Retrovirus-mediated gene expression in mammalian cells William R.A. Osborne University of W a s h i n g t o n , Seattle, W a s h i n g t o n , USA Significant advances have been made in precisely defining the elements in the Moloney murine leukemia virus genome responsible for tissue-restricted expression. This knowledge should lead to improved expression vectors for gene transfer in mammalian cells. In the past year, retrovirus-mediated gene expression in a diverse range of cell types has been reported. These cells have been used to study gene transfer relevant to a range of inherited diseases. Current Opinion in Biotechnology 1991, 2:708-712
Introduction The use of retroviral vectors to efficiently transfer genes to mammalian cells, both in vitro and in vivo, is now well established. The non-expression or inactivation of Moloney murine leukemia vires (M-MuLV) in early embryonic stem cells has, however, affected the use of retroviral vectors in gene transfer experiments in transgenic mice. The observation that transplanted skin fibroblasts and bone marrow stem cells, as well as embryonic stem cells, inactivate transduced genes has stimulated improvements in vector design. In this review, I will pay particular attention to recent developments identifying elements in the M-MuLV genome involved in tissue specific expression and their incorporation into improved vectors. Also, a definitive study of the dependence of infection upon host cell cycle and novel packaging cells is described. Most gene transfer studies concern genes of therapeutic relevance with the majority relating to the gene for adenosine deaminase (ADA). However, as the transfer of the ADA gene was comprehensively discussed last year in this joumal [1] and in a recent review [2], it will not be reconsidered in this article. Instead, examples of both in vitro and in vivo gene transfer that demonstrate the broad range of diseases and cell types that are currently candidates for gene transfer therapy will be discussed.
Vector design Most vectors used to obtain in vitro or in vivo gene expression are based on M-MuLV, which displays restricted tissue expression and does not, for example, productively infect early embryonic stem (ES) cells or murine embryonal carcinoma (EC) cells [3,4,5]. The block in the viral life cycle occurs after proviral integration at the
site of viral RNA production, i.e., it is transcription mediated. Although inactive transcriptional units in undifferentiated EC cells are subject to methylation, the kinetics of this post-integrational modification are inadequate to account for the observed vector suppression [6]. At least two c~s-acting elements have been implicated in the block in RNA transcription; the U 3 region in the M-MuLV enhancer [5,7] and a 5' untranslated region involving the tRNA primer-binding site (PBS) [8"',9"]. The inhibitor action of the intragenic PBS element is independent of orientation [ 8 " , 9 " ] , which also provides evidence for a transcriptional mechanism of inhibition. Two independent studies have shown the inhibitory, intragenic, 5' untranslated region spans the PBS. In these studies the M-MuLV PBS was replaced by either a mouse mammary tumor virus PBS (lysine tRNA) [8o,] or an endogenous provirus PBS (glutamine tRNA) [9"]. Permissive expression in EC cells was obtained from both recombinant vectors, indicating that restriction was specific for the M-MuLV sequence. Mutation experiments identified the repressor element as an 18bp segment ( + 146 to + 163) spanning the tRNA-binding site [8..]. Restricted expression was obtained when this element was moved upstream of the long terminal repeat (LTR), indicating that it was not position dependent. DNA-binding assays [9 "°] and exonuclease protection assays [8 ,°] employing nuclear extracts from EC cells, identified a protein that bound specifically to the wild-type PBS of M-MuLV and not to the alternative tRNA PBSs that provided permissive expression in EC cells. These data suggest that a transacting cellular repressor restricts M-MuLV expression in undifferentiated EC cells by interacting with a negative regulatory element at the tRNA binding site of proviral DNA. Repressors of enhancer activity and a lack of transcriptional activators have been implicated in the absence of LTR-mediated transcription in M-MuLV infected EC and
Abbreviations ADA--adenosine deaminase; CF---cysticfibrosis; CFTR--cystic fibrosis transmembraneconductance regulator; EC--murine embryonal carcinoma; ES~embryonic stem; LTR--Iong terminal repeat; LAD---leukocyte adherence deficiency; M-MuLV--Moloney murine leukemia virus; PBS--primer-binding site; SIN~self-inactivating vectors.
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Retrovirus-mediated gene expression in mammalian cells Osborne ES cells [3,4,5,7]. Recently, mutant viruses have been purified that permit expression in EC cell lines [10.']. Deletion analysis of these mutants defined three point mutations in the U3 enhancer region at positions - 3 4 5 , - 3 2 6 and - 1 6 6 , which correlated with expression in EC cells. A nuclear factor in EC cells bound strongly to a homologous region (positions - 354 to - 306) in wildtype M-MuLV but bound only weakly to corresponding sequences derived from the mutant viruses [10,.]. Thus, mutations in the LTR are essential for virus expression in EC cells and occur within the recognition site of an EC cell-specific DNA-binding factor. A vector designated the murine embryonic stem cell virus, incorporating both distinct sequence elements, the LTR enhancer and primer binding site mutations, has permitted LTR-mediated expression of retroviral genomes in ES cells [11.]. The promoter strength of the mutant LTR has yet to be compared with the wildtype M-MuLV in stably integrated cells. An alternative approach to obtaining vector expression in EC and ES cells is to construct vectors that delete the negative regulatory LTR elements [12,13]. This is accomplished by designing cloning vectors with U3 enhancer deletions that transfer to the 5' LTR during provirus integration. In these so called self-inactivating (SIN) vectors, reporter genes are expressed from internal promoters that can provide either tissue-specific or general expression. The uniformly low titers of SIN vectors has limited their usefulness [12]. High titer self-inactivating vectors that efficiently express genes from internal promoters in infected embryo stem cells have now been constructed and these may provide vectors for general use in undifferentiated cells [13]. These vectors, however, lack the viral promoter function that has proven to be one of the strongest promoters tested in mammalian cells. The non-expression of M-MuLV vectors is not confined to ES cells and has been documented in hematopoietic cells [2,14] and skin fibroblasts [15,.]. Murine bone marrow stem cells infected with a M-MuLV vector provided hematopoietic repopulation in recipient mice, but longterm high level expression of vector encoded genes was only observed in red blood cells [14]. Although vector sequences were clearly present in high copy numbers in white blood cells, vector expression was not detectable. In vivo studies of human clotting Factor IX expression in rats receiving autologous transduced skin fibroblasts did not show long-term therapeutic protein expression [16]. Vector inactivation has been implicated in these experiments. This phenomenon was investigated in rats by using retroviral vectors encoding human ADA and neomycin phosphotransferase [ 15..]. Monitoring human ADA, a cytosolic protein, prevented the antibody-mediated loss of activity shown in the studies of human factor IX, a secreted protein. Human ADA expression in transplanted skin fibroblasts decreased from high to undetectable levels after I month, while vector sequences persisted for at least 8.5 months after grafting. Cellularor antibody-mediated immune responses were not detected in animals that had transplants. The transplanted vector-infected cells were polyclonal populations, sug-
gesting that suppression was independent of the virus integration site. Both the viral LTR and an internal SV40 promoter were transcriptionally inactive and could not be reactivated by culturing in the presence of demethylating agents [15..]. It is very possible that the vector elements responsible for in vivo gene suppression in fibroblasts and hematopoietic cells may be those identified as causing vector repression in murine ES cells. Thus, the generation of viral mutants and the use of internal promoters as outlined above may be two alternative strategies to overcome expression restriction. Vectors providing permissive expression in transplanted fibroblasts are especially important as these cells are particularly attractive target tissues for gene therapy.
