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Gene expression using retroviral vectors
Gene expression using retroviral vectors Paul Tolstoshev and W. French Anderson* Genetic Therapy Incorporated, Gaithersburg, MD 20878, *Molecular Hematology Branch, National Health Lung and Blood Institute, National Institute of Health, Bethesda, MD 20892, USA Current Opinion in Biotechnology 1990, 1:55-61
Introduction The objective of this review is to consider the most significant advances that have been made in the past few years in the use of retroviral vectors for gene expression. Undoubtedly, one of the most significant of these has been the first sanctioned use, in May 1989, of such agents for human clinical studies. Significant progress has also been achieved in the in vivo expression of the adenosine deaminase (ADA) gene inserted into mouse bonemarrow cells and reintroduced via bone-marrow transplantation (BMT), in the retroviral-mediated expression of other genes of clinical relevance, and in retroviral vector technology itself, including construction of new types of vectors, attempts to increase the titers of retroviral supernatants, and some detailed safety studies particularly in primates. The next few years promise to yield substantial clinical evaluation of retroviral vectors for use not only in the correction of genetic defects, but also as a generalized drug-delivery system for protein therapeutics. In the latter application, genes expressed from retroviral vectors have the potential to solve some of the substantial problems associated with protein delivery using conventional delivery modalities.
Retroviral vectors and packaging systems The details of the ways in which retroviruses, in particular Moloney murine leukemia virus (MoMuLV), can be used as vectors to introduce foreign genes into cells will not be described, as they have been the subject of a number of very good recent reviews [1 °,2-4]. The concepts of replacement of internal viral function genes with the foreign gene(s), and provision of viral proteins via a packaging cell line that is itself unable to make packageable transcripts [5] are now very standard. Initial vectors incorporating a single selectable marker gene, such as the neomycin phosphotransferase (neoR) gene [6] or the human hypoxanthine phosphoribosyl transferase gene [7] were shortly followed by neoR-gene-containing vectors such as pZlPNeo [8] and then N2 [9]. In these
newer vectors, a second foreign gene is able to be introduced, downstream from the neoR gene, in the vector backbone. Such vectors have been used to express an impressively long list of foreign genes [1 ° ] for a wide variety of purposes. Major applications of MoMuLV~based vectors have been the introduction of genes into bonemarrow stem and progenitor cells as well as other cell types, their use as insertional mutagens in cultured cells or transgenic animals and their use as lineage markers for cells during development. Recent efforts in vector development have concentrated on minimizing the chances of recombination between the vector and the packaging-defective helper functions within packaging cells. Such modifications aim to minimize the chances of generating recombinant wild-type virus; their generation is undesirable for both research purposes and safety reasons in clinical applications. The modification of the N2 vector to form the LNL series [10] has resulted in vectors that, when used in conjunction with the PA317 packaging cell line [11], have a greatly reduced likelihood of regenerating wild-type virus. This is a result of modifications that include conversion of the gagATG start to a TAG stop codon, and the replacement of the region 5' to the authentic gag start with the same region from Moloney murine sarcoma virus. Both these changes greatly reduce the chance of the retroviral reading frames being expressed. For the LNL6/PA317 combination, two recombination events would be required to generate active wild-type virus. The safety features of this vector/packaging system are sufficient for it to have been approved for use in the first sanctioned clinical protocol (see below). A further modification of this vector set yielded the LN set of vectors [12 °'], which, in addition to the modifications already described for LNL6, have an additional deletion of all remaining env sequences at the 3' end. These vectors have been constructed as a set of four: LN, LNSX, LNCX and LXSN, in which the S designates a simian virus (SV)40 early promoter, the C a cytomegalovirus immediate early promoter, and X the site of a unique restriction site set, where a cDNA sequence for expression can be inserted into the vector. Clones
Abbreviations ADA--adenosine deaminase; BMT--bone-marrowtransplantation; cfu--colony-forming units; DC~ouble copy; GC~lucocerebrosidase; IL--interleukin; LTR--Iongterminal repeat; MoMuLV--Moloney murine leukemia virus; neoR--neomycin phosphotransferase;PK--phosphoglyceratekinase; SClD--severe combined immunodeficiency; SIN--self-inactivating; SV--simian virus; TIL--tumor-infiltrating lymphocytes; tPA--tissue plasminogen activator. © Current Biology Ltd ISSN 0958-1669
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Expressionsystems from the LN vector have been shown to yield high titers [as high as those achieved with N2, i.e. up to 4 x 107 colony-forming units (cfu)/ml] and, for three different clones, to remain negative for helper virus for up to eight sequential passages. An alternative approach to reducing the likelihood of recombInation events is to separate or 'split' the packaging functions in the packaging cell line. This concept was first elucidated and demonstrated for avian reticulo-endotheliosis virus [5] and has now been extended to MoMuLV packaging lines in the form of the ~CRIP and ~CRE lines [13], and the GP + E packaging cell line [14]. In the split packaging lines, and with the appropriate vectors, three recombinational events are required to regenerate wildtype virus. A general concern associated with expressing more than one gene in a retroviral vector is that the retroviral long terminal repeat (LTR) transcriptional unit will interfere with the expression of an internally promoted gene; however, there is little definitive data to support this. A recent attempt to circumvent this problem has led to a novel vector, called a double copy (DC) vector [15"]. In this construct, an ADA gene and its cellular promoter were inserted into the U3 region of the 3' viral LTR. Integration of this vector into a target cell results in the duplication of the ADA gene, and in its placement outside the retroviral transcriptional unit (at least for the 5' duplicated ADA gene---the second copy remains within the U3 region of the 3' LTR). Such a vector led to a 10-20fold increase in ADA transcripts and human ADA synthesis in NIH 3T3 cells, when compared with a conventional internally promoted ADA vector. Similar increases were also observed in two human lymphoid cell lines. Such vectors hold promise as they may substantially increase gene expression in gene transfer both in vivo and in vitro. Whether difficulties will be encountered in generating sufficiently high titers to allow widespread use of such vectors remains to be established. An earlier vector con cept, the self-inactivating (SIN) vector [16], where a deletion in the 3' LTR leads to the inactivation of the proviral transcriptional unit, has not achieved general utility, primarily because of problems in achieving acceptable vector titers. Of paramount importance is the titer of virus particles that can be achieved. Most consistently high and stable titers appear to be achieved when packaging cell lines are infected, rather than transfected. Such infected packaging lines will contain one, or only a small number of copies of the vector, and further infection by the same vector is blocked. One approach to increasing the titer of vector particles produced is to introduce larger numbers of vector genome copies via a vector packaged within a different envelope. This concept, most easily achieved by cocultivation of two different packaging cell lines each producing differently packaged vector (e.g. amphotropic and ecotropic), is now commonly known as a 'ping-pong' approach. It was initially used [ 17] to achieve an increase in the expression of genes introduced into target cells with retroviral vectors. A recent extension of this approach was reported by Bodine et al. [18 °'] who used a cocultivation approach with
an N2-vector-producing amphotropic producer cell line. In addition, this producer had been co-transfected with plasmids containing the gibbon interleukin (10-3, human IL-6 and hygromycin-resistance genes. A hygromycin-resistant population was isolated, shown to express the IL3 and 1I,-6 genes, and co-cultivated with the ecotropic GP + E86 cell line for 14 days. After this time, the initial viral titer had risen from 5 x 106 to 109 cfu/ml. Individual amphotropic clones could be reisolated from the mixed population by hygromycin selection, and, when these were screened by titer, four clones producing titers of greater than 109 cfu/ml were identified. The highest titer reported was 2 x 1010 cfu/ml and, in this clone, Southern blot analysis showed an approximate 20-fold increase in provirus copy number, and about a 1000-fold increase in production of retroviral RNA transcripts when compared with the parental cell line. This producer clone was then used to introduce the vector into rhesus bonemarrow cells, by cocultivation; the marrow was then reinfused into three sub-lethally irradiated animals. The vector genome was detectable in the blood and bone marrow of all three animals at 30-90 days post-transplantation. No gene transfer was detected in three animals where a lower titer (5 x 106 cfu/ml) vector producer was used. These preliminary, but encouraging, data reinforce the idea that high vector titers may be of great importance in introducing vectors into bone marrow, particularly into pluripotential stem cells. However, an important and intriguing question remains about the role of replicationcompetent virus in the success of this approach. The initial N2 amphotropic producer already generated low levels (less than 101 cfu/ml) of replication-competent virus, and in the high-titer producer, replication-competent virus was detected at about 104 cfu/ml. The cocultivation ping-pong approach is clearly well suited to increasing vastly the chances of occurrence of the recombination events leading to replication-competent virus production. In fact, this approach has been used to evaluate the safety of vector/packaging cell systems. For example, a series of cross-infection cycles was used to compare the generation of replication-competent virus using ~CRIP/~CRE and PA317/~CRE packaging systems [13]. A more recent study [19" ] used the cocultivation approach to evaluate a whole series of packaging cell/vector combinations. The time at which the replication-competent virus arose appeared to correlate well with the number of recombination events needed to generate the virus in each system. Interestingly, the very large increase in titer observed by Bodine et al. [18,.] was not seen in this study. However, the relative growth rates of the amphotropic and ecotropic producer lines will clearly influence the rate of amplification of vector copies in the genome. The basic question that remains concerns the role of replication-competent virus in the process of increasing the titer from producer cells; is it a prerequisite for, or a consequence of, the amplification process? An alternative hypothesis is that presence of helper, and its subsequent spread to both the ecotropic and amphotropic producers, will shut down the ping-pong process and, in
Gene expression using retroviral vectors Tolstoshev and Anderson fact, limit the increase in titer. These are clearly important questions to resolve, especially as the clinical utility of such high-titer producers generated by Bodine et aL may be less significant if wild-type virus cannot be eliminated. However, such elevated titer vector producers are extremely attractive and useful for studies of bone-marrow transduction, for optimization of conditions to transduce a variety of other cells, and for other research pur poses. The details of the binding of MoMuLV to the cells it infects are still poorly understood at the molecular level but should become clearer following the recent isolation [20] of a cDNA clone that may encode the receptor that binds to the ecotropic envelope of this retrovirus. Expression of this cDNA in human EJ cells resulted in a 106fold increase in MoMuLV infectivity. The cDNA sequence predicts an extremely hydrophobic protein with 14 potential membrane-spanning domains.
