Gene Therapy (1997) 4, 128–139 1997 Stockton Press All rights reserved 0969-7128/97 $12.00 High transdominant RevM10 protein levels are required to inhibit HIV-1 replication in cell lines and primary T cells: implication for gene therapy of AIDS I Plavec1, M Agarwal1,KEHo2, M Pineda1, J Auten1, J Baker1, H Matsuzaki3, S Escaich4, M Bonyhadi1 and E Bo¨ hnlein1 1Progenesys Program, SyStemix Inc, 3155 Porter Drive, Palo Alto, CA 94304, USA Expression of antiviral genes in CD4+ T cells has been pro- uniformly higher than from internal promoters (eg CMV, posed as a strategy for gene therapy of AIDS. Over the PGK). Analysis of selected vectors in acutely and chron- past years, we and others have developed retroviral vec- ically HIV-infected cell lines suggested that threshold levels tors encoding the RevM10 protein, a dominant-negative of RevM10 expression are required to achieve inhibition of mutant of the HIV-1 Rev trans-activator protein. We could HIV replication. LTR-driven RevM10 expression also demonstrate gene transfer and inhibition of HIV-1 repli- yielded high steady-state protein levels in activated primary cation in cultured T cell lines and primary T cells. However, T cells resulting in inhibition of HIV replication, and there little is known about the levels of the antiviral protein was no apparent difference between the MoMLV, MPSV required to achieve a therapeutic effect, particularly in pri- and MESV-LTR vectors. However, RevM10 expression mary cells. In this report, we compare different vector was down-regulated in resting primary cells and conse- designs with regard to expression of the antiviral gene to quently anti-HIV efficacy was significantly reduced. Taken develop an optimal vector for clinical applications. Our together, the data suggest that relatively high steady-state results demonstrate that intracellular steady-state RevM10 levels of RevM10 protein are required to achieve inhibition protein levels expressed from the Moloney murine leuke- of HIV replication and that the MPSV- and MESV-derived mia virus (MoMLV), myeloproliferative sarcoma virus retroviral vectors show no advantage over the MoMLV- (MPSV) or mouse embryonic stem cell virus (MESV) pro- based vectors for expression of anti-HIV genes in human moters located in the long terminal repeat (LTR) were T cells. Keywords: gene therapy; HIV-1; retroviral vectors; transgene expression; T cells Introduction of Rev function.9 Furthermore, a number of studies have demonstrated that RevM10 can inhibit viral replication in Human immunodeficiency virus type-1 (HIV-1) encodes HIV-infected T cell lines and primary T cells.10–15 several regulatory genes including Rev (reviewed in Refs Intracellular expression of genes which can suppress 1–3). Through binding to a structured RNA sequence, HIV-1 replication has been proposed as an alternative to termed the Rev-responsive element (RRE), Rev promotes standard therapies for the treatment of AIDS.16,17 A large 4,5 the export of RRE-containing mRNAs from the nucleus number of antiviral genes including RNA decoys, anti- leading to cytoplasmic accumulation of incompletely sense RNA, transdominant mutants (including RevM10), spliced HIV-1 transcripts. First, these transcripts serve as ribozymes and intracellular antibodies (reviewed in Ref. templates for translation of viral proteins gag, pol and env. 16) have been shown to inhibit effectively HIV-1 in vitro. Second, the nonspliced genomic mRNA is needed for The target cell population for delivery of anti-HIV genes infectious particle formation. HIV-1 Rev function requires could be peripheral blood mononuclear cells,18,19 or alter- 6–8 interaction with one or more cellular host proteins. natively hematopoietic stem cells.16 Retroviral vectors are RevM10, a mutant of Rev retains the ability to bind to presently the most widely used system to deliver genes RRE but it does not transactivate, possibly because of its into those cells.20 Irrespective of the target cell popu- 8,9 inability to interact with host factor(s). When co- lation, the success of gene therapy will depend on both expressed with wild-type Rev protein in transient trans- efficient gene transfer and persistent high-level gene fection assays, RevM10 acts as a transdominant inhibitor expression. Initially, efforts in developing retroviral vec- tors were mainly focused on improving virus titers to increase gene transfer and only recently more work is Correspondence: E Bo¨hnlein 2 being invested in optimizing vectors for efficient trans- Present addresses: Genentech Inc, 460 Point San Bruno Boulevard, South gene expression (reviewed in Refs 21–23). A wide variety San Francisco, California 94080-4990, USA; 3Affymetrix, 3380 Central Expressway, Santa Clara, California 95051, USA; 4Rhoˆne-Poulenc Rorer, of vector constructions have been described including 94403 Vitry-sur-Seine, France different promoters (retroviral LTRs, internal promoters), Received 15 August 1996; accepted 29 October 1996 orientation of the gene of interest (forward, reverse), self- RevM10 