Virus infection Infection studies of stationary phase cells have important implications for retroviral mediated gene transfer in stem cells [1,2]. Productive infection of cells is hostcell-cycle dependent; infection of stationary phase cells blocks vires infection before the production of unintegrated viral DNA [17,18,19".]. It has been reported that, although retrovims infection was inhibited in non-replicating cells, infection could occur when cells were stimulated to proliferate from 1 day to I week after infection [17,18], suggesting the formation of a persistent stable viral intermediate. These studies were complicated by the use of replicating competent virus, permitting spread in the target cells. Recently, use of a replication defective vector showed that gene transfer is inhibited in stationary phase cells [19"] in agreement with previous studies [17,18]. The block to infection, however, could not be relieved by stimulating cells to divide at times from 6 h to 10 days after infection, showing that stable retroviral intermediates are not formed in stationary phase cells. These findings are particularly germane to gene transfer involving bone marrow, stem cells, and invalidate the notion that quiescent stem cells might be productively infected if stimulated several days after exposure to virus. They also imply that quiescent hematopoietic stem cells cannot be productively infected and must be proliferating for gene transfer to occur. Paradoxically, if stem cells are stimulated and differentiate, their ability to provide long-term hematopoietic repopulation may be compromised. Thus, any growth factor that induces marrow stem cell self renewal without inducing differentiation could be of great value in bone marrow targeted gene therapy.
Packaging cells Cat and rabbit packaging cells have been investigated as an alternative to the ubiquitous murine producer cell lines: both amphotropic and xenotropic viruses have been generated [20.]. Cat amphotropic producer lines efficiently infected cat, mouse, sheep and beef cells, but
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Expressionsystems pig packaging cells were only mildly infected. Xenotropic retroviruses produced in cat and rabbit packaging cells transferred genes to human, cat, rabbit and sheep cells with the same efficiency as amphotropic vectors. These xenotropic retroviruses infected pig, beef and horse cells with a high efficiency. The most favorable infection of pig cells was obtained from cat packaging cells. These resuits are of interest in the production of transgenic farm animals, where techniques other than the microin]ection of DNA into eggs (for example, retrovirus gene transfer) are required in order to produce large numbers of transgenic animals. Although these studies were not extended to expression in embryonic stem cells, this could be accomplished using vectors incorporating the LTR and PBS mutations discussed above.
Expression of genes of clinical significance In vitro
studies
Gene therapy for hemoglobinopathies such as [3-thalassemia requires high level regulated expression of globin genes. In human ]3-globin gene transfer experiments in mice, the level of transduced [3-globin expression, although tissue specific, has only been 1-2% of that of the endogenous ~maj globin gene; a level unlikely to provide therapeutic benefit [21,22]. Efforts to address this problem have focused on vectors incorporating locus activating regions of the human ~-globin-like gene cluster, characterized by a group of four DNAse I hypersensitive sites [23,24]. Functional assays of these hypersensitive sites showed that transduced gene expression of human [3-globin in both transgenic mice and erythroid cells can approach normal levels. Experiments with murine erythroleukemia cells have shown high-level [3-globin expression, up to 132% of that of endogenous ~maj globin R_N_A,can be achieved by vectors encoding a 1267bp fragment of a hypersensitive site at 10.9kbp 5' of the ~-globin gene [25"]. The reduction of hypersensitive sites to fragments compatible with production of high-titer virus that retains the capacity to direct therapeutic levels of [3-globin expression, is a significant step towards the goal of gene therapy for [3-thalassemia. Cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians and is characterized by abnormalities in water and electrolyte transport in several organs, principally the lung, leading to the abnormal mucocilliary clearance and lung disease that are the major cause of morbidity and mortality in affected individuals. The defective gene in cystic fibrosis patients, the cystic fibrosis transmembrane conductance regulator (CFTR), has recently been cloned [26]. An amphotropic selectable retrovirus has been used to transduce a functional CFFR cDNA into cells from a patient with CF [27 "o]. Expression of the normal gene corrected the abnormal physiology of the patient cells. These results are significant in that they not only prove that the CFTR is the CF gene but also suggest a strategy for treating CF patients by retroviralmediated gene transfer of a functional CFTR gene.
Two independent studies have recently achieved in vitro correction of CD18 deficient lymphocytes by retrovirusmediated transfer of a normal CD18 subunit [28°,29"]. Defects in the CD18 subunit are associated with leukocyte adherence deficiency (LAD) which is characterized by severe recurring bacterial infections, primarily caused by defective neutrophil function. As a result of the CD18 subunit deficiency, three transmembrane glycoproteins that comprise heterodimers of CD18 and one of three different CDll subunits, fail to form. It may not be necessary to achieve vector-mediated regulation of CD18 expression for reconstitution of normal adherence function in patients because, during the inflammatory response, the majority of regulation is post-transcriptionally controlled in a lineage-specific manner during myeloid cell development. But the effect on normal hematopoiesis of inappropriate expression of CD18 is not known. These studies, together with the successful treatment of LAD by bone marrow transplantation, suggest that application of retroviral-mediated gene transfer to this disorder may be successful. In vivo studies Familial hypercholesterolemia is an inherited disease that is associated with coronary artery disease and results from a deficiency of the receptor that mediates the internalization of low density lipoprotein (LDL) [30]. As the liver is the primary organ responsible for the degradation of LDL,hepatocytes are considered the most appropriate target cell for gene therapy. In a study of hyperlipidernic rabbits, hepatocytes infected with a retrovirus encoding human LDL receptor were transplanted into the livers of diseased rabbits via the portal vein [31"]. Significant, albeit temporary (6 days), amelioration of hyperlipidemia was observed. Deterioration of in vivo LDL receptor function was probably caused by loss of transplanted cells as a result of allogeneic cell graft rejection; this could, perhaps, be circumvented by the use of autologous transduced cells. This study is noteworthy as it involves transfer of a receptor gene to a transplantable cultured liver cell, broadening the potential application of gene therapy.
