Phase I/II and Phase II Studies of Targeted Gene Delivery In Vivo: Intravenous Rexin-G for Chemotherapy-resistant Sarcoma and Osteosarcoma
Authors: Sant P Chawla, Victoria S Chua, Lita Fernandez, Doris Quon, Andreh Saralou, William C Blackwelder, Frederick L Hall, Erlinda M Gordon
Journal: Molecular Therapy (2009)

Rexin-G, a pathotropic nanoparticle bearing a cytocidal cyclin G1 construct was tested in a phase I/II study for chemotherapy-resistant sarcomas and a phase II study for chemotherapy-resistant osteosarcoma. Twenty sarcoma patients and 22 osteosarcoma patients received escalating doses of Rexin-G intravenously from 8 × 1011 to 24 × 1011 colony forming units (cfu)/cycle. Treatment was continued if there was ≤ grade 1 toxicity. No dose-limiting toxicity (DLT) was observed, and no vector DNA integration, replication-competent retrovirus (RCR) or vector-neutralizing antibodies were noted. In the phase I/II study, 3/6 patients had stable disease (SD) at the lowest dose; median progression-free survival (PFS) was 1.2 months, and overall survival (OS), 3.3 months. At higher doses, 10/14 patients had SD; median PFS was 3.7 months and median OS, 7.8 months. In this phase I/II study, a dose–response relationship with Rexin-G dosage was observed for progression-free and OS times (P = 0.02 and 0.005, respectively). In the phase II study, 10/17 evaluable patients had SD, median PFS was ≥3 months and median OS, 6.9 months. These studies suggest that Rexin-G is safe, may help control tumor growth, and may possibly improve survival in chemotherapy-resistant sarcoma and osteosarcoma. Rexin-G, a pathotropic nanoparticle bearing a cytocidal cyclin G1 construct was tested in a phase I/II study for chemotherapy-resistant sarcomas and a phase II study for chemotherapy-resistant osteosarcoma. Twenty sarcoma patients and 22 osteosarcoma patients received escalating doses of Rexin-G intravenously from 8 × 1011 to 24 × 1011 colony forming units (cfu)/cycle. Treatment was continued if there was ≤ grade 1 toxicity. No dose-limiting toxicity (DLT) was observed, and no vector DNA integration, replication-competent retrovirus (RCR) or vector-neutralizing antibodies were noted. In the phase I/II study, 3/6 patients had stable disease (SD) at the lowest dose; median progression-free survival (PFS) was 1.2 months, and overall survival (OS), 3.3 months. At higher doses, 10/14 patients had SD; median PFS was 3.7 months and median OS, 7.8 months. In this phase I/II study, a dose–response relationship with Rexin-G dosage was observed for progression-free and OS times (P = 0.02 and 0.005, respectively). In the phase II study, 10/17 evaluable patients had SD, median PFS was ≥3 months and median OS, 6.9 months. These studies suggest that Rexin-G is safe, may help control tumor growth, and may possibly improve survival in chemotherapy-resistant sarcoma and osteosarcoma. IntroductionOsteosarcoma is a rare malignant tumor of bone usually affecting adolescents and young adults.1Surveillance, Epidemiology, and End Results (SEER) Program Prevalence database: "US estimated 29-year LD counts on prevalence on 1/1/2004 by duration" for Bones and Joints Cancer. National Cancer Institute, DCCPS, Surveillance Research Program, Statistical Research and Applications Branch, based on the November 2006 SEER data submission. 2007; (www.seer.cancer.gov)Google Scholar Current combination chemotherapy, radiation therapy, and surgery, have significantly improved the survival of affected persons. Effective drugs include doxorubicin, cisplatin, methotrexate, and ifosfamide.2Longhi A Errani C De Paolis M Mercuri M Bacci G Primary bone osteosarcoma in the pediatric age: state of the art.Cancer Treat Rev. 2006; 32: 423-436Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar However, no standard second line therapy exists for those who relapse or fail to achieve a second remission with the best reported overall survival (OS) of 0.6 years.3Kempf-Bielack B Bielack SS Jürgens H Branscheid D Berdel WE Exner GU et al.Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS).J Clin Oncol. 