Manganese Superoxide Dismutase-plasmid/liposome Research Articles

660. Manganese Superoxide Dismutase Plasmid Liposomes (MnSOD-PL) Protects the Murine Oral Cavity from Irradiation Induced Mucositis

660. Manganese Superoxide Dismutase Plasmid Liposomes (MnSOD-PL) Protects the Murine Oral Cavity from Irradiation Induced Mucositis

Abstract 2502: Swallowed small molecule GS-nitroxide, JP-4-039, protects the irradiated esophagus

Abstract Irradiation induced esophagitis is a dose limiting factor in the treatment of lung cancer, and limits irradiation dose escalation. Intraesophageal manganese superoxide dismutase plasmid liposomes has been shown to protect against irradiation esophagitis (Tarhini, et.al. Human Gene Therapy, in press). A new small molecule drug recently has shown irradiation protection and mitigation in vitro. The new drug (JP4-039) is a nitroxide attached to a mitochondrial targeting sequence. Drug was delivered to the top of the esophagus and the mouse swallowed the F15 formulation, consisting of soy PC: Tween-80: L-glutamyl dioleylamide (4:1:1 w/w) and 8 mg/ml of JP4-039 in PBS. C57BL/6NHsd mice swallowed 100 µl water followed by 100 µl F15-JP4-039 (0.4 mg/mouse) ten minutes before 28 Gy to the upper body. F15-JP4-039 resulted in a significant increase in survival compared to irradiated controls. F15-JP4-039 produced a 30 day survival of 75% compared to 30% for irradiation control (p = 0.0315). The JP4-039 nitroxide signal, detected by EPR, allowed demonstration that drug was in whole esophagus and in the esophageal stem cells. F15-JP4-039 intraorally treated mice were sacrificed at 10 min, 30 min, 1 hr or 5 hr after swallowing the F15-JP4-039, esophagus removed, single cell suspensions made, frozen, and analyzed by EPR. At 10 min, 2971.3 fmoles of JP4-039/million cells were detected, and still detected out to 5 hours, 1081 fmoles/million cells, after irradiation. Sorted side population cells (SP), enriched for stem cells, and nonside population cells (NSP) isolated by flow cytometer were analyzed at 10 min after swallow, by EPR for JP4-039 uptake. The SP cells had 3666.7 fmoles per million cells and the NSP cells had 73.7 fmoles nitroxide per million cells demonstrating that drug had reached stem cells. Thus, intraesophageal administration of small molecule antioxidant GS-nitroxide reduces murine radiation esophagitis, and may be a valuable translational radiation protector to ameliorate clinical esophagitis in lung cancer patients. Supported by 2U19A1068021-06 and RO1-CA83876-06 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2502. doi:10.1158/1538-7445.AM2011-2502

A Phase I Study of Concurrent Chemotherapy (Paclitaxel and Carboplatin) and Thoracic Radiotherapy with Swallowed Manganese Superoxide Dismutase Plasmid Liposome Protection in Patients with Locally Advanced Stage III Non-Small-Cell Lung Cancer

Manganese superoxide dismutase (MnSOD) is a genetically engineered therapeutic DNA/liposome containing the human MnSOD transgene. Preclinical studies in mouse models have demonstrated that the expression of the human MnSOD transgene confers protection of normal tissues from ionizing irradiation damage. This is a phase I study of MnSOD plasmid liposome (PL) in combination with standard chemoradiation in surgically unresectable stage III non-small-cell lung cancer. Chemotherapy (carboplatin and paclitaxel) was given weekly (for 7 weeks), concurrently with radiation. MnSOD PL was swallowed twice a week (total 14 doses), at three dose levels: 0.3, 3, and 30 mg. Dose escalation followed a standard phase I design. Esophagoscopy was done at baseline, day 4, and 6 weeks after radiation with biopsies of the squamous lining cells. DNA was extracted and analyzed by PCR for the detection of the MnSOD transgene DNA. Ten patients with AJCC stage IIIA (three) and IIIB (seven) completed the course of therapy. Five had squamous histology, two adenocarcinoma, one large cell, and two not specified. Patients were treated in three cohorts at three dose levels of MnSOD PL: 0.3 (three patients), 3 (three patients), and 30 mg (four patients). The median dose of radiation was 77.7 Gy (range 63-79.10 Gy). Overall response rate for the standard chemoradiation regimen was 70% (n = 10). There were no dose-limiting toxicities reported in all three dosing tiers. It is concluded that the oral administration of MnSOD PL is feasible and safe. The phase II recommended dose is 30 mg.