Canine keratinocytes have been employed as a target cell for in vivo gene therapy [32"]. Transplanted autologous keratinocytes infected with a neomycinphosphotransferase-encoding virus showed G418 antibiotic resistance for up to 130 days. Of interest was the finding that keratinocytes, in contrast to transplanted fibroblasts, did not appear to inactivate transduced genes. This study demonstrates the potential of genetically modified keratinocytes to provide long-term expression of therapeutic proteins, for example clotting and growth factors [2]. The feasibility of retrovirus-mediated gene transfer as a tool for studying T-cell development has been demonstrated by the long-term expression of a T-cell receptor [3-chain in mice reconstituted with infected hematopoietic stem cells [33"]. Transplanted mice expressed the exogenous T-cell receptor [5-chain gene in lyrnphocytes
Retrovirus-mediated gene expression in mammalian cells Osborne and T-cells for at least 5 months. These studies suggest that vectors encoding one or more genes involved in T-cell development, for example specific T-cell receptors, CD4, CD8, or interleukins, may be introduced into different target cells, such as lineage-restricted stem cells, T-cell precursors or lymphocytes, enabling their development to be examined in diverse host environments.
6.
NIWA O, YOKOTA Y, ISHIDA H, SUGAHARA T: I n d e p e n d e n t Mechanisms Involved in Suppression of t h e Moloney Leukemia Virus G e n o m e During Differentiation o f Murine Teratocarcinoma Cells. Cell 1983, 32:1105-1113.
7.
H1LBERGF, STOCKING C, OSTERTAG W, GREZ M: Punctional Analysis of a Retroviral Host-range Mutant: Altered Long Terminal Repeat Sequences Allow Expression of Embryonal Carcinoma Cells. Proc Natl Acad Sci USA 1987, 84:5232-5236.
LOH TP, SIEVERT EL, SCOTF RW: Evidence for a Stem Cellspecific Repressor of Moloney Murine Leukemia Virus Expression in Embryonal Carcinoma Cells. Mol Cell Biol 1990, 10:4045-4057. A negative regulatory element mediating embryonal carcinoma cell repression of M-MuLV was precisely defined as an 18bp segment of the tRNA primer binding site. Binding of a protein derived from EC cell extracts correlated strongly with repression. 8. oo
Conclusion Studies of M-MuLV mutants have defined the elements of M-MuLV responsible for tissue-specific expression. This will lead to improved vectors designed to produce gene expression in embryonal and marrow stem cells and their progenitors, which will be of major benefit to both transgenic mouse and gene therapy studies. Although the M-MuLV elements causing restricted expression have been identified in experiments using embryonic carcinoma cells, it seems likely that the same sequences are involved in the vector inactivation observed in transplanted fibroblasts. During the past year, vector expression has been monitored in a broad range of cell types, including hepatocytes, lymphocytes, keratinocytes and fibroblasts. These cells were explored as vehicles for therapeutic gene transfer in a range of inherited diseases. In the future, improved vector design together with the increasingly diverse tissues that can be cultured, infected and transplanted should permit the application of retroviral vectors in novel systems for drug delivery.
PETERSENR, KEMPLER G, BARKLIS E: A Stem Cell-specific Silencer in the Primer-binding Site of a Retrovirus. Mol Cell Biol 1991, 11:1214-1221. An embryonal carcinoma cell-specific repressor binding element was mapped to MMuLV nucleotides 147-174 spanning the tRNA primer binding site and w~s shown to repress heterologous promoters from an upstream position. An EC cell nuclear protein specifically recognized the M-MuLV repressor element. 9. oo
10. •.
AKGUNE, Z1EGLERM, GREZ M: Determinants of Retrovirus Gene Expression in Embryonal Carcinoma Cells. J virol 1991, 65:382-388. M-MuLV mutants and their recombinants were studied in EC cells. Three point mutations in the U 3 enhancer region, and two point mutations in the PBS were defined as essential for EC cell retrovirus expression. The mutations in the enhancer region were associated with reduced binding of an EC cell factor. 11. •
GREZM, AKGUN E, HILBERGF, OSTERTAGW: Embryonic Stem Cell Virus, a Recombinant Murine Retrovirus w i t h Expression in Embryonic Stem Cells. Proc Natl Acad Sci USA 1990, 87:9202-9206, A recombinant virus that permitted permissive expression in ES cells was derived by incorporating M-MuLV enhancer and PBS mutations. 12.
Yu SF, VON RUDEN T, KANTOFF PW, GARBER C, SEIBERG M, RUTHER U, ANDERSONWE, WAGNER EF, GILBOA E: Self-inactivating Retroviral Vectors Designed for Transfer of Whole Genes into Mammalian Cells. Proc Natl Acad Sci USA 1986, 83:3194-3198.
13.
SOmANOP, FmEDPaCH G, LAW1NGERP: Promoter Interactions in Retrovirus Vectors Introduced into Fibroblasts and Embryonic Stem Cells. J Virol 1991, 65:2314-2319.
References and recommended reading
14.
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest oo of outstanding interest
KALEKOM, GARC1AJV, OSBORNE WRA, MILLERAD: Expression of H u m a n Adenosine Deaminase in Mice After Transplantation of Genetically-modified Bone marrow. Blood 1990, 75:1733-1741.
15. °°
Acknowledgements I thank Ramesh for his many helpful comments. Supported by NIH grant DK 38531.
1.
TOLSTOSHOVP, ANDERSON~F: Gene Expression using Retroviral Vectors. Curr Opin Biotechnol 1990, 1:55-61.
2.
MILLERAD: Progress Toward H u m a n G e n e Therapy. Blood 1990, 76:271-278.
3.
GORMANCM, RiGBY PWJ, LANE DP: Negative Regulation o f Viral Enhancers in Undifferentiated Embryonic Stem Cells. Cell 1985, 42:519-526.
4.
ROSENCA, HASELTnVEWA, LENZ J, RUPRECHT R, CLOYD MW: Tissue Selectivity of Murine Leukemia Virus Infection is Determined by Long Terminal Repeat Sequences. J Virol 1985, 55:862-866.
5.
WEII-IERH, BARKLISE, OSTERTAGW, JAENISCH R: T w o Distinct Sequence Elements Mediate Retroviral Gene Expression in Embryonal Carcinoma Cells. J Virol 1987, 61:2742-2746.
PALMERTD, ROSMANGJ, OSBORNEWRA, MILLERAD: Genetically Modified Skin Fibroblasts Persist Long After Transplantation b u t Gradually Inactivate Introduced Genes. Proc Natl Acad Sci USA 1991, 88:1330-1334. Demonstrates that in rats, transplanted transduced fibroblasts are subject to gene inactivation. Vector sequences persisted for up to 8.5 months in vivo but after 1 month vector expression was undetectable. 16.
