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Recent advances in retrovirus vector technology
Recent advances in retrovirus vector technology Kathleen A. Boris-Lawrie and Howard M. Temin University of Wisconsin, Madison, W i s c o n s i n , USA Retroviral vectors are widely used for the study of retroviral replication and to introduce DNA into somatic cells. An exciting new approach in retroviral vector technology is the use of internal ribosome entry sites from picornaviruses to provide stable expression of multiple genes. In addition, strategies are being developed that target the expression of retroviral vectors to specific cell populations. Current Opinion in Genetics and Development 1993, 3:102-109
Introduction Retroviruses are RNA viruses that replicate through a DNA intermediate. The DNA intermediate is integrated into the host chromosome as a provirus. After infection of a susceptible cell, reverse transcription of the retroviral genomic RNA to double-stranded DNA requires several c~acting viral sequences, including the primer-binding site (pbs), the repeat (R) region of the long terminal repeats (LTRs), and the polypurine tract (ppt) (see Fig. 1). Integration of the viral DNA into the host genome is mediated by the terminal attachment (art) sites. The provirus promoter within the unique 3' (U3) region of the 5' LTR governs transcription, and all viral transcripts initiate at the first nucleotide of the R region. The full-length transcript can be packaged or used for translation, either directly into Gag---Pol protein or, after splicing, into Env protein. The genomic RNA is packaged by viral proteins, which recognize viral RNA by the c/s-acting packaging or encapsiclation sequence, located between the unique 5' (U5) region and near the beginning of gag. The Gag, Pol and Env proteins, and viral RNA interact at the surface of the cell membrane, and mature virions are released by budding. The cell tropism of subsequent infection by the viral particles is determined primarily by the Env glycoprotein. Although the biology of retroviral replication is complex, knowledge of the c/s-acting viral sequences that control retroviml replication has enabled the construction of retroviml vectors and packaging cells using parts from retroviruses. Most retroviral vectors contain only the c~acting viral sequences required for packaging, reverse transcription, and integration of inserted for-
eign sequences. Packaging or helper cells provide the viral proteins in tram, but lack the packaging or integration signals. These packaging cells can be transfected with the retroviral vector, which then produces RNA that can be packaged and released as vector virus particles. These vector viruses can be used to introduce the foreign DNA stably into target cells, either in culture or direcdy in animal cells. The purpose of this review is to summarize recent advances in retrovirus vector technology.
Retroviruses as vectors There are several reasons why retrovimses are useful as vectors. Retroviral vectors provide efficient and stable transfer of DNA into replicating cells. Vector integration is precise, and because no viral proteins are encoded by the vector, there is no possibility of vires spread. The inserted DNA can be expressed from the LTR promoter and efficiently spliced, or expressed from an internal promoter. A diverse group of animal cells have been shown to be infectable by retroviruses as a result of the wide tropism of the various viral Env proteins. However, retroviral vectors also have some limitations. Infection with retroviral vectors derived from simpler retroviruses is limited to dividing cells, and the size limit of inserted foreign sequence is less than 8-10 kb. Because genetic variation occurs during retroviral replication, proviral clones need to be analyzed to determine that the provims has the expected structure. As proviral insertion is random, insertional mutagenesis is possible; however, in mouse models, insertional mutagenesis is
Abbreviations ALV--avian leukosis virus; araM~6*methoxypurine arabinonucleoside; aN--attachment; BPV--bovine papillomavirus; CAT--chloramphenicol acetyltransferase; CFU--colony forming unit; DHFR---clihydrofolate reductase; EMCV-~encephalornyocarditis virus; FH--familial hypercholesterolemia; GCV--gancicIovir; hGH--human growth hormone; HSV--herpes simplex virus; IRES~internal ribosome entry site; hADA--human adenosine deaminase; hGH--human growth hormone; LDLR--Iow density lipoprotein receptor; LTR--Iong terminal repeat; MDRl--multidrug resistance 1; MoMLV--Moloney murine leukemia virus; Mt--metallothionein; neo~neomycin phosphotransferase; ori--origin of replication; pbs--primer-binding site; ppt--polypurine tract; R--repeat; RCV--replication-cornpetent virus; RSV--Rous sarcoma virus; SNV--spleen necrosis virus; SV40~simian virus 40; tk--thymidine kinase; U3--unique 3'; US--unique 5'; UTR--untranslated region; VZV--varicella zoster virus. 102
(~ Current Biology Ltd ISSN 0959-437X
Recent advances in retrovirus vector technology Boris-Lawrie and Temin
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Fig. 1. The structure of wild-type retrovirus DNA and retrovirus vector DNA. (a) Wild-type spleen necrosis virus DNA with the gag, pol and env genes shown, and (b-f) retrovirus vector DNA containing different combinations of foreign genes (see text for details). The division of the long terminal repeats (LTRs) into the unique 3' (U3), repeat (R) and unique 5' (US) regions (light shading) of the viral DNA is shown, as are the attachment sites relative to the polarity of the provirus in the host chromosome (attL and attR for left and right, respectively). Also shown are the primer-binding site (pbs), splice donor (sd), cis-acting encapsidation sequence (E), splice acceptor (sa), and polypurine tract (ppt). IRES1 and IRES2 denote internal ribosome entry sites from the polio and encephalomyocarditis viruses, respectively. (b) In the splicing vector, the vector transcript is expressed from the LTR promoter, and is spliced and translated to produce two proteins [39]. (c) The internal promoter vector expresses the second protein from a second transcriptional unit [22]. (d) In this internal promoter vector, the second transcriptional unit is inserted within the 3' U3 region, outside of the retroviral transcriptional unit. After reverse transcription of the vector, the 3' U3 region is duplicated in both LTRs of the provirus [40]. (e,f} In the IRES vectors, a single vector transcript is translated to produce two or three proteins, respectively [27°°,28°'].
associated with a spreading virus infection. Replicationcompetent vires (RCV) can be generated by recombination between sequences of the retroviral vector and the packaging provirus. Therefore, packaging cell lines are constantly being redesigned to contain the minimum sequence overlap with vectors. As a vector safety feature, genes such as the herpes simplex virus (HSV) thymidine kinase (tk) gene, which confers ganciclovir (GCV) sensi-
tivity, can be used in vectors to enable selection against transduced cells if necessary [1]. Retrovirus vectors are useful for introducing DNA into cells for the study of retrovirus replication, for cell line'age studies, and for gene therapy. The predominant use of retroviral vectors is to study viral replication. For example, retroviral vectors are in-
103
104 Viralgenetics valuable reagents for studying the mechanism of reverse transcription [2] and dissecting events involved in the assembly and release of virions [3]. Vectors have been used to study the generation of genetic diversity during viral replication [4,5], and mechanisms by which retroviruses can mobilize cellular proto-oncogenes (J Zhang and HM Temin, unpublished data) [6]. Retroviral vectors-have been designed to screen for anti-retroviral compounds and to study the effect of drugs on viral replication [7]. See I Lemischka (this issue, pp 115-118) for a review on retroviruses as markers for cell lineage studies.