A Phase I Study Demonstrating Manganese Superoxide Dismutase Plasmid Liposome Complex (MnSOD-PL) Reduction of Esophagitis following Standard Chemoradiation in Surgically Unresectable Stage III NSCLC

Purpose/Objective(s): Esophageal toxicity has been accepted reluctantly, as a necessary side effect of the benefits of chemoradiotherapy for lung cancer. MnSOD-PL is a genetically engineered therapeutic DNA/liposome complex containing the human MnSOD transgene. Preclinical studies in mouse models have demonstrated that the expression of the human MnSOD transgene confers protection of normal tissues from ionizing irradiation damage. A phase I trial has been completed demonstrating MnSOD-PL may reduce esophagitis following chemoradiation in surgically unresectable Stage III non-small cell lung cancer.

A phase I study of concurrent chemotherapy (paclitaxel and carboplatin) and thoracic radiotherapy with swallowed manganese superoxide dismutase (MnSOD) plasmid liposome (PL) protection in patients with locally advanced stage III non-small cell lung cancer.

e17501 Background: Esophageal toxicity has been accepted reluctantly, as a necessary side effect of the benefits of chemoradiotherapy for lung cancer. MnSOD is a genetically engineered therapeutic DNA/liposome containing the human MnSOD transgene. Preclincial studies in mouse models have demonstrated that the expression of the human MnSOD transgene confers protection of normal tissues from ionizing irradiation damage. Methods: Chemotherapy (carboplatin AUC=2 and paclitaxel 45 mg/m2 intravenously) was given weekly (for 7 weeks), concurrently with radiation therapy for pts with locally advanced NSCLC. MnSOD PL was swallowed twice a week (total 14 doses), at 3 dose levels: 0.3, 3, and 30 mg. Dose escalation followed a standard phase I design. Esophagoscopy was done at baseline, day 4 and 6 weeks after radiation with biopsies of the squamous lining cells. DNA was extracted and analyzed by PCR for the detection of the MnSOD transgene DNA. Results: Nine patients with stage IIIA (2) and IIIB (7) completed the course of therapy per study protocol. Five had squamous histology, 2 adenocarcinoma, and 2 not specified. These patients were treated in 3 different cohorts at 3 dose levels of MnSOD PL: 0.3, 3, and 30 mg. The median dose of radiation was 77.7Gy (range 63-79.10Gy). Overall response rate for the standard chemo-radiation regimen was 67% (n=9). Grade 2 dyspepsia and a grade 1 rash in one patient was considered possibly related to MnSOD PL. None of the other reported toxicities were considered possibly, probably, or definitely related to MnSOD PL. There were no dose-limiting toxicities reported in all 3 dosing tiers. MnSOD PCR product was not detected in the esophageal samples. Conclusions: The oral administration of MnSOD PL is feasible and safe. The phase II recommended dose is 30 mg. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Valentis, Inc.

514. Nitric Oxide Synthase One Homologous Recombinant Negative (NOS1 -/-) Mice Are Protected from Thoracic Irradiation Induced Seizures by Esophageal Administration of Manganese Superoxide Dismutase Plasmid Liposome Complex (MnSOD-PL)

514. Nitric Oxide Synthase One Homologous Recombinant Negative (NOS1 -/-) Mice Are Protected from Thoracic Irradiation Induced Seizures by Esophageal Administration of Manganese Superoxide Dismutase Plasmid Liposome Complex (MnSOD-PL)

Radioprotection In Vitro and In Vivo by Minicircle Plasmid Carrying the Human Manganese Superoxide Dismutase Transgene

