Rad51 Promoter-Targeted Gene Therapy Is Effective for In Vivo Visualization and Treatment of Cancer

original article

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Rad51 Promoter-Targeted Gene Therapy Is Effective for In Vivo Visualization and Treatment of Cancer Christopher M Hine1,2, Andrei Seluanov1 and Vera Gorbunova1 1 Department of Biology, University of Rochester, Rochester, New York, USA; 2Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York, USA

Rad51 protein is overexpressed in a wide range of human cancers. Our previous in vitro studies demonstrated that a construct comprised Rad51 promoter driving expression of the diphtheria toxin A gene (pRad51-diphtheria toxin A (DTA)) destroys a variety of human cancer cell lines, with minimal to no toxicity to normal human cells. Here we delivered Rad51 promoter-based constructs in vivo using linear polyethylenimine nanoparticles, in vivo jetPEI, to visualize and treat tumors in mice with HeLa xenografts. For tumor detection, we used pRad51-Luc, a construct containing the firefly luciferase under the Rad51 promoter, administered by intraperitoneal (IP) injection. Tumors were detected with an in vivo bioluminescent camera. All mice with cancer displayed strong bioluminescence, while mice without cancer displayed no detectable bioluminescence. Treatment with pRad51DTA/jetPEI decreased tumor mass of subcutaneous (SC) and IP tumors by sixfold and fourfold, respectively, along with the strong reduction of malignant ascites. Fifty percent of the mice with SC tumors were cancer-free after six pRad51-DTA/jetPEI injections, and for the mice with IP tumors, mean survival time increased by 90% compared to control mice. This study demonstrates the clinical potential of pRad51-based constructs delivered by nanoparticles for the diagnostics and treatment of a wide range of cancers. Received 11 January 2011; accepted 13 September 2011; published online 18 October 2011. doi:10.1038/mt.2011.215

Introduction RAD51 is a recombinase protein essential in repairing DNA double-strand breaks (DSBs) and stalled replication forks by homologous recombination (HR).1,2 Due to the dangers of gross genomic instability caused by aberrant chromosomal recombination, Rad51 expression is tightly controlled in normal human cells.3,4 The majority of human tumor cells, including those of the prostate, pancreas, breast, lung, and cervix, overexpress Rad51.5–9 This overexpression of Rad51 protein is due to transcriptional and posttranscriptional deregulation of Rad51 expression.3,6 Cancer cells overexpressing Rad51 have a selective advantage due to

Rad51’s ability to support rapidly dividing cancer cells and eliminate DNA replicative stress.2,10 Overexpression of Rad51 in cancer cells leads to resistance to DSB-inducing therapies.11,12 Levels of Rad51 are positively correlated with the aggressiveness13 and increased invasiveness of cancers.14 Rad51 levels can also be used as a prognostic marker for patient survival time,15,16 as higher levels of Rad51 correlate with poorer survival times. Due to Rad51 having a crucial role in cancer progression, therapies targeting Rad51’s downregulation, either directly or indirectly, have been used to inhibit tumor growth and sensitize cancer cells to radioand chemotherapies.11,17–19 With Rad51 expression being regulated primarily transcriptionally and posttranscriptionally,3,6 it is promising to harness the power of Rad51 promoter to selectively drive expression of genes of interest in cancer cells while having minimal expression in normal cells. This approach, termed transcriptionally targeted anticancer gene therapy, employs a reporter and/or cytotoxic gene/ oncolytic virus under the transcriptional control of the cancer and/ or tissue-specific promoters (reviewed in refs 20–22). Promoters used include those active in many different tumor types, such as telomerase hTER and hTERT,23–25 and mesothelin,26 or those of a tumor’s tissue-specific origin, such as the prostate PSA,27,28 or ovaries HE4.26 The results of these studies are promising, especially with mesothelin promoter–driven constructs targeting difficult to treat ovarian, pancreatic, and lung cancers.26,29–31 It would be beneficial to identify additional strong cancer-specific promoters for treating a wide range of cancers while having minimal toxicity.22 We previously reported that when the Rad51 open reading frame (ORF) is replaced with another ORF, such as firefly luciferase ORF to form the plasmid pRad51-Luc, expression of the transgene is highly activated in cancer cells and repressed in the normal cells.3 The difference in Rad51 promoter activity between cancerous and noncancerous cells reached up to 12,500-fold, with an average difference of 800-fold.3 The cells assayed included several breast cancer cell lines, cervical cancer, fibrosarcoma3, and prostate cancer (Supplementary Figure S1 and Supplementary Materials and Methods). The dramatic difference in promoter activity between normal and cancer cells can be explained by negative posttranscriptional regulation of Rad51 expression being removed when the Rad51 ORF is replaced. This difference allows for the targeting of cancer cells with high efficacy and selectivity. Consequently, when