Recent experiments using retroviral vectors for gene therapy A major focus in retrovirus vector technology is the design of vectors and packaging cell lines for gene therapy. Original conceptions for gene therapy involved the correction of single gene disorders to cure a variety of congenital and acquired diseases. After the success of the first human gene-transfer experiment (study of tumor infiltrating lymphocytes transduced with vector virus expressing neomycin resistance [8]), the first clinical trial for human gene therapywas initiated in September 1990 (transfer of lymphocytes transduced with vector viruses expressing the gene encoding human adenosine deaminase (hADA) to correct ADA deficiency; see [9]). Recent strategies for gene therapy also include disease prevention and the expression of genes to aid in disease treatment. Here, we will summarize some of the experiments reported durhag the past year that used retroviral vectors to introduce DNA into somatic cell populations. Bone-marrow stem cells are an important target for retroviral vector transduction because the pluripotent hematopoetic stem cell is the progenitor of a complete hematopoetic system. Human ADA has been expressed in mice and, for short periods, in primates (less than 100 days). Van Beuseheum etaL [10o] transduced autologous bone-marrow stem cells from rhesus monkeys with vectors encoding hAD& Vector packaging cells were cocultured with the bone-marrow cells, and the co-culture was injected back into the monkeys. Stable hADAexpression was achieved for greater than one year. The use of selected growth factors for culturing the bone-marrow cells facilitated stable transduction of early hematopoetic progenitor cells (granulocytes). The presence of packaging virus in the injected cells may have increased the chance of generating RCV, but injection of the co-culture maximized the number of viable transduced cells. This first example of long-term hADA expression in primates is encouraging for transduction of hematopoetic progenitor cells in human gene therapy. Familial hypercholesterolemia (FH) is caused by a deficiency of low density lipoprotein receptors (LDLRs). Using a retroviral vector expressing LDLR, hepatocyte e x vivo gene therapy was therapeutic for FH in the FH animal model, the Watanabe heritable hyperlipidemic rabbit [11]. Autologous hepatocytes were iso-
lated from resected liver (30% of the liver), grown in culture, and transduced with recombinant retroviruses expressing rabbit LDLR. After injection into the autologous rabbit, LDLRexpression was stable for six months (the duration of the study), and serum cholesterol was reduced by 25% to yield a therapeutic level. No immune response to the recombinant LDLRprotein was detected. A number of potentially therapeutic proteins would need to be distributed systemically. Transduced muscle ceils have now been shown to provide systemic distribution of recombinant proteins [12,13]. Mouse myoblast cells transduced with a retroviral vector expressing human growth hormone (hGH) were re-injected into limb tissue [12]. The transduced myoblasts differentiated to myotubes and fused with the vascularized, multinucleated myofibers. The expression of hGH in serum was detected for the duration of the study (three months). No tumors were found in mice treated with the transduced cells [13]. Although these experiments have detected stable vector gene expression for up to one year, a major question remains as to the longevity of vector gene expression in transduced somatic cells. Vector transcription has been suggested to be subject to programed control of cellular gene expression [14]. In a study of engrafted transduced mouse fibroblasts, the housekeeping promoter, DHFR was placed within a retroviral vector and remained transcriptionally active at 60 days post-engraftment, whereas transcription from the LTR promoter or an internal cytomegalovirus immediate early promoter was suppressed [15]. As discussed below, the activity of an internal promoter in transduced somatic cells can not be predicted from experiments in culture [16]. New vector designs that use cell type specific promoters may be useful for providing sustained vector gene expression [17",18-].
Retroviral vectors for disease treatment Recently, gene-therapy strategies based on the ability of the simpler retroviruses to infect only actively growing cells have targeted the selective destruction of neoplastic cells. To destroy experimental brain tumors selectively in mice, glioma cells were injected with fibroblasts producing amphotropic vector viruses expressing the HSV tk gene [19*], which encodes sensitivity to GCV. After allowing for virus infection of the actively dividing glioma cells, the mice were treated with GCV. The tumors completely regressed, and neighboring tissue remained unaffected. No systemic cytotoxicity or spread of virus to other rapidly growing tissues was detected. Residual virus would be subject to lysis in serum by a complement-mediated pathway [20]. The results of this short term study (30 days) are encouraging, and it will be important to establish the effect of long-term treatment with these viruses. In another study, tumor cells were targeted by using vectors with internal tissue-specific promoters that transcribe
Recent advances in retrovirus vector technology Boris-kawrieand Temin 105 a cytotoxic gene [17"]. The varicella zoster virus (VZV) tk-encoded protein phosphorylates 6-methoxypurine arabinonucleoside (araM) to cytotoxic adenine arabinonucleoside triphosphate, while mammalian kinases do not have this activity. Expression of VZV tk was directed by liver cell specific promoters (hepatoma-associated a-fetoprotein or liver-associated albumin promoter). Hepatoma and other cell lines were transduced with the vectors and treated with araM. Cytotoxicity was detected in the hepatoma lines, but not other cell lines. In another approach to cancer therapy, the multidrug resistance 1 (MDR1) gene was expressed in bone-marrow cells to address the problem of bone-marrow toxicity during chemotherapy [21]. Bone-marrow ceils were transduced by co-culture with ecotropic packaging cells containing the murine sarcoma virus MDR1 vector virus and then used to reconstitute mouse bone marrow. After treatment with the drug taxol, the mean number of proviruses per peripheral blood leukocyte more than tripled, and the neutrophil count was more stable than in the control. Thus, MDR1 may be useful for the selection of drug-resistant cells in vivo. Studies of the therapeutic value of this approach are awaited.
A new approach in the design of retroviral vectors: internal ribosome entry sites Retroviruses have evolved three strategies to produce multiple proteins from a single mRNA: splicing provides Env protein; ribosomal frameshifting provides Pol protein; and proteolytic processing yields the capsid, matrix, protease, and reverse transcriptase proteins from the Gag-Pol polyprotein. Retroviral vectors use two strategies for protein expression, either splicing or use of an interhal promoter (see Fig. 1). In splicing vectors, the viral transcript is expressed from the LTR promoter and is spliced and translated to produce two proteins. TypicaUy, this arrangement results in higher production of the upstream protein relaUve to the downstream protein. In the second configuration, the LTR promoter expresses the upstream gene, and an internal promoter transcribes the downstream mRNA. For example, the promoter of an unselected gene may be suppressed as the result of an unknown epigenetic mechanism [22,23]. The second transcription unit may also be inserted within the 3' U3 region (Fig. 1). In this arrangement, the second transcription unit is duplicated in the 5' U3 region after reverse transcription. This strategy can provide more balanced protein production; however, a second promoter near the LTR promoter can lead to unstable transcription. The effect of viral regulatory sequences on the internal promoter can be detected independent of transcription from the LTR [24]. Importantly, because the markers on splicing vectors or vectors with internal promoters are n o t co-selected, stable expression of the markers is often problematic. A new approach for expression of multiple genes from retroviral vectors has been adapted from picornaviruses.