Expression of the ADA gene The first genetic disease to be treated by gene therapy will most likely be severe combined immunodeficiency (SCID), a disease that is caused by lack of ADA. A number of groups have constructed retroviral vectors containing the ADA gene, and have demonstrated expression of the gene in cells that have been transfected in culture. However, when such studies have been extended to the requisite target, the hematopoietic stem cell, and such cells reintroduced into animals by BMT, sustained expression of the ADA gene has been far more difficult to obtain. In mice, monkeys, dogs and cats, only low-level and transient expression had been observed. The first demonstration of long-term (approximately 1 month) expression in mice [21] also revealed replication-competent virus in the animals' plasma, raising the possibility that persistent reinfection of progenitor cells (rather than an initial stemcell transduction) is responsible for the observed activity. A series of recent studies [22 o.- 25 ..,26.,27,.] in mice have reported somewhat more success in achieving longterm in v i v o A D A expression. The first of these, by Moore et al. [22--], extended earlier studies but with the vector now packaged in a 'split' packaging line, GP + E86. In the absence of a selectable marker such as neoR, titers of this virus are estimated through its ability to transduce and express in a tissue-culture cell, with ADA expression measured immunohistochemically. Mouse bone marrow was transduced by cocultivation with producer cells, and then reintroduced into irradiated recipients. Human ADA expression was detected in total blood, and persisted best (five out of seven mice positive at 18-31 weeks) under conditions where a mixed conditioned medium was used during transduction. All 37 transplanted mice were ADA positive for at least 9 weeks. A detailed study of expression in a variety of tissues of hematopoietic origin showed great variability among the individual mice. All mice tested negative for replication-competent virus over the study period. Expression and presence of the vector in secondary recipient mice was, however, only limited and transient. Though efficacy of stem-cell transduction, and efficiency of expression were low in this study, it
did demonstrate that extended ADA expression in transduced bone :narrow can be obtained in mice. A similar study, also in mice using vectors without a selectable marker, was performed by Wilson et al. [23 "']. Two vectors were compared: one with the ADA gene driven from the viral LTR, the other with an internal chicken [3-actin promoter driving the ADA gene in a vector also containing a deletion of enhancer sequences from the 3' LTR (to reduce retroviral transcription after integration). Bone marrow was transduced by cocultivation and the cells were then transplanted into lethally-irradiated recipients. Analysis of human ADA expression in peripheral blood mononuclear cells revealed that by day 33 all of the nine animals reconstituted with the [3-actinpromoted vector expressed the gene, while only two out of seven receiving the LTR-driven ADA gene vector were positive. The pattern continued basically unchanged to 181 days. A study of the tissue distribution of activity among hematopoietic tissues in selected animals showed, once again, substantial variability, but this time the activity could be detected in virtually all tissues in each animal studied. The levels of human ADA relative to endogenous mouse ADA were quite variable, and reached 10-fold the endogenous level in the hemolysate of one animal. DNA analysis indicated high levels of intact provirus in all tissue fractions, with copy numbers ranging from 0.5 to 1.0 per cell for the vector with the LTR driving the gene, and less than 0.5 for the internally promoted ADA gene vector. No infectious virus, or transmission of packaging functions, could be detected in any animal. A third study in mice [24"',25"'] used vectors of the LN series, where either the LTR, the SV40 or the cytomegalovirus immediate early promoter controlled ADA gene expression. Procedures used were very similar to the two studies described above. On testing the peripheral blood of recipients at 6 months after transplantation, about a third of the animals expressed human ADA at levels of 1-5% of the endogenous enzyme; resuits were similar for all three vectors tested. In contrast, analysis of these vectors in culture with various human hematopoietic cell lines [26. ] revealed significant differences in expression when different promoters drove the ADA gene. Although in the in vivo studies [24..] helper virus was detected in the blood in three out of the four mice tested this had the characteristics of endogenous mouse xenotropic virus, most likely arising by activation of endogenous retrovirus. Confirmation of this source of the replication-competent virus was obtained in a subsequent study [25"']. Finally, Lim et al. [27"] have demonstrated stable long-term expression of human ADA in recipient mice of bone marrow transduced with a vector lacking a selectable marker, and where the ADA gene is driven by an internal phosphoglycerate kinase (PK) promoter. All recipients expressed the gene at 30 days post-transplantation, and about half maintained it until 4 months. A secondary transplant into a further recipient showed continued expression of the ADA gene. All of these studies provide encouragement that the longterm objective of treatment of ADA deficiency by gene transfer into bone-marrow stem cells will ultimately be
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58 Expressionsystems feasible in humans. Before this, however, studies similar to those described for mice will have to be performed in primates. An additional relevant study [28" ] has described substantial expression of human ADA (15-100% of normal) in the lymphoid and myeloid progeny of longterm cultures of bone-marrow cells from ADA-deficient patients, where the bone marrow was transduced with ADA-gene-containing retroviral vectors. Transduced cells derived from the marrow cultures exhibited restoration of their capacity to respond to phytohemagglutinin and IL-2. Finally, the study of Bodine et al. [29] showing increased preservation of stem cells in culture and higher genetransfer efficiency in primates using combinations of IL3 and IL-6 advances our understanding of the optimal in vitro conditions for handling bone marrow for gene transfer. In the shorter time-frame, however, successful transfer of marker genes via retroviruses to cancer patients' lymphocytes has confirmed the concept that the human ADA gene could be inserted into the peripheral lymphocytes of ADA-deficient patients. A clinical protocol to this effect received provisional approval from the National Institutes of Health (NIH) Recombinant DNA Committee's Human Gene Therapy Subcommittee in June, 1990.
Expression of other genes of clinical relevance A variety of other genetic diseases are candidates for gene therapy approaches. Two recent reviews [30",31 ,] consider the prospects for achieving such therapy, primarily with retroviral-based technologies. "While the initial focus is likely to be on gene introductions into lymphocytes for treatment of ADA deficiency and for certain forms of cancer, other clinical targets are clearly beginning to emerge. Bone-marrow based diseases are a major area of interest. Attempts to refine the procedures for long-term culturing of bone-marow for gene transfer continue [28. ,32,33], but questions still remain as to how relevant such studies are to gene insertion into true hematopoietic stem cells, rather than committed progenitor cells. Reconstitution by BMT still remains the only definitive way to demonstrate true totipotent stem-cell function. Gaucher disease, the most prevalent human lysosomal storage disorder, is caused by a deficiency of the enzyme glucocerebrosidase (GC) and is characterized by accumulation of complex glycolipids in macrophages. Allogenic BMT has been shown to be beneficial, but can be limited by lack of histocompatible sibling donors. This disease is clearly a candidate for gene therapy, and the human GC gene is now available as a cDNA clone. Two recent reports [34",35 " ] describe studies involving retroviral vectors expressing the human GC gene. In the first [ 3 4 " ] , the vector has been used to demonstrate persistent expression of the human GC gene in long-term bone-marrow cultures for over two months. The second study [35 " ] compared a set of four GC-gene-expressing vectors for their ability to transduce and express the GC gene in mouse multipotential hematopoietic progenitor cells. Both human GC-specific RNA, and GC protein were
detected. Two of the vectors were shown to direct human GC RNA transcription after transplantation of transformed bone marrow into lethally irradiated mice. Provision of clotting factors to patients with hemophilia is another potential gene-therapy application. Retroviral vectors expressing the Factor IX gene have been constructed and shown to express the biologically active product in fibroblasts both in vitro and transiently in vivo when fibroblasts are transplanted back into mice or rats [36" ,37]. Two recent studies [38" ,39" ] have reported the introduction of truncated forms of the human Factor VIII gene into retroviral vectors, and the generation of vectors that could transduce fibroblasts and generate detectable levels of Factor VIII. In both cases, however, titers of vector from producer cells were low. In the application of gene-therapy techniques to hemophilia, attention must be given to which is the most appropriate recipient cell to express the gene, and to strategies for the reintroduction or implantation of the transduced cells back into the patient. A candidate cell for a variety of genes and for Factor VIII in particular, is the vascular endothelial cell [40]. Retroviral-mediated gene transfer into vascular endothelial cells has been possible for some time, and two stud ies [41 ,-,42-.] have demonstrated in vivo expression of retrovirally transduced genes in such cells. The first [41 -.] used prosthetic vascular grafts seeded with transduced endothelial cells expressing a bacterial [3-galactosidase gene. Five weeks after implantation into dogs, cells that were expressing introduced genes could still be found. In the second study [42 ,-], a novel approach, using a balloon catheter, was used to introduce porcine endothelial cells expressing a recombinant fl-galactosi dase from a murine amphotopic vector into denuded iliofemoral arteries. Explants of such arterial segments yielded ~-galactosidase-expressing cells 2 4 weeks later. Finally, retroviral vectors expressing [3-galactosidase or human tissue plasminogen activator (tPA) have been introduced into cultured sheep endothelial cells [43"]. These cells were seeded onto stainless steel stents, where they continued to secrete tPA. This approach could improve stent function through localized delivery of an anticoagulant, thrombolytic or antiproliferature molecule.
Gene marking of human tumor-infiltrating lymphocytes Tumor-infiltrating lymphocytecell immunotherapy Tumor-infiltrating lymphocytes (TIL) cells are a lymphocyte population grown in vitro from tumor biopsy sampies of patients with metastatic melanoma, and are re-administered to patients, in combination with high doses of IL-2, as an adoptive immunotherapy approach to this form of cancer. The therapy, developed by Rosenberg and his associates [44] has shown a significant response (40%) in terminal melanoma patients. However, TIL cells are a mixed population of lymphocytes, and it has proven difficult to obtain clinical correlates of successful treatment. One approach is to use cell-marking studies, but
Gene expression using retroviral vectors Tolstoshev and Anderson conventional 111Indium labelling has such a short half-life (2.8 days) that it yields data for only a few days [45]. The idea of using a marker gene for neomycin resistance, delivered via a retroviral vector, to track TIL cells during therapy was conceived in early 1988. This was a logical extension of previous uses of retroviral vectors as genetic markers in cell lineage studies during embryonic development, tissue differentiation, and as somatic cell hybrid chromosome markers. Because of the presence of the acquired neomycin-resistance gene, such studies of TIL cells held the prospect of identifying active subpopulations in TIL cell populations, of establishing homing to tumor sites over an expanded time frame, of determining the half-lives of TIL cells in humans and of re-isolation of cell subpopulations.