Correspondence: Andrei Seluanov, University of Rochester, Department of Biology, River Campus Box 270211, Rochester, NY 14627, USA. E-mail: [email protected] Molecular Therapy vol. 20 no. 2, 347–355 feb. 2012

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the ORF for a suicide gene, DTA,32,33 was placed under the Rad51 promoter, forming the plasmid pRad51-DTA, and transfected into either cancer cells or normal cells of the same origin, the cancer cells were killed and there was no significant effect on the normal cell types.3 In summary, our previous in vitro work showed that Rad51based constructs kill cancer cells with high efficacy and specificity. In this study, we tested Rad51 promoter-based targeted cancer therapy in vivo. A major challenge to applying gene therapy in vivo is delivery. We used the clinically tested polyethylenimine-derived in vivo-jetPEI transfection reagent (Polyplus-Transfection, Illkirch, France) for nanoparticle-mediated delivery of therapeutic constructs. In vivo-jetPEI, a cationic linear polymer, efficiently transfects cells by combining plasmid DNA compaction with endosomolytic activity, resulting in endosomal escape and intracellular delivery of the plasmid DNA. 34–37 Here we show that pRad51-Luc/jetPEI injected into the peritoneal space is effective for detection and visualization of tumor xenografts. Furthermore, we show that subcutaneous (SC) or intraperitoneal (IP) injection of pRad51-DTA/jetPEI decreased tumor mass, alleviated malignant ascites, and either completely cured or extended life span in mice. This study demonstrates the clinical potential of Rad51 promoter-based nanoparticle anticancer technologies for the detection and treatment of cancer.

Results pRad51-Luc/jetPEI is effective for in vivo bioluminescent imaging of tumors To test whether pRad51-Luc construct (Figure  1a) could be used for detection of tumors in vivo, we complexed it with jetPEI polyethylenimine nanoparticles and injected into mice carrying human cervical cancer (HeLa) xenografts. We used in vivo-jetPEI nanoparticles because they are well tolerated and are currently in clinical trials.37 HeLa xenografts were chosen due to their highly aggressive growth, although our previous results3 indicate the majority of human cancer types could have been used for xenografts due to Rad51 promoter being highly expressed in the majority of human cancer cell lines. Tumor xenografts were established by injection of HeLa cells into the IP space of mice, while no cancer was given to the control mice (n = 4 for each group). Three weeks later, mice were injected IP with pRad51-Luc complexed with in vivo-jetPEI. Twenty-four, 48, 72, and 144 hours after injection, the mice were imaged for luciferase activity using a cooled charge coupled device (CCD) camera (Figure  1b,c). Mice with no DNA injected were used for background analysis. Bioluminescence was detectable at all time points in the mice with cancer, but no light counts above

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Figure 1  Visualization of IP xenografts using pRad51-Luc construct delivered by nanoparticles. (a) Diagram of pRad51-Luc construct, containing Rad51 promoter, including the 5’ UTR, first intron, and first 13 amino acids of the Rad51 ORF fused in-frame with the gene encoding firefly luciferase. (b) Representative images of a mouse with xenografts and a control mouse injected with pRad51-Luc. Athymic nude-Foxn1nu mice were injected IP with HeLa cancer cells or kept cancer free. Three weeks later; mice were injected IP with pRad51-Luc complexed with in vivo-jetPEI nanoparticles. At indicated times post pRad51-Luc injection, tumor-bearing and control mice were imaged for in vivo bioluminescence on a CCD camera at 1-minute exposures. Color scale bar indicates fluorescence intensity in photons per second. (c) Quantification of the in vivo bioluminescence data for mice with tumors, tumor-free mice injected with pRad51-Luc, and untreated tumor-free mice (n = 4, for each group). (d) Ex vivo bioluminescent imaging of organs and tumor xenografts from control mice, control mice injected with pRad51-Luc, and mice bearing IP xenografts injected IP with pRad51-Luc. Images were taken 48 hours after injection. ORF, open reading frame.