Picomavimses express several proteins from a polycistronic mRNA. The 5' end of this mRNA terminates with pUpU, instead of the typical eukaryotic m7GpppN cap, and the transcript contains long 5' untranslated regions (UTRs) with complex secondary structure and multiple AUG codons. These UTRs are c~acting sequences that promote cap-independent translation initiation at internal initiation codons. These sequences are designated internal ribosome entry sites (IRESs). IRES sequences cloned from poliovirus and encephalomyocarditis virus (EMCV) have been used in retroviral vectors to direct translation of LTR-driven polycistronic RNA. Adam et aL [25"*] constructed Moloney murine leukemia virus (MoMLV)-based retroviral vectors that positioned a selectable marker or a reporter gene either downstream of the 5' LTR, downstream of an IRES sequence, or downstream of an internal simian virus 40 (SV40) early promoter. Virus titers were similar among the vectors, suggesting the IRES did not alter virus replication. The level of reporter protein produced was similar when the gene was directly downstream of the LTR or an IRES. Expression of the selectable marker, neomycin phosphotransferase (neo) was similar when neo was positioned downstream from an IRES or the SV40 early promoter. The predicted polycistronic transcripts were detected by northern-blot analysis, suggesting the IRES sequence promoted internal initiation of translation. Another survey of IRES function used Rous sarcoma virus (RSV)-based vectors and also found virus titer was not affected by IRES [26]. Reporter gene activity from an EMCV IRES was twofold higher than from an internally promoted mRNA, and spliced transcripts provided only 5--10% the level of activity. Gene expression from splicing vectors or internal promoter vectors in long-term culture can be low-level or absent. With polycistronic IRES vectors, selection for one gene should ensure expression of the second gene. Koo et a t [27"'] constructed a dicistronic spleen necrosis virus (SNV) vector, pJD214HyBi+, which contained two selectable markers between the SNV LTRs, the genes conferring resistance to hygromycin B (byg), and G418 (neo). T h e EMCV IRES sequence was inserted upstream of neo (Fig. 1). Virus titers on target cells after selection with hygromycin, G418, or hygromycin and G418 were similar (3.4 x 105, 2.1 x 105, and 2.1 x 105 colony forming units (CFU)/ml, respectively), suggesting that both hyg and neo were expressed in the same cell. Two months after the initial drug selection, populations of cells selected with hygromycin, G418, or doubly selected were analyzed. In each population the pJD214HyBi+ proviruses remained intact, the predicted single vector transcript was detected using a neo~specilic probe, and the Neo protein was stably produced. The level of Neo protein produced from the IRES vector was similar to the level from a control vector that expressed neo directly from the SNV LTR. These results indicate that selection of either marker on a dicistronic RNA allows expression of both markers. The activity of an RSV LTR-/acZ-EMCV IRES--v-src-LTR vector was examined in ovo [26]. Virus stocks were in-
106 Viral genetics jected imo chicken embryos, and the embryos were harvested 3-6 days later. Inspection of LacZ-positive cells revealed morphological properties associated with expression ofv-sr~ Thus, in the short term, protein production was achieved from a retroviral IRES vector in viva The polio and EMCV IRESs were used in tandem to construct a tricistronic retroviral vector (MLV LTR-neo-polio IRES--cat-EMCV IRES-ada-LTR) (see Fig. 1) [28°']. Virus titer remained similar to either dicistronic vector (MLV LTR-neo-polio IRES-cat-LTR or MLV LTR-neoEMCV IRES-ada-~LTR). Vector proviruses were found to be structurally intact and to transcribe the predicted single tricistronic mRNA. Each of the three vector proteins were detected, although the levels were lower than from the corresponding dicistronic vector. Thus, retroviral vectors containing IRES s.equences arranged in tandem can express multiple genes from a polycistronic mRNA. In future experiments, translation levels may be increased by optimizing spacing between the EMCV start codon and the downstream gene, or by precisely positioning the inserted gene at the EMCV start codon. Clearly, the use of picomavirus IRES sequences is a promising advance for retroviral vector technology. Importantly, the selection of one marker ensures the expression of the other genes within the cistron. Among the possible applications are vectors producing multiple subunit proteins or the cellular co-expression of synergistic proteins.
Targeted vector transcription Retroviral vectors with intemal promoters that are tissuespecific can target vector gene expression. As discussed above, hepatoma-specific transcription from liver-cell promoters provided targeted expression of the cytotoxic VZV tkgene [17"]. The stringency of tissue-specific transcription from internal promoters was surveyed in transgenic chickens using avian leukosis virus (ALV) vectors [18o]. Chicken embryos were transduced with replication-competent ALV vector virus, and gene expression was studied in the progeny chicks. Chloramphenicol acetyitransferase (CAT) activity directed from the internal tissue-specific chicken skeletal muscle ~t-actin promoter was compared to CAT levels from the constitutive chicken IB-actin promoter. The skeletal muscle at-actin promoter-cat vector expressed high-level CAT reporter gene activity in muscle, but not in other tissues (71% versus 0--8% substrate conversion). Reporter gene activity from the 13-actin promoter vector was low in all tissues (3%). Similar results were obtained when the internal promoters were placed in the opposite orientation. The copy number of vector proviruses was similar in muscle and other tissues, and virus production in each tissue was equivalent as measured by immunoblot with a capsid protein antibody. Therefore, transcription from the internal skeletal muscle ct-actin promoter appeared to be tissue-specific, although as the authors note, RNA analysis is needed to conclude definitively muscle-specific transcription.
In culture, expression from the skeletal muscle 0t-actin promoter was not found to be tissue-specific. The authors suggest this may be the result of incomplete differentiation of the cells [18o]. Experiments by U et aL [16] similarly suggest that the efficiency of expression from internal promoters in primary cells can not be predicted from ceU-culture experiments. In this study, five different internal promoters were used to test the relative expression of a dihydrofolate reductase (DHFR) gene, which confers methotrexate resistance [16]. The vectors expressed neo directly from the MoMLV LTR and instead of inserting the promoter-DHFR sequences within the LTR transcription unit, a poly(A) signal was inserted within the U3 region of the 3' LTR. The second transcriptional unit (each promoter and DHFR) was then positioned downstream of the poly(A) sequence. After infection and reverse transcription, the second transcriptional unit was duplicated in the 5' LTR. In five cell lines, the relative level of methotrexate resistance from each vector matched the relative level of RNA expressed. However, the relative abundance of transcription among the promoters was inconsistent when the vectors were compared in cell lines and in transduced primary bone-marrow cells. These results are similar to the inconsistency in transcriptional specificity of the ALV vectors observed in culture and in vivo [18o]. Other groups have also reported that transcription from internal promoters in retroviral vectors can be unstable [14,15]. Therefore, once transduced into somatic cells, vectors need to be monitored for stable gene expression. Another approach to targeted vector gene expression is to regulate LTR promoters with trans-acting factors. Transcription from the HIV LTR is induced by the viral transacting protein, Tat, through the c/s-acting transactivation response site, TAR. Tat-inducible replication-defective HW vectors may be useful for anti-HW gene therapy by providing controlled expression of proteins that interfere with the viral replication cycle or are deleterious to infected cells. A recent example takes advantage of the stringent regulation of transcription from the HW LTR to target expression of the murine cytocidal influenza virus hemaglutinin (H5 HA) in cells upon infection with HIV [29]. H5 HA was detected by immunofluroscence only in cells expressing Tat, which suggested that this system may be useful for the expression of genes that elicit a cytocidal immune response to HIV infection. In vivo, potential spread of such a vector virus would be beneficial for further controlling the HIV infection. Using inducible vectors to target gene expression to virus-infected cells is a promising approach for controlling HIV infection.
Developments in the design of packaging cell lines Another approach to targeting retroviral vector expression is to pseudotype vector virus with envelope glycoproteins that have a specific host range. Cosset et aL [30*] constructed ALV-derived vectors bearing
Recent advances in retrovirus vector technology Boris-Lawrie and Temin 107 the e n v regions of ALV subgroups & B, C and E. The ALV subgroups are based on host range, cross interference of receptors, and antigenicity. The 5' terminus of the ALV e n v gene contains the subgroup-specific sequences. This region of e n v in packaging plasmids was replaced with the corresponding segment of each subgroup. Using interference assays, the subgroup vector viruses demonstrated resistance to superinfection by the same subgroup and were able to infect the predicted cell types. Cell-type specificity was also described: subtype A and B vectors both infected chicken embryos; subgroup A infected both cardiac and skin cells; and subgroup B infected cardiac but not skin cells. Thus, these packaging cells can be used to direct retroviral vectors to specific cell targets.