Manganese superoxide dismutase plasmid liposomes (MnSOD-PL) confer organ-specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new minicircle construct was tested. Minicircle MnSOD was purified and then cotransfected into 32D cl 3 murine interleukin-3-dependent hematopoietic progenitor cells along with another plasmid carrying the neo gene. Cells were selected in G418 (50 microg/ml) and cloned by limiting dilution. Biochemical analysis of minicircle MnSOD-transfected cells showed an MnSOD biochemical activity level of 5.8 +/- 0.5 U/mg compared with 2.7 +/- 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D-mc-MnSOD cells were as radioresistant as full-length MnSOD-PL transgene-expressing 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 +/- 0.2 and n = 4.6 +/- 0.2, respectively, compared with 1.5 +/- 0.5 for 32D cl 3 cells; p = 0.007). C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DsRed-PL control, full-length MnSOD-PL, or blank-PL and then were irradiated 24 hr later with 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL showed increased survival compared with control mice or mice treated with mc-DsRed-PL (p = 0.0003 and 0.039, respectively), and comparable to full-length MnSOD-PL. Intravenous, systemic administration of mc-MnSOD-PL protected mice from total body irradiation (9.75 Gy). Therefore, minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.

Radioprotection In Vitro and In Vivo by Mini Circle Plasmid Containing the Human Manganese Superoxide Dismutase (MnSOD) Transgene

Manganese superoxide dismutase-plasmid liposomes (MnSOD-PL) confer organ specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new mini circle construct was tested. Mini circle-MnSOD was purified and then co-transfected into 32D cl 3 murine IL-3 dependent hematopoietic progenitor cells along with another plasmid containing the neo gene. Cells were selected in G418 (50 μg/ml) and cloned by limiting dilution. Biochemical analysis of mini circle MnSOD transfected cells showed 5.8 ± 0.5 U/mg MnSOD biochemical activity compared to 2.7 ± 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D -MC-MnSOD cells were comparably radioresistant to full length MnSOD-PL transgene 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 ± 0.2 and n = 4.6 ± 0.2 respectively compared to 1.5 ± 0.5 for 32D cl 3, p = 0.007). C57BL/6NHsd mice received intra-oral MC-MnSOD-PL, MC-DS-red-PL control, full length MnSOD-PL, or blank-PL then were irradiated 24 hours later to 31 Gy to the esophagus. Mice receiving MC-MnSOD-PL showed increased survival compared to control mice or mice treated with MC-DS-red-PL (p = 0.0099, or 0.039, respectively), and comparable to full length MnSOD-PL. Intravenous, systemic administration of MC-MnSOD-PL protected mice from 9.75 Gy total body irradiation. Therefore, mini circle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.

Increased Engraftment of Bone Marrow Progenitors of Esophageal Stem Cells by Intraesophageal Administration of Manganese Superoxide Dismutase Plasmid/Liposomes (MnSOD-PL) 24 hr before Irradiation.