2005; 23: 559-568Crossref PubMed Scopus (505) Google Scholar Additionally, the long-term sequelae and secondary malignancies associated with toxic chemotherapy in children and adolescents augment the need for more effective and less toxic therapies.3Kempf-Bielack B Bielack SS Jürgens H Branscheid D Berdel WE Exner GU et al.Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS).J Clin Oncol. 2005; 23: 559-568Crossref PubMed Scopus (505) Google ScholarSoft tissue sarcoma is also a rare cancer of mesenchymal tissues.4Surveillance, Epidemiology, and End Results (SEER) Program Prevalence database: "US estimated 29-year LD counts on prevalence on 1/1/2004 by duration" for Soft Tissue including Heart Cancer.National Cancer Institute, DCCPS, Surveillance Research Program, Statistical Research and Applications Branch, based on the November 2006 SEER data submission. 2007; (www.seer.cancer.gov)Google Scholar Current treatment for soft tissue sarcoma includes surgical resection, radiotherapy, and chemotherapy.5Milano A Apice G Ferrari E Fazioli F de Rosa V de Luna AS et al.New emerging drugs in soft tissue sarcoma.Crit Rev Oncol Hematol. 2006; 59: 74-84Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Despite improvements in the control of local disease, a significant number of patients ultimately die of metastatic disease following radical surgery due to a lack of effective adjuvant treatments.6Mocellin S Rossi CR Brandes A Nitti D Adult soft tissue sarcomas: conventional therapies and molecularly targeted approaches.Cancer Treat Rev. 2006; 32: 9-27Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,7Geer RJ Woodruff J Casper ES Brennan MF Management of small soft-tissue sarcoma of the extremity in adults.Arch Surg. 1992; 127: 1285-1289Crossref PubMed Scopus (149) Google Scholar,8Karakousis CP Zografos GC Radiation therapy for high grade soft tissue sarcomas of the extremities treated with limb-preserving surgery.Eur J Surg Oncol. 2002; 28: 431-436Abstract Full Text PDF PubMed Scopus (32) Google Scholar,9Pisters PW Leung DH Woodruff J Shi W Brennan MF Analysis of prognostic factors in 1,041 patients with localized soft tissue sarcomas of the extremities.J Clin Oncol. 1996; 14: 1679-1689Crossref PubMed Scopus (1007) Google Scholar,10Cormier JN Pollock RE Soft tissue sarcomas.CA Cancer J Clin. 2004; 54: 94-109Crossref PubMed Scopus (407) Google Scholar Only three drugs—doxorubicin, dacarbazine, and ifosfamide—are consistently associated with response rates of ≥20%, and after failure of these drugs, patients with advanced soft tissue sarcoma have few therapeutic options.11Demetri GD Major developments in the understanding and treatment of soft-tissue sarcomas in adults.Curr Opin Oncol. 1998; 10: 343-347Crossref PubMed Scopus (11) Google ScholarAmong the leading alternatives to traditional chemotherapeutics, both cancer immunotherapy and cancer gene therapy strategies are currently under active clinical investigation.12Meyers PA Schwartz CL Krailo MD Healey JH Bernstein ML Betcher D et al.Osteosarcoma: the addition of muramyl tripeptide to chemotherapy improves overall survival–a report from the Children's Oncology Group.J Clin Oncol. 2008; 26: 633-638Crossref PubMed Scopus (571) Google Scholar,13Mori K Rédini F Gouin F Cherrier B Heymann D Osteosarcoma: current status of immunotherapy and future trends (Review).Oncol Rep. 2006; 15: 693-700PubMed Google Scholar,14Witlox MA Lamfers ML Wuisman PI Curiel DT Siegal GP Evolving gene therapy approaches for osteosarcoma using viral vectors: review.Bone. 2007; 40: 797-812Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Rexin-G, the first and, so far, only targeted gene therapy vector bearing a cytocidal dominant negative cyclin G1 construct,15Waehler R Russell SJ Curiel DT Engineering targeted viral vectors for gene therapy.Nat Rev Genet. 