In addition to cell tropism, key issues in the design of packaging cell lines include the production of high-titer vector virus and avoiding the generation of RCV (see [31 ] for a review). Recent packaging cell lines have attempted to reduce the opportunity for generation of RCV by limiting sequence overlap between the packaging provirus and the retroviral vector to be packaged (see [31] for a review). The original packaging cell lines (C3) contained a deleted packaging signal [32]. Alterations of packaging cell lines (~-CRIP and ~-CRE) include deletion of the packaging signal and expression of the gag--pol and e n v genes on separate plasmids [33]. In addition, portions of the 5' LTR have been deleted and the 3' LTR replaced with the SV40 poly(A) signal (PA317; [34] ), or the 5' LTR has been replaced with the metallothionein (Mt) promoter and the 3' LTR is left intact (clone 32; [35] ). In the packaging cell lines DAN and DSN, both LTRs were replaced, the packaging signal was deleted, and the g a g - p o l and e n v genes were expressed on separate plasmids [36]. Although these alterations reduce the chance of generating RCV, they can also reduce the titer from the packaging cell line. In a recent packaging cell line construction (designated ampli-GPE) [37"], the 5' LTR was replaced with the M t promoter, and the 3' LTR was replaced with a [3-globin poly(A) sequence. The packaging signal was deleted, and the g a g - p o l and e n v genes of MoMLV were placed on separate plasmids to further reduce the chance of recombination to form RCV. Then, to amplify the packaging plasmids, the bovine papillomavirus (BPV) gene for gene amplification was inserted. High-titer vector viruses were produced using ampli-GPE, 5 x 105 to 1 x 106CFU/ml, compared to the titer of 2.6 x 104 CFU/ml previously reported for the the parental helper plasmid that contained the 3' LTR and lacked the BPV gene for amplification. The copy number of the ampli-GPE sequences was estimated to be 20-50 per cell, although it was not clear if the DNA was in the proviral or plasmid state. After a month in culture, the virus titer remained stable, and no RCV was detected. Thus, cells derived from a packaging plasmid in which both LTRs were replaced and the BPV gene was inserted for amplification can provide high-titer vector virus. The titer of the ampli-GPE cell line is similar to other recent packaging cell lines. The ampli-GPE
packaging plasmids contain the neo gene, so retroviral vectors expressing neo can not be used with this system. The SV40 origin of replication (ori) was used on packaging cell plasmids to produce high-titer vector virus during transient transfection of Cos-7 cells [38°]. MLV gag--pol and various e n v sequences were expressed on two separate packaging plasmids, each containing the SV40 ori and a deletion of the packaging signal. Cos-7 cells were co-transfected with the packaging plasmids and a packageable vector plasmid. Virus tater on target cells was measured 72 h post-transfectaon. The average vector virus titer was 9 x 104 CFU/ml, and was 40-fold higher than that from packaging cells lacking the SV40 or/sequence. No RCV was detected by a reverse transcriptase assay after allowing 16 days for virus spread, nor by rescue of a Neo a MLV vector provirus in NIH3T3 cells. Thus, transient expression of SV40 or/packaging plasmids in Cos-7 was effective for producing vector virus. The likelihood of RCV may be further decreased using retroviral vectors with different LTRs that are transcriptionally active in Cos-7 cells.
Conclusion The most significant advance in retroviral vector technology during the past year has been the demonstration that IRES sequences from picomaviruses can express proteins from dicistronic and tricistronic retroviral mRNAs. Selection for any one marker within the cistron ensures the expression of the other genes. This approach will be parOcularly important in vectors for gene therapy, in which stable gene expression is often problematic. Several papers published during the past year have described systems for targeting vector virus expression to specific cell populations. The limitation of productive retroviral infection to dividing cells was used to target vector infection to tumor cells in brain tissue. Gene expression was restricted to muscle tissue of transgenic chickens using an internal tissue-specific promoter in an ALVvector. ALV packaging cell lines containing envgenes with different subtype tropisms were useful for targeting virus infection to the predicted cell type. The amplification of packaging plasmids using BPV or SV40 viral genes is an interesting approach to increasing titer from packaging cell lines. A continuing challenge remains the construction of vectors providing long-term gene expression in transduced cells. In addition to tissue-specific internal promoters, IRES sequences provide a new and exciting approach for the selection of long-term vector expression.
Acknowledgements We thank GaryPulsinelli,AmyRaduegeand ShiaolonYangfor critical reading of the manuscript.
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Viral genetics
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DHAWANJ, PAN IX:, P^VLATH GK, TRAVlS MA, LANCTOT AM, BL~U HM: Systemic Delivery of Human Growth Hormone by Injection of Genetically Engineered Myoblasts. Science 1991, 254:1509-1512.
14.
PALMERTD, ROSMANGJ, OSBORNEWRA, MILLERAD: Genetically Modified Skin Fibroblasts Persist Long After Transplantation but Gradually Inactivate Introduced Genes. Proc Naa Acad Sci USA 1991, 88:1330-1334.
15.
SCHARFMAI~ R, AXELROD JH, VERMA IM: Long-Term in Vivo Expression of Retrovirus-Mediated Gene Transfer in Mouse Fibroblast Implants. Proc Naa Acad Sci USA 1991, 88:4626-4630.
16.
1.1 M, I-IANTZOPOULOS PA, BANERJEE D, ZHAO SC, SCHWEITZER BI, Gn.BO^ E, BESTh~OJR: Comparison of the Expression of a Mutant Dihydrofolate Reductase under Control of Differ-
18. •
[ETROPOULOSCJ, PAYNE W, SALTEROW, HUGHES SH: Using Appropriate In Vivo Expression of a Muscle-Specific Promoter by Using Avian Retroviral Vectors for Gene Transfer. J Virol 1992, 66:3391-3397. An ALV vector containing an internal chicken skeletal muscle 0t-actin promoter selectively expressed CAT reponer-gene activity in striated muscle tissue, whereas a constitutive internal promoter expressed lowlevel CAT activity in a number of tissues. 19.
CULVERKW, RAMZ, WALLBRIDGES, ISHII H, OLDFIELDEH, BLAESE RM: In Vivo Gene Transfer with Retroviral Vector-Producer Cells for Treatment of Experimental Brain Tumors. Science 1992, 256:1550-1552. Fibroblasts transduced with a retroviral vector encoding the HSV tk gene were injected into implanted gliomas in mice. After treatment with GCV, complete regression of tumor growth was observed, while the normal, non-dividing brain tissue was unaffected. •
25. .•
ADAMMA, RAMESHN, MILLERAD, OSBOURNEWRA: Internal Initiation of Translation in Retroviral Vectors Carrying Picornavirus 5' Nontranslated Regions. J viro11991, 65:4895-4990. In this study, MoMLV-basedvectors containing IRESsequences of EMCV or polio virus were constructed. Similar levels of vector-encoded proteins were produced from vectors expressing genes directly from the LTR or downstream of the IRES.A single transcript was expressed from the IRESvectors, suggesting internal initiation of translation in retroviral VeCtOrs. 26.
GHA'I'rASIR, SANESJR, MAJORSJE: The Encephalomyocarditis Virus Internal Ribosome Entry Site Allows Efficient Coexpression of Two Genes from a Recombinant Provirus in Cultured Cell and in Embryos. Mol Cell Biol 1991, 11:5848-5859.
27.
KOO H-M, BROWN AMC, KAUFMANRJ, PROROCKCM, RON Y, DOUGHERTYJP: A Spleen Necrosis Virus-Based Retroviral Vector which Expresses Two Genes from a Dicistronic mRNA. Virology 1992, 186:669-675. Long-term coordinate expression of NeoR and HygR from a SNV-ECMV IRES vector was demonstrated. Two months after infection, similar levels of vector virus titer, RNA and protein were observed when cells were selected with G418, hygromycin B or both drugs simultaneously. • o
28. o•
MORGANCA, COUTURE L, El.ROY-STEIN O, RAGHEBJ, Moss B, ANDERSONWF: Retroviral Vectors Containing Putative Interhal Ribosome Entry Sites: Development of a Polycistronic Gene Transfer System and Appfications to Gene Therapy. Nucleic Acids Res 1992, 20:1293--1299.
