . 05 was taken as significant .
Data were expressed as means + 5 . D .

a) Tr~ns~ ntation o~ EPO-produc;n~r m~vobl~qts lnto nor~-l nudo m~-e After the transfection with pRC/CMV-EPO, the clones were screened by G418, and 23 clones were 20 randomly selected. Eleven of twenty-three C2 myoblast clones had measurable EPO as det-rm; nf.fl by RIA ranging from 0.18 to 32.8 U/ml/106 cells/day. Using an ~n vltro bioassay with EPO-dependent human leukemic cell line, it was confirmed that the EPO secreted from 25 these engineered muscle cells is fllnr~;on~l ly active.
Transplantation of the cells from C2-EPO9 yielded a marked hematocrit increase ~or at least three months in healthy normal nude mice, while mice transplanted with parental C2 cells did not show a significant 30 hematocrit change during the period, as summarized in Table 4.

~183551 WO 95/13376 PCTNS9~/13066 Table 4 Persistent Elematocrit Increase by Transplantation of E~uman EPO-producing Myoblasts into Non-uremic Nude Mice .~ -Time ( weeks ) n 2 4 ~ 1 control 44.8 47.0 47.2 48.8 49.0 C2 cells i 2 . 4 i 3 .1 i 208 i 3 . 0 i 2 . 3 ~n=6) C2--EPO9 44.6 71.2 72.2 67.8 58.0 (N=9) i 3 .0* i 7 . 9:1: i 7 . 9:1~ i 11.8~ i 8.1 Nude mice were transplanted with 4xlO 7 cells from either control C2 or C2-EPO9, and the microhematocrit was measured weekly by a retroorbital approach.
~Significantly different from *.
Significantly different from * and ~
b) rreatirln of r~nAl ~l lure I ~df l ln m~ce 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 ., Cl inl cal Irrununology and ~mmunopathology. 35:276-284 (1985)).
Survivors of the acute phase of surgery (17 mice) were followed weekly with hematocrit and BUN
determinations. After three weeks, the mice were divided into two groups. Group I included five mice that showed only transient anemia during the three week observation period and were followed without transplantation. 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.
In Group II, the mean hematocrit decreased from a preoperative level of 45.2i2.7 to 33.9i3.7 (%0) three weeks after the second nephrectomy. The Group I mice -WO 95/13376 ~ 1 8 3 ~ ~ PCT/IJS94/13066 did not develop further anemia, and the degree of BUN
increase was much lower than that of the Group II mice (52.6il3.2 vs. 95.4~16.5 three week3 after the second nephrectomy); presumably due to insufficient 5 nephrectomy.
Among Group II mice, eight mice were transplanted with C2-EPO9 cells, and three mice were followed without transplantation as a non-transplantation control (one mouse died ~ust before transplantation, 10 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. All of the Group II mice, except one, died between six and eleven weeks (8.2:t1.8 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., ~i~ney International.
30:433-436 ~1986) ) . The one long term survivor in Group I also had the lowest levels of BUN in that group. These data clearly demonstrate the feasibility and potential efficacy of a myoblast gene transfer system even in the face of severe renal failure in mice.
c) Serl-m PPO level To examine the secretion of EPO protein from the transplanted cells, serum human EPO concentration was measured using an EEISA. As previously <l~t~orm1 n~
this method did not detect a significant level of 2183~51 WO 95ll3376 PCTIUS94/13066 ~, mouge EPO (<2.5 mU/ml) in Sera of nude mice phlebotomlzed (150 mll 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 ~ust the human EPO
produced by the transplanted muscle cells and not endogenous EPO levels. A week after transplantation with C2-EPO9 cells, the serum EPO level was 87.3i22.1mU/ml in group II uremic mice. It declined to 53.8:t:18.7 mU/m at week 2, and a similar concentration was maintained thereafter until week 8.
Thus, the transplanted C2-EPO9 cells persistently produced human EPO at a steady rate for at least two months after transplantation into mice with severe renal failure.
d) The fate of tr;~n~lAnted EPO-secre~ ng myol~lA~ts ~n rPn;ll faill-re r ,IP1 To analyze the fate of transplanted myoblasts, C2-EPO9 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-EPO9 clone had already been maintalned ln the presence of G418, BAG-transduced clones were selected by positive X-gal stainlng. Cells from one X-gal posltive clone (clone 9-BAG) were .oxr~n~lP~ and transplanted lnto nude mice wlth renal fallure accordlng to the same protocol used for C2-EPO transplantatlon. These mlce also showed a marked hematocrit increase as observed ln the C2-EPO9 transplanted group II mlce (not shown). Slx weeks later, X-gal posltlve myofibers were detected in the entire area of transplantation. At some sites, most of the myofibers were X-gal positive, while at other sites, both X-gal posltlve and negative myofibers _ _ _ _ _ _ _ _ _ . _ _ _ _ _ _ _ _ _ _ _ _ WOg5111376 218 35S 1 PCT/US94113066 coexlsted. These results demonstrated that the transplanted EPO-secreting myoblasts differentiated by fusing with preexisting host myofibers or themselves and that the transgenes were actively expressed from 5 the transplanted cells for the duration of the experimental protocol.
