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Simian virus 40 vectors for pulmonary gene therapy.

Eid L, Bromberg Z, El-Latif MA, Zeira E, Oppenheim A, Weiss YG - Respir. Res. (2007)

Bottom Line: Moreover, our results showed vector presence in type II alveolar cells.The vector did not induce significant cellular immune response.These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology and Critical Care Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, 91120, Israel. luminita25@yahoo.com

ABSTRACT

Background: Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS). Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40) vectors for pulmonary gene therapy.

Methods: Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP). SV40 vectors carrying the luciferase reporter gene (SV/luc) were administered intratracheally immediately after sepsis induction. Sham operated (SO) as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C). Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector.

Results: Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response.

Conclusion: In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

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In vivo detection of luminescence. Detection was performed with a CCCD camera (exposure time 2 min) 48 hours following intratracheal administration of 300 μl of 1.25 × 108 IU/ml SV/luc. 2a: A typical 2CLP rat showing luc activity over right lung and tracheostomy areas (right), left lung and tracheostomy areas (middle), and SO rat showing very low luc activity over pulmonary area (left). The color scales values are indicated on the wedge.2b: Graphic representation of means and standard deviations of 2CLP + SV/luc treated rats. Rt – right lung, Lt – left lung, Trach – tracheostomy area. The values represent the mean for 8 animals and the bars represent standard deviations. The signal over the lung area was different from the signal of tracheal area at P < 0.03 for both left and right lungs.2c: Comparison of SV/luc and Ad/luc transduction (exposure time 2 min) at 48 hours in a rat given intratracheal 300 μl of 1.25 × 108IU/ml SV/luc (left) and a rat given intratracheal 300 μl of 1 × 109 IU/ml Ad/luc (right).2d: Graphic representation of means and standard deviations of 2CLP + SV/luc (8 treated rats, black bar) and 2CLP+Ad/luc (3 treated rats, gray bar).
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Figure 2: In vivo detection of luminescence. Detection was performed with a CCCD camera (exposure time 2 min) 48 hours following intratracheal administration of 300 μl of 1.25 × 108 IU/ml SV/luc. 2a: A typical 2CLP rat showing luc activity over right lung and tracheostomy areas (right), left lung and tracheostomy areas (middle), and SO rat showing very low luc activity over pulmonary area (left). The color scales values are indicated on the wedge.2b: Graphic representation of means and standard deviations of 2CLP + SV/luc treated rats. Rt – right lung, Lt – left lung, Trach – tracheostomy area. The values represent the mean for 8 animals and the bars represent standard deviations. The signal over the lung area was different from the signal of tracheal area at P < 0.03 for both left and right lungs.2c: Comparison of SV/luc and Ad/luc transduction (exposure time 2 min) at 48 hours in a rat given intratracheal 300 μl of 1.25 × 108IU/ml SV/luc (left) and a rat given intratracheal 300 μl of 1 × 109 IU/ml Ad/luc (right).2d: Graphic representation of means and standard deviations of 2CLP + SV/luc (8 treated rats, black bar) and 2CLP+Ad/luc (3 treated rats, gray bar).

Mentions: At 24 hours, Luciferase activity was not detected in any animal groups. We explain this finding by the time needed for the reporter gene to express. However, at 48 hours, luc activity (detected as luminescence by the CCCD camera) was seen over the lung areas in 2CLP animals given SV/luc (Figure 2a). Low levels of luc activity were detected in the tracheostomy region, confirming a degree of vector affinity for airway epithelium (Figure 2b). No luc activity was detected in rats given PBS. No significant in vivo luc activity was detected in the heart, liver, spleen or kidneys of the animals given SV/luc.


Simian virus 40 vectors for pulmonary gene therapy.

Eid L, Bromberg Z, El-Latif MA, Zeira E, Oppenheim A, Weiss YG - Respir. Res. (2007)

In vivo detection of luminescence. Detection was performed with a CCCD camera (exposure time 2 min) 48 hours following intratracheal administration of 300 μl of 1.25 × 108 IU/ml SV/luc. 2a: A typical 2CLP rat showing luc activity over right lung and tracheostomy areas (right), left lung and tracheostomy areas (middle), and SO rat showing very low luc activity over pulmonary area (left). The color scales values are indicated on the wedge.2b: Graphic representation of means and standard deviations of 2CLP + SV/luc treated rats. Rt – right lung, Lt – left lung, Trach – tracheostomy area. The values represent the mean for 8 animals and the bars represent standard deviations. The signal over the lung area was different from the signal of tracheal area at P < 0.03 for both left and right lungs.2c: Comparison of SV/luc and Ad/luc transduction (exposure time 2 min) at 48 hours in a rat given intratracheal 300 μl of 1.25 × 108IU/ml SV/luc (left) and a rat given intratracheal 300 μl of 1 × 109 IU/ml Ad/luc (right).2d: Graphic representation of means and standard deviations of 2CLP + SV/luc (8 treated rats, black bar) and 2CLP+Ad/luc (3 treated rats, gray bar).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2238754&req=5

Figure 2: In vivo detection of luminescence. Detection was performed with a CCCD camera (exposure time 2 min) 48 hours following intratracheal administration of 300 μl of 1.25 × 108 IU/ml SV/luc. 2a: A typical 2CLP rat showing luc activity over right lung and tracheostomy areas (right), left lung and tracheostomy areas (middle), and SO rat showing very low luc activity over pulmonary area (left). The color scales values are indicated on the wedge.2b: Graphic representation of means and standard deviations of 2CLP + SV/luc treated rats. Rt – right lung, Lt – left lung, Trach – tracheostomy area. The values represent the mean for 8 animals and the bars represent standard deviations. The signal over the lung area was different from the signal of tracheal area at P < 0.03 for both left and right lungs.2c: Comparison of SV/luc and Ad/luc transduction (exposure time 2 min) at 48 hours in a rat given intratracheal 300 μl of 1.25 × 108IU/ml SV/luc (left) and a rat given intratracheal 300 μl of 1 × 109 IU/ml Ad/luc (right).2d: Graphic representation of means and standard deviations of 2CLP + SV/luc (8 treated rats, black bar) and 2CLP+Ad/luc (3 treated rats, gray bar).
Mentions: At 24 hours, Luciferase activity was not detected in any animal groups. We explain this finding by the time needed for the reporter gene to express. However, at 48 hours, luc activity (detected as luminescence by the CCCD camera) was seen over the lung areas in 2CLP animals given SV/luc (Figure 2a). Low levels of luc activity were detected in the tracheostomy region, confirming a degree of vector affinity for airway epithelium (Figure 2b). No luc activity was detected in rats given PBS. No significant in vivo luc activity was detected in the heart, liver, spleen or kidneys of the animals given SV/luc.

Bottom Line: Moreover, our results showed vector presence in type II alveolar cells.The vector did not induce significant cellular immune response.These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology and Critical Care Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, 91120, Israel. luminita25@yahoo.com

ABSTRACT

Background: Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS). Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40) vectors for pulmonary gene therapy.

Methods: Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP). SV40 vectors carrying the luciferase reporter gene (SV/luc) were administered intratracheally immediately after sepsis induction. Sham operated (SO) as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C). Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector.

Results: Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response.

Conclusion: In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

Show MeSH
Related in: MedlinePlus