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Envelope-Targeted Retrovirus Vectors Transduce Melanoma Xenografts but Not Spleen or Liver
doi:10.1006/mthe.2002.0550, available online at http://www.idealibrary.com on IDEAL
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Envelope-Targeted Retrovirus Vectors Transduce Melanoma Xenografts but Not Spleen or Liver Francisco Martín,* Simon Chowdhury, Stuart Neil, Neil Phillipps, and Mary K. Collins Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Science, University College London, 46 Cleveland Street, London W1P 6DB, UK *To whom correspondence and reprint requests should be addressed. Fax: 44-207-679-9301. E-mail: [email protected].
Many cancer gene therapy applications would benefit from the development of targeted vectors that could deliver genes in vivo. We have previously achieved efficient in vitro targeting of retrovirus vectors to melanoma cells by fusion of a single chain antibody recognizing the highmolecular-weight melanoma-associated antigen (HMWMAA), followed by a blocking peptide and a matrix metalloprotease cleavage site, to the amino terminus of the murine leukemia virus amphotropic strain envelope. Here we report that up to 3% of cells within an HMWMAA-positive tumor xenograft were infected following a single injection of targeted vector into the tumor and up to 10% of tumor cells became infected when they were co-injected with viral producer cells. No infected cells were detected after delivery of targeted vectors to HMWMAA-negative tumor xenografts. Intraperitoneal injection of amphotropic vectors or producer cells resulted in transduction in spleen and liver, which was not detected when targeted vectors or producer cells were used. Our results demonstrate the feasibility of using targeted retroviral vectors for in vivo gene delivery to tumors and highlight the safety benefits of targeted vectors that do not infect other host tissues. Key Words: gene therapy, retrovirus vectors, in vivo targeting, melanoma, xenografts, vector spreading
INTRODUCTION The development of efficient, targeted vectors will be necessary for gene therapy applications that require in vivo gene delivery. Retrovirus vectors are attractive for clinical gene delivery because integration of the vector genome allows stable gene expression in the infected cell and its progeny. Also, because viral coding regions are deleted from the vector, viral proteins are not expressed in infected cells, avoiding stimulation of an inappropriate antiviral immune response. Thus far, in gene therapy clinical trials retrovirus vectors have been used for in vitro transduction followed by transfer of modified cells to the patient [1]. Such modification of cells is time-consuming, costly, and may not be appropriate for all applications. Development of efficient, targeted retrovirus vectors is therefore desirable, as gene delivery to non-target cells may be harmful and would deplete the pool of viral particles. The host range of retroviruses is partly determined by the surface (SU) domain of the envelope glycoprotein, which binds to a cell surface receptor [2]. Envelope modification to incorporate new ligands that specifically bind receptors expressed only in target cells is one strategy for retrovirus vector targeting [3,4]. We have previously
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described efficient retrovirus vector targeting to melanomas by limiting the host-range of amphotropic murine leukemia virus (MLV-A) to tumor cells that express highmolecular-weight melanoma-associated antigen (HMWMAA; also called melanoma-associated chondroitin sulfate proteoglycan, HSMCSP) [5,6]. This integral membrane proteoglycan is expressed in more than 90% of human melanomas, but not in most normal adult tissues [7]. Its expression by melanomas is associated with a poor prognosis [8]. HMWMAA is also known to be a good in vivo target for radioimaging [9] and immunotherapy of melanoma [10,11]. We fused a single-chain antibody (scFv) that recognizes HMWMAA [12] to the amino terminus of MLV-A SU, followed by a proline-rich linker [13] and a matrix metalloprotease-2 (MMP2) cleavage site. MMPs are highly expressed on the surface of cancer cells [14] and are critical for tumor invasion of normal tissue [15]. The rationale for this approach was that attachment of vectors to HMWMAA would lead to MMP removal of the scFv and proline spacer at the cell surface, allowing transduction following MLV-A interaction with its receptor PiT-2. Retrovirus vectors carrying these envelopes selectively and efficiently infected HMWMAA-positive cells in mixed cultures [16].
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FIG. 1. LPMA targeted vectors selectively infect HMWMAApositive tumors. Transduction efficiency was determined for LPMA and Ampho vectors on HMWMAA-positive and negative cells. HMWMAA-positive (A375m and TE671) or negative cells (Ecv304) were mixed with lethally irradiated producer cells expressing the unmodified amphotropic (Ampho), the HMWMAA-targeted envelope (LPMA), or no envelope (No Env) and injected into nude mice. Transduction efficiency is shown as a percentage of tumor cells expressing the -galactosidase marker gene after counting approximately 105 cells, and is the mean (± SD) of at least three separate experiments.
Here we demonstrate the feasibility of using retrovirus vectors carrying this envelope, designated LPMA, for in vivo delivery. We show that LPMA vector transduction of HMWMAA-positive tumors is at least three orders of magnitude higher than of HMWMAA-negative tumors. In addition LPMA vectors were at least 100 times less likely to spread to spleen and liver than unmodified amphotropic vectors.
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LPMA Targeted Retrovirus Vectors Specifically Transduce HMWMAA-Positive Tumors in Vivo We mixed three cancer cells lines, two HMWMAA-positive (A375m and TE671) and one HMWMAA-negative (Ecv304), with irradiated packaging cells producing amphotropic vectors, HMWMAA-targeted LPMA vectors, or unenveloped particles. We then injected the mixture intradermally into nude mice. After 2 weeks, or when the tumor had reached 6–8 mm in diameter, we excised and disaggregated them before analyzing approximately 105 cells for -galactosidase expression. Amphotropic vectors showed a very high efficiency of transduction (up to 95% of the tumor) for all three of the tumor cell lines, but LPMA vectors transduced only the A375m and TE671 tumors (Fig. 1). The efficiency of LPMA transduction was approximately 10% of that seen with wild-type amphotropic envelope. We also analyzed the specificity of transduction of targeted and non-targeted vectors by -galactosidase staining of tumor sections. We only detected transduction with LPMA vectors in the HMWMAA-positive tumors, TE671 and A375m, whereas amphotropic vectors showed a high level of transduction in all the tumors. Tumor sections showed uniform distribution of transduced cells (data not shown). No transduction in any tumor model was seen when no-Env producer was used in the co-injection experiments. These results indicate that LPMA vectors are able to maintain their specificity and efficiency in vivo.