Safety and feasibility studies Before the clinical protocol could be approved, a number of safety and feasibility issues had to be addressed. The issues of major concern were related to the possibilities of generating replication-competent virus, both in the producer cell line and in vivo. A variety of safety studies have been performed, and some of these have been published [31 .,46..]. The introduction of amphotropic retrovirus into rhesus monkeys has been studied [47 "] under a variety of different conditions, including immunosuppression of the animals, and continuous delivery of virus through implantation or intraperitoneal injection of virus-producing autologous fibroblasts. The general conclusion was that murine amphotropic retrovimses do not appear to pose an acute health risk, as no instance of clinical illness was seen in a follow-up time of 32 monkey-years. The question of infectious virus production by producer cells has been discussed earlier, and is reviewed by Miller [47 °]. The activation of oncogenes by insertional mutagenesis (or inactivation of suppressor genes) could theoretically trigger oncogenic events in recipient cells, but current data suggest that the frequency is likely to be low and that additional events are required before a malignancy is established. One of the requisites for approval of a clinical protocol was a demonstration that human TIL cell populations could be transduced with a retroviral marker, and that such a transduction did not significantly change the properties of these cells. Such a study was performed and has been published [48..]. It is interesting to note, however, that mouse TIL cells in a mouse model of this immunotherapy treatment were totally refractory to transduction with a murine retroviral vector, despite enormous efforts in several laboratories. Other animal studies, involving lymphoid cell lines of mice and primates studies, were therefore performed as part of the preclinical studies.
mittee and its Human Gene Therapy Subcommittee, and the Food and Drug Administration. Final approval was granted on January 10, 1989. The first patient received marked cells on May 22, 1989, and, as of this writing, eight patients have received gene-marked cells. Of the patients who have received gene-marked cells to date, five have been analysed in detail [50"']. Gene-marked TIL persist in the circulatory system of patients for about 3 weeks and a proportion of TIL can be tracked to sites of tumors. On March 30 1990, the NIH Recombinant DNA Advisory Committee approved an extension of these studies to an unlimited number of patients. No adverse effects of the administration of the gene-marked cells have been observed in any of the patients.
Further clinical protocols involving retroviral vectors The concept of lymphocytes as target cells for retroviralmediated gene transfer is now well established. For TIL therapy, the next logical extension is to use TIL cells to deliver lymphokines such as tumor necrosis factor, IL2 etc. to attempt to augment the therapy. For other purposes, however, lymphocytes are also being considered as target cells; notably to introduce into the lymphocytes of ADA patients vectors containing the ADA gene. A clinical protocol along these lines is currently under Recombinant DNA Advisory Committee review. Additional genemarking experiments may also be of clinical utility; for example, in childhood and adult leukemia, it may be possible to obtain information about the origin of cells responsible for relapse after autologous BMT treatment for these diseases.
Conclusion The last year has seen major advances in the use of retroviral vectors for gene expression. Most notable among these were the first sanctioned clinical trial, and significant advances in transducing and expressing genes stably in murine bone-marrow stem cells. Spurred by such achievements, the field of retroviral-mediated gene transfer will undoubtedly continue to develop rapidly.
Annotated references and recommended reading • ••
Of interest Of outstanding interest
1. ,
McLAUCHLINJR, CORNETrA K, EGLITIS MA, ANDERSON WF: Retroviral-mediated gene transfer. Prog Nucl Acid Res Mol Biol 1990, 18:91-135. A comprehensive review of retroviral constructs, and the genes that have been expressed in them.
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2.
VARMUSH: Retrovirus. Science 1988, 240:1429-1435.
A clinical protocol to genetically mark patient TIL cells with a retroviral vector was submitted in June 1988. The protocol has been published [49"'] as has a chronology of the review process, which involved NIH internal committees, the NIH Recombinant DNA Advisory Corn-
3.
EGL1TISMA, ANDERSONWF: Retroviral v e c t o r s for introduction of genes into mammalian cells. Biotechniques 1988, 6:608414.
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Expressionsystems TEMIN HM: Construction o f a helper cell line for avian reticulo-endotheliosis virus d o n i n g vectors. Mol Cell Biol 1983, 3:2241-2249.
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MILLERAD, JOLLY DJ, FRIEDMANTJ, VERMAIM: A transmissible retrovirus expressing h u m a n HPRT: g e n e transfer into cells obtained from h u m a n s deficient in HPRT. Proc Natl Acad Sci USA 1983, 80:47094713.
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BENDERMA, PALMERTD, GELINASRE, MILLERAD: Evidence that t h e packaging signal of Moloney m u r i n e leukemia virus e x t e n d s into the gag region. J Virol 1987, 61:1639-1646.
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MILLERAD, BtrlqTMORE C: Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 1986, 6:2895-2902.
generation of replication c o m p e t e n t virus. Virology 1990, 176:262-265. Amplification of vector sequences by co-culture of ecotropic and amphotropic packaging cell lines is studied to determine the host range, and time of appearance of replication-competent vires. The time required was consistent with the number of recombination events required for generating wild-type vires. This procedure can be used to assess the safety of a particular packaging line/vector combination. 20.
ALBRITTONLM, TSENG L, SCADDEND, CUNNINGHAMJM: A putative m u r i n e ecotropic retrovirus receptor gene e n c o d e s a multiple m e m b r a n e - s p a n n i n g protein and confers susceptibility to virus infection. Cell 1989, 57:659-666.
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BELMONTJW, MACGREGORGR, WAGER-SMITHK, FLETCHER FA, MOORE KA, HAWKINS D, VILLALON D, CHANG SMW, CASKEY CT: Expression of h u m a n adenosine deaminase in m u r i n e hematopoietic cells. Mol Cell Biol 1988, 8:5116-5125.
22. ••
MOOREKA, FLETCHER FA, VILtALON DK, UTrER AE, BELMONT JW: H u m a n adenosine deaminase expression in mice. Blood 1990, 10:2085-2092. This report describes bone-marrow transduction experiments with an ADA vector, but no sign of replication-competent virus production. Long-term survival of the ADA gene and expression in hematopoietic tissues of recipient mice for as long as 7 months were demonstrated and no evidence of wild type virus was found in the animals.
12. MILLERAD, ROSMANGJ: Improved retroviral vectors for g e n e •• transfer and expression. Biotechniques 1989, 7:980-990. This paper describes the structure, construction and properties of the LNL and LN series of MoMuLV retroviral vectors. These vectors, together with the PA317 packaging line, currently represent one of the safest vector systems and their vector products have recently been used in clinical gene-marking studies in humans.
WILSONJM, DANOS O, GROSSMAN M, RAULET DH, MUIMGAN RC: Expression of h u m a n adenosine deaminase in mice reconstituted w i t h retrovirus transduced hematopoietic s t e m cells. Proc Natl Acad Sci USA 1990, 87:439-443. The authors report the successful reconstitution of mice with bone marrow containing one of two kinds of retroviral vector for expressing the h u m a n ADA gene. Significant expression of the gene in hematopoietic cells was observed for up to 6 months post-transplant.
13.
DANOSO, MULLIGANDAz Expression of retroviral trans-acting functions from c o m p l e m e n t a r y crippled genomes: a syst e m for helper free packaging of retroviral vectors. J Cell Biochem 1988, 12:172.
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MARKOWlTZD, G-OFF S, BANK A: A safe packaging line for g e n e transfer: separating viral g e n e s o n two different plasraids. J virol 1988, 62:1120-1124.
15. ••
HANZOPOULOSPA, SULLENGERBA, UNGERS G, GItBOA E: Improved g e n e expression u p o n transfer of the adenosine deaminase minigene outside of t h e transcriptional unit of a retroviral vector. Proc Natl Acad Sci USA 1989, 86:3519-3523. This study describes DC vector, and shows the construction of one such vector, containing a h u m a n ADA gene. The inserted gene is indeed duplicated on integration into target cells, and expression of the functioning protein is significandy increased both in mouse fibroblasts and in h u m a n lymphoid cell lines. 16.
Yu S-F, VON RUDEN T, KANTOFF PW, GARBER C, SEIBERG M, RUTHER U, ANDERSON WF, WAGNER EF, GILBOA E: Serf-inactivating retroviral vectors designed for transfer of w h o l e g e n e s into mammalian cells. Proc Natl Acad Sci USA 1986, 83:3194-3198.
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BESTWICKRK, KOZAKSL, KABATD: Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes. Proc Natl Acad Sci USA 1988, 85:5404-5408.
18.
BODINEDM, MCDONAGH KT, BRANT SJ, NEY PA, AGRICOLAB, NIENHUIS AW: Development of a high-titer retrovirus p r o d u c e r cell line capable of gene transfer into r h e s u s m o n k e y hematopoietic s t e m cells. Proc Natl Acad Sci USA 1990, 87:373~3742. In this paper, the authors report the generation of producer clones generating vectors of extremely high titer ( > 1010 cfu/ml) by a cocultivation of ecotropic and amphotropic producer cell lines. A concomitant increase in proviral DNA copy n u m b e r and DNA transcripts is demonstrated, and such high-titer'vectors are shown to improve the efficiency of gene transfer to bone-marrow cells of primates in an in vivo BMT study. However, such high-titer vector supematants do contain measurable levels (up to 104 focus-forming units/ml) of replication competent virus, as might be expected from the process of generating such producers. • •
19. •
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MUENCHAU DD, FREEMAN SM, CORNETrA K, ZWIEBEL JA, ANDERSONWF: Analysis of retroviral packaging lines for
23. •.