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pRad51-DTA/jetPEI therapy destroys SC tumor xenografts To test therapeutic efficacy of pRad51-DTA (Figure 2a), we injected pRad51-DTA nanoparticles directly into SC HeLa xenografts. To facilitate the quantification of the growth and/or reduction of the tumors, HeLa-Luc cells constitutively expressing luciferase were made by stably integrating the firefly luciferase gene under the SV40 promoter into HeLa cells. As DTA inhibits protein synthesis and eventually causes apoptosis,32,33 the light counts emitted by the tumors are expected to decrease due to a reduction in tumor mass. HeLa-Luc cells were injected SC into the lower back of mice and allowed to grow for 3 weeks before the start of intratumoral therapy injections. Six injections of the therapeutic pRad51-DTA or the control pRad51-GFP nanoparticles were delivered over 28 days. Tumors were measured with calipers and mice imaged for in vivo bioluminescence (Figure 2). One week after the last gene delivery, the light counts given off from the control group’s tumors had increased by 2.6-fold from the start of the experiment (P = 0.0075) and were sevenfold higher than the therapy group (P = 0.0001). The therapy group’s light counts had decreased by 2.4fold from the start of the experiment (P = 0.0071) (Figure  2c). Physical measurements of the tumors gave similar results, as the tumors of the control group continued to increase throughout the experiment, resulting in a sixfold increase in volume compared to the starting point (P = 0.0001) and a sixfold increase compared to the therapy group (P = 0.0001) (Figure 2d). The average tumor volume of the therapy group remained unchanged throughout the experiment, as there was no statistically difference between day 1 and day 53 (P = 0.8725). No change in tumor volume but a twofold decrease in tumor bioluminescence is likely explained by the fact that caliper measurements include dead cancer cells and/or stroma of host mouse origin, as reported in previous SC tumor studies.40 In vivo bioluminescence measurements were stopped 35 days after the first treatment and physical measurements stopped 53 days after the first treatment. At day 53, tumors of all the 10 control

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background were seen in the cancer-free mice. Maximum light counts were recorded 48–72 hours after injection and continued to decrease after this time point. To further examine the tumor specificity and biodistribution of pRad51 constructs complexed with in vivo-jetPEI, we performed ex vivo bioluminescence imaging of organs and tumors. Mice were killed 48 hours after IP pRad51-Luc/jetPEI injection, and their organs and tumors were excised and imaged (Figure 1d). No observable bioluminescence was detected in the heart, lung, liver, stomach, spleen or kidney of cancer-free control mice injected with pRad51-Luc/jetPEI compared to mice given no injection. Bioluminescence was detected in the majority of resected tumors and in tumors that had grown into the stomach and spleen of mice with the tumor xenografts. These results demonstrate pRad51-Luc/jetPEI is able to efficiently enter the cells in vivo and specifically target cancer cells and not healthy tissues for transgene expression. pRad51-Luc or other reporter genes under the Rad51 promoter may allow detection and imaging of tumors in the clinic using bioluminescent,38 nuclear medicine or magnetic resonance imaging (MRI) techniques.39

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Figure 2  pRad51-DTA delivered by nanoparticles selectively destroys SC cancer xenografts. (a) Diagram of pRad51-DTA construct, containing Rad51 promoter, 5’ UTR, first intron, and first 13 amino acids of the Rad51 ORF fused in-frame to the gene encoding bacteria diphtheria toxin A. (b) HeLa cells stably expressing firefly luciferase were used to establish xenografts in athymic nude-Foxn1nu mice. Three weeks after xenograft inoculation, mice were given a series of six intratumoral injections of pRad51-DTA/jetPEI or pRad51-GFP/jetPEI (n = 10 for each group) over 28 days. Shown are representative images of mice bearing SC HeLa-Luc xenografts treated with pRad51-DTA or control pRad51-GFP at the start of the treatment and after six treatment injections. Images were taken at 2-second exposures using bioluminescent CCD camera. (c) Quantification of tumor load using the bioluminescent light counts. (d) Quantification of tumor volume measured by calipers. Arrows indicate the days of DNA/nanoparticle injections. ORF, open reading frame.