Di.qcucsion It is unknown how other abnormalities in the lO uremic syndrome including electrolyte disorders, metabolic acidosis (i . e ., glucose intolerance), gastrointestinal disorders, neurologic abnormalities, and metabolic disorders, might affect the outcome of myoblast transplantation. Furthermore, the erythroid 15 response to EPO is significantly reduced, and red blood celL survival is shortened in uremia. In the present experiment, however, it was demonstrated that the transplantation of transformed myoblasts could, even in the face of severe uremia, deliver more than a 20 suf f icient amount of human EPO to correct anemia due to renal failure. Furthermore, the therapeutic effects lasted for at least two months. l.onger periods of analysis were limited by animal death probably due to severe renal failure. However, wlth 25 concomitant treatment of renal failure, it ls likely the hematocrit increase would persist for more than two months, as was the case with non-uremic mice (Table 4) .
The observation that the serum EPO concentration 30 was still high at two months, together with the fact that the half-life of red blood cells in mice is 20-45 days, also suppgrts the likelihood that the increased hematocrit would have been sustained longer than two months, if the uremia had been corrected by dialysis.
35 The sustained high serum human EPO concentration due to transformed myoblast transplantation ~ nf; ~--~ that WO 95/13376 21 g 3 ~ ~ 1 PCT/US94/13066 the observed hematocrit lncrease was due to human EPO
derived from the transplanted~cells rather than from endogenous mouse EPO. The perslstent presence of X-gal positlve myofibers after clone 9-BAG
5 transplantation further supports the notion that the transplanted myoblasts differentiate into myofibers and become a stable source of EPO production in the face of renal failure.
End-stage renal failure patients as well as 10 patients with hypoproliferative anemia secondary to 3 ' -azido-3 ' deoxythymidine (AZT) administration are currently treated with 100-150 U of re~ '-;nAnt 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/106 cells/day, 2 . 6-3 . 9 x 107 cells would, in theory, be sufficlent to provlde 1286 U/day. The dellvery of this number of muscle cells appears to be ~R~hl~, since in a phase I clinical trial of myoblast transfer in Duchenne muscular dystrophy patients, as many as 108 myoblasts could be prepared from small muscle biop3y (0.5-l.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 a~elications. For example, the technique may be modified to include:
(1) the use of primary myoblasts and/or (2) the use of an implantable; ~,~ Rolation device. Although primary myoblasts might be transfected with EPO cDNA
to secrete EPO and increase hematocrit in mice, this approach would require customized preparation of cells for an individual patient to avoid immunore~ection.
In this regard, a stocked cell line with an immunoisolation device might be a more practical approach for a large population o~ patients. Withln 2183$51 WO 95/13376 PCTtUS94tl3n66 such a device transformed myoblasts appear to retain an ability to differentiate ~Liu et al., Numan Gene Therapy. 4 :291-301 (1993~ ) aQd are likely to become a stable source of recombinant protein production.
While mice appear to tolerate the unusually high hematocrit for several months, overproduction of EPO
could have potentially deleterious conse~uences including polycythemia. Although recombinant gene production can be controlled to some degree by the number of cells transplanted, regulated transgene expression could also be achieved, such as by the use of inducible promoters to drive genes of interest, as mentioned above.
The present study demonstrated (1) that myoblast gene transfer technology could correct a disease condition (correction of anemia) as a systemic response to E~O transgene expression, and (2) that myoblast gene trans~er 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.