PALMERTD, THOMPSON AR, MILLER AD: Production of Hum a n Factor IX in Animals by Genetically Modified Skin Fibroblasts: Potential Therapy for Hemophilia B. Blood 1989, 73:438-445.
17.
VARMUSHE, PADGETTT, HEASLEYS, SIMON G, BISHOPJM: Cellular Functions are Required for t h e Synthesis and Integration of Avian Sarcoma Virus-specific DNA. Cell 1977, 11:307-319.
18.
HARELJ, RASSARTE, JOLICOEUR P: Cell Cycle D e p e n d e n c e of Synthesis of Unintegrated Viral DNA in Mouse Cells Newly Infected with Murine Leukemia Virus. Virology 1981, 110:202-207.
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Expressionsystems 19. •.
MILLERDG, ADAM MA, MILLERAD: Gene Transfer by Retrovirus Vectors Occurs only in Cells that are Actively Replicating at the Time of Infection. Mol Cell Biol 1990, 10:4239-4242. Clear evidence that the block to retroviral infection could not be relieved by stimulating cells to divide between 6h and 10 days, post infection. Therefore, host cells must be replicating at the time of infection for successful proviral integration. This finding is relevant to gene transfer targeted at stem cells. 20.
DELOUISC, MILAND, L'HARIDANR, GIANGUINTOL, BONNEROTC, NICOLAS J-F: Xenotropic and Amphotropic Pseudotyped Recombinant Retrovirus Vectors to Transfer Genes into Cells from Various Species. Biochem Biophys Res Commun 1990, 169:8-14. Cat, rabbit and mouse packaging cell fines were compared for their ability to transfer amphotropic and xenotropic recombinant vectors to cells from a variety of species. •
21.
22.
IY/aERAKEA, PAPAYANNOPOULOUT, MULLIGANRC: Lineage Specific Expression of a Human J3-globin Gene in Murine Bone Marrow Transplant Recipients Reconstituted with Retrovirus-transduced Stem Cells. Nature 1988, 331:35-41. BENDERMA, GELINASRE, MILLERA_O:A Majority of Mice Show Long-term Expression of a Human ]]-globin Gene After Retrovirus Transfer into Hematopoietic Stem Cells. Mol Cell Biol 1989, 9:1426-1434.
23.
GROSVELDF, VAN ASSENDEIFF GB, GREAVES DR, KOLLIAS G: Position-independent, High-level Expression of the Human I]-globin Gene in Transgenic Mice. Cell 1987, 51:975-985.
24.
TALBOTD, COLLIS P, ANTONIOU M, VIDAL M, GROSVELD F, GREAVESDR: A Dominant Control Region from the Human 13-globin Locus Conferring Integration of Site-independent Gene Expression. Nature 1989, 338:352-355.
25. •,
NOVAKU, HARRISEAS, FORRESTERW, GROUDINE M, GELINASR: High-level 13-globin Expression after Retroviral Transfer o f Locus Activation Region-containing Human 13-globin Gene Derivatives into Murine Erythroleukemia Cells. Proc Natl Acad Sci USA 1990, 87:3386--3390. Expression of human ~-globin mRNA levels equivalent to endogenous murine IFnal globin mRNA was measured in murine et3Maroleukemia cells infected with a vector that contained a fragment of a hypersensitive site from the locus activating region of the human ~-globin-like gene cluster. An important result concerning gene transfer treatment of hemoglobin deficiencies, 26.
27. ••
ROMMENSJ i , IANNUZZIMC, KEREM B-S, DRUMM ML, MELMER G, DEAN M, ROZMAHELR, COLE JL, KENNEDYD, HIDAKAM, ET AL: Identification of the Cystic Fibrosis Gene: Chromosome Walking and Jumping. Science 1989, 245:1059-1065. DRUMMML, POPE HA, CLIFF WH, ROMMENSJM, MARVIN SPh
TsuI LC, COLLINS FS, FRIZZELLRA, WILSONJM: Correction of
the Cystic Fibrosis Defect In Vitro by Retrovirus-mediated Gene Transfer. Cell 1990, 62:1227-1233. Amphotropic retroviruses were used to transfer functional CFTR cDNA into cells from a CF patient. The expression of the normal gene conferred cAMP-dependent chloride channel regulation on CF epithelial cells. 28. •
WILSONJM, PING AJ, KRAUSSJC, MAYO-BOND L, ROGERS CE, ANDERSONn c , TODD RE: Correction o f CD18-deficient Lymphocytes by Retrovirus-mediated Gene Transfer. Science 1990, 248:1413-1416. Lymphocytes from a patient with CD18 deficiency were infected with a retrovirus encoding normal CD18 subunit. Transduced cells expressed normal surface levels of CD18-containing transmembrane protein with reconstitution of normal adherence function. 29.
BACKAL, KWOK WW, ADAM M, COLLINS SJ, H1CKSTEIN DD: Retroviral-mediated Gene Transfer of the Leukocyte Integrin CD18 Subunit. Biochem Biophys Res Com 1990, 171:787-795. Similar study and conclusions to [28"]. •
30.
GOLDSTEINJL, BROWN MS: Familial Hypercholesterolemia. In The Metabolic Basis of Inherited Disease edited by Scriver CR, Beaudeut SL, sly WS, Valle D [book]. New York: McGraw-Hill 1989, pp 1215-1250.
31.
WILSONJM, CHOWDHURYNR, GROSSMANM, WAJSMANR, EPSTEIN A, MULLIGAN RC, CHOWDHURYJR: Temporary Amelioration of Hyperlipidemia in Low Density Lipoprotein Receptordeficient Rabbits Transplanted with Genetically Modified Hepatocytes. Proc Natl Acad Sci USA 1990, 87:8437-43441. A novel in vivo transplantation of transduced hepatocytes demonstrated the potential of gene therapy in ameliorating hyperlipidemia associated with familial hypercholesterolemia. •
32. •
FLOWERSMED, STOCKSCHLAEDERMAR, SCHUENINGFG, NmDERMILLERAn, STORB R: Long-term Transplantation o f Canine Keratinocytes Made Resistant to G418 Through Retrovirus-mediated Gene Transfer. Proc NatlAcad Sci USA 1990, 87:2349-2353. Transplanted infected autologous keratinocytes expressed a foreign gene for at least 130 days. WIESER D , HACKMAN R,
33.
KANGJ, WITHER J, HOZUMI N: Long-term Expression of a T-cell Receptor 13-chain Gene in Mice Reconstituted with Retrovirus-infected Hematopoietic Stem Cells. Proc Natl Acad Sci USA 1990, 87:1903-1907. Mice transplanted with infected bone marrow expressed an exogenous T-cell receptor I~-chain on thymocytes and splenic T cells, suggesting that genes involved in T-cell development can be studied in genetically diverse host environments. •
WRA Osborne, Department of Pediatrics, RD-20 University of Washington, Seattle, Washington 98195, USA.
