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Retrovirus Molecular Conjugates

2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês

10.1074/jbc.m010318200

1083-351X

Qiu Zhong, Jay K. Kolls, Paul Schwarzenberger,

Viral Infectious Diseases and Gene Expression in Insects

Two significant barriers limit the use of amphotropic retrovirus for human gene transfer protocols: 1) low transduction efficiency in cells with low receptor expression and 2) safety concerns originating from the risk of formation and propagation of replication competent virus in vivo. In principle, if ecotropic retrovirus, which is incapable of infecting human cells, could be transiently modified to effectively transduce human cells, this safety risk could be alleviated. Here we demonstrate that formation of amphotropic retrovirus polylysine molecular conjugates (aMMLV-PL) enhanced gene transfer up to 10-fold in a variety of human cell lines over the equivalent of unconjugated vector (aMMLV). The polylysine modification and formation of ecotropic retrovirus molecular conjugates (eMMLV-PL) permitted effective and stable transduction of different human cell lines as well as primary human bone marrow stroma cells at frequencies of greater than 80%. It is conceivable that this novel ecotropic-based conjugate retrovirus vector could also potentially provide enhanced safety characteristics not only over amphotropic retrovirus vectors but also over genetically tropism-modified recombinant ecotropic vectors. In contrast to genetic modifications, physical or chemical modifications are not propagated. Thus, formation of replication competent eMMLV from conjugates would be self-limited and would not result in virus propagation in humans. Two significant barriers limit the use of amphotropic retrovirus for human gene transfer protocols: 1) low transduction efficiency in cells with low receptor expression and 2) safety concerns originating from the risk of formation and propagation of replication competent virus in vivo. In principle, if ecotropic retrovirus, which is incapable of infecting human cells, could be transiently modified to effectively transduce human cells, this safety risk could be alleviated. Here we demonstrate that formation of amphotropic retrovirus polylysine molecular conjugates (aMMLV-PL) enhanced gene transfer up to 10-fold in a variety of human cell lines over the equivalent of unconjugated vector (aMMLV). The polylysine modification and formation of ecotropic retrovirus molecular conjugates (eMMLV-PL) permitted effective and stable transduction of different human cell lines as well as primary human bone marrow stroma cells at frequencies of greater than 80%. It is conceivable that this novel ecotropic-based conjugate retrovirus vector could also potentially provide enhanced safety characteristics not only over amphotropic retrovirus vectors but also over genetically tropism-modified recombinant ecotropic vectors. In contrast to genetic modifications, physical or chemical modifications are not propagated. Thus, formation of replication competent eMMLV from conjugates would be self-limited and would not result in virus propagation in humans. murine moloney leukemia retrovirus amphotropic MMLV ecotropic MMLV phosphate-buffered saline polylysine 2-{[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid sulfo-N-hydroxysuccinimide biotin 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydro-chloride Hepes-buffered saline NeutrAvidin polymerase chain reaction kilobase(s) multiplicity of infection Gene therapy is emerging as a promising novel therapeutic approach to treat human disease (1Avalosse B. Dupont F. Burny A. Curr. Opin. Oncol. 1995; 7: 94Crossref PubMed Google Scholar, 2Culver K.W. Blaese M.R. Trends Genet. 1994; 10: 174Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 3Haddada H. Cordier L. Perricaudet M. Curr. Top. Microbiol. Immunol. 1995; 199: 297-306PubMed Google Scholar, 4Hanania E.G. Kavanagh J. Hortobagyi G. Giles R.E. Champlin R. Deisseroth A.B. Am. J. Med. 1995; 99: 537-552Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 5Miller A.D. Blood. 