HUMAN GENE THERAPY 12:1417–1428 (July 20, 2001) Mary Ann Liebert, Inc. Stable Integration of Transgenes Delivered by a Retrotransposon–Adenovirus Hybrid Vector HARRIS SOIFER, 1,2 COLLIN HIGO, 1,2 HAIG H. KAZAZIAN, JR., 3 JOHN V. MORAN, 4 KOHNOSUKE MITANI, 5 and NORIYUKI KASAHARA 1,2,6 ABSTRACT Helper-dependent adenoviruses show great promise as gene delivery vectors. However, because they do not integrate into the host chromosome, transgene expression cannot be maintained indefinitely. To overcome these limitations, we have inserted an L1 retrotransposon/transgene element into a helper-dependent adeno- virus to create a novel chimeric gene delivery vector. Efficient adenovirus-mediated delivery of the L1 ele- ment into cultured human cells results in subsequent retrotransposition and stable integration of the trans- gene. L1 retrotransposition frequency was found to correlate with increasing multiplicity of infection by the chimeric vector, and further retrotransposition from newly integrated elements was not observed on pro- longed culture. Therefore, this vector, which utilizes components of low immunogenic potential, represents a novel two-stage gene delivery system capable of achieving high titers via the initial helper-dependent adeno- virus stage and permanent transgene integration via the retrotransposition stage. OVERVIEW SUMMARY ing to achieve efficient gene transfer, a variety of viruses have been adapted for use as gene delivery vectors, including retro- A retrotransposition-competent LINE-1 element (RC-L1) virus, adenovirus, and adeno-associated virus. Of these, ade- was inserted into a helper-dependent adenovirus (HDAd) to noviral vectors are capable of achieving high titers and have generate a hybrid adenovirus–retrotransposon vector. The been shown to efficiently infect many cell types in vivo by di- hybrid vector could be amplified to high titer and was ca- rect injection. However, because the adenovirus life cycle is in- pable of efficiently infecting a variety of human cells as an herently episomal in nature, and lacks the machinery for effi- adenovirus. The RC-L1 within the hybrid vector then un- cient integration into host chromosomes, adenoviral integration derwent retrotransposition in target cells, resulting in sta- events are rare (Harui et al., 1999), and transgene expression ble integration of the linked transgene cassette. A linear in- is transient in duration. Moreover, the utility of adenovirus vec- crease in retrotransposition frequency was observed when tors is further limited by cellular and humoral immune re- cells were infected at increasing multiplicities of infection. sponses against the adenoviral gene products, which can be ex- Thus, we have developed a novel hybrid vector that can pressed at low levels in the transduced cells despite deletion of achieve efficient transduction via the HDAd stage and sta- the E1 regulatory region (Yang et al., 1996a,b; Kaplan et al., ble integration of transgenes via the RC-L1 stage. 1997; Sparer et al., 1997). As one approach to overcome these limitations, helper-de- pendent adenoviral vector systems, which lack all the coding INTRODUCTION sequences that could be toxic or immunogenic to the host (Mi- tani et al., 1995; Kochanek et al., 1996; Scheidner et al., 1998), HE LACK OFAN OPTIMAL METHOD for direct gene transfer have been developed. These modifications not only allow an Tand permanent transduction of tissues in vivo is a signifi- expanded cloning capacity with the ability to insert up to 38 kb cant impediment to the success of human gene therapy. In seek- of foreign DNA into the vector, but also result in prolonged ex- 1Institute for Genetic Medicine, University of Southern California, Los Angeles, CA 90033. 2Department of Biochemistry, University of Southern California, Los Angeles, CA 90033. 3Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104. 4Department of Human Genetics and Internal Medicine, University of Michigan, Ann Arbor, MI 48109. 5Department of Microbiology and Immunology, University of California at Los Angeles, Los Angeles, CA 90095. 6Department of Pathology, University of Southern California, Los Angeles, CA 90033. 