2007; 8: 573-587Crossref PubMed Scopus (540) Google Scholar is currently being tested simultaneously in three phase I/II clinical trials for chemotherapy-resistant metastatic sarcoma, pancreatic cancer, and breast cancer, and in one phase II study for chemotherapy-resistant metastatic osteosarcoma in the United States.In this article, we report on the results of two independent studies (i) evaluating the overall safety and potential efficacy of Rexin-G in chemotherapy-resistant sarcoma in a phase I/II study, and (ii) confirming the efficacy and overall safety of Rexin-G in chemotherapy-resistant osteosarcoma in a phase II study.ResultsPatient demographicsTable 1 shows the patient demographics for the phase I/II sarcoma study and the phase II osteosarcoma study. There were nine different types of sarcomas enrolled in the phase I/II study, including leiomyosarcoma (n = 5), osteosarcoma (n = 3), synovial cell sarcoma (n = 3), liposarcoma (n = 3), mixed malignant Mullerian tumor of ovary (n = 2), Ewing's sarcoma (n = 1), chondrosarcoma (n = 1), malignant fibrous histiocytoma (n = 1), and malignant spindle cell sarcoma (n = 1). Ninety-five percent of patients in the phase I/II sarcoma study had metastatic disease and a median of four previous chemotherapy regimens, whereas 100% of patients in the phase II osteosarcoma study had metastatic disease and a median of four previous chemotherapy regimens. Eastern Cooperative Oncology Group score was 0–1. Table 2 shows the performance scoring system employed.Table 1Patient demographicsPhase I/II sarcoma study (n = 20)Age, years Median50.5 Range(15.0-70.0)Gender Female8 (40%) Male12 (60%)Race White15 (75%) Black1 (5%) Hispanic3 (15%) Asian1 (5%)Disease stage Metastatic19 (95%) Nonmetastatic1 (5%)Performance score 120 (100%)No. of previous chemotherapy regimens Median4 Range1-10Phase II osteosarcoma study (n = 22)Age, years Median33 Range(7.0-68.0)Gender Female5 (23 %) Male17 (77%)Race White9 (41%) Black4 (18%) Hispanic7 (32%) Asian1 (4.5%) Others1 (4.5%)Disease stage Metastatic22 (100%)Performance score 122 (100%)No. of previous chemotherapy regimens Median4 Range1-9 Open table in a new tab Table 2Eastern Cooperative Oncology Group performance scoring systemDescriptionScoreFully active, able to carry on all predisease performance without restriction.0Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light housework, office work.1Ambulatory and capable of all self care but unable to carry out any work activities. Up and about >50% of waking hours.2Capable of only limited self care, confined to bed or chair >50% of waking hours.3Completely disabled. Cannot carry on any self care. Totally confined to bed or chair.4 Open table in a new tab Analysis of safetyTreatment-related adverse events are listed in Table 3. There was no dose-limiting toxicity (DLT) or organ-related toxicity. In the phase I/II study, at dose level 0, study drug-related adverse events included grade 1 chills in one patient, and grade 1–2 fatigue in two patients, whereas at dose level I–II, one patient had grade 1 presyncope. In the phase II study, one patient experienced grade 1 photophobia, and two patients had grade 1 fatigue which was considered possibly study drug-related.Table 3Treatment-related adverse eventsDose level (n)Adverse eventNo. of patients with grade 1 toxicityNo. of patients with grade 2 toxicityPhase I/II sarcoma study0 (6)Chills1−Fatigue11I-II (14)Presyncope1−Phase II osteosarcoma studyI-II (22)Photophobia1−Fatigue2− Open table in a new tab Correlative analysis showed no neutralizing antibodies detected in the patients' sera. However, 3/14 patients who received dose level II developed weakly positive antibodies against the gp70 envelope protein by western blot analysis, 3–4 months after treatment initiation. Further, all DNA samples from patients' peripheral blood lymphocytes (PBLs) were found to be negative for replication-competent retrovirus (RCR), and no vector DNA integration was detected.Analysis of efficacyIn the phase I/II study, six patients were treated at dose level I and 14 patients at dose level I–II (combined). Table 4 (Sarcoma study) shows the tumor responses using standard response evaluation criteria in solid tumors (RECIST), International positron emission tomography (PET), and CHOI criteria for each dose level.Table 4Effects of Rexin-G on tumor response by RECIST, PET, and CHOI criteria, on PFS and OSDose level (n)Tumor response by RECISTTumor response by PETTumor response by CHOIMedian PFS by RECIST (months)Median