1990; 76: 271Crossref PubMed Google Scholar). However, technical barriers related to the biology of vector cell interaction and patient safety remain the most critical end points in clinical trials. Therefore, the success of human gene therapy depends on effective transfer and expression of the desired genes into the target cell at the lowest possible risk for the individual. Gene therapy approaches most commonly utilize recombinant viral vectors, in which both cellular uptake and transgene expression depend on interactions between the viral vector and the target cell (3Haddada H. Cordier L. Perricaudet M. Curr. Top. Microbiol. Immunol. 1995; 199: 297-306PubMed Google Scholar,4Hanania E.G. Kavanagh J. Hortobagyi G. Giles R.E. Champlin R. Deisseroth A.B. Am. J. Med. 1995; 99: 537-552Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 6Jenks S. J. Natl. Cancer Inst. 1997; 89: 1182-1184PubMed Google Scholar). The best established vector system for achieving stable transduction in human cells is the amphotropic retrovirus vector, with its infectivity or cellular uptake at least in part determined by the presence of natural receptors on the target cells. Since the use of retrovirus vectors is restricted to specific receptor-expressing cells, transduction efficiency is largely determined by cell surface receptor density (7Chen L. Pulsipher M. Chen D. Sieff C. Elias A. Fine H.A. Kufe D.W. J. Clin. Invest. 1996; 98: 2539-2548Crossref PubMed Scopus (59) Google Scholar, 8Thoma S.J. Lamping C.P. Ziegler B.L. Blood. 1994; 83: 2103-2114Crossref PubMed Google Scholar, 9Crooks G.M. Kohn D.B. Blood. 1993; 82: 3290-3297Crossref PubMed Google Scholar). Due to the lack of ecotropic receptors on human cells, unmodified ecotropic retrovirus cannot be used for transduction of human cells. Currently, all clinical approaches for stable transduction of human cells rely on amphotropic retrovirus. The rare event of formation of replication-competent amphotropic retrovirus could result in systemic viremia of a treated individual, as amphotropic retrovirus can infect and replicate in most human cells. If technology would permit the effective use of ecotropic retrovirus for human gene therapy protocols, this crucial safety concern could be alleviated. Uncontrolled virus replication in humans with ecotropic virus would be unlikely, since the lack of natural receptors prevents infection of human cells. Here we have developed and evaluated a strategy to accomplish this goal by synthesizing ecotropic retrovirus molecular conjugates (eMMLV-PL).1 To prove feasibility for this concept, the first step would require physical or chemical modifications of ecotropic retrovirus, not only permitting infection of human cells but also the efficient cellular transduction with the transferred gene. The development of strategies to overcome ecotropic/amphotropic receptor-dependent vector uptake and enhancement of cellular transduction efficiency through circumventing the natural pathway of virus to cell entry has become a major goal of gene therapy research. We previously determined that recombinant adenovirus molecular conglomerate vectors substantially enhanced vector uptake and transgene expression after formation of polylysine-based conglomerates over recombinant adenovirus particles alone (10Schwarzenberger P. Hunt J.D. Robert E. Theodossiou C. Kolls J.K. J. Virol. 1997; 71: 8563-8571Crossref PubMed Google Scholar). Here, we investigate the hypothesis that polylysine-conjugated retrovirus results in increased uptake and gene expression over the equivalent of unconjugated virus (Fig. 1 A). Furthermore, we hypothesized that through this procedure, the tropism restriction of ecotropic virus could be overcome, and human cells would become amenable to transduction with ecotropic retrovirus vectors (Fig.1 B). If successful, this conjugate ecotropic vector would have an additional significant safety advantage over currently employed amphotropic systems. The human embryonic kidney cell 293 line was provided by Dr. Robert Gerard, University of Texas Southwestern, Dallas, TX (also available from ATCC ((Manassas, Virginia))) (11Graham F.L. Smiley J. Russell W.C. Nairn R. J. Gen. Virol. 