Introduction The use of retroviral vectors to efficiently transfer genes to mammalian cells, both in vitro and in vivo, is now well established. The non-expression or inactivation of Moloney murine leukemia vires (M-MuLV) in early embryonic stem cells has, however, affected the use of retroviral vectors in gene transfer experiments in transgenic mice. The observation that transplanted skin fibroblasts and bone marrow stem cells, as well as embryonic stem cells, inactivate transduced genes has stimulated improvements in vector design. In this review, I will pay particular attention to recent developments identifying elements in the M-MuLV genome involved in tissue specific expression and their incorporation into improved vectors. Also, a definitive study of the dependence of infection upon host cell cycle and novel packaging cells is described. Most gene transfer studies concern genes of therapeutic relevance with the majority relating to the gene for adenosine deaminase (ADA). However, as the transfer of the ADA gene was comprehensively discussed last year in this joumal [1] and in a recent review [2], it will not be reconsidered in this article. Instead, examples of both in vitro and in vivo gene transfer that demonstrate the broad range of diseases and cell types that are currently candidates for gene transfer therapy will be discussed.
Vector design Most vectors used to obtain in vitro or in vivo gene expression are based on M-MuLV, which displays restricted tissue expression and does not, for example, productively infect early embryonic stem (ES) cells or murine embryonal carcinoma (EC) cells [3,4,5]. The block in the viral life cycle occurs after proviral integration at the
site of viral RNA production, i.e., it is transcription mediated. Although inactive transcriptional units in undifferentiated EC cells are subject to methylation, the kinetics of this post-integrational modification are inadequate to account for the observed vector suppression [6]. At least two c~s-acting elements have been implicated in the block in RNA transcription; the U 3 region in the M-MuLV enhancer [5,7] and a 5' untranslated region involving the tRNA primer-binding site (PBS) [8"',9"]. The inhibitor action of the intragenic PBS element is independent of orientation [ 8 " , 9 " ] , which also provides evidence for a transcriptional mechanism of inhibition. Two independent studies have shown the inhibitory, intragenic, 5' untranslated region spans the PBS. In these studies the M-MuLV PBS was replaced by either a mouse mammary tumor virus PBS (lysine tRNA) [8o,] or an endogenous provirus PBS (glutamine tRNA) [9"]. Permissive expression in EC cells was obtained from both recombinant vectors, indicating that restriction was specific for the M-MuLV sequence. Mutation experiments identified the repressor element as an 18bp segment ( + 146 to + 163) spanning the tRNA-binding site [8..]. Restricted expression was obtained when this element was moved upstream of the long terminal repeat (LTR), indicating that it was not position dependent. DNA-binding assays [9 "°] and exonuclease protection assays [8 ,°] employing nuclear extracts from EC cells, identified a protein that bound specifically to the wild-type PBS of M-MuLV and not to the alternative tRNA PBSs that provided permissive expression in EC cells. These data suggest that a transacting cellular repressor restricts M-MuLV expression in undifferentiated EC cells by interacting with a negative regulatory element at the tRNA binding site of proviral DNA. Repressors of enhancer activity and a lack of transcriptional activators have been implicated in the absence of LTR-mediated transcription in M-MuLV infected EC and
Abbreviations ADA--adenosine deaminase; CF---cysticfibrosis; CFTR--cystic fibrosis transmembraneconductance regulator; EC--murine embryonal carcinoma; ES~embryonic stem; LTR--Iong terminal repeat; LAD---leukocyte adherence deficiency; M-MuLV--Moloney murine leukemia virus; PBS--primer-binding site; SIN~self-inactivating vectors.
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(~ Current Biology Ltd ISSN 0958-1669
Retrovirus-mediated gene expression in mammalian cells Osborne ES cells [3,4,5,7]. Recently, mutant viruses have been purified that permit expression in EC cell lines [10.']. Deletion analysis of these mutants defined three point mutations in the U3 enhancer region at positions - 3 4 5 , - 3 2 6 and - 1 6 6 , which correlated with expression in EC cells. A nuclear factor in EC cells bound strongly to a homologous region (positions - 354 to - 306) in wildtype M-MuLV but bound only weakly to corresponding sequences derived from the mutant viruses [10,.]. Thus, mutations in the LTR are essential for virus expression in EC cells and occur within the recognition site of an EC cell-specific DNA-binding factor. A vector designated the murine embryonic stem cell virus, incorporating both distinct sequence elements, the LTR enhancer and primer binding site mutations, has permitted LTR-mediated expression of retroviral genomes in ES cells [11.]. The promoter strength of the mutant LTR has yet to be compared with the wildtype M-MuLV in stably integrated cells. An alternative approach to obtaining vector expression in EC and ES cells is to construct vectors that delete the negative regulatory LTR elements [12,13]. This is accomplished by designing cloning vectors with U3 enhancer deletions that transfer to the 5' LTR during provirus integration. In these so called self-inactivating (SIN) vectors, reporter genes are expressed from internal promoters that can provide either tissue-specific or general expression. The uniformly low titers of SIN vectors has limited their usefulness [12]. High titer self-inactivating vectors that efficiently express genes from internal promoters in infected embryo stem cells have now been constructed and these may provide vectors for general use in undifferentiated cells [13]. These vectors, however, lack the viral promoter function that has proven to be one of the strongest promoters tested in mammalian cells. The non-expression of M-MuLV vectors is not confined to ES cells and has been documented in hematopoietic cells [2,14] and skin fibroblasts [15,.]. Murine bone marrow stem cells infected with a M-MuLV vector provided hematopoietic repopulation in recipient mice, but longterm high level expression of vector encoded genes was only observed in red blood cells [14]. Although vector sequences were clearly present in high copy numbers in white blood cells, vector expression was not detectable. In vivo studies of human clotting Factor IX expression in rats receiving autologous transduced skin fibroblasts did not show long-term therapeutic protein expression [16]. Vector inactivation has been implicated in these experiments. This phenomenon was investigated in rats by using retroviral vectors encoding human ADA and neomycin phosphotransferase [ 15..]. Monitoring human ADA, a cytosolic protein, prevented the antibody-mediated loss of activity shown in the studies of human factor IX, a secreted protein. Human ADA expression in transplanted skin fibroblasts decreased from high to undetectable levels after I month, while vector sequences persisted for at least 8.5 months after grafting. Cellularor antibody-mediated immune responses were not detected in animals that had transplants. The transplanted vector-infected cells were polyclonal populations, sug-
gesting that suppression was independent of the virus integration site. Both the viral LTR and an internal SV40 promoter were transcriptionally inactive and could not be reactivated by culturing in the presence of demethylating agents [15..]. It is very possible that the vector elements responsible for in vivo gene suppression in fibroblasts and hematopoietic cells may be those identified as causing vector repression in murine ES cells. Thus, the generation of viral mutants and the use of internal promoters as outlined above may be two alternative strategies to overcome expression restriction. Vectors providing permissive expression in transplanted fibroblasts are especially important as these cells are particularly attractive target tissues for gene therapy.