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To study targeted gene delivery more relevant to clinical application, we then grew tumors as xenografts in nude mice and injected retroviral vectors into the tumors when they reached 3–4 mm in diameter. Western blot analysis of the concentrated viral particles (Fig. 2A) demonstrated that LPMA and amphotropic vectors contain equal particle numbers as assessed by p30 capsid content, whereas the unenveloped preparation contains slightly more. One week after vector injection, we excised and disaggregated the tumors and analyzed them for -galactosidase expression. Approximately 3% of the tumor cells were transduced when HMWMAA-positive tumors (TE671 and HT1080) were injected with LPMA vectors, compared with about 10–20% when the same tumors were injected with the amphotropic vectors (Fig. 2B). No transduced cells were detected when the HMWMAA-negative tumor PAE was injected with LPMA vectors, whereas approximately 5% of the PAE tumor cells were transduced when amphotropic vector was used. Delivery of vector to these small tumors was subject to considerable experimental variation (Fig. 2B). Ecv304 could not be analyzed using this system because the small size of the tumors did not allow injection of the vectors. Transduction of injected targeted and non-targeted vectors was also analyzed by -galactosidase staining of sections from HMWMAA-positive and -negative tumors. Sections from tumors injected with LPMAtargeted vectors showed -galactosidase staining only in HMWMAA-positive tumors, TE671 and HT1080 (TE671 shown in Fig. 2C). In contrast, sections from all amphotropic vector-injected tumors showed high levels of transduction (Fig. 2C). These results indicate that LPMA vectors are able to maintain their specificity of transduction after intratumoral injection. It should be noted that in both in vivo experimental protocols, the co-injection of packaging cells and the intratumoral injection of vector, no polycation such as Polybrene or cationic liposomes was used to enhance infection. Our previous results in cell culture showed that the LPMA vector was extremely inefficient compared with amphotropic vector when incubated for 1 hour in the absence of such agents (infection undetectable; less than 0.1% of that seen with amphotropic) [16]. The relative efficient transduction by LPMA in vivo,
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approximately 10% of that seen with amphotropic in either protocol, was therefore an unexpected advantage as the use of cationic liposomes in vivo would be undesirable due to complement activation [17]. As Polybrene and cationic liposomes enhance the rate of virus attachment to target cells [18], our in vivo data may imply that the tumor cells in both protocols are exposed to LPMA vector for a prolonged period. However, the efficiency of transduction and the spread of the targeted vector through a solid tumor (Fig. 2C) must be improved if a practical clinical protocol is to be developed. LPMA-Targeted Vectors, Unlike Amphotropic Vectors, Are Not Detected in Spleen and Liver Initially, we examined spleens from the mice used in the above experiments to determine whether amphotropic and LPMA vectors differed in their spread to other tissues. We used DNA extracted from the spleens in a PCR reaction using LacZ1/LTR1 primers that amplify a vector fragment of 480 bp. Southern blots of the PCR samples were then probed for lacZ. However, any spleens in which the viral vector was detected were also positive when analyzed for the presence of human-specific sequences (human endogenous retrovirus gag; data not shown). These results implied that transduced tumor cells are likely to be detected as micrometastases and we were therefore unable to measure vector spread in tumor-bearing animals.
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FIG. 2. Intratumoral injections of LPMA-targeted vectors. HMWMAApositive (HT1080 and TE671) and negative (PAE) tumors were injected with unenveloped, unmodified (Ampho), or HMWMAA-targeted vectors (LPMA). (A) Western blot of concentrated supernatant preparations from the different producer cells expressing the HMWMAA-targeted envelope (LPMA), the 1040A amphotropic envelope (Ampho), or no envelope (No Env). Envelope SU and p30 capsid are detected by an anti-RLV antiserum. (B) Table showing transduction efficiency as a percentage of cells from individual injected tumors expressing the -galactosidase marker gene. (C) Tumor sections from TE671 (HMWMAA +ve) and PAE (HMWMAA -ve) stained for -galactosidase after intratumoral injection with unmodified vector (Ampho) or HMWMAA-targeted vectors (LPMA).
We therefore used intraperitoneal injection of vectors or irradiated producer cells to measure spread of amphotropic and LPMA vectors. We injected equal amounts of LPMA, amphotropic, and unenveloped particles, or equal numbers of LPMA, amphotropic, and unenveloped irradiated producer cells intraperitoneally in nude mice. Two weeks after injections, we sacrificed the mice and extracted spleen and liver DNA for nested PCR analysis. All five mice injected with amphotropic vectors and the two mice injected with irradiated amphotropic producer cells harbored proviral DNA in the spleen and liver (Fig. 3). However, none of the five animals injected with LPMA, the three injected with unenveloped virus and the two injected with either irradiated producer cells, showed detectable levels of proviral DNA in the spleen or liver (Fig. 3). Two additional bands can be observed in some liver samples from mice inoculated with LPMA supernatant. We think they are nonspecific because they are a different size to the MFGnlsLacZ-amplified fragment, and they also appeared in some liver samples from uninjected mice (data not shown). We carried out semiquantitative analysis of proviral content in spleen and liver to determine the level of amphotropic vector transduction in these organs. We used serial dilutions of DNA to carry out nested PCR using the same primers. The result for spleen and liver DNA from the AP1 mouse (injected with irradiated amphotropic
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producer cells) is shown in Fig. 4A, which can be used to calculate that they respectively contained 104 and 103 copies of proviral DNA per 106 cells. In a similar way, we analyzed all the spleen and liver DNA samples for proviral DNA content and plotted this in a table (Fig. 4B). We estimate that spleen and livers from mice injected with irradiated amphotropic producer cells contained from 10,000 to 1000 copies of proviral DNA per 106 cells, whereas spleen and liver from mice injected with amphotropic vectors supernatant harbor from 1000 to 10 copies per 106 cells. No proviral DNA was detected in any organ from mice injected with LPMA vector or producer cells (Fig. 4B, right). Based on these results and taking into account that the limit of detection is 10 copies per 106 cells (Fig. 3A), we argue that LPMA vectors are approximately 103 times less likely to spread than amphotropic vectors, although their lower infectivity on HMWMAA-positive tumors (approximately 10% of amphotropic) may account for part of this. Vector spreading is undesirable during in vivo gene therapy, as it may present a safety problem and also reduce the pool of vector available to transduce target cells. We have described previously a retroviral vector, LPMA, which achieved targeting to melanoma cells [16]. Here we have demonstrated that LPMA-targeted vectors specifically transduce tumors expressing the HMWMAA antigen in vivo. Targeted gene delivery was demonstrated both when viral producer cells were co-injected with tumor cells and when vectors were directly injected into tumors. In the co-injection model, local production of vector allowed more efficient tumor cell transduction,
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FIG. 3. LPMA-targeted vectors do not spread to normal tissues. A total of 7 mice were analyzed for each enveloped vector (LPMA and Ampho in (A) and (B)). Five mice were injected intraperitoneally with supernatant (1–5) and two with irradiated producer cell lines (p1 and p2). An additional five mice were used for unenveloped particles (No Env), three were injected intraperitoneally with supernatant (1–3), and two with unenveloped producer cells (p1 and p2). Proviral analysis was carried out by nested PCR using primers that amplify a 480-bp DNA fragment located between the 3⬘ end of the -galactosidase gene and the 3⬘ LTR of the integrated vector. DNA (10 g, approximately 106 cells) from spleen (A) or liver (B) from individual mice was analyzed for proviral content. First (LacZ1/LTR1) and second (LacZ2/LTR2) rounds of nested PCR reactions are shown as indicated. Plasmid DNA serial dilutions (from 104 to 1 plasmids) of an MFGnlslacZ vector were used to determine sensitivity (A).