OS (months)Phase I/II sarcoma study 0 (6) 1 × 1011 cfu two times a week3 SD, 3PD1 PR, 4 SD, 1 PD2 PR, 4 SD1.23.3 I-II (14) 1-2 × 1011 cfu three times a week10 SD, 4 PD4 PR, 9 SD, 1 PD7 PR, 7S D3.77.8 Phase II osteosarcoma study (17) 2 × 1011 cfu three times a week10 SD, 7 PD4 PR, 8 SD, 5 PD3 PR, 12 SD, 2 PD>36.9a22 Osteosarcoma patients were started on Rexin-G, 5 of whom had <1 treatment cycle or did not return for evaluation; all 5 of these patients died within 2 months of starting Rexin-G therapy. Median OS is shown for the 17 evaluable patients; median OS for all 22 enrolled patients was 6.0 months.Abbreviations: OS, overall survival; PD, progressive disease; PET, positron emission tomography; PFS, progression-free survival; RECIST, response evaluation criteria in solid tumors; SD, stable disease.a 22 Osteosarcoma patients were started on Rexin-G, 5 of whom had <1 treatment cycle or did not return for evaluation; all 5 of these patients died within 2 months of starting Rexin-G therapy. Median OS is shown for the 17 evaluable patients; median OS for all 22 enrolled patients was 6.0 months. Open table in a new tab In the phase I/II sarcoma study, at dose level 0, 3/6 patients had stable disease (SD) whereas at dose level I–II, 10/14 patients had SD by standard RECIST criteria. Interestingly, using the International PET criteria, at dose level 0, 1/6 patients had a partial response (PR), and 4/6 patients had SD, and at dose level I–II, 4/14 patients had PRs, and 9/14 patients had SD. Similarly, using CHOI criteria, at dose level 0, 2/6 patients had PRs, and 4/6 patients had SD, and at dose level I–II, 7/14 patients had PRs and 7/14 patients had SD. These findings suggest that the International PET and CHOI criteria may be more sensitive indicators of tumor responses to Rexin-G treatment than the standard RECIST.The median progression-free survival (PFS) times by RECIST was 1.2 months for dose level 0 versus 3.7 months for dose levels I–II (combined), and the corresponding median overall survival times were 3.3 months for dose level 0 versus 7.8 months for dose levels I–II (Table 4). Using the Cox regression model and Kaplan–Meier analysis,16Kaplan E Meier P Nonparametric estimation for incomplete observations.J Am Stat Assoc. 1958; 53: 457-481Crossref Scopus (47708) Google Scholar a dose–response relationship was observed between Rexin-G dosage and both PFS and OS (P = 0.02 for PFS by RECIST, 0.004 by PET and 0.003 by CHOI criteria; P = 0.005 for OS; Figures 1a–c and 2a).Figure 2Analyses of overall survival of patients in the phase I/II sarcoma study and the phase II osteosarcoma study. Kaplan-Meier analyses show the survival curves of patients in (a) the phase I/II sarcoma study and (b) the phase II osteosarcoma study.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the phase II study, 17 of 22 patients completed at least one treatment cycle and were considered evaluable. Five patients were taken off study without completing one treatment cycle due to disease-related complications or a personal decision to discontinue participation and return home. The tumor responses based on RECIST, International PET criteria and CHOI criteria are shown in Table 4. Using standard RECIST, 10/17 (59%) evaluable patients had SD, whereas using International PET criteria, 4/17 patients had PRs, and 8/17 patients had SD, totaling 70% of patients having PRs or SD. Using CHOI criteria, 3/17 had PRs and 12/17 had SD totaling 88% of patients having PRs or SD. Therefore, tumor responses were significantly higher in the Rexin-G treated group compared to those expected of historical controls (with ≤5% having a positive response if untreated; P < 0.025).3Kempf-Bielack B Bielack SS Jürgens H Branscheid D Berdel WE Exner GU et al.Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS).J Clin Oncol. 