Virus infection Infection studies of stationary phase cells have important implications for retroviral mediated gene transfer in stem cells [1,2]. Productive infection of cells is hostcell-cycle dependent; infection of stationary phase cells blocks vires infection before the production of unintegrated viral DNA [17,18,19".]. It has been reported that, although retrovims infection was inhibited in non-replicating cells, infection could occur when cells were stimulated to proliferate from 1 day to I week after infection [17,18], suggesting the formation of a persistent stable viral intermediate. These studies were complicated by the use of replicating competent virus, permitting spread in the target cells. Recently, use of a replication defective vector showed that gene transfer is inhibited in stationary phase cells [19"] in agreement with previous studies [17,18]. The block to infection, however, could not be relieved by stimulating cells to divide at times from 6 h to 10 days after infection, showing that stable retroviral intermediates are not formed in stationary phase cells. These findings are particularly germane to gene transfer involving bone marrow, stem cells, and invalidate the notion that quiescent stem cells might be productively infected if stimulated several days after exposure to virus. They also imply that quiescent hematopoietic stem cells cannot be productively infected and must be proliferating for gene transfer to occur. Paradoxically, if stem cells are stimulated and differentiate, their ability to provide long-term hematopoietic repopulation may be compromised. Thus, any growth factor that induces marrow stem cell self renewal without inducing differentiation could be of great value in bone marrow targeted gene therapy.
Packaging cells Cat and rabbit packaging cells have been investigated as an alternative to the ubiquitous murine producer cell lines: both amphotropic and xenotropic viruses have been generated [20.]. Cat amphotropic producer lines efficiently infected cat, mouse, sheep and beef cells, but
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Expressionsystems pig packaging cells were only mildly infected. Xenotropic retroviruses produced in cat and rabbit packaging cells transferred genes to human, cat, rabbit and sheep cells with the same efficiency as amphotropic vectors. These xenotropic retroviruses infected pig, beef and horse cells with a high efficiency. The most favorable infection of pig cells was obtained from cat packaging cells. These resuits are of interest in the production of transgenic farm animals, where techniques other than the microin]ection of DNA into eggs (for example, retrovirus gene transfer) are required in order to produce large numbers of transgenic animals. Although these studies were not extended to expression in embryonic stem cells, this could be accomplished using vectors incorporating the LTR and PBS mutations discussed above.
Expression of genes of clinical significance In vitro
studies
Gene therapy for hemoglobinopathies such as [3-thalassemia requires high level regulated expression of globin genes. In human ]3-globin gene transfer experiments in mice, the level of transduced [3-globin expression, although tissue specific, has only been 1-2% of that of the endogenous ~maj globin gene; a level unlikely to provide therapeutic benefit [21,22]. Efforts to address this problem have focused on vectors incorporating locus activating regions of the human ~-globin-like gene cluster, characterized by a group of four DNAse I hypersensitive sites [23,24]. Functional assays of these hypersensitive sites showed that transduced gene expression of human [3-globin in both transgenic mice and erythroid cells can approach normal levels. Experiments with murine erythroleukemia cells have shown high-level [3-globin expression, up to 132% of that of endogenous ~maj globin R_N_A,can be achieved by vectors encoding a 1267bp fragment of a hypersensitive site at 10.9kbp 5' of the ~-globin gene [25"]. The reduction of hypersensitive sites to fragments compatible with production of high-titer virus that retains the capacity to direct therapeutic levels of [3-globin expression, is a significant step towards the goal of gene therapy for [3-thalassemia. Cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians and is characterized by abnormalities in water and electrolyte transport in several organs, principally the lung, leading to the abnormal mucocilliary clearance and lung disease that are the major cause of morbidity and mortality in affected individuals. The defective gene in cystic fibrosis patients, the cystic fibrosis transmembrane conductance regulator (CFTR), has recently been cloned [26]. An amphotropic selectable retrovirus has been used to transduce a functional CFFR cDNA into cells from a patient with CF [27 "o]. Expression of the normal gene corrected the abnormal physiology of the patient cells. These results are significant in that they not only prove that the CFTR is the CF gene but also suggest a strategy for treating CF patients by retroviralmediated gene transfer of a functional CFTR gene.
Two independent studies have recently achieved in vitro correction of CD18 deficient lymphocytes by retrovirusmediated transfer of a normal CD18 subunit [28°,29"]. Defects in the CD18 subunit are associated with leukocyte adherence deficiency (LAD) which is characterized by severe recurring bacterial infections, primarily caused by defective neutrophil function. As a result of the CD18 subunit deficiency, three transmembrane glycoproteins that comprise heterodimers of CD18 and one of three different CDll subunits, fail to form. It may not be necessary to achieve vector-mediated regulation of CD18 expression for reconstitution of normal adherence function in patients because, during the inflammatory response, the majority of regulation is post-transcriptionally controlled in a lineage-specific manner during myeloid cell development. But the effect on normal hematopoiesis of inappropriate expression of CD18 is not known. These studies, together with the successful treatment of LAD by bone marrow transplantation, suggest that application of retroviral-mediated gene transfer to this disorder may be successful. In vivo studies Familial hypercholesterolemia is an inherited disease that is associated with coronary artery disease and results from a deficiency of the receptor that mediates the internalization of low density lipoprotein (LDL) [30]. As the liver is the primary organ responsible for the degradation of LDL,hepatocytes are considered the most appropriate target cell for gene therapy. In a study of hyperlipidernic rabbits, hepatocytes infected with a retrovirus encoding human LDL receptor were transplanted into the livers of diseased rabbits via the portal vein [31"]. Significant, albeit temporary (6 days), amelioration of hyperlipidemia was observed. Deterioration of in vivo LDL receptor function was probably caused by loss of transplanted cells as a result of allogeneic cell graft rejection; this could, perhaps, be circumvented by the use of autologous transduced cells. This study is noteworthy as it involves transfer of a receptor gene to a transplantable cultured liver cell, broadening the potential application of gene therapy.