We hypothesized that following irradiation the esophageal microenvironment would have increased reactive oxygen species (ROS) production and that antioxidant gene therapy would decrease ROS resulting in increased engraftment of esophageal stem cells. Decreased ROS production following irradiation has been shown to result from expression of Manganese Superoxide Dismutase (MnSOD) transgene product delivered by plasmid/liposome complexes. We tested whether increased expression of MnSOD following intraesophageal administration of Manganese Superoxide Dismutase Plasmid/Liposomes (MnSOD-PL) increased engraftment of esophageal stem cells. Female C57BL/6NHsd mice which had received MnSOD-PL (100 μg plasmid DNA) intraesophageally 24 hr earlier were irradiated along with control mice to 29 Gy to the esophagus. The mice were injected intravenously with bone marrow from male ROSA (Lac-Z, G418r transgenic mice) five days later (time of greatest esophageal basal cell apoptosis). Fourteen days later the mice (first generation) were sacrificed, esophagus removed, single cell suspensions prepared, stained with Hoescht dye and sorted by flow cytometer into side population (SP) and nonside population (NSP) cells. Half of the SP and NSP cells isolated from the MnSOD-PL treated mice were injected into a second generation of recipient MnSOD-PL treated as well as control mice. The remaining cells were grown in either methylcellulose or in 96 well plates in G418. Second generation recipient mice were sacrificed 14 days after transplant and the SP and NSP cells isolated and grown in either methylcellulose or in 96 well plates as described above. Tissue culture grown G418r colonies were stained for donor (Lac-Z+) cells. In first generation recipient mice, those pre-treated with MnSOD-PL had increased expression of Lac-Z+ SP and NSP cells per esophagus (38.4 ± 4.2% and 21.8 ± 5.5%, respectively) compared to the control irradiated mice (21.4 ± 4.4%, p=0.0105, and 5.2 ± 2.4%, p=0.0277, respectively). Second generation MnSOD-PL treated recipient mice, transplanted with esophageal SP cells from MnSOD-PL treated mice had more Lac-Z+ SP cells per esophagus (47.9 + 3.5%) than that detected in mice injected with NSP cells from the same donors (22.3 ± 2.4%, p < 0.0001), control irradiation only donor SP cells (22.3 ± 2.0, p = 0.0001) or control irradiation only donor NSP cells (9.6 ± 2.5%, p < 0.0001). To further demonstrate that MnSOD-PL pretreatment resulted in better engraftment of donor cells, the first and second generation sorted cells were analyzed for the presence of the Y chromosome. In the first generation, MnSOD-PL pretreatment resulted in an increased percent of Y+ SP and NSP cells (23.9 ± 4.6, p = 0.0318, and 11.4 ± 4.0, respectively) compared to the control SP and NSP (10.5 ± 3.5 or 7.9 ± 3.2, respectively). In the second generation recipient mice, MnSOD-PL pre-treatment prior to first generation MnSOD-PL SP cells resulted in more Y+ cells in the SP fraction compared to control irradiated recipients (15.2 ± 4.4 or 4.5 ± 2.3, respectively, p = 0.0467). Therefore, swallowed MnSOD-PL pretreatment improves esophageal engraftment of serially transferable marrow progenitors of esophageal stem cells.

Minicircle Plasmid Containing the Human Manganese Superoxide Dismutase (MnSOD) Transgene Confers Radioprotection to Hematopoietic Progenitor Cell Line 32Dcl3.

Manganese superoxide dismutase plasmid/liposomes (MnSOD-PL) delivered by intratracheal, intraesophageal, or intraoral routes in rodent models has been demonstrated to confer organ specific ionizing irradiation protection. In addition intravenous injections of MnSOD-PL protect mice from whole body irradiation. Currently a seven week phase I/II clinical trial is in progress in lung cancer patients consisting of twice weekly swallowed MnSOD-PL for protection of the esophagus from chemoradiotherapy damage. To prepare for a potential clinical trial of systemic MnSOD-PL for radioprotection in humans, plasmid bacterial sequences were removed to diminish the immune response. The human MnSOD transgene attached to a CMV promoter and a poly A tail was inserted in the site between Spe I and Xho I into a eukaryotic expression cassette located in the p2ØC31 plasmid. The plasmid contains an endonuclease I-SceI gene which can be cleaved resulting in the formation of two minicircle plasmids. The smaller minicircle contains the eukaryotic expression cassette but no bacterial sequences while the larger minicircle plasmid contains the plasmid bacterial backbone. The minicircle MnSOD was purified and then co-transfected into 32Dcl3 murine hematopoietic progenitor cells with a plasmid containing the neo gene. Cells were selected in G418 (50μg/ml G418) and cloned by limiting dilution into 96 well plates. The clones were expanded and analyzed by PCR for the presence of the human MnSOD transgene using primers specific for the human MnSOD. One clone was chosen and the MnSOD biochemical activity was determined. The 32Dcl3 cells had a specific MnSOD activity of 2.7 ± 0.1 U/mg protein compared to 5.8 ± 0.5 U/mg protein for the 32D-mc-MnSOD clone (p=0.0039). To determine if the MnSOD transgene in the minicircle DNA retained radioprotective capacity 32D-mc-MnSOD, a clone transfected with a pRK5 plasmid containing the human MnSOD transgene (2C6), and parent 32Dcl3 cells were irradiated to doses of 0–8 Gy then grown at 37° C for 7 days at which time colonies of greater than 50 cells were counted. The data was analyzed by linear quadratic and single-hit, multi-target models. The 32D-mc-MnSOD cells were more radioresistant than 32Dcl3 cells as demonstrated by an increased shoulder on the irradiation survival curve (n = 4.8 ± 0.2 compared to 1.5 ± 0.5, respectively, p = 0.0078). In contrast, there was no significant reduction in the shoulder of the survival curve comparing 32D-mc-MnSOD and 2C6 (n = 4.8 ± 0.2 and 4.6 ± 0.2, respectively). In vivo C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DS-red-PL, MnSOD-PL or Blank-PL, swallowed the plasmid/liposome complexes and were then irradiated 24 hr later along with control mice to 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL had increased survival compared to both the control mice or mice treated with mc-DS-red-PL (p = 0.0099 or 0.0391, respectively). There was significant and equivalent improved survival of mice injected with mc-MnSOD-PL compared to full length MnSOD-PL. Therefore minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection to cells in vitro and the esophagus in vivo compared to a fully intact plasmid containing the MnSOD transgene.