which led us to conclude that LPMA vector transduction of HMWMAA-positive tumors is at least 103 times more efficient than that of HMWMAA-negative tumors. The intratumoral injection model represents a more practical strategy for clinical cancer gene therapy. Although results were quite variable between individual tumors, we found up to 3% of HMWMAA-positive tumor cells transduced by LPMA vectors, compared with < 0.01% of HMWMAA-negative tumor cells. This result is encouraging for the future clinical use of such vectors, although improved efficiency and spread throughout the solid tumor would be desirable. We also demonstrated that LPMA vectors are less likely to spread to normal tissues than amphotropic vectors. All spleens and livers from mice injected intraperitoneally with amphotropic vector or producer cells harbored proviral DNA at up to 104 copies per 106 cells, whereas we could not detect any proviral DNA in mice injected with an equal amount of LPMA vector or producer cells (limit of detection 10 copies per 106 cells). Some of the proviral DNA detected in spleen and liver probably represents transduction of cycling hematopoietic cells, either in the tissue or in the peritoneum followed by migration to spleen and liver. For equal amounts of vector particles (determined by gag levels), LPMA vectors are 10 times less efficient than amphotropic vectors in the transduction of HMWMAApositive tumors. Therefore the targeting decreases the likelihood of spread of LPMA vectors to at least 1% of that seen with amphotropic vectors. Several strategies to target retroviruses to specific cells have been described based on either envelope protein modification or bridging virus vector with target cells by means of antibodies or ligands [19]. Although retargeted binding was achieved, transduction of target cells tended to be inefficient or have low selectivity. A few groups have analyzed the efficacy of these vectors for in vivo
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FIG. 4. Semi-quantitative analysis of proviral DNA content. We analyzed spleen and liver DNA from the mice injected intraperitoneally with supernatant or irradiated producer cells shown in Fig. 3. (A) Nested PCR reactions using serial dilution of spleen and liver DNA from Ampho p1 (AP1) of Fig. 3. The estimated cell number used for the PCR reaction is shown at the top of each sample. Serial dilutions of MFGnlslacZ vector plasmid DNA (from 105 to 1 plasmids) were added to 1 g spleen DNA from control mice and used to determine sensitivity. (B) Quantitation from all mice. Estimated proviral number was calculated based on the number of cells used in the last dilution where positive amplification was found and the sensitivity of detection of the nested PCR reaction.
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delivery using mice models. Jiang and Dornburg showed that spleen necrosis virus (SNV) based vectors targeted to human cells expressing the ERBB2 receptor could transduce these cells when vectors and cells were injected intraperitoneally into nude mice [20]. The main problems of this strategy were the low efficiency and the artificiality of the model used for in vivo delivery. Other targeting strategies tested in vivo are targeting through protease substrate interactions [21] where the authors reported strong selectivity for MMP-rich tumor xenografts in vivo. The same group showed that intravenous infusion of nontargeted HIV-1 vectors pseudotyped with amphotropic murine leukemia virus (MLV) envelope glycoprotein led to transduction in liver and spleen, whereas inverse targeted EGF-displaying vectors were able to transduce spleen but not liver [22]. Another promising in vivo gene delivery system is based on matrixtargeted retroviral vectors [23–25]. The authors demonstrated enhanced vector penetration and transduction of tumor nodules after regional or systemic delivery of matrix-targeted vectors. For the first time a targeted retrovirus system was more efficient that unmodified vector for in vivo gene delivery. However, in these studies vector spreading to other organs was not analyzed. Therefore, they demonstrated a gain in efficiency, but not in safety. To our knowledge, the experiments reported here are the first to show selective transduction of targeted cells and reduced non-target cell transduction after in vivo delivery of targeted retroviral vectors.