OSBORNEWRA, HOCK RA, KALEKO M, MILLERAD: Long-term expression of h u m a n adenosine deaminase in mice after transplantation o f bone m a r r o w infected with amphotropic retroviral vectors. H u m Gene Ther 1990, 1:31~41. Another study that successfully demonstrated long-term expression of h u m a n ADA in mice transplanted with bone marrow transduced with amphotropically packaged vector. 25. ••
KALEKOM, GARCIAJV, OSBORNE W f ~ MILLERAD: Expression of h u m a n adenosine deaminase in mice after transplantation of genetically-modified bone marrow. Blood 1990, 75:1733-1741. An extension of the studies described in [24 ••] of survival and expression of the h u m a n ADA gene in the LN series in long-term BMT studies in mice using ecotropically packaged vector. 26. •
HOCK RA, MILLERAD, OSBORNE WRA: Expression of h u m a n adenosine deaminase from various strong p r o m o t o r s after gene transfer into h u m a n hematopoietic cell lines. Blood 1989, 74:8764381. A comparative analysis of different LN vector constructs carrying the h u m a n ADA gene under the control of different promoters with regard to their ability to express the gene in h u m a n hematopoietic cell lines in vitro. 27. ••
L1MB, APPERLEYJF, ORKIN SH, WILLIAMSDA: Long-term expression of h u m a n adenosine deaminase in mice transplanted w i t h retrovirus-infected hematopoietic s t e m cells. Proc Natl Acad Sci USA 1989, 86:8892-8896. This study uses a retroviral vector lacking a selectable marker to transfer a human ADA gene into mice bone marrow, and shows long-term expression for up to 4 months. In addition, a secondary transplant continues to confer h u m a n ADA expression of the recipient mouse. 28. •
BORDIGNONC, YU S-F, SMITH CA, HANTZOPOULOSP, UNGERS GE, KEEVER CA, O'REILLYR, GILBOA E: Retroviral vectormediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADAdeficient m a r r o w cells. Proc Natl Acad Sci USA 1989, 86:6748-6752. Bone marrow from ADA-deficient patients was transduced with vectors containing h u m a n ADA genes. Lymphoid and myeloid progeny of cultures of such marrow expressed substantial levels of h u m a n ADA_ 29.
BODINEDM, KARLSSON S, NIENHUIS AW: Combination of interleukins 3 and 6 preserves s t e m cell function in culture and e n h a n c e s retrovirus-mediated g e n e transfer into hematopoietic s t e m cells. Proc Natl Acad Sci USA 1989, 86:8897-8901.
G e n e expression using retroviral vectors T o l s t o s h e v a n d A n d e r s o n 30.
FRIEDMANT: Progress toward h u m a n gene therapy. Science 1989, 244:1275-1281. ~his review considers the current state of retroviral vector technology directed towards gene therapy procedures for the major single-gene h u m a n genetic diseases. CORNETrAK, WIEDER R, ANDERSON WE: G e n e transfer into primates and prospects for g e n e therapy in humans. Prog Nucl Acid Res Mol Biol 1989, 36:311-322. A review that focuses o n the prospects of bone-marrow-directed gene therapy procedures and reviews safety data derived from primate studies with retroviral vectors and replication-competent helper virus. 31. •
32.
HUGHES PFD, EAVES CJ, HOGGE DE, HUMPHRIES RK: Highefficiency g e n e transfer to h u m a n hematopoietic cells maintained in long-term m a r r o w culture. Blood 1989, 74:1915-1922.
33.
SCHUENING FG, STORB R, STEAD RB, GOEHLE S, NASH R, MILLERaD: I m p r o v e d retroviral transfer of g e n e s into canine hematopoietic progenitor cells kept in long-term m a r r o w culture. Blood 1989, 74:152-155.
34. ••
NOTTAJA, SENDER IS, BARRANGERJA, KOHN DB: Expression of h u m a n glucocerebrosidase in murine long-term bonem a r r o w cultures after retroviral vector-mediated transfer. Blood 1990, 75:787-797. An N2-based retroviral vector containing the h u m a n GC gene was used to transduce m o u s e bone-marrow cells. Maintenance of bone marrow in long-tern1 culture showed persistence of GC expression for up to 2 months. 35. ••
CORRELLPH, FINK JK, BRADEYRO, PERRY IX, KARISSON S: Production of h u m a n glucocerebrosidase in mice after retroviral gene transfer into multipotential h e m a t o p o e t i c progenitor cells. Proc Natl Acad Sci USA 1989, 86:8912-8916. This study describes the use of a series of N2-based vectors containing the h u m a n GC gene to transduce m o u s e hematopoietic progenitor cells, as assayed by colony assays, and stem cells after BMT. 36. •
PALMERTO, THOMPSON AR, MILLERAn: Production of h u m a n Factor IX in animals by genetically modified skin fibroblasts: potential t h e r a p y for hemophilia B. Blood 1989, 73:438-445. The human Factor IX gene is expressed in an active form in fibroblasts in vitro after transduction with a variety of LN-based retroviral vectors containing the gene. When such rat fibroblasts are reintroduced back into animals, a transient production of h u m a n Factor IX was detected in plasma. 37.
ST LOUIS D, VERMA IM: An alternative approach to somatic ceR g e n e therapy. Proc Natl Acad Sci USA 1988, 85:3150-3154.
38. •
ISRAELDI, KAUFMANRJ: Retroviral mediated transfer and amplification of a functional h u m a n Factor VIII gene. Blood 1990, 75:1074-1080. A retroviral vector containing both a h u m a n ADA gene, as a selectable and amplifiable marker, and a truncated h u m a n Factor VIII gene is described. Expression of both genes was demonstrable in mouse fibroblasts in vitro, viral titer, ADA expression and Factor VIII expression were increased by selection for ADA. 39. •
HOEBENRC, VAN DER JAGT RCM, SCHOUTE F, VAN TILBERG NM, VERBEETMP, BIET E, VAN ORMONDT M, VANDER EB AJ: Expression of functional Factor VIII in primary h u m a n skin fibroblasts after retrovirus-mediated g e n e transfer. J Biol Chem 1990, 265:7318-7323. A neoR/Factor VIII (truncated gene) retroviral construct was shown to be able to transduce murine and primary h u m a n fibroblasts and express biologically active h u m a n Factor VIII activity. 40.
ZWEIBELJA, FREEMAN SM, KANTOFF PW, CORNE'ITA K, RYAN US, ANDERSONWF: High-level r e c o m b i n a n t g e n e expression in rabbit endothelial cells transduced by retroviral vectors. Science 1989, 243:220-222.
41. ••
WILSON JM, BIRINY1 IX, SOLOMON RN, LIBBY P, CALLOW AD, MULLIGAN RC: Implantation of vascular grafts lined
w i t h genetically modified endothelial cells. Science 1989, 244:1344-1346. Vascular endothelial cells of dogs were transduced with a retrovims containing a 13-galactosidase gene, and then seeded onto vascular grafts in dogs in vivo. After 5 weeks, grafts were recovered and the endothelial cells were shown to contain biologically active 13 -galactosidase. 42. , •
NABELEG, PLAUTZ G, BOYCE FM, STANLEYJC, NABEL GJ: Rec o m b i n a n t gene expression in vivo within endothelial cells of t h e arterial wall. Science 1989, 244:1342-1344. A report of reintroduction of retrovirally transduced porcine endothelial cells, expressing a 13-galactosidase gene and their subsequent reintroduction into denuded arteries of syngenic pigs. Explants 2~4 weeks later still contained cells expressing the 13-galactosidase gene. 43. DICHEK DA, NEVILLE RF, ZWIEBEL JA, FREEMAN SM, LEON • MB, ANDERSON WE: Seeding of intravascular stents with genetically engineered endothelial cells. Circulation 1989, 80:134~1353. Vascular endothelial cells from sheep were transduced with retroviral vectors containing genes for either ~-galactosidase or tPA. They were seeded onto stainless steel stents in vitro and grown till the stents were covered. Cells remained attached to the stents after balloon inllation, and were shown to express 13 galactosidase and tPA both before and after seeding onto the stents. 44.
ROSENBERG SA, PACKARD BS, AEBERSOLD PM, SOLOMAN D, TOPALIAN SL, TOY ST, SIMON P, LOTZE MT, WANG JC, SEIPP c a , SIMPSON C, CARTER C, BOCK S, SCHWARTZENTRUBERD, WEI JP, WHITE DE: Use of tumor-infiltrating lymphocytes and interleukin-2 in the i m m u n o t h e r a p y o f patients with metastatic melanoma. New EnglJ Med 1988, 319:167~1680. 45. FISHERB, PACKARDBS, READ EJ, CARRASQUIttOJA, CARTERCS, TOPAUAN SL, YANO JC, YOtLES SM, ROSENBERG SA: T u m o r localization of adoptively transferred I n d i u m - I l l labeled tum o r infiltrating lymphocytes in patients w i t h metastatic melanoma. J Clin Onc 1989, 7:25(~261. 46. CORNETTAK, MOEN PC, CULVER K, MORGAN RA, MCLAUCHLIN .. JR, STURN S, SELEGUEJ, LONDON W, BLAESE RM, ANDERSONWE: Amphotropic murine leukemia retrovirus is not an acute p a t h o g e n for primates. H u m Gene 7her 1990, 1:15-30. This paper reports safety studies involving the administration of amphotropic murine retrovims to five rhesus monkeys with a variety of delivery routes. The conclusion is that such retrovimses do not appar to pose an acute health risk. 47.
MILLERAD: Retrovirus packaging cells. H u m Gene 7her 1990, 1:5-14. review of currently available packaging systems and the properties of each as they relate to the possibility of producing helper vires. KASlDA, MORECKI S, AEBERSOLD P, CORNETrA K, CULVER K, FREEMANS, DIRECTOR E, LOTZE MT, BLAESE RM, ANDERSON ~(7F, ROSENBERG sa: H u m a n g e n e transfer: characterization of h u m a n tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man. Proc Natl Acad Sci USA 1990, 87:4432477. A study that combines transducing h u m a n TIL cells with the N2-retroviral vector, and then analyses the properties and characteristics of the transduced cell populations. 48. ••
49. NEWSAND COMMENT: The N2-TIL h u m a n gene transfer cllni.. cal protocol. H u m Gene Ther 1990, 1:73-92. Published here are the clinical protocol, the informed consent documents, a chronological listing of the review process, the members of the Recombinant DNA Advisory Committee and its Human Gene Therapy Subcommittee, and the am~ouncement of the final approval of the protocol. 50. ROSENBERGSA, AEBERSOtD P, CORNETrA K, KASlD A, MORGAN •, RA, MOEN R, KARSON EM, LOTZE MT, WANGJC, TOPALIAN SL, MERINO mJ, CULVERK, MILLERAD, BLAESE RM, ANDERSONWF: Gene transfer into humans: i m m u n o t h e r a p y of patients w i t h advanced m e l a n o m a using t u m o r infiltrating lymphocytes modified by retroviral gene transduction. New Engl J Med 1990, 323:570--578. Analysis of samples from advanced melanoma patients receiving TIL therapy with gene-marked TIL.