mice were very large causing ulceration of the skin, and animal distress, commanding euthanasia. At the same time, we also euthanized five of the treated mice with larger tumors. Tumors were excised and weighed. It is important to note that none of the treated mice had tumors large enough to require euthanasia; these mice were killed for the sole reason of comparing the tumors to untreated group. The remaining five treated mice with smaller 349

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Figure 3 Treatment with pRad51-DTA nanoparticles efficiently reduces or eliminates SC HeLa-Luc cancer xenografts. (a) Tumors from control (pRad51-GFP) and pRad51-DTA-treated mice. The tumors were excised 25 days after the last therapy injection, imaged with a digital camera and then weighed. The largest tumors from the treated mice are shown, as the tumors in the remaining five mice were undetectable by calipers, and these mice were left alive to observe tumor recurrence. (b) Tumor mass of the tumors excised from the control and treated mice 25 days after the last injection. The star indicates five mice whose tumors became undetectable after therapy. (c) Five of the treated mice with undetectable tumors. A 1-minute exposure 100 days after the last pRad51-DTA/jetPEI injection reveals no detectable luminescence from HeLa-Luc cells. At 12 months after therapy, the mice did not show any tumor recurrence. (d) H&E staining of tumors excised from mice after six injections of pRad51-Luc (control) or pRad51-DTA (treatment). Arrows indicate locations of blood vessels where angiogenesis has occurred. ORF, open reading frame.

tumors were kept alive for 9 months to monitor for tumor relapse. Figure 3a shows five representative tumors from control mice that received pRad51-GFP/jetPEI and five of the largest tumors from the therapy mice that received pRad51-DTA/jetPEI. It can be noted that extensive presence of blood vessel vasculature in the larger tumors from control mice that is not seen in the tumors from the mice that received therapy (Figure 3a,d). The average mass of the tumors from the 10 control mice was threefold larger compared to the five largest tumors taken from the Rad51-DTA-treated mice (P = 0.0051) (Figure 3b). The five mice with undetectable tumors were then imaged 3 months after the last therapy injection for in vivo bioluminescence at 1-minute exposure times (Figure 3c). No bioluminescence was detected even at this longer exposure time. At Twelve months after the last therapy treatment, this group of mice was alive with no detectable tumors (Supplementary Figure S2) and no detectable side effects of therapy. Ex vivo hematoxylin and eosin (H&E) staining of tumors from treated and control mice indicates that the treatment suppressed angiogenesis and resulted in large necrotic areas (Figure 3d). In summary, the results from this SC tumor model show that pRad51-DTA/jetPEI is effective for in vivo cancer-targeted suicide gene therapy by intratumoral injection.

pRad51-DTA/jetPEI therapy reduces tumor burden and malignant ascites while extending survival in mice with IP tumor xenografts We next tested pRad51-DTA/jetPEI therapy in the more clinically relevant IP cancer xenograft model. In this model, tumors form throughout the peritoneal cavity and for the treatment to be 350

effective the IP-injected nanoparticles must find there way into the tumors and selectively eliminate cancer cells. Furthermore, as a frequent complication of the IP tumors is accumulation of malignant ascites, we were able to measure the effect of pRad51DTA/jetPEI on ascites volume. HeLa-Luc cancer cells suspended in phosphate-buffered saline (PBS) were injected intraperitoneally into mice and allowed to establish for 1 week before the start of the therapy. Six IP injections of pRad51-DTA/jetPEI, or control pRad51-GFP/jetPEI nanoparticles were performed over 15 days. Mice were analyzed by bioluminescence on days 0, 9, 17, and 21 of the treatment (Figure 4). Tumors in mice injected with the control nanoparticles increased sixfold between day 0 and day 21 (P = 0.0002, n = 8), while the tumors injected with Rad51-DTA nanoparticles decreased by threefold (P = 0.0177, n = 8). Weight of mice was monitored throughout the experiment to determine the change in mass due to the growth of tumors and malignant ascites or possible negative effects of the pRad51-DTA/ jetPEI therapy (Figure 5). There was no significant difference in mass between the pRad51-DTA/jetPEI-treated tumor-bearing mice and a control group of mice of the same age with no cancer (P = 0.3998). pRad51-DTA/jetPEI-treated mice increased in mass by an average of 18% and healthy, cancer-free-untreated mice increased by an average of 15% from day 0 to day 21, while mice with cancer given pRad51-GFP/jetPEI increased in mass by over 49% (P = 0.0003) (Figure 5a,b). This significant increase in mass over healthy untreated mice and pRad51-DTA/jetPEItreated mice with cancer can be attributed to the increase in tumor growth and development of malignant ascites. On day 21, www.moleculartherapy.org vol. 20 no. 2 feb. 2012

