Gene Therapy (1999) 6, 660–666 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt Inhibition of HIV-1 by an anti-integrase single-chain variable fragment (SFv): delivery by SV40 provides durable protection against HIV-1 and does not require selection M BouHamdan1, L-X Duan1, RJ Pomerantz1 and DS Strayer1,2 1The Dorrance H Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Department of Medicine; and 2Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA Human immunodeficiency virus type I (HIV-1) encodes the SFv-IN was confirmed by Western blotting and several proteins that are packaged into virus particles. Inte- immunofluorescence staining, which showed that Ͼ90% of grase (IN) is an essential retroviral enzyme, which has SupT1 T-lymphocytic cells treated with SV(Aw) expressed been a target for developing agents to inhibit virus repli- the SFv-IN protein without selection. When challenged, cation. In previous studies, we showed that intracellular HIV-1 replication, as measured by HIV-1 p24 antigen expression of single-chain variable antibody fragments expression and syncytium formation, was potently inhibited (SFvs) that bind IN, delivered via retroviral expression vec- in cells expressing SV40-delivered SFv-IN. Levels of inhi- tors, provided resistance to productive HIV-1 infection in bition of HIV-1 infection achieved using this approach were T-lymphocytic cells. In the current studies, we evaluated comparable to those achieved using murine leukemia virus simian-virus 40 (SV40) as a delivery vehicle for anti-IN (MLV) as a transduction vector, the major difference being therapy of HIV-1 infection. Prior work suggested that deliv- that transduction using SV40 did not require selection in ery using SV40 might provide a high enough level of trans- culture whereas transduction with MLV did require selec- duction that selection of transduced cells might be tion. Therefore, the SV40 vector as gene delivery system unnecessary. In these studies, an SV40 expression vector represents a novel therapeutic strategy for gene therapy to was developed to deliver SFv-IN (SV(Aw)). Expression of target HIV-1 proteins and interfere with HIV-1 replication. Keywords: HIV-1; SV-40; gene therapy; intracellular immunization Introduction of cell types from humans and other mammals and expresses its genes in them. Recombinant, replication- Various techniques have been developed to express deficient SV40-derived vectors may express transgenes recombinant constructs within cells, in culture, in animal stably in cell lines, in primary cultures, and in vivo.4–6 The 1 models and in humans. The application of molecular virus is stable to manipulation and can be made to high genetics to human biology and disease has improved our titer (у1010 infectious units (IU)/ml), and further concen- understanding of and ability to treat a variety of disease trated if needed. states. Retroviral gene delivery vectors based on onco- HIV-1, as a member of the lentivirus family, has a com- retroviruses, such as Moloney murine leukemia virus plex viral life cycle and utilizes multiple cellular and (MLV), have been the most commonly used vectors for virally encoded regulatory proteins to tightly control its 2,3 gene transfer into the host cell genome. MLV has been replication.7 The essential retroviral enzymes, reverse used to deliver therapies for diverse diseases, including transcriptase (RT), ribonuclease H (RNaseH), protease cancer, inherited genetic disorders and infection with (PR) and integrase (IN), lack cellular counterparts and HIV-1. However, onco-retroviral vectors have limitations have been used as targets for developing agents that that often include a need to select cultured cells to enrich inhibit virus replication.8–10 Despite considerable for those cells expressing the transgene. Such selection advances in anti-RT and PR chemotherapy, genetic generally necessitates transduction ex vivo, followed by changes in the virus can confer drug resistance.11 This cumbersome selection in culture. problem has led to proposals for alternative therapeutic We have devised a gene transfer system based on sim- strategies in which host cells may be genetically altered 4 ian virus-40 (SV40) as a vector. SV40 infects a wide range or engineered to confer long-lasting protection against virus infection or replication.12,13 Several such strategies are currently being reported and applied to the inhibition Correspondence: DS Strayer, Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadel- of HIV-1 replication. They include exploitation of trans- phia, Pennsylvania 19107, USA dominant-negative mutant HIV-1 protein expression, Received 14 July 1998; accepted 28 October 1998 viral antisense oligonucleotide sequences, specific Inhibition of HIV-1 by SV40-delivered anti-integrase SFv M BouHamdan et al 661 ribozymes, HIV-1 transactivated suicide genes and intra- cellular