Intraesophageal Administration of Manganese Superoxide Dismutase Plasmid/liposomes (MnSOD-PL) Pre-irradiation Results in Increased Engraftment of Bone Marrow Progenitors of Esophageal Stem Cells

Intraesophageal Administration of Manganese Superoxide Dismutase Plasmid/liposomes (MnSOD-PL) Pre-irradiation Results in Increased Engraftment of Bone Marrow Progenitors of Esophageal Stem Cells

Gene therapy approaches for stem cell protection

Cytotoxic exposure of bone marrow and other non-hematopoietic organs containing self-renewing stem cell populations is associated with damage to the supportive microenvironment. Recent evidence indicates that radical oxygen species resulting from the initial oxidative stress persist for months after ionizing irradiation exposure of tissues including oral cavity, esophagus, lung and bone marrow. Antioxidant gene therapy using manganese superoxide dismutase plasmid liposomes has provided organ-specific radiation protection associated with delay or prevention of acute and late toxicity. Recent evidence has suggested that manganese superoxide dismutase transgene expression in cells of the organ microenvironment contributes significantly to the mechanism of protection. Incorporating this knowledge into designs of novel approaches for stem cell protection is addressed in the present review.

349. Pretreatment of the Esophagus with Manganese Superoxide Dismutase-Plasmid Liposome (MnSOD-PL) Complex Increases Migration and Proliferation of Marrow Stem Cell Progenitors of Esophageal Squamous Epithelium

Following irradiation of the murine esophagus there is detectable migration of marrow origin stem cells into the esophagus associated with recovery from irradiation-induced damage. Irradiation of the esophagus or the bone marrow results in stably increased detectable production of reactive oxygen species (ROS) for weeks. We hypothesized that reducing the production of ROS following irradiation may allow for increased stable recruitment of stem cells into the irradiated areas. C57BL/6NHsd female mice were administered MnSOD-PL (100 |[mu]|g plasmid DNA) by intraesophageal injection 24 hours before irradiation to 29 Gy. Control mice were irradiated to 29 Gy. The mice were then injected I.V. with bone marrow cells isolated from ROSA (LacZ+) male mice. Fourteen days after irradiation mice were sacrificed and the esophagus excised and sorted into side population (SP) and non-SP cells. In mice that received MnSOD-PL prior to irradiation, 40% of the non-SP cells were LacZ+ compared to 10% in mice that received irradiation only. Sorting of esophageal SP cells which include multilineage stem cells showed 20% from MnSOD-PL pretreated then irradiated mice were LacZ+ compared to 8% of the SP cells from control irradiated mice. In addition, mice that received MnSOD-PL intraesophageally 24 hours before irradiation also had decreased apoptotic cells. At day 5 following irradiation, 20% of control irradiated esophageal cells were undergoing apoptosis; whereas, in mice that were pre-treated with MnSOD-PL there was less than 1% apoptosis. In conclusion, administration of MnSOD-PL results in increased migration of stem cells from the bone marrow into the esophagus and decreased apoptosis in the esophagus. The data suggest that MnSOD-PL pre- irradiation treatment of the esophagus results in increased marrow cell engraftment due to reduction of ROS.