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(according to the University of Rochester Animal Care and Use Committee protocol). Fifty percent of pRad51-GFP/jetPEI control mice survived to 34 days after xenograft establishment while 50% of the pRad51-DTA/jetPEI-treated mice survived to 65 days, resulting in a 90% increase in mean survival time for mice treated with pRad51-DTA/jetPEI therapy compared to the pRad51-GFP/ jetPEI controls (P = 0.0027) (Figure 7). One of the treated mice had no detectable tumor by day 17 and day 21 of treatment. This mouse appeared to be cured and was alive and tumor free 9 months after therapy. In summary, the treatment with pRad51-DTA/­ jetPEI significantly reduced tumor burden, malignant ascites, and prolonged survival of mice with IP tumor xenografts.

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Figure 4 Treatment with pRad51-DTA nanoparticles reduces tumor burden in mice with IP HeLa-Luc xenografts. HeLa cells stably expressing firefly luciferase were used to establish xenografts in athymic nudeFoxn1nu mice. One week after xenograft inoculation, mice were given a series of six IP injections of pRad51-DTA/jetPEI or pRad51-GFP/jetPEI (n = 8 for each group) over 15 days. (a) Representative images of mice bearing IP HeLa-Luc xenografts treated with IP injections of pRad51-DTA or control pRad51-GFP at the start of the treatment and after the six treatment injections. Images were taken at 5-second exposures using bioluminescent CCD camera. (b) Quantification of tumor load using the bioluminescent light counts. Arrows indicate the days of DNA/nanoparticle injections.

four representative mice from the pRad51-DTA/jetPEI and four mice from pRad51-GFP/jetPEI groups were killed for dissection to determine the total tumor mass and volume of ascites. Tumor mass was determined by removing all tumors and weighing them (Figure  5c). The control cancer mice had a 3.7-fold increase in tumor mass compared to the mice given pRad51-DTA/jetPEI treatment (P = 0.0003). Ascites volume was measured by siphoning out the fluid with a syringe before the peritoneum was cut (Figure 5d). We found that mice given pRad51-GFP/jetPEI had a 32.9-fold increase in ascites volume compared to mice given pRad51-DTA/jetPEI (P < 0.0001). Similar to previous studies,26 we examined if pRad51-DTA/ jetPEI peritoneal administration induces any observable toxicity to noncancerous tissues by H&E staining. Tissues from the heart, lung, liver, stomach, spleen, and kidney did not display any signs of pathology such as fibrosis or liver cell drop out or inflammation as a result of six injections of pRad51-DTA/jetPEI as compared to the control group, which received pRad51-Luc/jetPEI (Figure 6). To determine if the treatment with pRad51-DTA prolongs life, we followed the survival of the remaining mice that received pRad51-DTA/jetPEI or pRad51-GFP/jetPEI injections. The end points were reached when a mouse died or was euthanized after becoming moribund due to a large volume of tumors and ascites Molecular Therapy vol. 20 no. 2 feb. 2012