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Introduction The objective of this review is to consider the most significant advances that have been made in the past few years in the use of retroviral vectors for gene expression. Undoubtedly, one of the most significant of these has been the first sanctioned use, in May 1989, of such agents for human clinical studies. Significant progress has also been achieved in the in vivo expression of the adenosine deaminase (ADA) gene inserted into mouse bonemarrow cells and reintroduced via bone-marrow transplantation (BMT), in the retroviral-mediated expression of other genes of clinical relevance, and in retroviral vector technology itself, including construction of new types of vectors, attempts to increase the titers of retroviral supernatants, and some detailed safety studies particularly in primates. The next few years promise to yield substantial clinical evaluation of retroviral vectors for use not only in the correction of genetic defects, but also as a generalized drug-delivery system for protein therapeutics. In the latter application, genes expressed from retroviral vectors have the potential to solve some of the substantial problems associated with protein delivery using conventional delivery modalities.
Retroviral vectors and packaging systems The details of the ways in which retroviruses, in particular Moloney murine leukemia virus (MoMuLV), can be used as vectors to introduce foreign genes into cells will not be described, as they have been the subject of a number of very good recent reviews [1 °,2-4]. The concepts of replacement of internal viral function genes with the foreign gene(s), and provision of viral proteins via a packaging cell line that is itself unable to make packageable transcripts [5] are now very standard. Initial vectors incorporating a single selectable marker gene, such as the neomycin phosphotransferase (neoR) gene [6] or the human hypoxanthine phosphoribosyl transferase gene [7] were shortly followed by neoR-gene-containing vectors such as pZlPNeo [8] and then N2 [9]. In these
newer vectors, a second foreign gene is able to be introduced, downstream from the neoR gene, in the vector backbone. Such vectors have been used to express an impressively long list of foreign genes [1 ° ] for a wide variety of purposes. Major applications of MoMuLV~based vectors have been the introduction of genes into bonemarrow stem and progenitor cells as well as other cell types, their use as insertional mutagens in cultured cells or transgenic animals and their use as lineage markers for cells during development. Recent efforts in vector development have concentrated on minimizing the chances of recombination between the vector and the packaging-defective helper functions within packaging cells. Such modifications aim to minimize the chances of generating recombinant wild-type virus; their generation is undesirable for both research purposes and safety reasons in clinical applications. The modification of the N2 vector to form the LNL series [10] has resulted in vectors that, when used in conjunction with the PA317 packaging cell line [11], have a greatly reduced likelihood of regenerating wild-type virus. This is a result of modifications that include conversion of the gagATG start to a TAG stop codon, and the replacement of the region 5' to the authentic gag start with the same region from Moloney murine sarcoma virus. Both these changes greatly reduce the chance of the retroviral reading frames being expressed. For the LNL6/PA317 combination, two recombination events would be required to generate active wild-type virus. The safety features of this vector/packaging system are sufficient for it to have been approved for use in the first sanctioned clinical protocol (see below). A further modification of this vector set yielded the LN set of vectors [12 °'], which, in addition to the modifications already described for LNL6, have an additional deletion of all remaining env sequences at the 3' end. These vectors have been constructed as a set of four: LN, LNSX, LNCX and LXSN, in which the S designates a simian virus (SV)40 early promoter, the C a cytomegalovirus immediate early promoter, and X the site of a unique restriction site set, where a cDNA sequence for expression can be inserted into the vector. Clones
Abbreviations ADA--adenosine deaminase; BMT--bone-marrowtransplantation; cfu--colony-forming units; DC~ouble copy; GC~lucocerebrosidase; IL--interleukin; LTR--Iongterminal repeat; MoMuLV--Moloney murine leukemia virus; neoR--neomycin phosphotransferase;PK--phosphoglyceratekinase; SClD--severe combined immunodeficiency; SIN--self-inactivating; SV--simian virus; TIL--tumor-infiltrating lymphocytes; tPA--tissue plasminogen activator. © Current Biology Ltd ISSN 0958-1669
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Expressionsystems from the LN vector have been shown to yield high titers [as high as those achieved with N2, i.e. up to 4 x 107 colony-forming units (cfu)/ml] and, for three different clones, to remain negative for helper virus for up to eight sequential passages. An alternative approach to reducing the likelihood of recombInation events is to separate or 'split' the packaging functions in the packaging cell line. This concept was first elucidated and demonstrated for avian reticulo-endotheliosis virus [5] and has now been extended to MoMuLV packaging lines in the form of the ~CRIP and ~CRE lines [13], and the GP + E packaging cell line [14]. In the split packaging lines, and with the appropriate vectors, three recombinational events are required to regenerate wildtype virus. A general concern associated with expressing more than one gene in a retroviral vector is that the retroviral long terminal repeat (LTR) transcriptional unit will interfere with the expression of an internally promoted gene; however, there is little definitive data to support this. A recent attempt to circumvent this problem has led to a novel vector, called a double copy (DC) vector [15"]. In this construct, an ADA gene and its cellular promoter were inserted into the U3 region of the 3' viral LTR. Integration of this vector into a target cell results in the duplication of the ADA gene, and in its placement outside the retroviral transcriptional unit (at least for the 5' duplicated ADA gene---the second copy remains within the U3 region of the 3' LTR). Such a vector led to a 10-20fold increase in ADA transcripts and human ADA synthesis in NIH 3T3 cells, when compared with a conventional internally promoted ADA vector. Similar increases were also observed in two human lymphoid cell lines. Such vectors hold promise as they may substantially increase gene expression in gene transfer both in vivo and in vitro. Whether difficulties will be encountered in generating sufficiently high titers to allow widespread use of such vectors remains to be established. An earlier vector con cept, the self-inactivating (SIN) vector [16], where a deletion in the 3' LTR leads to the inactivation of the proviral transcriptional unit, has not achieved general utility, primarily because of problems in achieving acceptable vector titers. Of paramount importance is the titer of virus particles that can be achieved. Most consistently high and stable titers appear to be achieved when packaging cell lines are infected, rather than transfected. Such infected packaging lines will contain one, or only a small number of copies of the vector, and further infection by the same vector is blocked. One approach to increasing the titer of vector particles produced is to introduce larger numbers of vector genome copies via a vector packaged within a different envelope. This concept, most easily achieved by cocultivation of two different packaging cell lines each producing differently packaged vector (e.g. amphotropic and ecotropic), is now commonly known as a 'ping-pong' approach. It was initially used [ 17] to achieve an increase in the expression of genes introduced into target cells with retroviral vectors. A recent extension of this approach was reported by Bodine et al. [18 °'] who used a cocultivation approach with
an N2-vector-producing amphotropic producer cell line. In addition, this producer had been co-transfected with plasmids containing the gibbon interleukin (10-3, human IL-6 and hygromycin-resistance genes. A hygromycin-resistant population was isolated, shown to express the IL3 and 1I,-6 genes, and co-cultivated with the ecotropic GP + E86 cell line for 14 days. After this time, the initial viral titer had risen from 5 x 106 to 109 cfu/ml. Individual amphotropic clones could be reisolated from the mixed population by hygromycin selection, and, when these were screened by titer, four clones producing titers of greater than 109 cfu/ml were identified. The highest titer reported was 2 x 1010 cfu/ml and, in this clone, Southern blot analysis showed an approximate 20-fold increase in provirus copy number, and about a 1000-fold increase in production of retroviral RNA transcripts when compared with the parental cell line. This producer clone was then used to introduce the vector into rhesus bonemarrow cells, by cocultivation; the marrow was then reinfused into three sub-lethally irradiated animals. The vector genome was detectable in the blood and bone marrow of all three animals at 30-90 days post-transplantation. No gene transfer was detected in three animals where a lower titer (5 x 106 cfu/ml) vector producer was used. These preliminary, but encouraging, data reinforce the idea that high vector titers may be of great importance in introducing vectors into bone marrow, particularly into pluripotential stem cells. However, an important and intriguing question remains about the role of replicationcompetent virus in the success of this approach. The initial N2 amphotropic producer already generated low levels (less than 101 cfu/ml) of replication-competent virus, and in the high-titer producer, replication-competent virus was detected at about 104 cfu/ml. The cocultivation ping-pong approach is clearly well suited to increasing vastly the chances of occurrence of the recombination events leading to replication-competent virus production. In fact, this approach has been used to evaluate the safety of vector/packaging cell systems. For example, a series of cross-infection cycles was used to compare the generation of replication-competent virus using ~CRIP/~CRE and PA317/~CRE packaging systems [13]. A more recent study [19" ] used the cocultivation approach to evaluate a whole series of packaging cell/vector combinations. The time at which the replication-competent virus arose appeared to correlate well with the number of recombination events needed to generate the virus in each system. Interestingly, the very large increase in titer observed by Bodine et al. [18,.] was not seen in this study. However, the relative growth rates of the amphotropic and ecotropic producer lines will clearly influence the rate of amplification of vector copies in the genome. The basic question that remains concerns the role of replication-competent virus in the process of increasing the titer from producer cells; is it a prerequisite for, or a consequence of, the amplification process? An alternative hypothesis is that presence of helper, and its subsequent spread to both the ecotropic and amphotropic producers, will shut down the ping-pong process and, in
Gene expression using retroviral vectors Tolstoshev and Anderson fact, limit the increase in titer. These are clearly important questions to resolve, especially as the clinical utility of such high-titer producers generated by Bodine et aL may be less significant if wild-type virus cannot be eliminated. However, such elevated titer vector producers are extremely attractive and useful for studies of bone-marrow transduction, for optimization of conditions to transduce a variety of other cells, and for other research pur poses. The details of the binding of MoMuLV to the cells it infects are still poorly understood at the molecular level but should become clearer following the recent isolation [20] of a cDNA clone that may encode the receptor that binds to the ecotropic envelope of this retrovirus. Expression of this cDNA in human EJ cells resulted in a 106fold increase in MoMuLV infectivity. The cDNA sequence predicts an extremely hydrophobic protein with 14 potential membrane-spanning domains.