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Cell culture. TELCeB6 cells are derived from the TE671 cell line (ATCC CRL-8805, now known to be identical to the human rhabdomyosarcoma
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cell line RD CCL-136) and harbor the MFGnlslacZ vector genome and an MLV-Gag-Pol expression plasmid, CeB [26]. A375m (ATCC CRL-1619) is a human melanoma cell line. Ecv304 is a spontaneously transformed human endothelial cell line (ATCC CRL-1998) and PAE cells are porcine aorta endothelial cells [27]. HT1080 is a human fibrosarcoma cell line (ATCC CCL-121). All cells were grown in DMEM (Gibco BRL) supplemented with 10% fetal calf serum (FCS) at 37⬚C and 10% CO2. Vector production, concentration, and titer. The TELCeB6/A cell line producing amphotropic vectors and the TELCeB6/LPMA cell line producing HMWMAA-targeted vectors have been described [16]. To harvest vectors, producer cells were grown at 37⬚C until they became confluent and then cultured at 32⬚C for 3–5 days feeding fresh DMEM supplemented with 10% FCS after 2 days. The medium was then replaced with serum-free Optimen (Gibco BRL) and supernatant was collected 12–16 hours later. The vector harvests were filtered through 0.45-m filters and concentrated by centrifugation at 2500g at 4⬚C for 12 hours then resuspended in Optimen. Concentrated vector was kept frozen at –70⬚C. Viral protein content in the concentrated preparations was assessed by western blot analysis using antigoat antiserum raised against Rauscher leukemia virus, which detects MLV gp70 and p30 capsid as described [16]. Vector titers were determined as follows. Target cells were seeded in 24-well plates at a density of 105 cells/well 24 hours before incubation with the vector supernatants. Cells were then incubated in the presence of the vector supernatant for 12 hours at 37°C, washed once in Optimen, and cultured for 24–48 hours. X-gal staining was carried out as described [28] Growth of tumor xenograft models in nude mice. For co-injection models, 1 ⫻ 106 tumor cells were mixed with 5 ⫻ 106 irradiated (40Gy) producer cells and then injected in 0.2 ml HBSS (Gibco) intradermally into a nude mouse. Tumors were explanted after 2 weeks, or when the tumor reached approximately 0.5 cm3, mashed, and incubated with 2 volumes of 5 mg/ml collagenase Ia (Sigma) for 2 hours at 37⬚C. After separation of the tumor cells was achieved, the suspension cells were pelleted, washed, and resuspended in DMEM supplemented with 10% FCS and plated in a tissue culture dish. After 2 hours of incubation, nonadherent cells were discarded and adherent tumor cells were incubated overnight in fresh medium before staining for -galactosidase expression. An average of 106 tumor cells were analyzed for blue nuclear staining as an indication of tumor transduction. For intratumoral injection, 5 ⫻ 106 tumor cells were resuspended in 0.2 ml HBSS and injected subcutaneously into the left side of the abdomen. Unmodified and targeted vectors were injected in 0.1 ml Optimen when tumors reached 3–4 mm3. Four days post viral injection, or when the tumor
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reached 0.5 cm3, animals were sacrificed and the tumor explanted. Half of each tumor was then embedded in OCT compound (BDH Laboratories, UK), snap-frozen in liquid nitrogen, and 10-m sections were stained with X-gal. The other half was disaggregated and stained as above. Analysis of vector spreading into normal organs. Mice were injected intraperitoneally with either 107 irradiated (40Gy) producer cells in 0.2 ml HBSS or 0.2 ml concentrated supernatant from the same cells. Animals were sacrificed 2 weeks after injection and spleens and livers were removed and snap-frozen at –70⬚C. DNA was extracted from 5–10 mg of tissue using a DNAeasy tissue kit (Qiagen). Proviral analysis was done by nested PCR using primers that amplify a fragment located between the 3⬘ end of the -galactosidase gene and the 3⬘ LTR of the MFGnlsLacZ integrated vector. External primers were LacZ1, 5⬘-GCACATGGCTGAATATCGACGG-3⬘ (beginning 78 bp 5⬘ of the EcoRI site within lacZ), and LTR1, 5⬘GCTTCAGCTGGTGATATTGTTGAG-3⬘ (spanning the PvuII site in the retrovirus), and internal primers were LacZ2, 5⬘-ATTGGTGGCGACGACTCCTG3⬘, and LTR2, 5⬘-AGCCTGGACCACTGATATCCTG-3⬘. PCR conditions were 35 cycles of 30 seconds at 94⬚C, 1 minute at 60⬚C, and 1 minute at 72⬚C. For qualitative analysis, DNA was quantified by OD 260/280 nm in a spectrophotometer and 10 g (approximately 106 mouse cell equivalents) were used for the first PCR reaction (LTR1/LacZ1) and 1/10 of this reaction was used as template for the second round of the nested PCR (LTR2/LacZ2). Semi-quantitative PCR was carried out by serial dilution of the DNA samples. Plasmid DNA harboring one copy of the MFGnlslacZ vector genome was used to determine the sensitivity of the system (1 plasmid = 0.1 fg).
ACKNOWLEDGMENTS We thank Colin Porter, Yasuhiro Takeuchi, and Yasuhiro Ikeda for technical advice and all the people from the group for support. This work was supported by the Medical Research Council, UK, and the Cancer Research Campaign, UK. RECEIVED FOR PUBLICATION NOVEMBER 6, 2001; ACCEPTED JANUARY 22, 2002.
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antigen mimicry by an anti-idiotypic antibody: characterization of the immunogenicity and the immune response to the mouse monoclonal antibody IMel-1. Cancer Res. 51: 6045–6051. 9. Lamki, L. M., et al. (1990). Radioimaging of melanoma using 99mTc-labeled Fab fragment reactive with a high molecular weight melanoma antigen. Cancer Res. 50: 904s–908s. 10. Bender, H., Grapow, M., Schomburg, A., Reinhold, U., and Biersack, H. J. (1997). Effects of diagnostic application of monoclonal antibody on survival in melanoma patients. Hybridoma 16: 65–68. 11. Mittelman, A., Chen, Z. J., Liu, C. C., Hirai, S., and Ferrone, S. (1994). Kinetics of the immune response and regression of metastatic lesions following development of humoral anti-high molecular weight-melanoma associated antigen immunity in three patients with advanced malignant melanoma immunized with mouse antiidiotypic monoclonal antibody MK2-23. Cancer Res. 54: 415–421. 12. Kupsch, J. M., et al. (1995). Generation and selection of monoclonal antibodies, singlechain Fv and antibody fusion phage specific for human melanoma-associated antigens. Melanoma Res. 5: 403–411. 13. Valsesia-Wittmann, S., Morling, F. J., Hatziioannou, T., Russell, S. J., and Cosset, F. L. (1997). Receptor co-operation in retrovirus entry: recruitment of an auxiliary entry mechanism after retargeted binding. EMBO J. 16: 1214–1223. 14. Brooks, P. C., et al. (1996). Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin ␣ v  3. Cell 85: 683–693. 15. Werb, Z. (1997). ECM and cell surface proteolysis: regulating cellular ecology. Cell 91: 439–442. 16. Martin, F., et al (1999). Retrovirus targeting by tropism restriction to melanoma cells. J. Virol. 73: 6923–6929. 