Our previous cell culture work established that Rad51-DTA construct destroys a wide range of cancer cell types with high efficacy and specificity. In this report, we have demonstrated that Rad51based constructs are effective at targeting tumors in vivo when delivered by nanoparticles. pRad51-Luc construct complexed with linear jetPEI and injected IP was able to “locate” the tumors disseminated in the peritoneal cavity and was expressed in the tumors with high specificity. This technique opens a way for development of a new diagnostic tool, where patients can be injected with pRad51-Luc nanoparticles and the tumors then visualized using a sensitive bioluminescent CCD camera. Since the level of Rad51 expression is correlated with tumor aggressiveness,13,14 the amount of bioluminescence can be used to distinguish benign and malignant tumors. This would be a unique nonradioactive in vivo diagnostic tool based on tumor biology rather than on its physical characteristics. Luciferase-mediated bioluminescent imaging may have its own clinical limitations, such as low tissue penetrance and unknown toxicity of large doses of luciferin. Therefore, alternative imaging tools such as MRI and nuclear medicine position emission tomography (PET) reporter genes39,41 under the Rad51 promoter should be further tested. Furthermore, Rad51 promoter could be fused to other reporters such as secreted human placental alkaline phosphatase, which can be measured in a blood sample. This could be developed into an inexpensive and noninvasive screening tool for the presence of cancer in the body. pRad51-DTA was effective at destroying tumors. We observed ~ sixfold and fourfold reductions in tumor mass for both SC and IP xenografts, respectively. Cancer was cured in 50% of the mice with SC tumors after six pRad51-DTA/jetPEI treatments. The mean survival time of the mice with IP tumors treated with pRad51-DTA/jetPEI increased by 90% compared to control mice. One mouse with IP tumors was cured while the remaining mice eventually succumbed to cancer. Low cure rate for IP xenografts is likely caused by incomplete delivery of pRad51-DTA into all tumor cells. Figure  1d shows that although most tumors displayed luciferase signal, some small areas of tumors did not. The areas that did not receive the therapeutic construct could give rise to recurrence. It should be noted that we had only given a course of six injections of pRad51-DTA/jetPEI. Because the therapy was specific to the tumor tissue and well tolerated, it would be possible to give longer courses or higher doses of pRad51-DTA/jetPEI to increase the cure rate. Although we did not observe apparent toxicity of commercial jetPEI preparations that we used, PEI toxicity 351

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Figure 5  IP injections of pRad51-DTA nanoparticles reduce tumor burden and malignant ascites in mice carrying IP xenografts. (a) Representative images of untreated tumor-free mice, and mice with IP xenografts that received the pRad51-GFP control or pRad51-DTA treatment. (b) Changes in body mass of mice receiving treatments or of the control tumor-free mice of the same age. Body mass of the cancer-free mice increases slightly because of animal growth. Body mass of the tumor-bearing mice receiving pRad51-GFP increased because of tumor growth and development of ascites. The increase in body mass in the pRad51-GFP-treated group was 2.7-fold greater than that of the increase in body mass in the pRad51DTA group (P = 0.0003). There was no significant difference (P = 0.3998) in body mass between cancer-free mice and pRad51-DTA-treated cancer mice indicating that the treatment efficiently reduced tumor growth and ascites. Furthermore, there was no weight loss in the treated group indicating low toxicity. (c) Tumors and (d) ascites fluid were removed on day 21 from pRad51-DTA-treated (n = 4) and pRad51-GFP control (n = 4) mice. Mice treated with pRad51-DTA had 3.7-fold lower tumor mass (P = 0.0003) and 32.9-fold less ascites fluid (P > 0.0001) than pRad51-GFP control mice.

has been noted previously. Alternative nanoparticle DNA carriers such as poly(β-amino esters),42 or one of the many polymer-, dendrimer-, lipid-, metal- or silica-based carriers43 or modified PEI44 could be considered to improve delivery and biodistribution of pRad51-DTA. Another alternative would be a viral-mediated delivery of Rad51-DTA constructs. It would be important to test pRad51-DTA in an immunocompetent mouse model. It is possible that therapeutic effect will be enhanced as the immune system will accelerate clearance of DTA-poisoned cells. pRad51-DTA/jetPEI treatment significantly reduced the volume of ascites in mice with IP tumors. Malignant ascites are a serious clinical problem as patients with malignant ascites have a mean survival of only 20 weeks.45 Importantly, repeated administration of pRad51-DTA did not cause noticeable side effects, weight loss or signs of toxicity to vital organs. The treatment with IP-injected pRad51-DTA/jetPEI can benefit patients with ovarian, pancreatic, and other cancers located in the peritoneal cavity. Notably, IP administration of chemotherapy in advanced ovarian cancer patients gives better results than intravenous therapy alone.46 352