Recent advances in retrovirus vector technology Boris-Lawrie and Temin Tricistronic retroviral vectors containing both the EMCVand polio virus IRES were shown to produce the three vector proteins and express a single vector RNA-The titer of the tricistronic IRES vector on NIH3T3 cells was 3 x 10 5CFU/ml.
36.
29.
37. •
BUCHSCHACHERGL, PANGANmAN AT: Human Immunodeficiency Virus Vectors for Inducible Expression of Foreign Genes. J Virol 1992, 66:2731-2739.
30.
COSSETF-L, RONFORT C, MOLINA R-M, FLAMANTF, DRYNDAA, BENCHAIBI M, VAkSESIAS, NIGON V-N, VERDIER G: Packaging Cells for Avian Leukosis Virus-Based Vectors with Various Host Ranges. J Virol 1992, 66:5671-5676. In this work, ALVpackaging cells were constructed that express the Env protein of ALV subgroups A, B, C and E. Infection by packaged vector partic[es was directed to specific cell targets based on the tropism of each subgroup Env.
DOUGHERTYJP, WtSNIEWSK]R, YANGS, RHODE BW, TEMIN HM: New Retrovirus Helper Cells with Almost no Nucleotide Sequence Homology to Retrovirus Vectors. J virol 1989, 63:3209-3213.
TAKAHARAY, HAMADAK, HOUSMAN DE: A New Retrovirus Packaging Cell for Gene Transfer Constructed from Amplified Long Terminal Repeat-Free Chimeric Proviral Genes. J virol 1992, 66:3725--3732. High-titer vector virus (5 × 105 to 1 x 106CFU/ml) was produced by a new packaging line, ampli-GPE. The packaging plasmids contain the BPV gene for gene amplification, the Mt promoter was substituted for the 5' LTR, the packaging signal was deleted, the 3' LTR was replaced with a J~-globin poly(A) sequence, and the gag-pol and env genes of MoMLVwere placed on separate plasmids. 38. •
LANDAUNR, LrrrMAN DR: Packaging System for Rapid Production of Murine Leukemia Virus Vectors with Variable Tropism. J virol 1992, 66:5110-5113. Transient co.transfection of packaging cell plasmids containing the SV40 on" sequence and a vector plasmid into Cos-7 cells produced a vector virus titer of 9 x 104 CFU/ml, 40-fold higher than the titer observed from cells without the SV40 or/sequence.
31.
. MILLERAD: Retrovirus Packaging Cells. Hum Gene 7herapy 1990, 1:5-14.
32.
WATANABE.% TEMIN HM: Construction of a Helper Cell Line for Avian Reticuloendotheliosis Virus Cloning Vectors. Mol Cell Biol 1983, 3:2241-2249.
33.
DANOSO, MULUGANRC: Safe and Efficient Generation of Recombinant Retroviruses with Amphotropic and Ecotropic Host Ranges. Proc Naa Acad Sci USA 1988, 85:6460--6464.
39.
DOUGHERTYJP, TEMIN HM: High Mutation Rate of a Spleen Necrosis Virus-Based Retroviros Vector. Mol Cell Biol 1986, 6:4387-4395.
34.
MILLERAD, BUTnMORE C: Redesign of Retrovirus Packaging Cells to Avoid Recombination Leading to Helper Virus Production. Mol Cell Biol 1986, 6:2895-2902.
40.
35.
BOSSELMANRA, HSU R-Y, BRUSZEWSKI J, HU S, MARTIN F, NICOLSON M: Replication-Defective Chimeric Helper Proviruses and Factors Affecting Generation of Competent Virus: Expression of Moloney Murine Leukemia Virus Structural Genes via the Metallothionein Promoter. Mol Cell Btol 1987, 7:1797-1806.
HANTZOPOULOSPA, SULLENGERBA, UNGERSG, GII.BOA E: Improved Gene Expression Upon Transfer of the Adenosine Deaminase Minigene Outside the Transcriptional Unit of a Retroviral Vector. Proc Natl Acad Sct USA 1989, 86:3519-3523.
KA Boris.Lawrie and HM Temin, McArdle Laboratory for Cancer Research, 1400 University Avenue, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
109
Introduction Retroviruses are RNA viruses that replicate through a DNA intermediate. The DNA intermediate is integrated into the host chromosome as a provirus. After infection of a susceptible cell, reverse transcription of the retroviral genomic RNA to double-stranded DNA requires several c~acting viral sequences, including the primer-binding site (pbs), the repeat (R) region of the long terminal repeats (LTRs), and the polypurine tract (ppt) (see Fig. 1). Integration of the viral DNA into the host genome is mediated by the terminal attachment (art) sites. The provirus promoter within the unique 3' (U3) region of the 5' LTR governs transcription, and all viral transcripts initiate at the first nucleotide of the R region. The full-length transcript can be packaged or used for translation, either directly into Gag---Pol protein or, after splicing, into Env protein. The genomic RNA is packaged by viral proteins, which recognize viral RNA by the c/s-acting packaging or encapsiclation sequence, located between the unique 5' (U5) region and near the beginning of gag. The Gag, Pol and Env proteins, and viral RNA interact at the surface of the cell membrane, and mature virions are released by budding. The cell tropism of subsequent infection by the viral particles is determined primarily by the Env glycoprotein. Although the biology of retroviral replication is complex, knowledge of the c/s-acting viral sequences that control retroviml replication has enabled the construction of retroviml vectors and packaging cells using parts from retroviruses. Most retroviral vectors contain only the c~acting viral sequences required for packaging, reverse transcription, and integration of inserted for-
eign sequences. Packaging or helper cells provide the viral proteins in tram, but lack the packaging or integration signals. These packaging cells can be transfected with the retroviral vector, which then produces RNA that can be packaged and released as vector virus particles. These vector viruses can be used to introduce the foreign DNA stably into target cells, either in culture or direcdy in animal cells. The purpose of this review is to summarize recent advances in retrovirus vector technology.
Retroviruses as vectors There are several reasons why retrovimses are useful as vectors. Retroviral vectors provide efficient and stable transfer of DNA into replicating cells. Vector integration is precise, and because no viral proteins are encoded by the vector, there is no possibility of vires spread. The inserted DNA can be expressed from the LTR promoter and efficiently spliced, or expressed from an internal promoter. A diverse group of animal cells have been shown to be infectable by retroviruses as a result of the wide tropism of the various viral Env proteins. However, retroviral vectors also have some limitations. Infection with retroviral vectors derived from simpler retroviruses is limited to dividing cells, and the size limit of inserted foreign sequence is less than 8-10 kb. Because genetic variation occurs during retroviral replication, proviral clones need to be analyzed to determine that the provims has the expected structure. As proviral insertion is random, insertional mutagenesis is possible; however, in mouse models, insertional mutagenesis is
Abbreviations ALV--avian leukosis virus; araM~6*methoxypurine arabinonucleoside; aN--attachment; BPV--bovine papillomavirus; CAT--chloramphenicol acetyltransferase; CFU--colony forming unit; DHFR---clihydrofolate reductase; EMCV-~encephalornyocarditis virus; FH--familial hypercholesterolemia; GCV--gancicIovir; hGH--human growth hormone; HSV--herpes simplex virus; IRES~internal ribosome entry site; hADA--human adenosine deaminase; hGH--human growth hormone; LDLR--Iow density lipoprotein receptor; LTR--Iong terminal repeat; MDRl--multidrug resistance 1; MoMLV--Moloney murine leukemia virus; Mt--metallothionein; neo~neomycin phosphotransferase; ori--origin of replication; pbs--primer-binding site; ppt--polypurine tract; R--repeat; RCV--replication-cornpetent virus; RSV--Rous sarcoma virus; SNV--spleen necrosis virus; SV40~simian virus 40; tk--thymidine kinase; U3--unique 3'; US--unique 5'; UTR--untranslated region; VZV--varicella zoster virus. 102
(~ Current Biology Ltd ISSN 0959-437X
Recent advances in retrovirus vector technology Boris-Lawrie and Temin
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Fig. 1. The structure of wild-type retrovirus DNA and retrovirus vector DNA. (a) Wild-type spleen necrosis virus DNA with the gag, pol and env genes shown, and (b-f) retrovirus vector DNA containing different combinations of foreign genes (see text for details). The division of the long terminal repeats (LTRs) into the unique 3' (U3), repeat (R) and unique 5' (US) regions (light shading) of the viral DNA is shown, as are the attachment sites relative to the polarity of the provirus in the host chromosome (attL and attR for left and right, respectively). Also shown are the primer-binding site (pbs), splice donor (sd), cis-acting encapsidation sequence (E), splice acceptor (sa), and polypurine tract (ppt). IRES1 and IRES2 denote internal ribosome entry sites from the polio and encephalomyocarditis viruses, respectively. (b) In the splicing vector, the vector transcript is expressed from the LTR promoter, and is spliced and translated to produce two proteins [39]. (c) The internal promoter vector expresses the second protein from a second transcriptional unit [22]. (d) In this internal promoter vector, the second transcriptional unit is inserted within the 3' U3 region, outside of the retroviral transcriptional unit. After reverse transcription of the vector, the 3' U3 region is duplicated in both LTRs of the provirus [40]. (e,f} In the IRES vectors, a single vector transcript is translated to produce two or three proteins, respectively [27°°,28°'].