expression in cell lines and primary T cells I Plavec et al 129 inactivating LTR vectors and other LTR modifications, driven transcript (Figure 2, lower left panel) resulting in and insertion of an expression cassette into the LTR RevM10 protein levels approximately 10-fold higher than (double-copy vectors) (reviewed in Refs 21, 23). Despite internal promoter vectors (Figure 3). No apparent differ- all these studies, the optimal vector design for the ence between the MoMLV-LTR and MPSV-LTR vectors expression of a particular gene needs to be determined could be detected in CEMSS cells and the different empirically and the final result may depend on the com- primer binding sites also had no measurable impact. To bination of both gene and vector sequences.24–26 further improve expression of the LTR-RevM10 vectors, As part of our efforts to develop clinical protocols for the tk-Neo sequence was replaced by the SV40-Puro tran- gene therapy of AIDS we have tested a number of retro- scription unit (LMSPL vector; Figure 1). This modifi- viral vector designs to optimally express the transdomi- cation resulted in a two- to three-fold increase in RevM10 nant RevM10 protein. In particular, we carefully ana- protein levels in CEMSS cells (compare LMTNL with lyzed vector expression in primary T cells at various LMSPL, Figure 3). stages of activation. We also evaluated the anti-HIV In all vectors (Figure 1; numbers 4–8), the packaging potency of selected vectors in chronically and acutely signal is encoded by a short retroviral sequence13,36 and HIV-infected cell lines as well as in primary T cells. The the RevM10 protein is synthesized from an unspliced results suggest that the extent of inhibition of HIV-1 repli- full-length messenger RNA. The presence of an intron in cation is a function of the relative expression of RevM10 a transcription unit can improve the expression of some protein in those cells, supporting the hypothesis that cDNAs.37 To test this possibility, we constructed RevM10 RevM10 acts as an intracellular competitive inhibitor.9 vectors based on the LXSN38 (construct LMSPL-INT1; Implications of these findings for the development of Figure 1, No. 9) and MFG design39 (construct LMSPL- effective retrovirus vector-based anti-HIV gene therapies INT2; Figure 1, No. 10) in which protein expression relies are discussed. on the efficient splicing of the primary transcript.39,40 The ratio of spliced to unspliced transcripts was higher for the LMSPL-INT2 vector compared to the LMSPL-INT1 Results (Figure 2, lower right panel). This finding was not sur- prising since the splicing in the MFG vector had been Retroviral vector LTR-driven expression yields high optimized for protein expression.39 However, both vec- steady-state levels of RevM10 protein in transduced tors yielded similar steady-state intracellular RevM10 T cells protein levels and these were not significantly higher We started our study by testing vectors in which RevM10 when compared to the intronless LMSPL vector (Figure expression is driven by an internal promoter using the 2). Thus, inclusion of an intron did not further improve immediate–early promoter of the human cytomegalo- RevM10 protein expression. An unexpected short Rev- virus27 (LNCML vector; Figure 1) and the mouse phos- specific transcript was detected in the LMSPL RNA phoglycerate kinase gene promoter28 (LNMPL and (Figure 2). This RNA is a splice product between the LPMPL vectors; Figure 1). Human CEMSS T lymphoid splice donor in the retroviral packaging sequence and a cells were transduced with amphotropic retroviral vec- splice acceptor located in the RevM10 coding sequence tors harvested from PA317-based producer cells and and does not encode functional RevM10 protein (data not selected either with G418 or puromycin to yield homo- shown). As expected, a 10-fold higher virus titer was genous populations of transduced cells. RevM10 protein obtained with the LMSPL-INT1 vector because of the and RNA expression were analyzed by Rev-specific extended packaging sequence38 (Figure 1). immunofluorescence29 and Northern blotting, respect- ively. Regardless of the internal promoter used, low RevM10 protein threshold level is required for inhibition RevM10 RNA expression levels were observed in trans- of HIV-1 replication in chronically infected T cell lines duced cells (Figure 2) which resulted in intracellular We have used an assay system based on chronically HIV- RevM10 protein levels barely detectable above back- infected A3.01/ProHygro T cells to compare the anti-HIV ground (Figure 3). Since it has been reported that the neo- efficacy of the different vectors expressing various levels mycin phosphotransferase gene (Neo) gene sequence can of the RevM10 protein.13 The A3.01/ProHygro cells were act as a transcriptional silencer,30 we attempted to transduced by co-cultivation with the PA317-derived improve RevM10 expression by replacing the Neo- retroviral vector producer cells followed by selection in resistance gene with the puromycin-resistance gene G418 and hygromycin.