2005; 23: 559-568Crossref PubMed Scopus (505) Google Scholar Median PFS was ≥3 months, and OS was 6.9 months (6 months for all 22 enrolled patients).Two patients are disease-free after surgical resection of residual tumors, with three cycles of Rexin-G given before and 3–4 cycles after surgery. These patients enjoy sustained surgical remissions 10.7 and 9.7 months after treatment initiation, and >6 months after surgery. In one patient, histopathologic examination of two resected tumor nodules showed a 2.5 cystic nodule and rare osteosarcoma cells and a 0.9 cm calcified nodule and residual osteosarcoma cells with rare mitosis (Figure 3). Of note, the 2.5 cm tumor had increased in size and was diagnosed as progressive disease (PD) by RECIST but a PR by PET criteria. This finding suggests that RECIST criteria may not be a reliable indicator of tumor response to Rexin-G treatment in osteosarcoma.Figure 3Histopathological findings in the lung metastases of a patient with chemotherapy-resistant osteosarcoma after Rexin-G treatment. Representative H&E stained tissue sections from resected residual lung nodules in a Rexin-G treated patient. (a) 2.5 cm cystic mass with organized clot and rare osteosarcoma cells; (b) 0.9 cm calcified tumor mass with reparative fibrosis, immune infiltrate and osteosarcoma cells with rare mitosis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionRexin-G is a pathotropic nanoparticle incorporating a collagen-matrix binding motif on its surface and bearing a dominant negative cyclin G1 construct as its genetic payload. When injected intravenously, Rexin-G accumulates in cancerous lesions wherein collagen is exposed by tumor invasion, neoangiogenesis or extracellular remodeling, thus increasing the effective local concentration in tumors in the vicinity of target cancer cells. Rexin-G causes cell death by blocking the cell cycle in G1 phase and inducing apoptosis of proliferative cancer cells and associated neovasculature in preclinical studies.17Skotzko M Wu L Anderson WF Gordon EM Hall FL Retroviral vector-mediated gene transfer of antisense cyclin G1 (CYCG1) inhibits proliferation of human osteogenic sarcoma cells.Cancer Res. 1995; 55: 5493-5498PubMed Google Scholar,18Gordon EM Chen ZH Liu L Whitley M Liu L Wei D et al.Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice.Hum Gene Ther. 2001; 12: 193-204Crossref PubMed Scopus (47) Google ScholarIn the phase I/II sarcoma study, tumor control was suggested by longer PFS and OS in the higher dose cohorts compared to the low dose cohort. In the phase II osteosarcoma study, a significant proportion of patients had SD by RECIST or PRs/SD by PET and CHOI criteria, suggesting increased sensitivity of PET and CHOI criteria for early detection of tumor responses to Rexin-G treatment. No DLT or organ-related toxicity was observed with prolonged use of Rexin-G. Correlative analysis showed no vector-neutralizing antibodies detected in serum, and no evidence of vector DNA integration nor RCR in PBLs, further attesting to the overall safety of Rexin-G. Surgical resection of residual tumors following Rexin-G treatment—which enabled histological examination, as well as strategic tumor debulking—followed by the administration of additional postoperative treatment, serves to underscore the potential clinical benefit of Rexin-G in neoadjuvant/adjuvant protocols.In conclusion, the objectives of the phase I/II Study have been met. The overall safety of Rexin-G administered within the defined dose ranges was clearly established by the absence of DLT or vector safety concerns. Further, the objectives of the phase II study for osteosarcoma have also been met, wherein tumor responses by RECIST of 10 SD of 17 evaluable patients (59%; 95% confidence interval, 33–82%) and a median PFS of ≥3 months and a median OS of 6.9 months in patients treated with at least 1 cycle, and 6 months in all enrolled patients, were demonstrated. Taken together, the results of these two independent well-defined phase I/II and phase II studies suggest that Rexin-G may help control tumor growth, and may possibly improve progression-free and OS times in chemotherapy-resistant sarcoma and osteosarcoma, thus hopefully providing the required elements for consideration of accelerated approval for osteosarcoma by the US Food and Drug Administration.19Oncologic "Threshold of Credibility" Paradigm being explored at FDA (2004): "The Pink Sheet" Prescription Pharmaceuticals and Biotechnology 66: p. 15Google Scholar,20Guidance for industry clinical trial endpoints for the approval of cancer drugs and biologics DHHS, FDA, CDER, CBER 2007 1 19Google ScholarMaterials and MethodsStudy design. The phase I/II sarcoma study employed a modified cohort of three design.21Storer BE Design and analysis of phase I clinical trials.Biometrics. 