associated with a spreading virus infection. Replicationcompetent vires (RCV) can be generated by recombination between sequences of the retroviral vector and the packaging provirus. Therefore, packaging cell lines are constantly being redesigned to contain the minimum sequence overlap with vectors. As a vector safety feature, genes such as the herpes simplex virus (HSV) thymidine kinase (tk) gene, which confers ganciclovir (GCV) sensi-
tivity, can be used in vectors to enable selection against transduced cells if necessary [1]. Retrovirus vectors are useful for introducing DNA into cells for the study of retrovirus replication, for cell line'age studies, and for gene therapy. The predominant use of retroviral vectors is to study viral replication. For example, retroviral vectors are in-
103
104 Viralgenetics valuable reagents for studying the mechanism of reverse transcription [2] and dissecting events involved in the assembly and release of virions [3]. Vectors have been used to study the generation of genetic diversity during viral replication [4,5], and mechanisms by which retroviruses can mobilize cellular proto-oncogenes (J Zhang and HM Temin, unpublished data) [6]. Retroviral vectors-have been designed to screen for anti-retroviral compounds and to study the effect of drugs on viral replication [7]. See I Lemischka (this issue, pp 115-118) for a review on retroviruses as markers for cell lineage studies.
Recent experiments using retroviral vectors for gene therapy A major focus in retrovirus vector technology is the design of vectors and packaging cell lines for gene therapy. Original conceptions for gene therapy involved the correction of single gene disorders to cure a variety of congenital and acquired diseases. After the success of the first human gene-transfer experiment (study of tumor infiltrating lymphocytes transduced with vector virus expressing neomycin resistance [8]), the first clinical trial for human gene therapywas initiated in September 1990 (transfer of lymphocytes transduced with vector viruses expressing the gene encoding human adenosine deaminase (hADA) to correct ADA deficiency; see [9]). Recent strategies for gene therapy also include disease prevention and the expression of genes to aid in disease treatment. Here, we will summarize some of the experiments reported durhag the past year that used retroviral vectors to introduce DNA into somatic cell populations. Bone-marrow stem cells are an important target for retroviral vector transduction because the pluripotent hematopoetic stem cell is the progenitor of a complete hematopoetic system. Human ADA has been expressed in mice and, for short periods, in primates (less than 100 days). Van Beuseheum etaL [10o] transduced autologous bone-marrow stem cells from rhesus monkeys with vectors encoding hAD& Vector packaging cells were cocultured with the bone-marrow cells, and the co-culture was injected back into the monkeys. Stable hADAexpression was achieved for greater than one year. The use of selected growth factors for culturing the bone-marrow cells facilitated stable transduction of early hematopoetic progenitor cells (granulocytes). The presence of packaging virus in the injected cells may have increased the chance of generating RCV, but injection of the co-culture maximized the number of viable transduced cells. This first example of long-term hADA expression in primates is encouraging for transduction of hematopoetic progenitor cells in human gene therapy. Familial hypercholesterolemia (FH) is caused by a deficiency of low density lipoprotein receptors (LDLRs). Using a retroviral vector expressing LDLR, hepatocyte e x vivo gene therapy was therapeutic for FH in the FH animal model, the Watanabe heritable hyperlipidemic rabbit [11]. Autologous hepatocytes were iso-
lated from resected liver (30% of the liver), grown in culture, and transduced with recombinant retroviruses expressing rabbit LDLR. After injection into the autologous rabbit, LDLRexpression was stable for six months (the duration of the study), and serum cholesterol was reduced by 25% to yield a therapeutic level. No immune response to the recombinant LDLRprotein was detected. A number of potentially therapeutic proteins would need to be distributed systemically. Transduced muscle ceils have now been shown to provide systemic distribution of recombinant proteins [12,13]. Mouse myoblast cells transduced with a retroviral vector expressing human growth hormone (hGH) were re-injected into limb tissue [12]. The transduced myoblasts differentiated to myotubes and fused with the vascularized, multinucleated myofibers. The expression of hGH in serum was detected for the duration of the study (three months). No tumors were found in mice treated with the transduced cells [13]. Although these experiments have detected stable vector gene expression for up to one year, a major question remains as to the longevity of vector gene expression in transduced somatic cells. Vector transcription has been suggested to be subject to programed control of cellular gene expression [14]. In a study of engrafted transduced mouse fibroblasts, the housekeeping promoter, DHFR was placed within a retroviral vector and remained transcriptionally active at 60 days post-engraftment, whereas transcription from the LTR promoter or an internal cytomegalovirus immediate early promoter was suppressed [15]. As discussed below, the activity of an internal promoter in transduced somatic cells can not be predicted from experiments in culture [16]. New vector designs that use cell type specific promoters may be useful for providing sustained vector gene expression [17",18-].
Retroviral vectors for disease treatment Recently, gene-therapy strategies based on the ability of the simpler retroviruses to infect only actively growing cells have targeted the selective destruction of neoplastic cells. To destroy experimental brain tumors selectively in mice, glioma cells were injected with fibroblasts producing amphotropic vector viruses expressing the HSV tk gene [19*], which encodes sensitivity to GCV. After allowing for virus infection of the actively dividing glioma cells, the mice were treated with GCV. The tumors completely regressed, and neighboring tissue remained unaffected. No systemic cytotoxicity or spread of virus to other rapidly growing tissues was detected. Residual virus would be subject to lysis in serum by a complement-mediated pathway [20]. The results of this short term study (30 days) are encouraging, and it will be important to establish the effect of long-term treatment with these viruses. In another study, tumor cells were targeted by using vectors with internal tissue-specific promoters that transcribe
Recent advances in retrovirus vector technology Boris-kawrieand Temin 105 a cytotoxic gene [17"]. The varicella zoster virus (VZV) tk-encoded protein phosphorylates 6-methoxypurine arabinonucleoside (araM) to cytotoxic adenine arabinonucleoside triphosphate, while mammalian kinases do not have this activity. Expression of VZV tk was directed by liver cell specific promoters (hepatoma-associated a-fetoprotein or liver-associated albumin promoter). Hepatoma and other cell lines were transduced with the vectors and treated with araM. Cytotoxicity was detected in the hepatoma lines, but not other cell lines. In another approach to cancer therapy, the multidrug resistance 1 (MDR1) gene was expressed in bone-marrow cells to address the problem of bone-marrow toxicity during chemotherapy [21]. Bone-marrow ceils were transduced by co-culture with ecotropic packaging cells containing the murine sarcoma virus MDR1 vector virus and then used to reconstitute mouse bone marrow. After treatment with the drug taxol, the mean number of proviruses per peripheral blood leukocyte more than tripled, and the neutrophil count was more stable than in the control. Thus, MDR1 may be useful for the selection of drug-resistant cells in vivo. Studies of the therapeutic value of this approach are awaited.