antibodies against several different HIV-1- specific proteins.14–26 Recently, we reported that intracellular expression of SFv moieties targeted to RT, IN and Rev strongly inhibited HIV-1 replication in human cells.15–20,23,24,27 An early event in the life cycle of all retroviruses, including HIV-1, is integration of a double-stranded proviral DNA into the host cell genome. This step is necessary for pro- ductive viral replication.28 In natural infection, linear viral DNA contained within pre-integration complexes is the direct precursor of the integrated proviral genome.29 In previous studies, we demonstrated that anti-IN SFv #33, driven by the cytomegalovirus immediate–early pro- moter (CMV-IEP) and delivered by MLV to selected human T-lymphocytes, effectively inhibited HIV-1 repli- cation.20 To assess intracellular immunization as a tool for gene therapy of HIV-1 infection further, IN was tar- geted for specific blockade by the same anti-IN intracellu- lar SFv expression construct, but delivered using an SV40 vector. The effectiveness of this construct in inhibiting HIV-1 was tested in a CD4+ human T lymphoma cell line. Figure 1 Schematic map of the SV40-derivative construct used to express We show here that an SV40-derived vector can trans- SFvIN#33. The genome of SV(Aw), the SV40-derivative used to transduce duce human T-lymphocytic cells to express the protein the SFvIN#33, is illustrated here. This virus was constructed as described SFv-IN effectively without selection. Furthermore, HIV-1 in Materials and methods, and in general according to the approach out- replication, as measured by HIV-1 p24 antigen expression lined in previous publications.4 Briefly, in this virus, the SFvIN#33 cDNA and syncytia formation, was inhibited in SupT1 is driven by the CMV-IEP, which is in turn immediately downstream T-lymphocytic cells expressing SFv-IN. from the SV40-EP. The latter overlaps the SV40 origin of replication, and thus cannot be entirely deleted from the viral genome. The late virus genes (VP1, VP2, VP3) are intact in this construct, as are the SV40 late pro- moter, enhancer and early and late polyadenylation signals. The construc- Results tion of SV(HBS), the control SV40 virus used in these studies, was reported previously.30 Construction of viruses expressing SFvIN#33 and HBsAg proteins A map of SV(HBS) has been reported.30 This recombinant SV40 virus was used as a negative control. Construction of pSLXCMV-SFvIN#33, which expresses SFvIN#33, has been described previously.20 Briefly, sequences encoding the variable light (V1) and variable heavy (Vh) chains of the anti-IN monoclonal antibody (Mab) were cloned from a murine hybridoma cell-line’s RNA. After ligation of V1 and Vh chains into a single fragment, by utilizing the flexible linker (GGGGS)3, the SFv fragment was cloned into an SV40 expression vector downstream of cytomega- lovirus immediate–early promoter (CMV-IEP). The orien- tations and structures of open reading frames were veri- fied by DNA sequencing. A map of the SV40 vector containing the SFvIN#33 (SV(Aw)) driven by CMV-IEP, and used for all these studies, is shown in Figure 1. Detection of SFvIN#33 by Western blotting Lysates were prepared from 106 SupT1 cells transduced as described with SV(Aw) or with MLV-SFvIN#33, or mock-transduced. These proteins were electrophoresed using SDS-PAGE, blotted to PVDF membranes, probed with anti-mouse IgG antibody and visualized as described in Materials and methods. As a positive con- Figure 2 Expression of anti-IN SFv in SV(Aw)- and MLV-SFvIN#33- trol, recombinant SFvIN#33 produced in E. coli was used. transduced cells. SupT1 cells were treated with SV(Aw), once at MOI Results demonstrated that both selected MLV-SFvIN#33- of 10, without selection. Protein from 106 cells, harvested 4 days after transduced SupT1 cells and unselected SV(Aw)-trans- transduction, was electrophoresed on SDS-PAGE, blotted to PVDF mem- # branes and probed with anti-mouse IgG to visualize expression of the duced cells expressed the SFvIN 33 transgene (Figure 2). # Levels of transgene expression were comparable in SFvIN 33. In parallel, protein from an equal number of selected, MLV- SFvIN#33-transduced cells, was electrophoresed, blotted and visualized unselected SV(Aw)-transduced cells and in selected similarly. For both preparations, SFvIN#33 expressed in E. coli was the MLV-SFvIN#33-transduced cells (Figure 2). The larger positive control. Equal numbers of non-transduced SupT1 cells were the molecular size bands seen here in the extracts from negative control. Inhibition of HIV-1 by SV40-delivered anti-integrase SFv M BouHamdan et al 662 SV(Aw)-transduced cells may represent either splice vari- cells, compared to the same SFv delivered by MLV- ants of the original transcript or ribosomal reading SFVIN#33. HIV-1 challenge studies were performed through translational stop signals.