1997; 36: 59-74Crossref Scopus (3512) Google Scholar). The human retinoblastoma cell line 911 was provided by Dr. Bout (12Fallaux F.J. Kranenburg O. Cramer S.J. Houweling A. Van Ormondt H. Hoeben R.C. Van Der Eb A.J. Hum. Gene Ther. 1996; 7: 215-222Crossref PubMed Scopus (443) Google Scholar). The human pulmonary epithelial carcinoma-derived cell line A549 was purchased from ATCC. All cell lines were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and penicillin/streptomycin (Cellgro Mediatech, Herndon, VA) and maintained at 37 °C under a 5% CO2atmosphere in a humidified incubator (standard cell culture conditions). Primary human bone marrow cells were obtained by standard aspiration from the iliac crest of healthy voluntary donors and collected in MyeloCult H5100 medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 250 units/ml heparin. The institutional review board at Louisiana State University Health Sciences Center of New Orleans, LA approved this study involving human subjects. For erythrocyte lysis, specimens were incubated for 1 min in 0.747% NH4Cl solution. After two washing steps with phosphate-buffered saline (PBS), 1×107 cells were seeded in 10-cm dishes (Nunc, Naperville, IL) and cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum and penicillin/streptomycin and maintained under standard conditions. Non-adherent cells were decanted with media exchange 2–3 times/week. A near confluent bone marrow stroma layer of fibroblast morphology was established after 2–3 weeks. Gene transfer experiments in these cells were performed at 70–80% confluence. The amphotropic retroviral helper cell line PA317 or the ecotropic retroviral packaging cell line GP+E86 were transfected with the plasmid pLZ12 using calcium phosphate (13Graham F.L. Abrahams P.J. Mulder C. Heijneker H.L. Warnaar S.O. De V.F. Fiers W., Van Der E.A. Cold Spring Harbor Symp. Quant. Biol. 1975; 39: 637-650Crossref PubMed Google Scholar, 14Markowitz D. Goff S. Bank A. J. Virol. 1988; 62: 1120-1124Crossref PubMed Google Scholar). Plasmid pLZ12 was described by Galileo et al. (15Galileo D.S. Gray G.E. Owens G.C. Majors J. Sanes J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 458-462Crossref PubMed Scopus (210) Google Scholar) and is a modification from plasmids originally made by Casadaban et al. (16Casadaban M.J. Chou J. Cohen S.N. J. Bacteriol. 1980; 143: 971-980Crossref PubMed Google Scholar). Plasmid pLZ121 encodes the lacZreporter gene under control of the Rous sarcoma virus promoter and has a 5′ nuclear localization signal (Fig. 1). The retroviral helper cell line was cultured under standard conditions (14Markowitz D. Goff S. Bank A. J. Virol. 1988; 62: 1120-1124Crossref PubMed Google Scholar). Supernatants were collected from stable vector-producing cells, and retrovirus purification was performed after a procedure previously described by Akatsuka et al. (17Akatsuka Y. Emi N. Kato H. Abe A. Tanimoto M. Lupton S.D. Saito H. Int. J. Hematol. 1994; 60: 251-261PubMed Google Scholar). Briefly, a 30% (w/w) stock solution of polyethylene glycol 8000 (Sigma) was prepared in double-distilled water and stored in aliquots at 4 °C. Viral supernatants were gently mixed with polyethylene glycol in 250-ml polystyrene tubes to achieve a final 8% polyethylene glycol solution. The mixture was maintained overnight at 4 °C. After centrifugation at 1500 × g for 45 min, the precipitate was dissolved in 3 ml of TES buffer (10 mm Tris-HCl, pH 7.2, 2 mm EDTA, 150 mm NaCl). The titer of virus preparations was determined as previously described on NIH3T3 monolayers (18;19). Retrovirus was covalently linked to poly-l-lysine (PL) (Sigma), molecular mass 30–70 kDa, using the heterobifunctional chemical linker 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Pierce). Briefly, 3.6 ml of retrovirus (7 × 107colony-forming units/ml) was resuspended in Hepes-buffered saline (HBS; 5 mm HEPES pH 7.8, 150 mm NaCl) and 0.4 ml of PL (6 mg/ml in HBS). 