Systemic Intravenous (IV) as Well as Local Administration of Manganese Superoxide Dismutase-Plasmid/Liposome (MnSOD-PL) Displays No Detectable Toxicity While Offering Protection From Irradiation-Induced Damage

Systemic Intravenous (IV) as Well as Local Administration of Manganese Superoxide Dismutase-Plasmid/Liposome (MnSOD-PL) Displays No Detectable Toxicity While Offering Protection From Irradiation-Induced Damage

In a Model of Irradiation Retreatment of the Lung (Simulating Radiotherapy for Lung Cancer Local Recurrence), Normal Lung Tolerance is Increased by Administration of Manganese Superoxide Dismutase-Plasmid/Liposome (MnSOD-PL)

Purpose/Objective: In 30–50% of unresectable non-small cell lung cancer patients treated initially with irradiation, locoregional recurrence necessitates a second course of irradiation. The retreatment dose delivered must be reduced to maintain normal tissue tolerance. We have previously demonstrated that intratracheal injection of MnSOD-PL prevents irradiation damage following initial irradiation treatments. Now we demonstrate that administration of MnSOD-PL is effective in preventing irradiation-induced damage to the lung during retreatment.

Inhalation delivery of manganese superoxide dismutase-plasmid/liposomes protects the murine lung from irradiation damage

Intratracheal injection of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) complexes has been demonstrated to delay the onset and reduce the extent of ionizing irradiation-induced murine pulmonary organizing alveolitis/fibrosis. To facilitate translation of this modality to clinical fractionated radiotherapy, inhalation delivery of MnSOD-PL was developed using an ultrasonic nebulizer. Transgene product was quantitated by immunohistochemical quantitation and pulmonary tissue levels of MnSOD biochemical activity. C57BL/6NHsd female mice demonstrated a plasmid dose-dependent increased expression of MnSOD transgene product over the range of 250 microg-2.5 mg of MnSOD-PL administered over a constant 5 min interval. Delivery of a constant concentration of 500 microg of MnSOD-PL with varying times of administration ranging from 0.5 to 10 min demonstrated optimal MnSOD expression at 5 min. Mice pretreated by inhalation delivery of MnSOD-PL demonstrated significantly improved survival after 20 Gy single fraction irradiation to both lungs compared to LacZ-PL inhalation-treated or irradiated control mice. Mice receiving 10 fractions of 3.5 cGy demonstrated increased pulmonary MnSOD transgene product activity by a protocol of every Monday-Wednesday or daily inhalation of MnSOD-PL. Thus, inhalation radioprotective gene therapy using MnSOD-PL provides a practical and effective method for delivery of lung-specific radioprotection during fractionated radiotherapy protocols in a mouse model.

Cell phenotype specific kinetics of expression of intratracheally injected manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) during lung radioprotective gene therapy.

Intratracheal (IT) injection of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) complexes prior to whole lung irradiation of C57BL/6J mice provides significant protection from acute and chronic irradiation damage. We determined the duration of increased MnSOD biochemical activity and differential expression of a hemagglutinin (HA) epitope-tagged MnSOD transgene. HA-MnSOD-PL was IT injected at doses of 0-1000 microg, and mice were killed 1,2,3 or 4 days later. Other groups of mice were irradiated to 20 Gy to the pulmonary cavity 24 h after injection and killed at the same time points as non-irradiated mice. Both non-irradiated and irradiated groups of mice showed increased MnSOD biochemical activity with plasmid dose that plateaued at 100 microg of MnSOD plasmid DNA. In control mice, MnSOD biochemical activity decreased at 2, 3 or 4 days after injection. In irradiated mice, MnSOD biochemical activity decreased at day 2 but increased on days 3 and 4. HA-MnSOD expression decreased in broncheoalveolar macrophages and alveolar type-II cells 3 days after injection in non-irradiated and irradiated mice, but remained elevated in endothelial and epithelial cells past 4 days. The data provide a rationale for every second-day administration of intrapulmonary MnSOD-PL in clinical trials of radioprotective gene therapy. This should be sufficient to provide radioprotection during radiation treatments.