Canine keratinocytes have been employed as a target cell for in vivo gene therapy [32"]. Transplanted autologous keratinocytes infected with a neomycinphosphotransferase-encoding virus showed G418 antibiotic resistance for up to 130 days. Of interest was the finding that keratinocytes, in contrast to transplanted fibroblasts, did not appear to inactivate transduced genes. This study demonstrates the potential of genetically modified keratinocytes to provide long-term expression of therapeutic proteins, for example clotting and growth factors [2]. The feasibility of retrovirus-mediated gene transfer as a tool for studying T-cell development has been demonstrated by the long-term expression of a T-cell receptor [3-chain in mice reconstituted with infected hematopoietic stem cells [33"]. Transplanted mice expressed the exogenous T-cell receptor [5-chain gene in lyrnphocytes
Retrovirus-mediated gene expression in mammalian cells Osborne and T-cells for at least 5 months. These studies suggest that vectors encoding one or more genes involved in T-cell development, for example specific T-cell receptors, CD4, CD8, or interleukins, may be introduced into different target cells, such as lineage-restricted stem cells, T-cell precursors or lymphocytes, enabling their development to be examined in diverse host environments.
6.
NIWA O, YOKOTA Y, ISHIDA H, SUGAHARA T: I n d e p e n d e n t Mechanisms Involved in Suppression of t h e Moloney Leukemia Virus G e n o m e During Differentiation o f Murine Teratocarcinoma Cells. Cell 1983, 32:1105-1113.
7.
H1LBERGF, STOCKING C, OSTERTAG W, GREZ M: Punctional Analysis of a Retroviral Host-range Mutant: Altered Long Terminal Repeat Sequences Allow Expression of Embryonal Carcinoma Cells. Proc Natl Acad Sci USA 1987, 84:5232-5236.
LOH TP, SIEVERT EL, SCOTF RW: Evidence for a Stem Cellspecific Repressor of Moloney Murine Leukemia Virus Expression in Embryonal Carcinoma Cells. Mol Cell Biol 1990, 10:4045-4057. A negative regulatory element mediating embryonal carcinoma cell repression of M-MuLV was precisely defined as an 18bp segment of the tRNA primer binding site. Binding of a protein derived from EC cell extracts correlated strongly with repression. 8. oo
Conclusion Studies of M-MuLV mutants have defined the elements of M-MuLV responsible for tissue-specific expression. This will lead to improved vectors designed to produce gene expression in embryonal and marrow stem cells and their progenitors, which will be of major benefit to both transgenic mouse and gene therapy studies. Although the M-MuLV elements causing restricted expression have been identified in experiments using embryonic carcinoma cells, it seems likely that the same sequences are involved in the vector inactivation observed in transplanted fibroblasts. During the past year, vector expression has been monitored in a broad range of cell types, including hepatocytes, lymphocytes, keratinocytes and fibroblasts. These cells were explored as vehicles for therapeutic gene transfer in a range of inherited diseases. In the future, improved vector design together with the increasingly diverse tissues that can be cultured, infected and transplanted should permit the application of retroviral vectors in novel systems for drug delivery.
PETERSENR, KEMPLER G, BARKLIS E: A Stem Cell-specific Silencer in the Primer-binding Site of a Retrovirus. Mol Cell Biol 1991, 11:1214-1221. An embryonal carcinoma cell-specific repressor binding element was mapped to MMuLV nucleotides 147-174 spanning the tRNA primer binding site and w~s shown to repress heterologous promoters from an upstream position. An EC cell nuclear protein specifically recognized the M-MuLV repressor element. 9. oo
10. •.
AKGUNE, Z1EGLERM, GREZ M: Determinants of Retrovirus Gene Expression in Embryonal Carcinoma Cells. J virol 1991, 65:382-388. M-MuLV mutants and their recombinants were studied in EC cells. Three point mutations in the U 3 enhancer region, and two point mutations in the PBS were defined as essential for EC cell retrovirus expression. The mutations in the enhancer region were associated with reduced binding of an EC cell factor. 11. •
GREZM, AKGUN E, HILBERGF, OSTERTAGW: Embryonic Stem Cell Virus, a Recombinant Murine Retrovirus w i t h Expression in Embryonic Stem Cells. Proc Natl Acad Sci USA 1990, 87:9202-9206, A recombinant virus that permitted permissive expression in ES cells was derived by incorporating M-MuLV enhancer and PBS mutations. 12.
Yu SF, VON RUDEN T, KANTOFF PW, GARBER C, SEIBERG M, RUTHER U, ANDERSONWE, WAGNER EF, GILBOA E: Self-inactivating Retroviral Vectors Designed for Transfer of Whole Genes into Mammalian Cells. Proc Natl Acad Sci USA 1986, 83:3194-3198.
13.
SOmANOP, FmEDPaCH G, LAW1NGERP: Promoter Interactions in Retrovirus Vectors Introduced into Fibroblasts and Embryonic Stem Cells. J Virol 1991, 65:2314-2319.
References and recommended reading
14.
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest oo of outstanding interest
KALEKOM, GARC1AJV, OSBORNE WRA, MILLERAD: Expression of H u m a n Adenosine Deaminase in Mice After Transplantation of Genetically-modified Bone marrow. Blood 1990, 75:1733-1741.
15. °°
Acknowledgements I thank Ramesh for his many helpful comments. Supported by NIH grant DK 38531.
1.
TOLSTOSHOVP, ANDERSON~F: Gene Expression using Retroviral Vectors. Curr Opin Biotechnol 1990, 1:55-61.
2.
MILLERAD: Progress Toward H u m a n G e n e Therapy. Blood 1990, 76:271-278.
3.
GORMANCM, RiGBY PWJ, LANE DP: Negative Regulation o f Viral Enhancers in Undifferentiated Embryonic Stem Cells. Cell 1985, 42:519-526.
4.
ROSENCA, HASELTnVEWA, LENZ J, RUPRECHT R, CLOYD MW: Tissue Selectivity of Murine Leukemia Virus Infection is Determined by Long Terminal Repeat Sequences. J Virol 1985, 55:862-866.
5.
WEII-IERH, BARKLISE, OSTERTAGW, JAENISCH R: T w o Distinct Sequence Elements Mediate Retroviral Gene Expression in Embryonal Carcinoma Cells. J Virol 1987, 61:2742-2746.
PALMERTD, ROSMANGJ, OSBORNEWRA, MILLERAD: Genetically Modified Skin Fibroblasts Persist Long After Transplantation b u t Gradually Inactivate Introduced Genes. Proc Natl Acad Sci USA 1991, 88:1330-1334. Demonstrates that in rats, transplanted transduced fibroblasts are subject to gene inactivation. Vector sequences persisted for up to 8.5 months in vivo but after 1 month vector expression was undetectable. 16.
PALMERTD, THOMPSON AR, MILLER AD: Production of Hum a n Factor IX in Animals by Genetically Modified Skin Fibroblasts: Potential Therapy for Hemophilia B. Blood 1989, 73:438-445.