Expression of the ADA gene The first genetic disease to be treated by gene therapy will most likely be severe combined immunodeficiency (SCID), a disease that is caused by lack of ADA. A number of groups have constructed retroviral vectors containing the ADA gene, and have demonstrated expression of the gene in cells that have been transfected in culture. However, when such studies have been extended to the requisite target, the hematopoietic stem cell, and such cells reintroduced into animals by BMT, sustained expression of the ADA gene has been far more difficult to obtain. In mice, monkeys, dogs and cats, only low-level and transient expression had been observed. The first demonstration of long-term (approximately 1 month) expression in mice [21] also revealed replication-competent virus in the animals' plasma, raising the possibility that persistent reinfection of progenitor cells (rather than an initial stemcell transduction) is responsible for the observed activity. A series of recent studies [22 o.- 25 ..,26.,27,.] in mice have reported somewhat more success in achieving longterm in v i v o A D A expression. The first of these, by Moore et al. [22--], extended earlier studies but with the vector now packaged in a 'split' packaging line, GP + E86. In the absence of a selectable marker such as neoR, titers of this virus are estimated through its ability to transduce and express in a tissue-culture cell, with ADA expression measured immunohistochemically. Mouse bone marrow was transduced by cocultivation with producer cells, and then reintroduced into irradiated recipients. Human ADA expression was detected in total blood, and persisted best (five out of seven mice positive at 18-31 weeks) under conditions where a mixed conditioned medium was used during transduction. All 37 transplanted mice were ADA positive for at least 9 weeks. A detailed study of expression in a variety of tissues of hematopoietic origin showed great variability among the individual mice. All mice tested negative for replication-competent virus over the study period. Expression and presence of the vector in secondary recipient mice was, however, only limited and transient. Though efficacy of stem-cell transduction, and efficiency of expression were low in this study, it
did demonstrate that extended ADA expression in transduced bone :narrow can be obtained in mice. A similar study, also in mice using vectors without a selectable marker, was performed by Wilson et al. [23 "']. Two vectors were compared: one with the ADA gene driven from the viral LTR, the other with an internal chicken [3-actin promoter driving the ADA gene in a vector also containing a deletion of enhancer sequences from the 3' LTR (to reduce retroviral transcription after integration). Bone marrow was transduced by cocultivation and the cells were then transplanted into lethally-irradiated recipients. Analysis of human ADA expression in peripheral blood mononuclear cells revealed that by day 33 all of the nine animals reconstituted with the [3-actinpromoted vector expressed the gene, while only two out of seven receiving the LTR-driven ADA gene vector were positive. The pattern continued basically unchanged to 181 days. A study of the tissue distribution of activity among hematopoietic tissues in selected animals showed, once again, substantial variability, but this time the activity could be detected in virtually all tissues in each animal studied. The levels of human ADA relative to endogenous mouse ADA were quite variable, and reached 10-fold the endogenous level in the hemolysate of one animal. DNA analysis indicated high levels of intact provirus in all tissue fractions, with copy numbers ranging from 0.5 to 1.0 per cell for the vector with the LTR driving the gene, and less than 0.5 for the internally promoted ADA gene vector. No infectious virus, or transmission of packaging functions, could be detected in any animal. A third study in mice [24"',25"'] used vectors of the LN series, where either the LTR, the SV40 or the cytomegalovirus immediate early promoter controlled ADA gene expression. Procedures used were very similar to the two studies described above. On testing the peripheral blood of recipients at 6 months after transplantation, about a third of the animals expressed human ADA at levels of 1-5% of the endogenous enzyme; resuits were similar for all three vectors tested. In contrast, analysis of these vectors in culture with various human hematopoietic cell lines [26. ] revealed significant differences in expression when different promoters drove the ADA gene. Although in the in vivo studies [24..] helper virus was detected in the blood in three out of the four mice tested this had the characteristics of endogenous mouse xenotropic virus, most likely arising by activation of endogenous retrovirus. Confirmation of this source of the replication-competent virus was obtained in a subsequent study [25"']. Finally, Lim et al. [27"] have demonstrated stable long-term expression of human ADA in recipient mice of bone marrow transduced with a vector lacking a selectable marker, and where the ADA gene is driven by an internal phosphoglycerate kinase (PK) promoter. All recipients expressed the gene at 30 days post-transplantation, and about half maintained it until 4 months. A secondary transplant into a further recipient showed continued expression of the ADA gene. All of these studies provide encouragement that the longterm objective of treatment of ADA deficiency by gene transfer into bone-marrow stem cells will ultimately be
57
58 Expressionsystems feasible in humans. Before this, however, studies similar to those described for mice will have to be performed in primates. An additional relevant study [28" ] has described substantial expression of human ADA (15-100% of normal) in the lymphoid and myeloid progeny of longterm cultures of bone-marrow cells from ADA-deficient patients, where the bone marrow was transduced with ADA-gene-containing retroviral vectors. Transduced cells derived from the marrow cultures exhibited restoration of their capacity to respond to phytohemagglutinin and IL-2. Finally, the study of Bodine et al. [29] showing increased preservation of stem cells in culture and higher genetransfer efficiency in primates using combinations of IL3 and IL-6 advances our understanding of the optimal in vitro conditions for handling bone marrow for gene transfer. In the shorter time-frame, however, successful transfer of marker genes via retroviruses to cancer patients' lymphocytes has confirmed the concept that the human ADA gene could be inserted into the peripheral lymphocytes of ADA-deficient patients. A clinical protocol to this effect received provisional approval from the National Institutes of Health (NIH) Recombinant DNA Committee's Human Gene Therapy Subcommittee in June, 1990.
Expression of other genes of clinical relevance A variety of other genetic diseases are candidates for gene therapy approaches. Two recent reviews [30",31 ,] consider the prospects for achieving such therapy, primarily with retroviral-based technologies. "While the initial focus is likely to be on gene introductions into lymphocytes for treatment of ADA deficiency and for certain forms of cancer, other clinical targets are clearly beginning to emerge. Bone-marrow based diseases are a major area of interest. Attempts to refine the procedures for long-term culturing of bone-marow for gene transfer continue [28. ,32,33], but questions still remain as to how relevant such studies are to gene insertion into true hematopoietic stem cells, rather than committed progenitor cells. Reconstitution by BMT still remains the only definitive way to demonstrate true totipotent stem-cell function. Gaucher disease, the most prevalent human lysosomal storage disorder, is caused by a deficiency of the enzyme glucocerebrosidase (GC) and is characterized by accumulation of complex glycolipids in macrophages. Allogenic BMT has been shown to be beneficial, but can be limited by lack of histocompatible sibling donors. This disease is clearly a candidate for gene therapy, and the human GC gene is now available as a cDNA clone. Two recent reports [34",35 " ] describe studies involving retroviral vectors expressing the human GC gene. In the first [ 3 4 " ] , the vector has been used to demonstrate persistent expression of the human GC gene in long-term bone-marrow cultures for over two months. The second study [35 " ] compared a set of four GC-gene-expressing vectors for their ability to transduce and express the GC gene in mouse multipotential hematopoietic progenitor cells. Both human GC-specific RNA, and GC protein were
detected. Two of the vectors were shown to direct human GC RNA transcription after transplantation of transformed bone marrow into lethally irradiated mice. Provision of clotting factors to patients with hemophilia is another potential gene-therapy application. Retroviral vectors expressing the Factor IX gene have been constructed and shown to express the biologically active product in fibroblasts both in vitro and transiently in vivo when fibroblasts are transplanted back into mice or rats [36" ,37]. Two recent studies [38" ,39" ] have reported the introduction of truncated forms of the human Factor VIII gene into retroviral vectors, and the generation of vectors that could transduce fibroblasts and generate detectable levels of Factor VIII. In both cases, however, titers of vector from producer cells were low. In the application of gene-therapy techniques to hemophilia, attention must be given to which is the most appropriate recipient cell to express the gene, and to strategies for the reintroduction or implantation of the transduced cells back into the patient. A candidate cell for a variety of genes and for Factor VIII in particular, is the vascular endothelial cell [40]. Retroviral-mediated gene transfer into vascular endothelial cells has been possible for some time, and two stud ies [41 ,-,42-.] have demonstrated in vivo expression of retrovirally transduced genes in such cells. The first [41 -.] used prosthetic vascular grafts seeded with transduced endothelial cells expressing a bacterial [3-galactosidase gene. Five weeks after implantation into dogs, cells that were expressing introduced genes could still be found. In the second study [42 ,-], a novel approach, using a balloon catheter, was used to introduce porcine endothelial cells expressing a recombinant fl-galactosi dase from a murine amphotopic vector into denuded iliofemoral arteries. Explants of such arterial segments yielded ~-galactosidase-expressing cells 2 4 weeks later. Finally, retroviral vectors expressing [3-galactosidase or human tissue plasminogen activator (tPA) have been introduced into cultured sheep endothelial cells [43"]. These cells were seeded onto stainless steel stents, where they continued to secrete tPA. This approach could improve stent function through localized delivery of an anticoagulant, thrombolytic or antiproliferature molecule.
Gene marking of human tumor-infiltrating lymphocytes Tumor-infiltrating lymphocytecell immunotherapy Tumor-infiltrating lymphocytes (TIL) cells are a lymphocyte population grown in vitro from tumor biopsy sampies of patients with metastatic melanoma, and are re-administered to patients, in combination with high doses of IL-2, as an adoptive immunotherapy approach to this form of cancer. The therapy, developed by Rosenberg and his associates [44] has shown a significant response (40%) in terminal melanoma patients. However, TIL cells are a mixed population of lymphocytes, and it has proven difficult to obtain clinical correlates of successful treatment. One approach is to use cell-marking studies, but
Gene expression using retroviral vectors Tolstoshev and Anderson conventional 111Indium labelling has such a short half-life (2.8 days) that it yields data for only a few days [45]. The idea of using a marker gene for neomycin resistance, delivered via a retroviral vector, to track TIL cells during therapy was conceived in early 1988. This was a logical extension of previous uses of retroviral vectors as genetic markers in cell lineage studies during embryonic development, tissue differentiation, and as somatic cell hybrid chromosome markers. Because of the presence of the acquired neomycin-resistance gene, such studies of TIL cells held the prospect of identifying active subpopulations in TIL cell populations, of establishing homing to tumor sites over an expanded time frame, of determining the half-lives of TIL cells in humans and of re-isolation of cell subpopulations.