17. Chonn, A., Cullis, P. R., and Devine, D. V. (1991). The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes. J. Immunol. 15: 4235–4241. 18. Porter, C. D., Lukacs, K. V., Box, G., Takeuchi, Y., and Collins, M. K. (1998). Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo. J. Virol. 72: 4832–4840. 19. Russell, S. J., and Cosset, F. L. (1999). Modifying the host range properties of retroviral vectors. J. Gene Med. 1: 300–311. 20. Jiang, A., and Dornburg, R. (1999). In vivo cell type-specific gene delivery with retroviral vectors that display single chain antibodies. Gene Ther. 6: 1982–1987. 21. Peng, K. W., Vile, R., Cosset, F. L., and Russell, S. (1999). Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors. Gene Ther. 6: 1552–1557. 22. Peng, K. W., et al. (2001). Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors. Gene Ther. 8: 1456–1463. 23. Hall, F. L., et al. (2000). Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor. Hum. Gene Ther. 11: 983–993. 24. Gordon, E. M., et al. (2000). Inhibition of metastatic tumor growth in nude mice by portal vein infusions of matrix-targeted retroviral vectors bearing a cytocidal cyclin G1 construct. Cancer Res. 60: 3343–3347. 25. Gordon, E. M., et al. (2001). Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice. Hum. Gene Ther. 12: 193–204. 26. Cosset, F. L., Takeuchi, Y., Battini, J. L., Weiss, R. A., and Collins, M. K. (1995). Hightiter packaging cells producing recombinant retroviruses resistant to human serum. J. Virol. 69: 7430–7436. 27. Miyazono, K., Okabe, T., Urabe, A., Takaku, F., and Heldin, C. H. (1987). Purification and properties of an endothelial cell growth factor from human platelets. J. Biol. Chem. 262: 4098–4103. 28. Takeuchi, Y., et al. (1994). Type C retrovirus inactivation by human complement is determined by both the viral genome and the producer cell. J. Virol. 68: 8001–8007.
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Envelope-Targeted Retrovirus Vectors Transduce Melanoma Xenografts but Not Spleen or Liver Francisco Martín,* Simon Chowdhury, Stuart Neil, Neil Phillipps, and Mary K. Collins Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Science, University College London, 46 Cleveland Street, London W1P 6DB, UK *To whom correspondence and reprint requests should be addressed. Fax: 44-207-679-9301. E-mail: [email protected].
Many cancer gene therapy applications would benefit from the development of targeted vectors that could deliver genes in vivo. We have previously achieved efficient in vitro targeting of retrovirus vectors to melanoma cells by fusion of a single chain antibody recognizing the highmolecular-weight melanoma-associated antigen (HMWMAA), followed by a blocking peptide and a matrix metalloprotease cleavage site, to the amino terminus of the murine leukemia virus amphotropic strain envelope. Here we report that up to 3% of cells within an HMWMAA-positive tumor xenograft were infected following a single injection of targeted vector into the tumor and up to 10% of tumor cells became infected when they were co-injected with viral producer cells. No infected cells were detected after delivery of targeted vectors to HMWMAA-negative tumor xenografts. Intraperitoneal injection of amphotropic vectors or producer cells resulted in transduction in spleen and liver, which was not detected when targeted vectors or producer cells were used. Our results demonstrate the feasibility of using targeted retroviral vectors for in vivo gene delivery to tumors and highlight the safety benefits of targeted vectors that do not infect other host tissues. Key Words: gene therapy, retrovirus vectors, in vivo targeting, melanoma, xenografts, vector spreading
INTRODUCTION The development of efficient, targeted vectors will be necessary for gene therapy applications that require in vivo gene delivery. Retrovirus vectors are attractive for clinical gene delivery because integration of the vector genome allows stable gene expression in the infected cell and its progeny. Also, because viral coding regions are deleted from the vector, viral proteins are not expressed in infected cells, avoiding stimulation of an inappropriate antiviral immune response. Thus far, in gene therapy clinical trials retrovirus vectors have been used for in vitro transduction followed by transfer of modified cells to the patient [1]. Such modification of cells is time-consuming, costly, and may not be appropriate for all applications. Development of efficient, targeted retrovirus vectors is therefore desirable, as gene delivery to non-target cells may be harmful and would deplete the pool of viral particles. The host range of retroviruses is partly determined by the surface (SU) domain of the envelope glycoprotein, which binds to a cell surface receptor [2]. Envelope modification to incorporate new ligands that specifically bind receptors expressed only in target cells is one strategy for retrovirus vector targeting [3,4]. We have previously
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described efficient retrovirus vector targeting to melanomas by limiting the host-range of amphotropic murine leukemia virus (MLV-A) to tumor cells that express highmolecular-weight melanoma-associated antigen (HMWMAA; also called melanoma-associated chondroitin sulfate proteoglycan, HSMCSP) [5,6]. This integral membrane proteoglycan is expressed in more than 90% of human melanomas, but not in most normal adult tissues [7]. Its expression by melanomas is associated with a poor prognosis [8]. HMWMAA is also known to be a good in vivo target for radioimaging [9] and immunotherapy of melanoma [10,11]. We fused a single-chain antibody (scFv) that recognizes HMWMAA [12] to the amino terminus of MLV-A SU, followed by a proline-rich linker [13] and a matrix metalloprotease-2 (MMP2) cleavage site. MMPs are highly expressed on the surface of cancer cells [14] and are critical for tumor invasion of normal tissue [15]. The rationale for this approach was that attachment of vectors to HMWMAA would lead to MMP removal of the scFv and proline spacer at the cell surface, allowing transduction following MLV-A interaction with its receptor PiT-2. Retrovirus vectors carrying these envelopes selectively and efficiently infected HMWMAA-positive cells in mixed cultures [16].
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FIG. 1. LPMA targeted vectors selectively infect HMWMAApositive tumors. Transduction efficiency was determined for LPMA and Ampho vectors on HMWMAA-positive and negative cells. HMWMAA-positive (A375m and TE671) or negative cells (Ecv304) were mixed with lethally irradiated producer cells expressing the unmodified amphotropic (Ampho), the HMWMAA-targeted envelope (LPMA), or no envelope (No Env) and injected into nude mice. Transduction efficiency is shown as a percentage of tumor cells expressing the -galactosidase marker gene after counting approximately 105 cells, and is the mean (± SD) of at least three separate experiments.