0.04 ml of freshly made EDC solution (250 mg/ml in dH2O) was added, and the reaction mixture was incubated on ice for 4 h. This ratio of reagents was found to be optimal, as determined in several pilot experiments in different human and murine cell lines (data not shown). The product was then dialyzed overnight at 4 °C against a large volume of TES using 12–14-kDa cutoff membrane tubing (Spectra/Por, Laguna Hills, CA). The final product, retrovirus-polylysine (eMMLV-PL) was separated into aliquots in TES and stored at −80 °C. NeutrAvidin (NA) (Pierce) was covalently linked to PL, molecular mass 30–70 kDa, using the heterobifunctional chemical linker EDC. Briefly, 10 mg/ml NA was resuspended in HBS. 1.5 ml of NA solution was gently mixed with 0.5 ml of PL (6 mg/ml in HBS) before 0.04 ml of freshly made EDC solution (250 mg/ml in dH2O) was added. The reaction mixture was incubated on ice for 4 h. The product was then dialyzed overnight at 4 °C against a large volume of PBS using 12–14-kDa cutoff membrane tubing (Spectra/Por, Laguna Hills, CA). The final product, avidin-polylysine was separated into aliquots in PBS and stored at −20 °C. Retrovirus was covalently linked to sulfo-N-hydroxysuccinimide-biotin (biotin) following the instructions of the manufacturer (Pierce). Briefly, 0.2 ml of biotin (10 mg/ml in Me2SO) was mixed with 2.8 ml of retrovirus in PBS (7×107 colony-forming units/ml). The reaction mixture was incubated on ice for 2 h. The product (eMMLV-B) was then exchanged in TES buffer (0.01 mTris-HCl, pH 7.2, 0.002 m EDTA, 0.15 m NaCl) over EP 10 DG columns (Bio-Rad). EMMLV-B was separated into aliquots and stored in TES at −80 °C. The titer of eMMLV-B was determined as previously described on NIH3T3 monolayers (20Mann R. Mulligan R.C. Baltimore D. Cell. 1983; 33: 153-159Abstract Full Text PDF PubMed Scopus (1120) Google Scholar). LacZ staining was performed as previously described (21Kolls J.K. Peppel K. Silvia M. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 215-219Crossref PubMed Scopus (212) Google Scholar). Briefly, cells were fixed in 0.05% glutaraldehyde phosphate buffer (150 mm sodium chloride, 15 mm sodium phosphate, pH 7.3) (Sigma) for 10 min at room temperature and rinsed with PBS. Cells were stained for lacZexpression overnight at 37 °C using freshly made X-gal solution buffer (1 mg/ml X-gal, 20 mm potassium ferrocyanide, 20 mm potassium ferricyanide, and 2 mmMgCl2 in PBS). After rinsing with 70% ethanol, cells were analyzed by microscopy for blue cells. Genomic DNA was extracted from cell pellets using a DNA extraction kit (Qiagen, Valencia, CA). A unique and specific 1,036-base pair sequence of the Escherichia coli lacZ gene was amplified using the primer pair 5′-GCC-GAC-CGC-ACG-CCG-CAT-CCA-GC-3′ (sense) and 5′-GCG-GCG-CGG-TGA-CCA-CAC-CC-GG-3′ (antisense) using Taqpolymerase (Promega, Madison, WI). Amplification conditions consisted of a 1-min denaturing cycle at 94 °C, a primer annealing step at 65 °C for 1 min, and a primer extension step at 72 °C for 2 min for a total of 35 cycles. A final elongation cycle was performed for 10 min at 72 °C (MJ Research, Peltier Thermal Cycler, Watertown, MA). The product was analyzed after electrophoresis on a 1% agarose gel after ethidium bromide staining under UV fluorescence. For quantitative REAL-time PCR, a unique and specific 84-base pair sequence of the E. coli lacZ gene was amplified using the primer pair E. coli B-gal-f 5′-ATG-CGC-CCA-TCT-ACA-CCA-A-3′ (sense) and E. coliB-gal-rev 5′-AGA-AAC-AAC-CCG-TCG-GAT-TCT-C-3′ (antisense). Product labeling was performed using the fluorochrome SYBR® Green following the instructions recommended by the manufacturer (PE Biosystems, Foster City, CA). Amplification conditions consisted of a 10-min AmpliTaq Gold polymerase activation step at 95 °C followed by a denaturing step at 95 °C for 15 s and a combined annealing and extension step at 60 °C for 1 min in a thermocycler (GeneAmp®5700 sequence detection system, PE Applied Biosystems). A total of 40 cycles of sequential denaturing/annealing and extension