A new approach in the design of retroviral vectors: internal ribosome entry sites Retroviruses have evolved three strategies to produce multiple proteins from a single mRNA: splicing provides Env protein; ribosomal frameshifting provides Pol protein; and proteolytic processing yields the capsid, matrix, protease, and reverse transcriptase proteins from the Gag-Pol polyprotein. Retroviral vectors use two strategies for protein expression, either splicing or use of an interhal promoter (see Fig. 1). In splicing vectors, the viral transcript is expressed from the LTR promoter and is spliced and translated to produce two proteins. TypicaUy, this arrangement results in higher production of the upstream protein relaUve to the downstream protein. In the second configuration, the LTR promoter expresses the upstream gene, and an internal promoter transcribes the downstream mRNA. For example, the promoter of an unselected gene may be suppressed as the result of an unknown epigenetic mechanism [22,23]. The second transcription unit may also be inserted within the 3' U3 region (Fig. 1). In this arrangement, the second transcription unit is duplicated in the 5' U3 region after reverse transcription. This strategy can provide more balanced protein production; however, a second promoter near the LTR promoter can lead to unstable transcription. The effect of viral regulatory sequences on the internal promoter can be detected independent of transcription from the LTR [24]. Importantly, because the markers on splicing vectors or vectors with internal promoters are n o t co-selected, stable expression of the markers is often problematic. A new approach for expression of multiple genes from retroviral vectors has been adapted from picornaviruses.
Picomavimses express several proteins from a polycistronic mRNA. The 5' end of this mRNA terminates with pUpU, instead of the typical eukaryotic m7GpppN cap, and the transcript contains long 5' untranslated regions (UTRs) with complex secondary structure and multiple AUG codons. These UTRs are c~acting sequences that promote cap-independent translation initiation at internal initiation codons. These sequences are designated internal ribosome entry sites (IRESs). IRES sequences cloned from poliovirus and encephalomyocarditis virus (EMCV) have been used in retroviral vectors to direct translation of LTR-driven polycistronic RNA. Adam et aL [25"*] constructed Moloney murine leukemia virus (MoMLV)-based retroviral vectors that positioned a selectable marker or a reporter gene either downstream of the 5' LTR, downstream of an IRES sequence, or downstream of an internal simian virus 40 (SV40) early promoter. Virus titers were similar among the vectors, suggesting the IRES did not alter virus replication. The level of reporter protein produced was similar when the gene was directly downstream of the LTR or an IRES. Expression of the selectable marker, neomycin phosphotransferase (neo) was similar when neo was positioned downstream from an IRES or the SV40 early promoter. The predicted polycistronic transcripts were detected by northern-blot analysis, suggesting the IRES sequence promoted internal initiation of translation. Another survey of IRES function used Rous sarcoma virus (RSV)-based vectors and also found virus titer was not affected by IRES [26]. Reporter gene activity from an EMCV IRES was twofold higher than from an internally promoted mRNA, and spliced transcripts provided only 5--10% the level of activity. Gene expression from splicing vectors or internal promoter vectors in long-term culture can be low-level or absent. With polycistronic IRES vectors, selection for one gene should ensure expression of the second gene. Koo et a t [27"'] constructed a dicistronic spleen necrosis virus (SNV) vector, pJD214HyBi+, which contained two selectable markers between the SNV LTRs, the genes conferring resistance to hygromycin B (byg), and G418 (neo). T h e EMCV IRES sequence was inserted upstream of neo (Fig. 1). Virus titers on target cells after selection with hygromycin, G418, or hygromycin and G418 were similar (3.4 x 105, 2.1 x 105, and 2.1 x 105 colony forming units (CFU)/ml, respectively), suggesting that both hyg and neo were expressed in the same cell. Two months after the initial drug selection, populations of cells selected with hygromycin, G418, or doubly selected were analyzed. In each population the pJD214HyBi+ proviruses remained intact, the predicted single vector transcript was detected using a neo~specilic probe, and the Neo protein was stably produced. The level of Neo protein produced from the IRES vector was similar to the level from a control vector that expressed neo directly from the SNV LTR. These results indicate that selection of either marker on a dicistronic RNA allows expression of both markers. The activity of an RSV LTR-/acZ-EMCV IRES--v-src-LTR vector was examined in ovo [26]. Virus stocks were in-
106 Viral genetics jected imo chicken embryos, and the embryos were harvested 3-6 days later. Inspection of LacZ-positive cells revealed morphological properties associated with expression ofv-sr~ Thus, in the short term, protein production was achieved from a retroviral IRES vector in viva The polio and EMCV IRESs were used in tandem to construct a tricistronic retroviral vector (MLV LTR-neo-polio IRES--cat-EMCV IRES-ada-LTR) (see Fig. 1) [28°']. Virus titer remained similar to either dicistronic vector (MLV LTR-neo-polio IRES-cat-LTR or MLV LTR-neoEMCV IRES-ada-~LTR). Vector proviruses were found to be structurally intact and to transcribe the predicted single tricistronic mRNA. Each of the three vector proteins were detected, although the levels were lower than from the corresponding dicistronic vector. Thus, retroviral vectors containing IRES s.equences arranged in tandem can express multiple genes from a polycistronic mRNA. In future experiments, translation levels may be increased by optimizing spacing between the EMCV start codon and the downstream gene, or by precisely positioning the inserted gene at the EMCV start codon. Clearly, the use of picomavirus IRES sequences is a promising advance for retroviral vector technology. Importantly, the selection of one marker ensures the expression of the other genes within the cistron. Among the possible applications are vectors producing multiple subunit proteins or the cellular co-expression of synergistic proteins.
Targeted vector transcription Retroviral vectors with intemal promoters that are tissuespecific can target vector gene expression. As discussed above, hepatoma-specific transcription from liver-cell promoters provided targeted expression of the cytotoxic VZV tkgene [17"]. The stringency of tissue-specific transcription from internal promoters was surveyed in transgenic chickens using avian leukosis virus (ALV) vectors [18o]. Chicken embryos were transduced with replication-competent ALV vector virus, and gene expression was studied in the progeny chicks. Chloramphenicol acetyitransferase (CAT) activity directed from the internal tissue-specific chicken skeletal muscle ~t-actin promoter was compared to CAT levels from the constitutive chicken IB-actin promoter. The skeletal muscle at-actin promoter-cat vector expressed high-level CAT reporter gene activity in muscle, but not in other tissues (71% versus 0--8% substrate conversion). Reporter gene activity from the 13-actin promoter vector was low in all tissues (3%). Similar results were obtained when the internal promoters were placed in the opposite orientation. The copy number of vector proviruses was similar in muscle and other tissues, and virus production in each tissue was equivalent as measured by immunoblot with a capsid protein antibody. Therefore, transcription from the internal skeletal muscle ct-actin promoter appeared to be tissue-specific, although as the authors note, RNA analysis is needed to conclude definitively muscle-specific transcription.