Here we demonstrate the feasibility of using retrovirus vectors carrying this envelope, designated LPMA, for in vivo delivery. We show that LPMA vector transduction of HMWMAA-positive tumors is at least three orders of magnitude higher than of HMWMAA-negative tumors. In addition LPMA vectors were at least 100 times less likely to spread to spleen and liver than unmodified amphotropic vectors.
RESULTS
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DISCUSSION
LPMA Targeted Retrovirus Vectors Specifically Transduce HMWMAA-Positive Tumors in Vivo We mixed three cancer cells lines, two HMWMAA-positive (A375m and TE671) and one HMWMAA-negative (Ecv304), with irradiated packaging cells producing amphotropic vectors, HMWMAA-targeted LPMA vectors, or unenveloped particles. We then injected the mixture intradermally into nude mice. After 2 weeks, or when the tumor had reached 6–8 mm in diameter, we excised and disaggregated them before analyzing approximately 105 cells for -galactosidase expression. Amphotropic vectors showed a very high efficiency of transduction (up to 95% of the tumor) for all three of the tumor cell lines, but LPMA vectors transduced only the A375m and TE671 tumors (Fig. 1). The efficiency of LPMA transduction was approximately 10% of that seen with wild-type amphotropic envelope. We also analyzed the specificity of transduction of targeted and non-targeted vectors by -galactosidase staining of tumor sections. We only detected transduction with LPMA vectors in the HMWMAA-positive tumors, TE671 and A375m, whereas amphotropic vectors showed a high level of transduction in all the tumors. Tumor sections showed uniform distribution of transduced cells (data not shown). No transduction in any tumor model was seen when no-Env producer was used in the co-injection experiments. These results indicate that LPMA vectors are able to maintain their specificity and efficiency in vivo.
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To study targeted gene delivery more relevant to clinical application, we then grew tumors as xenografts in nude mice and injected retroviral vectors into the tumors when they reached 3–4 mm in diameter. Western blot analysis of the concentrated viral particles (Fig. 2A) demonstrated that LPMA and amphotropic vectors contain equal particle numbers as assessed by p30 capsid content, whereas the unenveloped preparation contains slightly more. One week after vector injection, we excised and disaggregated the tumors and analyzed them for -galactosidase expression. Approximately 3% of the tumor cells were transduced when HMWMAA-positive tumors (TE671 and HT1080) were injected with LPMA vectors, compared with about 10–20% when the same tumors were injected with the amphotropic vectors (Fig. 2B). No transduced cells were detected when the HMWMAA-negative tumor PAE was injected with LPMA vectors, whereas approximately 5% of the PAE tumor cells were transduced when amphotropic vector was used. Delivery of vector to these small tumors was subject to considerable experimental variation (Fig. 2B). Ecv304 could not be analyzed using this system because the small size of the tumors did not allow injection of the vectors. Transduction of injected targeted and non-targeted vectors was also analyzed by -galactosidase staining of sections from HMWMAA-positive and -negative tumors. Sections from tumors injected with LPMAtargeted vectors showed -galactosidase staining only in HMWMAA-positive tumors, TE671 and HT1080 (TE671 shown in Fig. 2C). In contrast, sections from all amphotropic vector-injected tumors showed high levels of transduction (Fig. 2C). These results indicate that LPMA vectors are able to maintain their specificity of transduction after intratumoral injection. It should be noted that in both in vivo experimental protocols, the co-injection of packaging cells and the intratumoral injection of vector, no polycation such as Polybrene or cationic liposomes was used to enhance infection. Our previous results in cell culture showed that the LPMA vector was extremely inefficient compared with amphotropic vector when incubated for 1 hour in the absence of such agents (infection undetectable; less than 0.1% of that seen with amphotropic) [16]. The relative efficient transduction by LPMA in vivo,
MOLECULAR THERAPY Vol. 5, No. 3, March 2002 Copyright © The American Society of Gene Therapy
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approximately 10% of that seen with amphotropic in either protocol, was therefore an unexpected advantage as the use of cationic liposomes in vivo would be undesirable due to complement activation [17]. As Polybrene and cationic liposomes enhance the rate of virus attachment to target cells [18], our in vivo data may imply that the tumor cells in both protocols are exposed to LPMA vector for a prolonged period. However, the efficiency of transduction and the spread of the targeted vector through a solid tumor (Fig. 2C) must be improved if a practical clinical protocol is to be developed. LPMA-Targeted Vectors, Unlike Amphotropic Vectors, Are Not Detected in Spleen and Liver Initially, we examined spleens from the mice used in the above experiments to determine whether amphotropic and LPMA vectors differed in their spread to other tissues. We used DNA extracted from the spleens in a PCR reaction using LacZ1/LTR1 primers that amplify a vector fragment of 480 bp. Southern blots of the PCR samples were then probed for lacZ. However, any spleens in which the viral vector was detected were also positive when analyzed for the presence of human-specific sequences (human endogenous retrovirus gag; data not shown). These results implied that transduced tumor cells are likely to be detected as micrometastases and we were therefore unable to measure vector spread in tumor-bearing animals.
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FIG. 2. Intratumoral injections of LPMA-targeted vectors. HMWMAApositive (HT1080 and TE671) and negative (PAE) tumors were injected with unenveloped, unmodified (Ampho), or HMWMAA-targeted vectors (LPMA). (A) Western blot of concentrated supernatant preparations from the different producer cells expressing the HMWMAA-targeted envelope (LPMA), the 1040A amphotropic envelope (Ampho), or no envelope (No Env). Envelope SU and p30 capsid are detected by an anti-RLV antiserum. (B) Table showing transduction efficiency as a percentage of cells from individual injected tumors expressing the -galactosidase marker gene. (C) Tumor sections from TE671 (HMWMAA +ve) and PAE (HMWMAA -ve) stained for -galactosidase after intratumoral injection with unmodified vector (Ampho) or HMWMAA-targeted vectors (LPMA).