was performed. A standard curve was established by concomitant amplification of DNA extracted from a retrovirally stably transduced NIH3T3 clone. The samples were analyzed with the 5700 software detection system and corrected for total DNA content using a spectrophotometer (DU640B, Beckman Instruments). AnR of >0.99 was required for standard curves. The sensitivity of the assay was determined to be 10 copies/sample. Copy number per cell was determined by determining sample LacZ copy from a standard curve. Genomic DNA was extracted form different lacZ-expressing 911 single cell clones previously transduced with eMMLV-PL using a commercial DNA extraction kit (Qiagen, Chatsworth, CA). 40 μg of restriction-digested genomic DNA and standards established with the lacZ gene derived from theNotI-digested control plasmid pACCMVLacZ (21Kolls J.K. Peppel K. Silvia M. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 215-219Crossref PubMed Scopus (212) Google Scholar) were loaded on a 0.8% agarose gel and separated by electrophoresis following previously described procedures (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The DNA was transferred to positively charged nylon membranes (MSI, Westborough, MA), which was subsequently hybridized with a 32P-radiolabeled probe specific for the lacZ sequence (5′-GCC-GAC-CGC-ACG-CCG-CAT-CCA-GC-3′). After removal of nonhybridized probe by washing, the membrane was exposed to radiographic film and developed. Restriction digestion of pACCMVLacZ with EcoRI linearized the plasmid, yielding a 12-kb fragment; digestion withNotI released a 4.3-kb cassette encoding the lacZgene (21Kolls J.K. Peppel K. Silvia M. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 215-219Crossref PubMed Scopus (212) Google Scholar). For demonstration of integration by junction fragments, genomic DNA was digested with XbaI. There is a uniqueXbaI site in the retrovirus construct just 3′ of thelacZ reporter gene (15Galileo D.S. Gray G.E. Owens G.C. Majors J. Sanes J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 458-462Crossref PubMed Scopus (210) Google Scholar, 16Casadaban M.J. Chou J. Cohen S.N. J. Bacteriol. 1980; 143: 971-980Crossref PubMed Google Scholar). The frequency ofXbaI restriction sites in human genomic is approximately 1/4 kb. For evaluation of lacZ copy number integrated into host cells, genomic DNA was digested with XhoI andXbaI to release a 4.4-kb fragment encoding thelacZ gene within the retrovirus construct (15Galileo D.S. Gray G.E. Owens G.C. Majors J. Sanes J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 458-462Crossref PubMed Scopus (210) Google Scholar, 16Casadaban M.J. Chou J. Cohen S.N. J. Bacteriol. 1980; 143: 971-980Crossref PubMed Google Scholar). GenomicXhoI- and XbaI-digested DNA was analyzed by Southern blot together with different dilutions of purifiedNotI-digested pACCMVLacZ plasmid DNA fragments to establish a standard. The membranes were probed with the above-describedlacZ-specific probe, and the radiographic intensities were determined using a chemiluminescent 4000 low light imaging system (Alpha Innotech Corp., San Leandro, CA). Copy number per cell was calculated using previously published formulas where the band intensity of the transduced clones were compared with intensities from the standard curve (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 23Southern E.M. Anal. Biochem. 1979; 100: 319-323Crossref PubMed Scopus (288) Google Scholar, 24Elder J.K. Southern E.M. Anal. Biochem. 