The Rad51-based construct can also be used to treat many other cancers if delivered intravenously, as Rad51 is overexpressed in the majority of cancers.5–9 We did not observe efficient gene delivery using intravenously injected jetPEI nanoparticles, but as mentioned previously, improved formulations of PEI or the use of alternative nanoparticles or viral delivery vehicles to increase biodistribution and transfection efficiency and reduce toxicity should be able to extend the utility of Rad51-based therapy to a wide spectrum of cancers through systemic delivery. Ovarian and pancreatic cancers are among the deadliest, and there is an urgent need for a better therapy. Therapies for these cancers fail because they either do not respond to initial therapy or develop recurrent disease. There has been great progress recently in the development of nanotechnology for cancer therapy,47 and in particular the use of nanoparticles for delivery of transcriptionally targeted suicide constructs.26,42,48 The ability to express suicide genes in the tumor cells only, offers a degree of specificity that cannot be achieved with traditional chemotherapy. Promoters employed in these studies were from HE4 and MSLN genes that are overexpressed in ovarian and pancreatic cancer,26,29 or from tissue-specific genes such as PSA for prostate www.moleculartherapy.org vol. 20 no. 2 feb. 2012

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cancer.42,48 Rad51 gene promoter adds to this arsenal and offers an important advantage over tissue-specific promoters. As tumor survival depends on Rad51 overexpression,10 and expression levels positively correlate with disease progression,13,14 the appearance of resistance is less likely than with tissue-specific gene expression that may become lost in advanced disease. Furthermore, Rad51 expression is tumorspecific and is tightly controlled in normal tissues. Thus, Rad51-based therapy is likely to have very low toxicity. Hence, we did not observe any unspecific expression or toxicity of Rad51 promoter constructs. In summary, our study suggests that it should be possible to use Rad51-based constructs delivered by nanoparticles for diagnosis and treatment of cancer. Considering widespread reliance of cancer cells on Rad51 overexpression, anticancer therapy based on Rad51 promoter may become a universal treatment for a wide variety of human cancers.

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(ATCC) was maintained in DMEM (Gibco, Grand Island, NY) supplemented with 10% FBS (Gibco), 1 × Pen/Strep (Gibco, Grand Island, NY), and 1× nonessential amino acids (Gibco). Cells were grown on treated polystyrene cell culture dishes (Corning) at 37 °C in 3% O2, 5% CO2, and 97% relative humidity in HERA Cell 240 incubators. Cells were split when they reached ~85% confluence. HeLa cells stably expressing firefly luciferase, termed HeLa-Luc, were made by transfecting HeLa cells at 50% confluence with 2 µg of Acl I (New England Biolabs, Ipswich, MA) linearized pCDNA3-Lucferase (Addgene plasmid 18964, William G. Kaelin) using

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Figure 7 Treatment with pRad51-DTA extends survival of mice with IP HeLa xenografts. Mice were injected IP with pRad51-DTA or the control pRad51-GFP nanoparticles. Arrows indicate the days of injection. The 50% survival point for pRad51-DTA-treated mice is extended by 90% over the control group (P = 0.0027). By day 42, all of the control mice had died (n = 8), while 80% of the pRad51-DTA-treated mice were alive (n = 6). *One pRad51-DTA-treated mouse was tumor-free after the treatment and remained tumor free until it was euthanized 9 months later.

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Fugene 6 transfection agent (Roche). Twenty-four hours after transfection, cells were selected for antibiotic resistance by replacing the media with new media containing Geneticin at a final concentrating of 2 mg/ml. After two weeks on selection, antibiotic resistant clones with strong luciferase activity were selected using the Luciferase Assay System (Promega, Madison, WI) on a GloMax 20/20 Luminometer (Promega). Mice. We used 8- to 12-week-old female athymic nude-Foxn1nu mice50 to

establish xenografts.

Xenografts. For SC xenografts, HeLa-Luc cells were harvested, counted on a

of six IP injections of 100 µg of therapy (pRad51-DTA; n = 8) or control (pRad51-GFP; n = 8) combined with 16 µl of in vivo-jetPEI transfection reagent in 500 µl of 5% glucose over a period of 15 days. Before, during and after these injections, mice were weighed and imaged for bioluminescence as previously described, with exposure times set at 5 seconds. Also, after six injections, tissues from the heart, lung, liver, stomach, spleen, and kidney were removed for histopathologic examination by H&E staining. End points of in vivo measurements were determined by University of Rochester UCAR and Animal Resources protocols when tumor burden caused visible signs of pain, discomfort, constipation, and inability to ambulate.