We therefore used intraperitoneal injection of vectors or irradiated producer cells to measure spread of amphotropic and LPMA vectors. We injected equal amounts of LPMA, amphotropic, and unenveloped particles, or equal numbers of LPMA, amphotropic, and unenveloped irradiated producer cells intraperitoneally in nude mice. Two weeks after injections, we sacrificed the mice and extracted spleen and liver DNA for nested PCR analysis. All five mice injected with amphotropic vectors and the two mice injected with irradiated amphotropic producer cells harbored proviral DNA in the spleen and liver (Fig. 3). However, none of the five animals injected with LPMA, the three injected with unenveloped virus and the two injected with either irradiated producer cells, showed detectable levels of proviral DNA in the spleen or liver (Fig. 3). Two additional bands can be observed in some liver samples from mice inoculated with LPMA supernatant. We think they are nonspecific because they are a different size to the MFGnlsLacZ-amplified fragment, and they also appeared in some liver samples from uninjected mice (data not shown). We carried out semiquantitative analysis of proviral content in spleen and liver to determine the level of amphotropic vector transduction in these organs. We used serial dilutions of DNA to carry out nested PCR using the same primers. The result for spleen and liver DNA from the AP1 mouse (injected with irradiated amphotropic
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producer cells) is shown in Fig. 4A, which can be used to calculate that they respectively contained 104 and 103 copies of proviral DNA per 106 cells. In a similar way, we analyzed all the spleen and liver DNA samples for proviral DNA content and plotted this in a table (Fig. 4B). We estimate that spleen and livers from mice injected with irradiated amphotropic producer cells contained from 10,000 to 1000 copies of proviral DNA per 106 cells, whereas spleen and liver from mice injected with amphotropic vectors supernatant harbor from 1000 to 10 copies per 106 cells. No proviral DNA was detected in any organ from mice injected with LPMA vector or producer cells (Fig. 4B, right). Based on these results and taking into account that the limit of detection is 10 copies per 106 cells (Fig. 3A), we argue that LPMA vectors are approximately 103 times less likely to spread than amphotropic vectors, although their lower infectivity on HMWMAA-positive tumors (approximately 10% of amphotropic) may account for part of this. Vector spreading is undesirable during in vivo gene therapy, as it may present a safety problem and also reduce the pool of vector available to transduce target cells. We have described previously a retroviral vector, LPMA, which achieved targeting to melanoma cells [16]. Here we have demonstrated that LPMA-targeted vectors specifically transduce tumors expressing the HMWMAA antigen in vivo. Targeted gene delivery was demonstrated both when viral producer cells were co-injected with tumor cells and when vectors were directly injected into tumors. In the co-injection model, local production of vector allowed more efficient tumor cell transduction,
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FIG. 3. LPMA-targeted vectors do not spread to normal tissues. A total of 7 mice were analyzed for each enveloped vector (LPMA and Ampho in (A) and (B)). Five mice were injected intraperitoneally with supernatant (1–5) and two with irradiated producer cell lines (p1 and p2). An additional five mice were used for unenveloped particles (No Env), three were injected intraperitoneally with supernatant (1–3), and two with unenveloped producer cells (p1 and p2). Proviral analysis was carried out by nested PCR using primers that amplify a 480-bp DNA fragment located between the 3⬘ end of the -galactosidase gene and the 3⬘ LTR of the integrated vector. DNA (10 g, approximately 106 cells) from spleen (A) or liver (B) from individual mice was analyzed for proviral content. First (LacZ1/LTR1) and second (LacZ2/LTR2) rounds of nested PCR reactions are shown as indicated. Plasmid DNA serial dilutions (from 104 to 1 plasmids) of an MFGnlslacZ vector were used to determine sensitivity (A).
which led us to conclude that LPMA vector transduction of HMWMAA-positive tumors is at least 103 times more efficient than that of HMWMAA-negative tumors. The intratumoral injection model represents a more practical strategy for clinical cancer gene therapy. Although results were quite variable between individual tumors, we found up to 3% of HMWMAA-positive tumor cells transduced by LPMA vectors, compared with < 0.01% of HMWMAA-negative tumor cells. This result is encouraging for the future clinical use of such vectors, although improved efficiency and spread throughout the solid tumor would be desirable. We also demonstrated that LPMA vectors are less likely to spread to normal tissues than amphotropic vectors. All spleens and livers from mice injected intraperitoneally with amphotropic vector or producer cells harbored proviral DNA at up to 104 copies per 106 cells, whereas we could not detect any proviral DNA in mice injected with an equal amount of LPMA vector or producer cells (limit of detection 10 copies per 106 cells). Some of the proviral DNA detected in spleen and liver probably represents transduction of cycling hematopoietic cells, either in the tissue or in the peritoneum followed by migration to spleen and liver. For equal amounts of vector particles (determined by gag levels), LPMA vectors are 10 times less efficient than amphotropic vectors in the transduction of HMWMAApositive tumors. Therefore the targeting decreases the likelihood of spread of LPMA vectors to at least 1% of that seen with amphotropic vectors. Several strategies to target retroviruses to specific cells have been described based on either envelope protein modification or bridging virus vector with target cells by means of antibodies or ligands [19]. Although retargeted binding was achieved, transduction of target cells tended to be inefficient or have low selectivity. A few groups have analyzed the efficacy of these vectors for in vivo
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FIG. 4. Semi-quantitative analysis of proviral DNA content. We analyzed spleen and liver DNA from the mice injected intraperitoneally with supernatant or irradiated producer cells shown in Fig. 3. (A) Nested PCR reactions using serial dilution of spleen and liver DNA from Ampho p1 (AP1) of Fig. 3. The estimated cell number used for the PCR reaction is shown at the top of each sample. Serial dilutions of MFGnlslacZ vector plasmid DNA (from 105 to 1 plasmids) were added to 1 g spleen DNA from control mice and used to determine sensitivity. (B) Quantitation from all mice. Estimated proviral number was calculated based on the number of cells used in the last dilution where positive amplification was found and the sensitivity of detection of the nested PCR reaction.