1989; 45: 925-937Crossref PubMed Scopus (607) Google Scholar Three patients were treated at each dose level with expansion to six patients per cohort if DLT was observed in one of the three first patients at each dose level. Each cohort was also expanded to six or seven patients if significant biologic activity was noted. Maximum tolerated dose was defined as the highest safely tolerated dose, where ≤1 patient experienced DLT, with the next higher dose level having at least two patients who experienced DLT. DLT was defined as any grade 3, 4, or 5 adverse event considered possibly, probably, or definitely related to the study drug, excluding grade 3 absolute neutrophil count lasting <72 hours, grade 3 alopecia, or any grade 3 or worse nausea, vomiting, or diarrhea where the patient did not receive maximal supportive care.22The NCI Common Terminology Criteria for Adverse Events Version 3 Cancer Therapy Evaluation Program DCTD, NCI, NIH, DHHS. 2003; (http://ctep.cancer.gov): 1-72Google ScholarAdaptive Trial Design: A phase II efficacy component was incorporated in the phase I/II sarcoma study by allowing additional treatment cycles to be given if the patient had ≤grade 1 toxicity. Further, across the board dose escalation was allowed by the Food and Drug Administration when the safety of preceding dose levels had been documented. The principal investigator, S.P.C., was also allowed to recommend surgical resection/debulking after at least one treatment cycle had been administered.For the phase II osteosarcoma study, the optimal dose was derived from the results of the phase I/II sarcoma study. The number of patients enrolled was deduced from a statistical projection of 25% of patients achieving SD or better by International PET criteria and/or RECIST, assuming that ≥95% of patients with chemo-resistant osteosarcoma will progress if untreated.3Kempf-Bielack B Bielack SS Jürgens H Branscheid D Berdel WE Exner GU et al.Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS).J Clin Oncol. 2005; 23: 559-568Crossref PubMed Scopus (505) Google Scholar Clinical toxicity was also reported according to severity and relatedness to the study drug.Clinical objectives/endpoints. The primary objective of the phase I/II study for sarcoma is to determine the clinical toxicity of escalating doses of Rexin-G as defined by patient performance status, toxicity assessment score, hematologic, and metabolic profiles. The secondary objectives include (i) evaluation of the potential of Rexin-G for evoking an immune response, recombination events and/or unwanted vector integration in nontarget organs, and (ii) identification of an antitumor response to Rexin-G.The primary objective of the phase II study for osteosarcoma is to confirm the efficacy of Rexin-G in terms of tumor responses (complete response (CR), PR, or SD) by International PET criteria and by RECIST. A favorable tumor response was recorded if the patient had a CR, PR, or SD. The secondary objectives are (i) to confirm the efficacy of Rexin-G in terms of a median PFS of ≥1 month and a median OS of ≥6 months, and (ii) to evaluate the safety/toxicity of continued use of Rexin-G and the potential of Rexin-G for evoking an immune response, recombination events and/or unwanted vector integration in nontarget organs (PBLs).Patient population. The phase I/II study included patients with a pathologic diagnosis of sarcoma who were refractory to standard chemotherapy, and the phase II study, patients with recurrent or metastatic osteosarcoma who were considered refractory to known therapies. Histologic or cytologic confirmation at diagnosis or recurrence was required. Inclusion criteria consisted of an Eastern Cooperative Oncology Group performance score of 0–1 and adequate hematologic, hepatic, and kidney