Safety and feasibility studies Before the clinical protocol could be approved, a number of safety and feasibility issues had to be addressed. The issues of major concern were related to the possibilities of generating replication-competent virus, both in the producer cell line and in vivo. A variety of safety studies have been performed, and some of these have been published [31 .,46..]. The introduction of amphotropic retrovirus into rhesus monkeys has been studied [47 "] under a variety of different conditions, including immunosuppression of the animals, and continuous delivery of virus through implantation or intraperitoneal injection of virus-producing autologous fibroblasts. The general conclusion was that murine amphotropic retrovimses do not appear to pose an acute health risk, as no instance of clinical illness was seen in a follow-up time of 32 monkey-years. The question of infectious virus production by producer cells has been discussed earlier, and is reviewed by Miller [47 °]. The activation of oncogenes by insertional mutagenesis (or inactivation of suppressor genes) could theoretically trigger oncogenic events in recipient cells, but current data suggest that the frequency is likely to be low and that additional events are required before a malignancy is established. One of the requisites for approval of a clinical protocol was a demonstration that human TIL cell populations could be transduced with a retroviral marker, and that such a transduction did not significantly change the properties of these cells. Such a study was performed and has been published [48..]. It is interesting to note, however, that mouse TIL cells in a mouse model of this immunotherapy treatment were totally refractory to transduction with a murine retroviral vector, despite enormous efforts in several laboratories. Other animal studies, involving lymphoid cell lines of mice and primates studies, were therefore performed as part of the preclinical studies.
mittee and its Human Gene Therapy Subcommittee, and the Food and Drug Administration. Final approval was granted on January 10, 1989. The first patient received marked cells on May 22, 1989, and, as of this writing, eight patients have received gene-marked cells. Of the patients who have received gene-marked cells to date, five have been analysed in detail [50"']. Gene-marked TIL persist in the circulatory system of patients for about 3 weeks and a proportion of TIL can be tracked to sites of tumors. On March 30 1990, the NIH Recombinant DNA Advisory Committee approved an extension of these studies to an unlimited number of patients. No adverse effects of the administration of the gene-marked cells have been observed in any of the patients.
Further clinical protocols involving retroviral vectors The concept of lymphocytes as target cells for retroviralmediated gene transfer is now well established. For TIL therapy, the next logical extension is to use TIL cells to deliver lymphokines such as tumor necrosis factor, IL2 etc. to attempt to augment the therapy. For other purposes, however, lymphocytes are also being considered as target cells; notably to introduce into the lymphocytes of ADA patients vectors containing the ADA gene. A clinical protocol along these lines is currently under Recombinant DNA Advisory Committee review. Additional genemarking experiments may also be of clinical utility; for example, in childhood and adult leukemia, it may be possible to obtain information about the origin of cells responsible for relapse after autologous BMT treatment for these diseases.
Conclusion The last year has seen major advances in the use of retroviral vectors for gene expression. Most notable among these were the first sanctioned clinical trial, and significant advances in transducing and expressing genes stably in murine bone-marrow stem cells. Spurred by such achievements, the field of retroviral-mediated gene transfer will undoubtedly continue to develop rapidly.
Annotated references and recommended reading • ••
Of interest Of outstanding interest
1. ,
McLAUCHLINJR, CORNETrA K, EGLITIS MA, ANDERSON WF: Retroviral-mediated gene transfer. Prog Nucl Acid Res Mol Biol 1990, 18:91-135. A comprehensive review of retroviral constructs, and the genes that have been expressed in them.
Approval and results of a clinical protocol
2.
VARMUSH: Retrovirus. Science 1988, 240:1429-1435.
A clinical protocol to genetically mark patient TIL cells with a retroviral vector was submitted in June 1988. The protocol has been published [49"'] as has a chronology of the review process, which involved NIH internal committees, the NIH Recombinant DNA Advisory Corn-
3.
EGL1TISMA, ANDERSONWF: Retroviral v e c t o r s for introduction of genes into mammalian cells. Biotechniques 1988, 6:608414.
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GILBOAE, EGL1TISMA, KANTOFF PW, ANDERSONWF: Transfer and expression of cloned genes using retroviral vectors. Biotechniques 1986, 4:504-512.
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MILLERAD, JOLLY DJ, FRIEDMANTJ, VERMAIM: A transmissible retrovirus expressing h u m a n HPRT: g e n e transfer into cells obtained from h u m a n s deficient in HPRT. Proc Natl Acad Sci USA 1983, 80:47094713.
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CEPKOCL, ROBERTSBE, MUIMGANRC: Construction and applications of a highly transmissible m u r i n e retrovirus shuttle vector. Cell 1984, 37:1053-1062.
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BENDERMA, PALMERTD, GELINASRE, MILLERAD: Evidence that t h e packaging signal of Moloney m u r i n e leukemia virus e x t e n d s into the gag region. J Virol 1987, 61:1639-1646.
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MILLERAD, BtrlqTMORE C: Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 1986, 6:2895-2902.
generation of replication c o m p e t e n t virus. Virology 1990, 176:262-265. Amplification of vector sequences by co-culture of ecotropic and amphotropic packaging cell lines is studied to determine the host range, and time of appearance of replication-competent vires. The time required was consistent with the number of recombination events required for generating wild-type vires. This procedure can be used to assess the safety of a particular packaging line/vector combination. 20.
ALBRITTONLM, TSENG L, SCADDEND, CUNNINGHAMJM: A putative m u r i n e ecotropic retrovirus receptor gene e n c o d e s a multiple m e m b r a n e - s p a n n i n g protein and confers susceptibility to virus infection. Cell 1989, 57:659-666.
21.
BELMONTJW, MACGREGORGR, WAGER-SMITHK, FLETCHER FA, MOORE KA, HAWKINS D, VILLALON D, CHANG SMW, CASKEY CT: Expression of h u m a n adenosine deaminase in m u r i n e hematopoietic cells. Mol Cell Biol 1988, 8:5116-5125.
22. ••
MOOREKA, FLETCHER FA, VILtALON DK, UTrER AE, BELMONT JW: H u m a n adenosine deaminase expression in mice. Blood 1990, 10:2085-2092. This report describes bone-marrow transduction experiments with an ADA vector, but no sign of replication-competent virus production. Long-term survival of the ADA gene and expression in hematopoietic tissues of recipient mice for as long as 7 months were demonstrated and no evidence of wild type virus was found in the animals.
12. MILLERAD, ROSMANGJ: Improved retroviral vectors for g e n e •• transfer and expression. Biotechniques 1989, 7:980-990. This paper describes the structure, construction and properties of the LNL and LN series of MoMuLV retroviral vectors. These vectors, together with the PA317 packaging line, currently represent one of the safest vector systems and their vector products have recently been used in clinical gene-marking studies in humans.
WILSONJM, DANOS O, GROSSMAN M, RAULET DH, MUIMGAN RC: Expression of h u m a n adenosine deaminase in mice reconstituted w i t h retrovirus transduced hematopoietic s t e m cells. Proc Natl Acad Sci USA 1990, 87:439-443. The authors report the successful reconstitution of mice with bone marrow containing one of two kinds of retroviral vector for expressing the h u m a n ADA gene. Significant expression of the gene in hematopoietic cells was observed for up to 6 months post-transplant.
13.
DANOSO, MULLIGANDAz Expression of retroviral trans-acting functions from c o m p l e m e n t a r y crippled genomes: a syst e m for helper free packaging of retroviral vectors. J Cell Biochem 1988, 12:172.
24. ••
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MARKOWlTZD, G-OFF S, BANK A: A safe packaging line for g e n e transfer: separating viral g e n e s o n two different plasraids. J virol 1988, 62:1120-1124.
15. ••
HANZOPOULOSPA, SULLENGERBA, UNGERS G, GItBOA E: Improved g e n e expression u p o n transfer of the adenosine deaminase minigene outside of t h e transcriptional unit of a retroviral vector. Proc Natl Acad Sci USA 1989, 86:3519-3523. This study describes DC vector, and shows the construction of one such vector, containing a h u m a n ADA gene. The inserted gene is indeed duplicated on integration into target cells, and expression of the functioning protein is significandy increased both in mouse fibroblasts and in h u m a n lymphoid cell lines. 16.
Yu S-F, VON RUDEN T, KANTOFF PW, GARBER C, SEIBERG M, RUTHER U, ANDERSON WF, WAGNER EF, GILBOA E: Serf-inactivating retroviral vectors designed for transfer of w h o l e g e n e s into mammalian cells. Proc Natl Acad Sci USA 1986, 83:3194-3198.
17.
BESTWICKRK, KOZAKSL, KABATD: Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes. Proc Natl Acad Sci USA 1988, 85:5404-5408.
18.
BODINEDM, MCDONAGH KT, BRANT SJ, NEY PA, AGRICOLAB, NIENHUIS AW: Development of a high-titer retrovirus p r o d u c e r cell line capable of gene transfer into r h e s u s m o n k e y hematopoietic s t e m cells. Proc Natl Acad Sci USA 1990, 87:373~3742. In this paper, the authors report the generation of producer clones generating vectors of extremely high titer ( > 1010 cfu/ml) by a cocultivation of ecotropic and amphotropic producer cell lines. A concomitant increase in proviral DNA copy n u m b e r and DNA transcripts is demonstrated, and such high-titer'vectors are shown to improve the efficiency of gene transfer to bone-marrow cells of primates in an in vivo BMT study. However, such high-titer vector supematants do contain measurable levels (up to 104 focus-forming units/ml) of replication competent virus, as might be expected from the process of generating such producers. • •
19. •
BYRNE E,
MUENCHAU DD, FREEMAN SM, CORNETrA K, ZWIEBEL JA, ANDERSONWF: Analysis of retroviral packaging lines for
23. •.