1417 1418 SOIFER ET AL. pression of the transgene compared with standard E1-deleted described previously (Sassaman et al., 1997). Plasmids were adenovirus vectors (Morral et al., 1998, 1999; Morsy et al., purified with Qiagen (Valencia, CA) Maxiprep columns ac- 1998; Scheidner et al., 1998), presumably because host immune cording the manufacturer instructions. responses against the vector are reduced. Nevertheless, it is un- likely that the transferred genes would persist permanently. Construction of helper-dependent Human retrotransposons (i.e., long interspersed nuclear ele- adenovirus–retrotransposon hybrid vector ments; LINE-1s or L1s) also have the potential to serve as gene A cytomegalovirus (CMV)-driven green fluorescent protein delivery vectors. Retrotransposition-competent L1s (RC-L1s) (GFP) marker gene cassette was inserted between BamHI sites are 6.0 kb and contain a 5 9 untranslated region (5 9 UTR) har- in plasmid pSTK120 to generate an intermediate helper-de- boring an internal promoter (Swergold, 1990; Minakami et al., pendent adenovirus construct, designated pSMK-GFP. A 1992), two nonoverlapping open reading frames (ORF 1 and ScaI–ApaI restriction fragment containing the CMV-driven ORF 2), and a 3 9 UTR that ends in a polyadenylic acid tail. L1.3mneoI sequence was then excised from pJM130 (Sassaman ORF 1 encodes a 40-kDa nucleic acid-binding protein that dis- et al., 1997), and ligated into the pSMK-GFP backbone after plays nucleic acid chaperone activity in vitro (Holmes et al., digestion with EcoRV and ApaI; this procedure also results in 1992; Hohjoh and Singer, 1996; Martin and Bushman, 2001). removal of the unstable Alu repeats in the HPRT intron se- ORF 2 encodes a protein with reverse transcriptase (RT) and quence (Sandig et al., 2000). The resultant plasmid, pSMK- endonuclease (EN) activities (Feng et al., 1996). Transgene cas- L1.3mneoI, thus contains a 14.7-kb helper-dependent adenovi- settes can be inserted into the 3 9 UTR of RC-L1s, and the re- rus (HDAd) vector construct that encodes two independent sultant constructs can retrotranspose from an extrachromoso- cassettes, CMV-L1.3mneoI and CMV-GFP. mal self-replicating plasmid episome into genomic DNA at a high frequency, resulting in stable integration of the transgene HDAd vector production. The helper virus AdLC8cluc, a re- (Moran et al., 1996). However, a rate-limiting step in further combinant Ad5 vector containing loxP sites flanking the pack- developing L1s as an integrating vector system has been their aging signal, was used to propagate the helper-dependent vec- inefficient delivery to target cells. tor in 293Cre4 cells (Chen et al., 1996). After transfection of Here we describe a novel hybrid vector, consisting of an RC- PmeI-linearized pSMK-L1.3mneoI, the resultant helper-depen- L1 encoded by a helper-dependent adenovirus vector. After ini- dent virus HDAd-L1.3mneoI was propagated by serial passage tial transduction of cultured human cells by the adenovirus car- on 293Cre4 cells with addition of the AdLC8cluc helper virus rier, expression of the L1 and its subsequent retrotransposition at each stage, and purified by double cesium chloride gradient result in stable integration of the transgene cassette. Thus, this ultracentrifugation (Parks et al., 1996). Helper virus contami- L1–adenovirus hybrid vector simultaneously overcomes the nation levels were determined by median tissue culture infec- problems of transient adenovirus-mediated transgene expres- tive dose (TCID ) and plaque assays on 293A cells. The helper- sion and low efficiency of retrotransposon vector delivery. It 50 dependent adenovirus was titered (transducing units, TU/ml) by represents a novel vector system that is capable of achieving flow cytometric analysis of GFP expression from cells infected high-titer gene transfer as well as stable integration and per- with limiting dilutions of purified vector. manent transduction. Southern blot analysis of adenovirus DNA. Approximately 10 7 TU of HDAd-L1.3mneoI virion DNA was incubated with 200 MATERIALS AND METHODS ml of capsid digestion buffer (50 m M Tris-HCl [pH 8.0], 1.0 m M EDTA [pH 8.0], 0.5% sodium dodecyl sulfate [SDS], and pro- Cells and plasmids teinase K [1.0 mg/ml]) at 50°C for 1 hr (Fisher et al., 1996a). Reactions were cooled to room temperature, extracted once with Human embryonic kidney (HEK) 293A cells (Quantum an equal volume of phenol–chloroform– isoamyl alcohol Biotechnologies, Laval, Quebec, Canada); 293Cre4 cells, which (25:24:1), and precipitated with ethanol, and the virion DNA was stably express the Cre recombinase (Chen et al., 1996) (Merck, resuspended in 10 m M Tris-HCl, 0.1 m M EDTA. Virion DNA Rahway, NJ); HeLa human cervical carcinoma cells (American was digested with restriction enzymes BstZ1107I (New England Type Culture Collection [ATCC], Rockville, MD); and BioLabs, Beverly, MA), EcoRI (GIBCO, Rockville, MD), and CFBE41o2 immortalized human bronchial epithelial cells (gen- NotI (New England BioLabs), fractionated on a 0.7% agarose erously provided by D. Gruenert, University of California at gel, blotted, and probed with a 32P-labeled PmeI restriction frag- San Francisco, San Francisco, CA) were cultured in Dulbecco’s ment from pSMK-L1.3mneoI (Decaprime II; Ambion, Austin, modified Eagle’s medium (DMEM) supplemented with 10% TX). Southern blots were analyzed by