17.
VARMUSHE, PADGETTT, HEASLEYS, SIMON G, BISHOPJM: Cellular Functions are Required for t h e Synthesis and Integration of Avian Sarcoma Virus-specific DNA. Cell 1977, 11:307-319.
18.
HARELJ, RASSARTE, JOLICOEUR P: Cell Cycle D e p e n d e n c e of Synthesis of Unintegrated Viral DNA in Mouse Cells Newly Infected with Murine Leukemia Virus. Virology 1981, 110:202-207.
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Expressionsystems 19. •.
MILLERDG, ADAM MA, MILLERAD: Gene Transfer by Retrovirus Vectors Occurs only in Cells that are Actively Replicating at the Time of Infection. Mol Cell Biol 1990, 10:4239-4242. Clear evidence that the block to retroviral infection could not be relieved by stimulating cells to divide between 6h and 10 days, post infection. Therefore, host cells must be replicating at the time of infection for successful proviral integration. This finding is relevant to gene transfer targeted at stem cells. 20.
DELOUISC, MILAND, L'HARIDANR, GIANGUINTOL, BONNEROTC, NICOLAS J-F: Xenotropic and Amphotropic Pseudotyped Recombinant Retrovirus Vectors to Transfer Genes into Cells from Various Species. Biochem Biophys Res Commun 1990, 169:8-14. Cat, rabbit and mouse packaging cell fines were compared for their ability to transfer amphotropic and xenotropic recombinant vectors to cells from a variety of species. •
21.
22.
IY/aERAKEA, PAPAYANNOPOULOUT, MULLIGANRC: Lineage Specific Expression of a Human J3-globin Gene in Murine Bone Marrow Transplant Recipients Reconstituted with Retrovirus-transduced Stem Cells. Nature 1988, 331:35-41. BENDERMA, GELINASRE, MILLERA_O:A Majority of Mice Show Long-term Expression of a Human ]]-globin Gene After Retrovirus Transfer into Hematopoietic Stem Cells. Mol Cell Biol 1989, 9:1426-1434.
23.
GROSVELDF, VAN ASSENDEIFF GB, GREAVES DR, KOLLIAS G: Position-independent, High-level Expression of the Human I]-globin Gene in Transgenic Mice. Cell 1987, 51:975-985.
24.
TALBOTD, COLLIS P, ANTONIOU M, VIDAL M, GROSVELD F, GREAVESDR: A Dominant Control Region from the Human 13-globin Locus Conferring Integration of Site-independent Gene Expression. Nature 1989, 338:352-355.
25. •,
NOVAKU, HARRISEAS, FORRESTERW, GROUDINE M, GELINASR: High-level 13-globin Expression after Retroviral Transfer o f Locus Activation Region-containing Human 13-globin Gene Derivatives into Murine Erythroleukemia Cells. Proc Natl Acad Sci USA 1990, 87:3386--3390. Expression of human ~-globin mRNA levels equivalent to endogenous murine IFnal globin mRNA was measured in murine et3Maroleukemia cells infected with a vector that contained a fragment of a hypersensitive site from the locus activating region of the human ~-globin-like gene cluster. An important result concerning gene transfer treatment of hemoglobin deficiencies, 26.
27. ••
ROMMENSJ i , IANNUZZIMC, KEREM B-S, DRUMM ML, MELMER G, DEAN M, ROZMAHELR, COLE JL, KENNEDYD, HIDAKAM, ET AL: Identification of the Cystic Fibrosis Gene: Chromosome Walking and Jumping. Science 1989, 245:1059-1065. DRUMMML, POPE HA, CLIFF WH, ROMMENSJM, MARVIN SPh
TsuI LC, COLLINS FS, FRIZZELLRA, WILSONJM: Correction of
the Cystic Fibrosis Defect In Vitro by Retrovirus-mediated Gene Transfer. Cell 1990, 62:1227-1233. Amphotropic retroviruses were used to transfer functional CFTR cDNA into cells from a CF patient. The expression of the normal gene conferred cAMP-dependent chloride channel regulation on CF epithelial cells. 28. •
WILSONJM, PING AJ, KRAUSSJC, MAYO-BOND L, ROGERS CE, ANDERSONn c , TODD RE: Correction o f CD18-deficient Lymphocytes by Retrovirus-mediated Gene Transfer. Science 1990, 248:1413-1416. Lymphocytes from a patient with CD18 deficiency were infected with a retrovirus encoding normal CD18 subunit. Transduced cells expressed normal surface levels of CD18-containing transmembrane protein with reconstitution of normal adherence function. 29.
BACKAL, KWOK WW, ADAM M, COLLINS SJ, H1CKSTEIN DD: Retroviral-mediated Gene Transfer of the Leukocyte Integrin CD18 Subunit. Biochem Biophys Res Com 1990, 171:787-795. Similar study and conclusions to [28"]. •
30.
GOLDSTEINJL, BROWN MS: Familial Hypercholesterolemia. In The Metabolic Basis of Inherited Disease edited by Scriver CR, Beaudeut SL, sly WS, Valle D [book]. New York: McGraw-Hill 1989, pp 1215-1250.
31.
WILSONJM, CHOWDHURYNR, GROSSMANM, WAJSMANR, EPSTEIN A, MULLIGAN RC, CHOWDHURYJR: Temporary Amelioration of Hyperlipidemia in Low Density Lipoprotein Receptordeficient Rabbits Transplanted with Genetically Modified Hepatocytes. Proc Natl Acad Sci USA 1990, 87:8437-43441. A novel in vivo transplantation of transduced hepatocytes demonstrated the potential of gene therapy in ameliorating hyperlipidemia associated with familial hypercholesterolemia. •
32. •
FLOWERSMED, STOCKSCHLAEDERMAR, SCHUENINGFG, NmDERMILLERAn, STORB R: Long-term Transplantation o f Canine Keratinocytes Made Resistant to G418 Through Retrovirus-mediated Gene Transfer. Proc NatlAcad Sci USA 1990, 87:2349-2353. Transplanted infected autologous keratinocytes expressed a foreign gene for at least 130 days. WIESER D , HACKMAN R,
33.
KANGJ, WITHER J, HOZUMI N: Long-term Expression of a T-cell Receptor 13-chain Gene in Mice Reconstituted with Retrovirus-infected Hematopoietic Stem Cells. Proc Natl Acad Sci USA 1990, 87:1903-1907. Mice transplanted with infected bone marrow expressed an exogenous T-cell receptor I~-chain on thymocytes and splenic T cells, suggesting that genes involved in T-cell development can be studied in genetically diverse host environments. •
WRA Osborne, Department of Pediatrics, RD-20 University of Washington, Seattle, Washington 98195, USA.