OSBORNEWRA, HOCK RA, KALEKO M, MILLERAD: Long-term expression of h u m a n adenosine deaminase in mice after transplantation o f bone m a r r o w infected with amphotropic retroviral vectors. H u m Gene Ther 1990, 1:31~41. Another study that successfully demonstrated long-term expression of h u m a n ADA in mice transplanted with bone marrow transduced with amphotropically packaged vector. 25. ••
KALEKOM, GARCIAJV, OSBORNE W f ~ MILLERAD: Expression of h u m a n adenosine deaminase in mice after transplantation of genetically-modified bone marrow. Blood 1990, 75:1733-1741. An extension of the studies described in [24 ••] of survival and expression of the h u m a n ADA gene in the LN series in long-term BMT studies in mice using ecotropically packaged vector. 26. •
HOCK RA, MILLERAD, OSBORNE WRA: Expression of h u m a n adenosine deaminase from various strong p r o m o t o r s after gene transfer into h u m a n hematopoietic cell lines. Blood 1989, 74:8764381. A comparative analysis of different LN vector constructs carrying the h u m a n ADA gene under the control of different promoters with regard to their ability to express the gene in h u m a n hematopoietic cell lines in vitro. 27. ••
L1MB, APPERLEYJF, ORKIN SH, WILLIAMSDA: Long-term expression of h u m a n adenosine deaminase in mice transplanted w i t h retrovirus-infected hematopoietic s t e m cells. Proc Natl Acad Sci USA 1989, 86:8892-8896. This study uses a retroviral vector lacking a selectable marker to transfer a human ADA gene into mice bone marrow, and shows long-term expression for up to 4 months. In addition, a secondary transplant continues to confer h u m a n ADA expression of the recipient mouse. 28. •
BORDIGNONC, YU S-F, SMITH CA, HANTZOPOULOSP, UNGERS GE, KEEVER CA, O'REILLYR, GILBOA E: Retroviral vectormediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADAdeficient m a r r o w cells. Proc Natl Acad Sci USA 1989, 86:6748-6752. Bone marrow from ADA-deficient patients was transduced with vectors containing h u m a n ADA genes. Lymphoid and myeloid progeny of cultures of such marrow expressed substantial levels of h u m a n ADA_ 29.
BODINEDM, KARLSSON S, NIENHUIS AW: Combination of interleukins 3 and 6 preserves s t e m cell function in culture and e n h a n c e s retrovirus-mediated g e n e transfer into hematopoietic s t e m cells. Proc Natl Acad Sci USA 1989, 86:8897-8901.
G e n e expression using retroviral vectors T o l s t o s h e v a n d A n d e r s o n 30.
FRIEDMANT: Progress toward h u m a n gene therapy. Science 1989, 244:1275-1281. ~his review considers the current state of retroviral vector technology directed towards gene therapy procedures for the major single-gene h u m a n genetic diseases. CORNETrAK, WIEDER R, ANDERSON WE: G e n e transfer into primates and prospects for g e n e therapy in humans. Prog Nucl Acid Res Mol Biol 1989, 36:311-322. A review that focuses o n the prospects of bone-marrow-directed gene therapy procedures and reviews safety data derived from primate studies with retroviral vectors and replication-competent helper virus. 31. •
32.
HUGHES PFD, EAVES CJ, HOGGE DE, HUMPHRIES RK: Highefficiency g e n e transfer to h u m a n hematopoietic cells maintained in long-term m a r r o w culture. Blood 1989, 74:1915-1922.
33.
SCHUENING FG, STORB R, STEAD RB, GOEHLE S, NASH R, MILLERaD: I m p r o v e d retroviral transfer of g e n e s into canine hematopoietic progenitor cells kept in long-term m a r r o w culture. Blood 1989, 74:152-155.
34. ••
NOTTAJA, SENDER IS, BARRANGERJA, KOHN DB: Expression of h u m a n glucocerebrosidase in murine long-term bonem a r r o w cultures after retroviral vector-mediated transfer. Blood 1990, 75:787-797. An N2-based retroviral vector containing the h u m a n GC gene was used to transduce m o u s e bone-marrow cells. Maintenance of bone marrow in long-tern1 culture showed persistence of GC expression for up to 2 months. 35. ••
CORRELLPH, FINK JK, BRADEYRO, PERRY IX, KARISSON S: Production of h u m a n glucocerebrosidase in mice after retroviral gene transfer into multipotential h e m a t o p o e t i c progenitor cells. Proc Natl Acad Sci USA 1989, 86:8912-8916. This study describes the use of a series of N2-based vectors containing the h u m a n GC gene to transduce m o u s e hematopoietic progenitor cells, as assayed by colony assays, and stem cells after BMT. 36. •
PALMERTO, THOMPSON AR, MILLERAn: Production of h u m a n Factor IX in animals by genetically modified skin fibroblasts: potential t h e r a p y for hemophilia B. Blood 1989, 73:438-445. The human Factor IX gene is expressed in an active form in fibroblasts in vitro after transduction with a variety of LN-based retroviral vectors containing the gene. When such rat fibroblasts are reintroduced back into animals, a transient production of h u m a n Factor IX was detected in plasma. 37.
ST LOUIS D, VERMA IM: An alternative approach to somatic ceR g e n e therapy. Proc Natl Acad Sci USA 1988, 85:3150-3154.
38. •
ISRAELDI, KAUFMANRJ: Retroviral mediated transfer and amplification of a functional h u m a n Factor VIII gene. Blood 1990, 75:1074-1080. A retroviral vector containing both a h u m a n ADA gene, as a selectable and amplifiable marker, and a truncated h u m a n Factor VIII gene is described. Expression of both genes was demonstrable in mouse fibroblasts in vitro, viral titer, ADA expression and Factor VIII expression were increased by selection for ADA. 39. •
HOEBENRC, VAN DER JAGT RCM, SCHOUTE F, VAN TILBERG NM, VERBEETMP, BIET E, VAN ORMONDT M, VANDER EB AJ: Expression of functional Factor VIII in primary h u m a n skin fibroblasts after retrovirus-mediated g e n e transfer. J Biol Chem 1990, 265:7318-7323. A neoR/Factor VIII (truncated gene) retroviral construct was shown to be able to transduce murine and primary h u m a n fibroblasts and express biologically active h u m a n Factor VIII activity. 40.
ZWEIBELJA, FREEMAN SM, KANTOFF PW, CORNE'ITA K, RYAN US, ANDERSONWF: High-level r e c o m b i n a n t g e n e expression in rabbit endothelial cells transduced by retroviral vectors. Science 1989, 243:220-222.
41. ••
WILSON JM, BIRINY1 IX, SOLOMON RN, LIBBY P, CALLOW AD, MULLIGAN RC: Implantation of vascular grafts lined
w i t h genetically modified endothelial cells. Science 1989, 244:1344-1346. Vascular endothelial cells of dogs were transduced with a retrovims containing a 13-galactosidase gene, and then seeded onto vascular grafts in dogs in vivo. After 5 weeks, grafts were recovered and the endothelial cells were shown to contain biologically active 13 -galactosidase. 42. , •
NABELEG, PLAUTZ G, BOYCE FM, STANLEYJC, NABEL GJ: Rec o m b i n a n t gene expression in vivo within endothelial cells of t h e arterial wall. Science 1989, 244:1342-1344. A report of reintroduction of retrovirally transduced porcine endothelial cells, expressing a 13-galactosidase gene and their subsequent reintroduction into denuded arteries of syngenic pigs. Explants 2~4 weeks later still contained cells expressing the 13-galactosidase gene. 43. DICHEK DA, NEVILLE RF, ZWIEBEL JA, FREEMAN SM, LEON • MB, ANDERSON WE: Seeding of intravascular stents with genetically engineered endothelial cells. Circulation 1989, 80:134~1353. Vascular endothelial cells from sheep were transduced with retroviral vectors containing genes for either ~-galactosidase or tPA. They were seeded onto stainless steel stents in vitro and grown till the stents were covered. Cells remained attached to the stents after balloon inllation, and were shown to express 13 galactosidase and tPA both before and after seeding onto the stents. 44.
ROSENBERG SA, PACKARD BS, AEBERSOLD PM, SOLOMAN D, TOPALIAN SL, TOY ST, SIMON P, LOTZE MT, WANG JC, SEIPP c a , SIMPSON C, CARTER C, BOCK S, SCHWARTZENTRUBERD, WEI JP, WHITE DE: Use of tumor-infiltrating lymphocytes and interleukin-2 in the i m m u n o t h e r a p y o f patients with metastatic melanoma. New EnglJ Med 1988, 319:167~1680. 45. FISHERB, PACKARDBS, READ EJ, CARRASQUIttOJA, CARTERCS, TOPAUAN SL, YANO JC, YOtLES SM, ROSENBERG SA: T u m o r localization of adoptively transferred I n d i u m - I l l labeled tum o r infiltrating lymphocytes in patients w i t h metastatic melanoma. J Clin Onc 1989, 7:25(~261. 46. CORNETTAK, MOEN PC, CULVER K, MORGAN RA, MCLAUCHLIN .. JR, STURN S, SELEGUEJ, LONDON W, BLAESE RM, ANDERSONWE: Amphotropic murine leukemia retrovirus is not an acute p a t h o g e n for primates. H u m Gene 7her 1990, 1:15-30. This paper reports safety studies involving the administration of amphotropic murine retrovims to five rhesus monkeys with a variety of delivery routes. The conclusion is that such retrovimses do not appar to pose an acute health risk. 47.
MILLERAD: Retrovirus packaging cells. H u m Gene 7her 1990, 1:5-14. review of currently available packaging systems and the properties of each as they relate to the possibility of producing helper vires. KASlDA, MORECKI S, AEBERSOLD P, CORNETrA K, CULVER K, FREEMANS, DIRECTOR E, LOTZE MT, BLAESE RM, ANDERSON ~(7F, ROSENBERG sa: H u m a n g e n e transfer: characterization of h u m a n tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man. Proc Natl Acad Sci USA 1990, 87:4432477. A study that combines transducing h u m a n TIL cells with the N2-retroviral vector, and then analyses the properties and characteristics of the transduced cell populations. 48. ••
49. NEWSAND COMMENT: The N2-TIL h u m a n gene transfer cllni.. cal protocol. H u m Gene Ther 1990, 1:73-92. Published here are the clinical protocol, the informed consent documents, a chronological listing of the review process, the members of the Recombinant DNA Advisory Committee and its Human Gene Therapy Subcommittee, and the am~ouncement of the final approval of the protocol. 50. ROSENBERGSA, AEBERSOtD P, CORNETrA K, KASlD A, MORGAN •, RA, MOEN R, KARSON EM, LOTZE MT, WANGJC, TOPALIAN SL, MERINO mJ, CULVERK, MILLERAD, BLAESE RM, ANDERSONWF: Gene transfer into humans: i m m u n o t h e r a p y of patients w i t h advanced m e l a n o m a using t u m o r infiltrating lymphocytes modified by retroviral gene transduction. New Engl J Med 1990, 323:570--578. Analysis of samples from advanced melanoma patients receiving TIL therapy with gene-marked TIL.
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