function. Exclusion criteria included human immunodeficiency virus, hepatitis B virus, or hepatitis C virus positivity, clinically significant ascites, medical, or psychiatric conditions that could compromise successful adherence to the protocol, and unwillingness to employ effective contraception during treatment with Rexin-G and for 6 weeks following treatment completion. The clinical protocols (C07-103 and C07-110) were reviewed and approved by the Western Institutional Review Board, Olympia WA 98502 and the Biosafety Committee of Epeius Clinical Research Unit, San Marino CA 91108.Patient recruitment and assignment. The phase I/II and phase II studies using Rexin-G for sarcoma and osteosarcoma were registered on www.clinicaltrials.gov (NCT00505713 and NCT00572130, respectively) within 1 week of study initiation, and patients were recruited on a first come first served basis after appropriate screening procedures were conducted. Written informed consent, in accordance with the Declaration of Helsinski protocols, was obtained from each patient at the time of enrollment. The phase I/II sarcoma study is an open label study using escalating doses of Rexin-G. Six patients were enrolled at dose level 0 followed by a dose escalation in seven patients at dose level I and another seven patients at dose level II when safety/toxicity data in at least three patients at lower dose levels had been recorded and no DLT was encountered. The phase II osteosarcoma study enrolled 22 patients at dose level II. Table 1 shows the demographics of all enrolled patients.Treatment. Rexin-G is a nonreplicative "pathotropic" (pathology-targeted) retroviral vector bearing a collagen-binding motif on its envelope protein23Hall FL Liu L Zhu NL Stapfer M Anderson WF Beart RW et al.Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor.Hum Gene Ther. 2000; 11: 983-993Crossref PubMed Scopus (51) Google Scholar and encoding an N-terminal deletion mutant construct of human cyclin G124Xu F Prescott MF Liu PX Chen ZH Liau G Gordon EM et al.Long term inhibition of neointima formation in balloon-injured rat arteries by intraluminal instillation of a matrix-targeted retroviral vector bearing a cytocidal mutant cyclin G1 construct.Int J Mol Med. 2001; 8: 19-30PubMed Google Scholar under the control of the moloney murine leukemia virus long terminal repeat promoter. The Rexin-G vector is produced by transient co-transfection of three separate plasmids in 293T cells (human kidney 293 cells transformed with the SV40 large T antigen) maintained as a fully validated master cell bank.17Skotzko M Wu L Anderson WF Gordon EM Hall FL Retroviral vector-mediated gene transfer of antisense cyclin G1 (CYCG1) inhibits proliferation of human osteogenic sarcoma cells.Cancer Res. 1995; 55: 5493-5498PubMed Google Scholar,25Gordon EM Cornelio GH Lorenzo CC Levy JP Reed RA Liu L et al.First clinical experience using a 'pathotropic' injectable retroviral vector (Rexin-G) as intervention for stage IV pancreatic cancer.Int J Oncol. 2004; 24: 177-185PubMed Google Scholar,26Gordon EM Lopez FF Cornelio GH Lorenzo CC Levy JP Reed RA et al.Pathotropic nanoparticles for cancer gene therapy Rexin-G IV: three-year clinical experience.Int J Oncol. 2006; 29: 1053-1064PubMed Google Scholar The final product exhibits a viral titer of 5 × 109 colony forming units (cfu) per milliliter, a biologic potency of 50–70% growth inhibitory activity in A375 melanoma cells, <550 bp residual DNA, no detectable E1A or SV40 large T antigen, and no detectable RCR.27Guidance for Industry: Supplemental guidance on testing for replication competent retrovirus in retroviral vector based gene therapy products and during follow-up of patients in clinical trials using retroviral vectors (2006) UUSPHS, FDA, CBER 1-13Google Scholar The clinical vector is stored in volumes of 23 ml in 30 ml vials or 40 ml in 150 ml cryobags in a −80 ± 10 °C freezer marked Biohazard. Preparation of the Rexin-G vector for patient administration consisted of rapid thawing of the vector in the vial or cryobag in a 34 °C waterbath. The vector was thawed 15–30 minutes prior to infusion into the patient, and was infus