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(A) NAME/REY: CDS
(B) LOCATION: 625.. 1203 (Yi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AAGCTTCTGG GCTTCCAGAC CCAGCTACTT TGCGGAACTC Arr~rcrAr GCATCTCTGA 60 AAr.Arcrr.r.A Tr~rcrrcrAr~ GGGAGGTGTC rr7GnAr~crA ~ ,A 120 r.ATArrAr~ TCCGCCAGTC cr~ Trr r7rAArcr~r-rT GCACTCCCCT rrrr,rr.Arrr 180 Arr"3~CCGr7r7 Ar~rAr~rcrrc ATr:ArrrArA cr7rArr~TrTr~ rAr~rAr~crrr GCTCACGCCC 240 WO 95/13376 ~ 1 8 ~ PCT/US94113066 rrGrr~Ar~rrT rAArcrAr~r~r ~ ,C TGCTCTGACC ~ ~l CCTACCCCTG 300 GCGACCCCTC Arfir.ArArAr. C~ ArrrcrArrr GrGrArGrAr ACATGCAGAT 360 ~rArrcrrG Arrcrcr.r.CC Ar.Arrrr,rAr. AGTCCCTGGG rrArccrr~r~c C~ 7c, 420 ACCGCGCTGT CCTCCCGGAG rcr-r~Arrr~r~ rrArrGrrr ~ 480 0 Crr.ArArrGr ~ .A rA~:rrrrrrT CTCCTCTAGG r~ a 540 rrr,rrrAr.rT TCcrrGr.ATr. Ar~rGrccrrr~ r~TrTGr~TrAr ~ CCAGGTCGCT 600 ~Arrr.ArCcr r~rrAr~rGr GGAG ATG GGG GTG CAC GAA TGT CCT GCC TGG 651 Met Gly Val His Glu Cy~ Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lya Glu Ala Glu Asn Ile Thr Thr Gly Cy:l Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu Hi3 Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala A~ip Thr Phe Arg Lys Leu Phe 2183~1 Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cya Arg Thr Gly Asp Ar~
CCACCTCCCT CACCAACATT ~ A CACCCTCCCC CGCCACTT GAACCCCGTC 1293 GAGGGGCTCT r~rrTr~-rr, rr~rr~TrTr CCATGGACAC TCCAGTGCCA GCAATGACAT 1353 15 AACTTGAGGG rcr~r.~rr~r. GAAGCATTCA r~r~r.r~rT TTAAACTCAG G~:~r~r~r~rr 1473 ATGCTGGGAA GACGTGAG CTCACTCGGC AcccTGcAaA ATTTGATGCC ~r~r~r~rG~T 1533 TTGGAGGCGA TTTACCTGTT TTCGCACCTA rrATr~t3~ CAGGATGACC TGGAGAACTT 1593 x~ ~ CACCGGGGTG GTGGGAACCA TGAAGACAGG ATGGGGGCTG ~.~X, I C~ 1713 25 CTCATGGGGT CCAAGTTTTG TGTATTCTTC AACCTCATTG ACAAGAACTG A~rrArr~ 1773 I~Al~A~AAI~ AAAAAA 1789 30 (3) INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE r~ ;S
~A) LENGTH: 193 amlno acids ~i3) TYPE: amino acid ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein ~xi) SEQUENCE DESCRIPTION: SEQ ID No:2:
~5et Gly Val ili3 Glu Cy8 Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu Ile Cy3 Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 50 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu llis Cys Ser Leu Asn Glu Asn Ile Thr val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg et Glu Val Gly Gln Gln Ala Val GIu Val Trp Gln Gly Leu Ala Leu 2183~51 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val AYn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu EliY Val AYp LyY Ala Val Ser Gly 115 120 lZ5 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Ly3 Glu 0 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155. 160 Thr Ala AYp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser AYn Phe Leu Arg Gly LyY Leu Lys Leu Tyr Thr Gly Glu Ala CYB Arg Thr Gly AYP