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delivery using mice models. Jiang and Dornburg showed that spleen necrosis virus (SNV) based vectors targeted to human cells expressing the ERBB2 receptor could transduce these cells when vectors and cells were injected intraperitoneally into nude mice [20]. The main problems of this strategy were the low efficiency and the artificiality of the model used for in vivo delivery. Other targeting strategies tested in vivo are targeting through protease substrate interactions [21] where the authors reported strong selectivity for MMP-rich tumor xenografts in vivo. The same group showed that intravenous infusion of nontargeted HIV-1 vectors pseudotyped with amphotropic murine leukemia virus (MLV) envelope glycoprotein led to transduction in liver and spleen, whereas inverse targeted EGF-displaying vectors were able to transduce spleen but not liver [22]. Another promising in vivo gene delivery system is based on matrixtargeted retroviral vectors [23–25]. The authors demonstrated enhanced vector penetration and transduction of tumor nodules after regional or systemic delivery of matrix-targeted vectors. For the first time a targeted retrovirus system was more efficient that unmodified vector for in vivo gene delivery. However, in these studies vector spreading to other organs was not analyzed. Therefore, they demonstrated a gain in efficiency, but not in safety. To our knowledge, the experiments reported here are the first to show selective transduction of targeted cells and reduced non-target cell transduction after in vivo delivery of targeted retroviral vectors.
MATERIALS
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METHODS
Cell culture. TELCeB6 cells are derived from the TE671 cell line (ATCC CRL-8805, now known to be identical to the human rhabdomyosarcoma
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cell line RD CCL-136) and harbor the MFGnlslacZ vector genome and an MLV-Gag-Pol expression plasmid, CeB [26]. A375m (ATCC CRL-1619) is a human melanoma cell line. Ecv304 is a spontaneously transformed human endothelial cell line (ATCC CRL-1998) and PAE cells are porcine aorta endothelial cells [27]. HT1080 is a human fibrosarcoma cell line (ATCC CCL-121). All cells were grown in DMEM (Gibco BRL) supplemented with 10% fetal calf serum (FCS) at 37⬚C and 10% CO2. Vector production, concentration, and titer. The TELCeB6/A cell line producing amphotropic vectors and the TELCeB6/LPMA cell line producing HMWMAA-targeted vectors have been described [16]. To harvest vectors, producer cells were grown at 37⬚C until they became confluent and then cultured at 32⬚C for 3–5 days feeding fresh DMEM supplemented with 10% FCS after 2 days. The medium was then replaced with serum-free Optimen (Gibco BRL) and supernatant was collected 12–16 hours later. The vector harvests were filtered through 0.45-m filters and concentrated by centrifugation at 2500g at 4⬚C for 12 hours then resuspended in Optimen. Concentrated vector was kept frozen at –70⬚C. Viral protein content in the concentrated preparations was assessed by western blot analysis using antigoat antiserum raised against Rauscher leukemia virus, which detects MLV gp70 and p30 capsid as described [16]. Vector titers were determined as follows. Target cells were seeded in 24-well plates at a density of 105 cells/well 24 hours before incubation with the vector supernatants. Cells were then incubated in the presence of the vector supernatant for 12 hours at 37°C, washed once in Optimen, and cultured for 24–48 hours. X-gal staining was carried out as described [28] Growth of tumor xenograft models in nude mice. For co-injection models, 1 ⫻ 106 tumor cells were mixed with 5 ⫻ 106 irradiated (40Gy) producer cells and then injected in 0.2 ml HBSS (Gibco) intradermally into a nude mouse. Tumors were explanted after 2 weeks, or when the tumor reached approximately 0.5 cm3, mashed, and incubated with 2 volumes of 5 mg/ml collagenase Ia (Sigma) for 2 hours at 37⬚C. After separation of the tumor cells was achieved, the suspension cells were pelleted, washed, and resuspended in DMEM supplemented with 10% FCS and plated in a tissue culture dish. After 2 hours of incubation, nonadherent cells were discarded and adherent tumor cells were incubated overnight in fresh medium before staining for -galactosidase expression. An average of 106 tumor cells were analyzed for blue nuclear staining as an indication of tumor transduction. For intratumoral injection, 5 ⫻ 106 tumor cells were resuspended in 0.2 ml HBSS and injected subcutaneously into the left side of the abdomen. Unmodified and targeted vectors were injected in 0.1 ml Optimen when tumors reached 3–4 mm3. Four days post viral injection, or when the tumor
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reached 0.5 cm3, animals were sacrificed and the tumor explanted. Half of each tumor was then embedded in OCT compound (BDH Laboratories, UK), snap-frozen in liquid nitrogen, and 10-m sections were stained with X-gal. The other half was disaggregated and stained as above. Analysis of vector spreading into normal organs. Mice were injected intraperitoneally with either 107 irradiated (40Gy) producer cells in 0.2 ml HBSS or 0.2 ml concentrated supernatant from the same cells. Animals were sacrificed 2 weeks after injection and spleens and livers were removed and snap-frozen at –70⬚C. DNA was extracted from 5–10 mg of tissue using a DNAeasy tissue kit (Qiagen). Proviral analysis was done by nested PCR using primers that amplify a fragment located between the 3⬘ end of the -galactosidase gene and the 3⬘ LTR of the MFGnlsLacZ integrated vector. External primers were LacZ1, 5⬘-GCACATGGCTGAATATCGACGG-3⬘ (beginning 78 bp 5⬘ of the EcoRI site within lacZ), and LTR1, 5⬘GCTTCAGCTGGTGATATTGTTGAG-3⬘ (spanning the PvuII site in the retrovirus), and internal primers were LacZ2, 5⬘-ATTGGTGGCGACGACTCCTG3⬘, and LTR2, 5⬘-AGCCTGGACCACTGATATCCTG-3⬘. PCR conditions were 35 cycles of 30 seconds at 94⬚C, 1 minute at 60⬚C, and 1 minute at 72⬚C. For qualitative analysis, DNA was quantified by OD 260/280 nm in a spectrophotometer and 10 g (approximately 106 mouse cell equivalents) were used for the first PCR reaction (LTR1/LacZ1) and 1/10 of this reaction was used as template for the second round of the nested PCR (LTR2/LacZ2). Semi-quantitative PCR was carried out by serial dilution of the DNA samples. Plasmid DNA harboring one copy of the MFGnlslacZ vector genome was used to determine the sensitivity of the system (1 plasmid = 0.1 fg).
ACKNOWLEDGMENTS We thank Colin Porter, Yasuhiro Takeuchi, and Yasuhiro Ikeda for technical advice and all the people from the group for support. This work was supported by the Medical Research Council, UK, and the Cancer Research Campaign, UK. RECEIVED FOR PUBLICATION NOVEMBER 6, 2001; ACCEPTED JANUARY 22, 2002.
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