1983; 128: 227-231Crossref PubMed Scopus (68) Google Scholar). To test the hypothesis that formation of polylysine-based retrovirus molecular conglomerates would improve transduction efficiency over retrovirus particles, biotinylated aMMLV encoding the lacZ reporter gene (aMMLV-B) and avidinylated polylysine (PL-NA) were synthesized. The biotinylation procedure did not significantly reduce the infectivity and transgene expression of aMMLV-B over unmodified virus (aMMLV) in murine NIH3T3 cells (data not shown). Human 911 cells were infected with aMMLV-B with or without Polybrene at increasing m.o.i. (0.05–1.5) or the equivalent amount of aMMLV-B as molecular conglomerate by adding PL-NA (aMMLV-PL). Formation of retrovirus conglomerates significantly increased transgene expression at low m.o.i. by up to 5-fold. At higher m.o.i., transduction efficiency reached 100% for all vectors as expected (Fig.2 A). To investigate the ubiquity of this transduction enhancement, other cell lines, human 293, A549, and murine NIH3T3 cells, were infected with aMMLV-B (with or without Polybrene) or the equivalent amount of retrovirus as aMMLV-PL. At low m.o.i. (0.2), formation of aMMLV-PL resulted in significant, 5-fold-increased transgene expression in the examined cell lines (Fig.2 B). To study the hypothesis that formation of polylysine-based conglomerates overcome the tropism restriction of ecotropic retroviruses, biotinylated eMMLV encoding the lacZreporter gene (eMMLV-B) was joined to PL-NA to form eMMLV-PL. Similar to aMMLV-B, the biotin modification did not significantly decrease infectivity of eMMLV (data not shown). Human embryonal kidney 293 cells were infected with different vector and control constructs at an m.o.i. of 0.4 or 1.5 and analyzed for reporter gene expression. As expected, no transgene expression was observed with unmodified ecotropic virus (eMMLV) or eMMLV-B. However, physical linkage of eMMLV-B to PL-NA with conglomerate formation of eMMLV-PL resulted in significant transgene expression of 25 and 84%, respectively (Fig.3). The human retinoblastoma cell 911, the human lung cancer cell line A549, and freshly isolated primary human bone marrow-derived adherent stroma cells were transfected with eMMLV or eMMLV-PL at the m.o.i. of 0.5 and 1.5. Cells were not cultured under selective conditions favoring proliferation of transduced cells. Transgene expression was only seen in cells transfected with eMMLV-PL with 32 and 87% for 911 cells, 25 and 82% for A549 cells, and 30 and 92% for primary human stroma cells (Figs.4 and5).Figure 5Transgene expression in human 293 and primary bone marrow stroma cells after eMMLV-PL lacZ gene transfer. Human 293 cells (A and B) and primary human bone marrow stroma cells (C and D) were transfected with eMMLV or eMMLV-PL, respectively. Cells were analyzed for lacZ transgene expression as outlined under “Materials and Methods” and visualized by transmission light microscopy.View Large Image Figure ViewerDownload (PPT) Integration oflacZ gene into the host genome was demonstrated by genomic DNA restriction digestion of five 911 single cell clones using the enzyme XbaI. Different sized junction fragments were identified in all clones examined for the presence of lacZ (Fig.6). The copy number of the transferred reporter gene in transduced 911 cells was measured by REAL-time PCR and by Southern blot. A copy number of 0.91/cell (0.87–0.95) was quantified by PCR, and a copy number of 0.83/cell was quantified by Southern blot (Fig. 7, A andB). The combined evidence for these experiments indicates that eMMLV-PL results in integration of the transgene at 1 copy/cell.Figure 7Determination of transgene copy number after retrovirus molecular conjugates (eMMLV-PL) gene transfer.After transduction of 911 cells with eMMLV-PL, lacZ-positive single cell clone cells were established. REAL-time PCR was performed using a specific probe for lacZ on two clones (denoted by triangles and squares) and compared to a standard curve established with the lacZ-encoding plasmid pACCMVLacZ (dots). Cycle threshold (CT) values are plotted against lacZ copy number (A). For quantitative Southern blot analysis, genomic DNA was restriction-digested with XhoI and XbaIto release the 4.4-kb lacZ cassette from the integrated retrovirus construct. Plasmid pACCMVLacZ was restriction-digested with the enzyme NotI, releasing the 4.3-kb lacZ gene to establish a standard. The membrane was hybridized using a probe specific for the lacZ gene as outlined under Materials and Methods.