Arg

Claims (19)

- 42 - What is claimed is:

1. A method for increasing red blood cell production, comprising the steps of:
transforming muscle cells with a nucleic acid construct comprising an expression control sequence and an erythropoietin gene which is operatively linked to said control sequence, wherein said muscle cells are transformed in vivo, or wherein said muscle cells are transformed in vitro and subsequently inserted into muscle tissue; and expressing erythropoietin protein in said transformed muscle cells in an amount sufficient to increase red blood cell production over pretreatment levels.

2. The method according to Claim 1, wherein said expression control sequence comprises a human cytomegalovirus promoter.

3. The method according to Claim 1, wherein said expression control sequence comprises a muscle-specific promoter.

4. The method according to Claim 1, wherein said expression control sequence comprises an inducible promoter.

5. The method according to Claim 1, wherein said muscle cells transformed in vitro are placed in an implantable immunoisolation device.

6. A nucleic acid construct for modifying muscle cells to produce erythropoietin, comprising:

a recombinant vector comprising an expression control sequence and an erythropoietin gene which is operatively linked to said expression control sequence, wherein said vector transforms muscle cells to express erythropoietin in an amount sufficient to increase circulating red blood cells.

7. The construct according to Claim 6, wherein said expression control sequence contains an inducible promoter.

8. The construct according to Claim 6, wherein said expression control sequence contains a muscle-specific promoter.

9. A pharmaceutical composition for the modification of muscle cells to produce erythropoietin, comprising:
a nucleic acid construct comprising an expression control sequence and an erythropoietin gene which is operatively linked to said control sequence, wherein upon transfer to a muscle cell said construct elicits the expression of erythropoietin in an amount sufficient to increase circulating red blood cells as compared to pretreatment levels; and a carrier capable of promoting uptake of said construct by said muscle cells.

10. The composition according to Claim 9, wherein said expression control sequence comprises an inducible promoter.

11. The composition according to Claim 9, wherein said expression control sequence comprises a muscle-specific promoter.

12. Modified muscle cells which expresses an erythropoietin gene following transformation with a nucleic acid construct comprising an expression control sequence and an erythropoietin gene which is operatively linked to said control sequence, wherein said muscle cells are transformed in vivo, or wherein said muscle cells are transformed in vitro and subsequently implanted, and wherein the modified cells express sufficient erythropoietin to increase the recipient's red blood cell production as compared to pretreatment levels.

13. The method according to Claim 12, wherein said muscle cells transformed in vitro are placed in an implantable immunoisolation device prior to implantation.

14. A method for increasing red blood cell production, comprising:
transfecting myoblasts in vitro with a recombinant viral vector comprising an expression promoter and an erythropoietin gene which is operatively linked to said promoter thereby forming transfected myoblasts; and implanting said transfected myoblasts into muscle tissue, wherein said transfected myoblasts fuse to form muscle cells which produce and release erythropoietin at a level sufficient to increase red blood cell production as compared to pretreatment levels.

15. A method for enhancing the production of red blood cells, comprising the steps of:
transforming skeletal muscle cells with a nucleic acid construct comprising an expression control sequence and an erythropoietin gene which is operatively linked to said control sequence; and expressing erythropoietin protein in said transformed muscle cells in an amount sufficient to increase red blood cell production.

16. The method according to Claim 15, wherein said expression control sequence comprises an inducible promoter.

17. A genetically modified muscle cell which expresses erythropoietin following the introduction of a nucleic acid construct into said cell, said nucleic acid construct comprising an expression control sequence and an erythropoietin gene which is operatively linked to said control sequence.

18. The modified muscle cell according to Claim 17, wherein said expression control sequence comprises an inducible promoter.

19. The modified muscle cell according to Claim 17, wherein said expression control sequence comprises a transcriptional enhancer element.