Pre-but not post irradiation intratracheal injection of manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) protects the lung from irradiation damage

Pre-but not post irradiation intratracheal injection of manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) protects the lung from irradiation damage

Pulmonary irradiation-induced expression of VCAM-I and ICAM-I is decreased by manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) gene therapy.

Pulmonary toxicity is a major complication of total body irradiation used in preparation of patients for bone marrow transplantation. The mechanism of the late pulmonary damage manifested by fibrosis is unknown. In C57BL/6NHsd mice, manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) intratracheal injection 24 hours prior to 20 Gy single-fraction irradiation to both lungs significantly reduced late irradiation damage. Single intratracheal injections of MnSOD-PL, at concentrations as low as 250 microg of plasmid DNA, in a constant volume of 78 microL of liposomes, reduced late damage. To determine whether a slowly proliferating population of cells in the lung was responsible for initiation of fibrosis and was altered by MnSOD-PL therapy, 20 Gy total lung-irradiated mice were examined at serial time points for bromodeoxyuridine (BrdU) uptake in sites of cell division. There was low-level, but nonsignificant, increased cell proliferation detected at 80 days, with a significant increase at 100 days, 120 days, and at the time of death. Immunohistochemical assay for up-regulation of adhesion molecules associated with recruitment, transendothelial migration, and proliferation of bronchoalveolar macrophages revealed significant up-regulation of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) at 100 days with further increases up to the time of death. Increases were first detected in endothelin-positive endothelial cells. MnSOD-PL administration prior to irradiation decreased both BrdU incorporation and delayed expression of VCAM-1 and ICAM-1. The data indicate that the appearance of late irradiation-induced pulmonary fibrosis is associated with the up-regulation of adhesion molecules and suggest that potential targets for intervention may focus on the pulmonary vascular endothelium.

Manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) administration protects mice from esophagitis associated with fractionated radiation.

Intraesophageal administration of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) prior to single fraction radiation has been shown to protect mice from lethal esophagitis. In our study, C3H/HeNsd mice received fractionated radiation in two protocols: (i) 18 Gy daily for four days with MnSOD-PL administration 24 hr prior to the first and third fraction, or (ii) 12 Gy daily for six days with MnSOD-PL 24 hr prior to the first, third, and fifth fraction. Control radiated mice received either no liposomes only or LacZ (bacterial beta-galactosidase gene)-plasmid/liposome (LacZ-PL) by the same schedules. We measured thiol depletion and lipid peroxidation (LP) in whole esophagus and tested the effectiveness of a new plasmid, hemagglutinin (HA) epitope-tagged MnSOD (HA-MnSOD). In fractionation protocols, mice receiving MnSOD-PL, but not LacZ-PL (200 microl of plasmid/liposomes containing 200 microg of plasmid DNA), showed a significant reduction in morbidity, decreased weight loss, and improved survival. Four and seven days after 37 Gy single fraction radiation, the esophagus demonstrated a significant increase in peroxidized lipids and reduction in overall antioxidant levels, reduced thiols, and decreased glutathione (GSH). These reductions were modulated by MnSOD-PL administration. The HA-MnSOD plasmid product was detected in the basal layers of the esophageal epithelium 24 hr after administration and provided significant radiation protection compared to glutathione peroxidase-plasmid/liposome (GPX-PL), or liposomes containing MnSOD protein, vitamin E, co-enzyme Q10, or 21-aminosteroid. Thus, MnSOD-PL administration significantly improved tolerance to fractionated radiation and modulated radiation effects on levels of GSH and lipid peroxidation (LP). These studies provide further support for translation of MnSOD-PL treatment into human esophageal radiation protection.