“ Signal intensity was used to calculate copy number per cell (B).View Large Image Figure ViewerDownload (PPT) Our findings demonstrate that ecotropic retrovirus could be modified with a conjugate technique to accomplish effective transduction of human cells. If successful, this procedure could potentially provide additional safety features for use in humans. Ecotropic virus is chemically linked within the conjugate, so it remains genetically unaltered. The theoretical risk of in vivo propagation is substantially less for a transient tropism modification compared with a genetically tropism-modified ecotropic retrovirus vector such as vesicular stomatitis virus-pseudotyped envelopes. In the event that transiently modified ecotropic virus becomes replication competent, its progeny would be ecotropic retrovirus, which is incapable of propagating in humans. On the other hand, progeny arising from a genetically engineered, tropism-modified virus could continue to maintain its tropism-modified form and, thus, would remain infectious to human cells, posing a significant safety risk. First, we had to proof that the polylysine conjugation does indeed enhance the transduction efficiency of retrovirus. Although previous studies showed in principle feasibility of retrovirus retargeting via alternate cell surface receptors, several retargeting studies were associated with significant reduction or even disappearance of cellular transduction (25Roux P. Jeanteur P. Piechaczyk M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9079-9083Crossref PubMed Scopus (109) Google Scholar) (26Neda H. Wu C.H. Wu G.Y. J. Biol. Chem. 1991; 266: 14143-14146Abstract Full Text PDF PubMed Google Scholar, 27Kasahara N. Dozy A.M. Kan Y.W. Science. 1994; 266: 1373Crossref PubMed Scopus (389) Google Scholar, 28Fielding A.K. Maurice M. Morling F.J. Cosset F.L. Russell S.J. Blood. 1998; 91: 1802-1809Crossref PubMed Google Scholar). In contrast, greatly enhanced transduction efficiency was reported for retrovirus vectors redirected by pseudotyping with the vesicular stomatitis virus envelope (29Emi N. Friedmann T. Yee J.K. J. Virol. 1991; 65: 1201-1202Crossref Google Scholar,30Yee J.K. Friedmann T. Burns J.C. Methods Cell Biol. 1994; 43: 99-112Crossref PubMed Scopus (397) Google Scholar). To investigate the hypothesis that polylysine conjugation would enhance retrovirus transduction, retrovirus molecular conglomerates were synthesized by biotinylation and linking to avidinylated polylysine. Our data demonstrate that at low m.o.i., formation of molecular conjugates synthesized with amphotropic virus (aMMLV-PL) substantially enhances transgene expression over the equivalent amount of free amphotropic retrovirus (aMMLV-B). The human cell lines tested were permissive to infection with amphotropic retrovirus; thus, increasing m.o.i. eventually resulted in nearly 100% transduction efficiency with both virus vectors. Therefore, the advantage in this particular experimental setting was limited to a transduction enhancement observed at low m.o.i. The mechanism of the increased transduction efficiency of amphotropic conjugates could be explained through enhancement of virus uptake via their natural receptors or the utilization of an alternate pathway for virus uptake. The former concept has been proposed for fibronectin-meditated retrovirus transduction augmentation (31Moritz T. Dutt P. Xiao X. Hanenberg H. Carstanjen D. Vik T. Williams D.A. Blood. 1996; 88: 855-862Crossref PubMed Google Scholar, 32MacNeill E.C. Hanenberg H. Pollok K.E. van der Loo J.C. Bierhuizen M.F. Wagemaker G. Williams D.A. J. Virol. 1999; 73: 3960-3967Crossref PubMed Google Scholar). This model proposes that fibronectin facilitates colocalization of retrovirus particles and target cells with a large increase in local virus titer presented to the cell (31Moritz T. Dutt P. Xiao X. Hanenberg H. Carstanjen D. Vik T. Williams D.A. Blood. 1996; 88: 855-862Crossref PubMed Google Scholar, 33Asada K. Uemori T. Ueno T. Hashino K. Koyama N. Kawamura A. Kato I. J. Biochem. ( Tokyo ). 1998; 123: 1041-1047Crossref PubMed Scopus (12) Google Scholar, 34Saunders S. Bernfield M. J. Cell. Biochem. 