Z2 Particle Counter (Beckman Coulter) and resuspended at 5 × 105 cells/200 µl of 20% matrigel (BD Bioscience, Franklin Lakes, NJ) in PBS (Gibco). This 200 µl solution containing cells and matrigel was injected SC into the hind leg/lower back region of mice. SC xenografts were allowed to grow for 2 weeks in mice before imaging, measurements or treatments commenced. For IP xenografts, HeLa or HeLa-Luc cells were harvested and counted as above and resuspended at 5 × 105 cells/500 µl of PBS. Mice were injected twice with 250 µl of this cell solution into the lower right and lower left peritoneum to facilitate even dispersal of the 5 × 105 cells throughout the abdominal region. IP xenografts were allowed to grow 1–3 weeks before detection experiments were performed and 1 week before the start of the treatment.

Statistical Analysis. Graph Pad Software (http://www.graphpad.com) was

Tumor detection with pRad51-Luc/jetPEI. To detect the IP tumors with

ACKNOWLEDGMENTS

pRad51-Luc/jetPEI and determine potential toxicity, mice with IP xenografts were injected with 100 µg of pRad51-Luc combined with 16 µl of in vivo-jetPEI (Polyplus-Transfection) in a total volume of 500 µl of 5% glucose solution and incubated for at least 20 minutes at room temperature. Mice with and without cancer (n = 4, for each group) were then injected with 500 µl of the nanoparticle solution into the lower peritoneal cavity and prepared for imaging 24, 48, 72, and 144 hours after injection. Imaging was done using a Xenogen IVIS 100 Bioluminescent cooled CCD camera and images analyzed for light counts by Xenogen/Caliper Living Image Software Caliper Life Science, Hopkinton, MA. On each day of imaging, mice were first injected with 400 µl of 1.25% tribromoethanol anesthetic and then with 200 µl of 15 µg/µl D-luciferin (Caliper Life Sciences, Hopkinton, MA) in PBS. Five minutes after the injection of the D-luciferin, images were taken at 1-minute exposures and light counts analyzed by the Living Image Software (Xenogen/Caliper). Ex vivo imaging of organs and tumors was performed similar to above, except imaging was done 48 hours after pRad51-Luc/jetPEI injection. After injection of D-luciferin, the mice were killed and the abdominal and chest cavity opened in order to remove heart, lung, liver, stomach, spleen, kidney, and where appropriate, tumors. Organs and tumors were placed on black construction paper and imaged exactly as described above. This was completed on four mice with IP xenografts and four cancer-free mice for IP delivery. Treatment of SC tumors with pRad51-DTA/jetPEI. After HeLa-Luc xeno-

grafts were established as previously described, mice were given a total of six intratumoral injections of 20 µg of therapy (pRad51-DTA; n = 10) or control (pRad51-GFP; n = 10) combined with 3.2 µl of in vivo-jetPEI transfection reagent in 100 µl of 5% glucose over a period of 28 days. pRad51-GFP was selected as the negative control due to this construct being identical to pRad51-DTA except for cytotoxic DTA expression being replaced with inert GFP expression. Tumors were measured with calipers to determine tumor volume along with mice being imaged for in vivo bioluminescence at 2-second exposure times, during and after the course of six gene therapy injections. Tumors were removed for visual observations, mass determination, and H&E staining. End points of in vivo measurements were determined by the University of Rochester Animal Resources protocols when tumor burden in mice caused signs of severe distress or tumors caused ulceration of the skin.

Treatment of IP tumors with pRad51-DTA/jetPEI. After HeLa-Luc xenografts were established as previously described, mice were given a total

354

used to determine P values and statistical significance using the t-test calculator. Microsoft Excel was used to calculate mean and standard deviations.

SUPPLEMENTARY MATERIAL Figure S1.  Rad51 protein levels and promoter activity are increased in human prostate cancer cells. Figure S2.No recurrence of subcutaneous tumors 12 months after the final pRad51-DTA/jetPEI injection. Materials and Methods We thank Mitra Azadniv and Mathew Au for technical support with in vivo imaging and David Hicks and Diana Scott for help with histopathologic staining and interpretation. This work was supported by grants the National Institutes of Health AG031227 and AG27237 to V.G. Authors declare no conflict of interest.

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