In culture, expression from the skeletal muscle 0t-actin promoter was not found to be tissue-specific. The authors suggest this may be the result of incomplete differentiation of the cells [18o]. Experiments by U et aL [16] similarly suggest that the efficiency of expression from internal promoters in primary cells can not be predicted from ceU-culture experiments. In this study, five different internal promoters were used to test the relative expression of a dihydrofolate reductase (DHFR) gene, which confers methotrexate resistance [16]. The vectors expressed neo directly from the MoMLV LTR and instead of inserting the promoter-DHFR sequences within the LTR transcription unit, a poly(A) signal was inserted within the U3 region of the 3' LTR. The second transcriptional unit (each promoter and DHFR) was then positioned downstream of the poly(A) sequence. After infection and reverse transcription, the second transcriptional unit was duplicated in the 5' LTR. In five cell lines, the relative level of methotrexate resistance from each vector matched the relative level of RNA expressed. However, the relative abundance of transcription among the promoters was inconsistent when the vectors were compared in cell lines and in transduced primary bone-marrow cells. These results are similar to the inconsistency in transcriptional specificity of the ALV vectors observed in culture and in vivo [18o]. Other groups have also reported that transcription from internal promoters in retroviral vectors can be unstable [14,15]. Therefore, once transduced into somatic cells, vectors need to be monitored for stable gene expression. Another approach to targeted vector gene expression is to regulate LTR promoters with trans-acting factors. Transcription from the HIV LTR is induced by the viral transacting protein, Tat, through the c/s-acting transactivation response site, TAR. Tat-inducible replication-defective HW vectors may be useful for anti-HW gene therapy by providing controlled expression of proteins that interfere with the viral replication cycle or are deleterious to infected cells. A recent example takes advantage of the stringent regulation of transcription from the HW LTR to target expression of the murine cytocidal influenza virus hemaglutinin (H5 HA) in cells upon infection with HIV [29]. H5 HA was detected by immunofluroscence only in cells expressing Tat, which suggested that this system may be useful for the expression of genes that elicit a cytocidal immune response to HIV infection. In vivo, potential spread of such a vector virus would be beneficial for further controlling the HIV infection. Using inducible vectors to target gene expression to virus-infected cells is a promising approach for controlling HIV infection.
Developments in the design of packaging cell lines Another approach to targeting retroviral vector expression is to pseudotype vector virus with envelope glycoproteins that have a specific host range. Cosset et aL [30*] constructed ALV-derived vectors bearing
Recent advances in retrovirus vector technology Boris-Lawrie and Temin 107 the e n v regions of ALV subgroups & B, C and E. The ALV subgroups are based on host range, cross interference of receptors, and antigenicity. The 5' terminus of the ALV e n v gene contains the subgroup-specific sequences. This region of e n v in packaging plasmids was replaced with the corresponding segment of each subgroup. Using interference assays, the subgroup vector viruses demonstrated resistance to superinfection by the same subgroup and were able to infect the predicted cell types. Cell-type specificity was also described: subtype A and B vectors both infected chicken embryos; subgroup A infected both cardiac and skin cells; and subgroup B infected cardiac but not skin cells. Thus, these packaging cells can be used to direct retroviral vectors to specific cell targets.
In addition to cell tropism, key issues in the design of packaging cell lines include the production of high-titer vector virus and avoiding the generation of RCV (see [31 ] for a review). Recent packaging cell lines have attempted to reduce the opportunity for generation of RCV by limiting sequence overlap between the packaging provirus and the retroviral vector to be packaged (see [31] for a review). The original packaging cell lines (C3) contained a deleted packaging signal [32]. Alterations of packaging cell lines (~-CRIP and ~-CRE) include deletion of the packaging signal and expression of the gag--pol and e n v genes on separate plasmids [33]. In addition, portions of the 5' LTR have been deleted and the 3' LTR replaced with the SV40 poly(A) signal (PA317; [34] ), or the 5' LTR has been replaced with the metallothionein (Mt) promoter and the 3' LTR is left intact (clone 32; [35] ). In the packaging cell lines DAN and DSN, both LTRs were replaced, the packaging signal was deleted, and the g a g - p o l and e n v genes were expressed on separate plasmids [36]. Although these alterations reduce the chance of generating RCV, they can also reduce the titer from the packaging cell line. In a recent packaging cell line construction (designated ampli-GPE) [37"], the 5' LTR was replaced with the M t promoter, and the 3' LTR was replaced with a [3-globin poly(A) sequence. The packaging signal was deleted, and the g a g - p o l and e n v genes of MoMLV were placed on separate plasmids to further reduce the chance of recombination to form RCV. Then, to amplify the packaging plasmids, the bovine papillomavirus (BPV) gene for gene amplification was inserted. High-titer vector viruses were produced using ampli-GPE, 5 x 105 to 1 x 106CFU/ml, compared to the titer of 2.6 x 104 CFU/ml previously reported for the the parental helper plasmid that contained the 3' LTR and lacked the BPV gene for amplification. The copy number of the ampli-GPE sequences was estimated to be 20-50 per cell, although it was not clear if the DNA was in the proviral or plasmid state. After a month in culture, the virus titer remained stable, and no RCV was detected. Thus, cells derived from a packaging plasmid in which both LTRs were replaced and the BPV gene was inserted for amplification can provide high-titer vector virus. The titer of the ampli-GPE cell line is similar to other recent packaging cell lines. The ampli-GPE
packaging plasmids contain the neo gene, so retroviral vectors expressing neo can not be used with this system. The SV40 origin of replication (ori) was used on packaging cell plasmids to produce high-titer vector virus during transient transfection of Cos-7 cells [38°]. MLV gag--pol and various e n v sequences were expressed on two separate packaging plasmids, each containing the SV40 ori and a deletion of the packaging signal. Cos-7 cells were co-transfected with the packaging plasmids and a packageable vector plasmid. Virus tater on target cells was measured 72 h post-transfectaon. The average vector virus titer was 9 x 104 CFU/ml, and was 40-fold higher than that from packaging cells lacking the SV40 or/sequence. No RCV was detected by a reverse transcriptase assay after allowing 16 days for virus spread, nor by rescue of a Neo a MLV vector provirus in NIH3T3 cells. Thus, transient expression of SV40 or/packaging plasmids in Cos-7 was effective for producing vector virus. The likelihood of RCV may be further decreased using retroviral vectors with different LTRs that are transcriptionally active in Cos-7 cells.
Conclusion The most significant advance in retroviral vector technology during the past year has been the demonstration that IRES sequences from picomaviruses can express proteins from dicistronic and tricistronic retroviral mRNAs. Selection for any one marker within the cistron ensures the expression of the other genes. This approach will be parOcularly important in vectors for gene therapy, in which stable gene expression is often problematic. Several papers published during the past year have described systems for targeting vector virus expression to specific cell populations. The limitation of productive retroviral infection to dividing cells was used to target vector infection to tumor cells in brain tissue. Gene expression was restricted to muscle tissue of transgenic chickens using an internal tissue-specific promoter in an ALVvector. ALV packaging cell lines containing envgenes with different subtype tropisms were useful for targeting virus infection to the predicted cell type. The amplification of packaging plasmids using BPV or SV40 viral genes is an interesting approach to increasing titer from packaging cell lines. A continuing challenge remains the construction of vectors providing long-term gene expression in transduced cells. In addition to tissue-specific internal promoters, IRES sequences provide a new and exciting approach for the selection of long-term vector expression.
Acknowledgements We thank GaryPulsinelli,AmyRaduegeand ShiaolonYangfor critical reading of the manuscript.
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Viral genetics
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KA Boris.Lawrie and HM Temin, McArdle Laboratory for Cancer Research, 1400 University Avenue, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
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