1988; 106: 423-430Google Scholar). Similarly, the adjunct use of the positively charged polycations such as Polybrene in retroviral gene transfer procedures has long been known to nonspecifically promote retrovirus transduction efficiency and is now used for many protocols (35Cornetta K. Anderson W.F. J. Virol. Methods. 1989; 23: 187-194Crossref PubMed Scopus (139) Google Scholar). To proof that the entry mechanisms of retrovirus-polylysine conjugates are independent of natural receptors and are entirely polylysine-mediated, experiments were designed using ecotropic retrovirus constructs in human cells. We found that only the physical binding of eMMLV to polylysine accomplished cellular transduction with efficiencies of 80–90% in different human cell lines and primary cells. Although polycations have previously been reported to facilitate infectivity and to enhance transduction efficiency (36Kierney P.C. Dorshkind K. Blood. 1998; 70: 1418-1424Crossref Google Scholar), our data indicate that this effect can not only be greatly enhanced by chemical or physical linkage of virus and polycation, but this linkage allows an ability to completely overcome receptor-dependent virus uptake. This phenomenon could be explained by the ability of positively charged polylysine to nonspecifically penetrate cell surface membranes and enter the cytosol (37Skutelsky E. Danon D. J. Cell Biol. 1976; 71: 232-241Crossref PubMed Scopus (105) Google Scholar, 38Wickham T.J. Roelvink P.W. Brough D.E. Kovesdi I. Nat. Biotechnol. 1996; 14: 1570-1573Crossref PubMed Scopus (290) Google Scholar). Compared with individually retargeted retrovirus particles, conglomerates consist of multiple virus particles bound to polylysine, thus providing a higher likelihood of delivering one intact virus to the nucleus. This explanation of an added survival benefit is supported by studies where DNA bound to polylysine was found to have significantly enhanced resistance to intracellular degradation over free DNA (39Chiou H.C. Tangco M.V. Levine S.M. Robertson D. Kormis K. Wu C.H. Wu G.Y. Nucleic Acids Res. 1994; 22: 5439-5446Crossref PubMed Scopus (130) Google Scholar). Similar to DNA, the retrovirus particles may be relatively protected from nuclease digestion within the polylysine conglomerate formation. Therefore, the improved transduction efficiency could result as the combined effect of both polylysine-mediated enhanced uptake and also improved retrovirus survival from cytosolic degradation. Transduction with eMMLV-PL in human cells resulted in a similar genomic copy number as eMMLV in the murine cell line NIH3T3. This implies that the formation of retrovirus conglomerates does not change the fundamental principal established for retrovirus/cell interactions of only a single integration event with one retroviral copy per cell (40Varmus H. Science. 1988; 240: 1427-1435Crossref PubMed Scopus (443) Google Scholar). In summary, this novel retrovirus-based gene transfer system has the potential to improve transduction efficiency that comes from low receptor expression, such as for instance in hematopoietic cells (8Thoma S.J. Lamping C.P. Ziegler B.L. Blood. 1994; 83: 2103-2114Crossref PubMed Google Scholar,9Crooks G.M. Kohn D.B. Blood. 1993; 82: 3290-3297Crossref PubMed Google Scholar). Moreover, this system would provide additional safety features over amphotropic retrovirus vectors currently used in human trials. Even the case of formation of replication-competent ecotropic virus would be theoretically a self-limited problem, as progeny of transiently modified ecotropic virus could not further propagate in humans due to its tropism restriction. Last, the polylysine-based retrovirus system may enable specific receptor retargeting by formation of receptor-targeted retrovirus molecular conglomerate vectors (10Schwarzenberger P. Hunt J.D. Robert E. Theodossiou C. Kolls J.K. J. Virol. 1997; 71: 8563-8571Crossref PubMed Google Scholar,41Schwarzenberger P. Spence S.E. Gooya J.M. Michiel D.M. Ruscetti F.W. Keller J.R. Blood. 1996; 87: 472-478Crossref PubMed Google Scholar). We thank Dr. Jakob Reiser for the critical review of our studies.