Lee Zourelias

Ultrasound-assisted nonviral gene transfer of AQP1 to the irradiated minipig parotid gland restores fluid secretion

Xerostomia is a common side effect of ionizing radiation used to treat head and neck cancer. A groundbreaking Phase I human clinical trial using Adenoviral gene transfer of Aquaporin-1 (AQP1) to a single salivary gland of individuals suffering from radiation-induced xerostomia has recently been reported. Unfortunately, the limitations of the Adenoviral vector system used in this pioneering trial preclude its advancement to a Phase II trial, and we have thus undertaken to evaluate the therapeutic potential of ultrasound-assisted nonviral gene transfer (UAGT) as an alternative means of delivering AQP1 gene therapy to the salivary gland by comparing head-to-head with the canonical Adenoviral vector in a swine model. Swine irradiated unilaterally with a 10-Gy electron beam targeted at the parotid gland suffered from significant, sustained hyposalivation that was bilateral, despite irradiation being confined to the targeted gland. Unilateral AQP1 gene therapy with UAGT resulted in bilateral restoration of stimulated salivary flow at 48 h and 1 week post treatment (1.62±0.48 ml and 1.87±0.45 ml) to preinjury levels (1.34±0.14 ml) in a manner comparable to Adenoviral delivery (2.32±0.6 ml and 1.33±0.97 ml). UAGT can replace the Adenoviral vector as a means of delivering AQP1 gene therapy in the irradiated swine model, and it is a candidate for advancement to a Phase I human clinical trial.

α-Galactosidase A Expressed in the Salivary Glands Partially Corrects Organ Biochemical Deficits in the Fabry Mouse Through Endocrine Trafficking

Fabry disease is caused by an X-linked deficiency of the lysosomal enzyme α-galactosidase A (GLA) and has been treated successfully with enzyme replacement therapy (ERT). Gene therapy has been proposed as an alternative to ERT due to the presumed advantages of continuous, endogenous production of the therapeutic enzyme. GLA production in the liver and its therapeutic efficacy in the Fabry mouse have been demonstrated previously with various viral vector systems. In consideration of the potential advantages of using the salivary glands as endogenous GLA biosynthesis sites, we explored the feasibility of this approach in the Fabry mouse. GLA -/0 or -/- mice received an adenoviral vector (2 × 10(10) or 1 × 10(9) viral particles) expressing GLA to the right submandibular gland via oral cannulation of the submandibular duct. Four days later, animals were sacrificed; saliva, plasma, kidney, liver, and brain were collected and assayed using ELISA, Western blot, and a GLA enzymatic activity assay using both traditional fluorescence methods and isotope dilution mass spectrometry by following the U.S. EPA Method 6800. GLA activity was significantly elevated in the serum and liver of both treatment groups, and improvement in the kidney was marginally significant (P < 0.069) in the high-dose group. Notably, we found that liver and salivary gland produce different glycoforms of the GLA transgene. Only small numbers of adenoviral genomes were observed in the livers of treated animals, but in four of 14 in the high-dose groups, liver levels of adenovirus exceeded 20 copies/μg, indicating that the sequestration in the salivary gland was imperfect at high doses. Taken together, these results indicate that the salivary gland-based gene therapy for Fabry disease is promising, and further studies with advanced viral vector gene delivery systems (e.g., adeno-associated virus) for long-term treatment appear to be warranted.

Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles.

In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis.

IL-17 sequestration via salivary gland gene therapy in a mouse model of Sjogren’s syndrome suppresses disease-associated expression of the putative autoantigen Klk1b22

IntroductionIL-17 has a putative role in the pathophysiology of Sjogren’s syndrome (SS) and has been shown to be upregulated in the salivary glands of affected individuals. Sequestration of IL-17 with Adenoviral-mediated gene therapy has previously shown a benefit upon the SS-like phenotype in the Aec1/Aec2 mouse model. We sought to understand the proteomic consequences of IL-17 sequestration in the salivary gland of this mouse model as a means of illuminating the role of IL-17 in SS-like disease.MethodsUltrasound-assisted gene transfer (UAGT) was utilized to express a fusion protein composed of the extracellular portion of the IL-17 receptor fused to fragment of crystallization (Fc) in the submandibular glands of Aec1/Aec2 mice at 8 weeks of age. After confirming expression of the fusion protein and local and systemic sequestration of IL-17, proteomic profiling was performed on submandibular glands of a treated cohort of Aec1/Aec2 animals relative to the background strain and sham-treated animals.ResultsThe most notable proteomic signatures of IL-17 sequestration on SS-like disease-related proteins were Kallikrein-related peptidases, including the putative autoantigen Klk1b22. IL-17 sequestration also notably led to an isoelectric shift, but not a molecular weight shift, of Kallikrein-1, attributed to phosphorylation.ConclusionNon-viral IL-17 sequestration gene therapy in the salivary gland is feasible and downregulates expression of a putative SS autoantigen in the Aec1/Aec2 mouse.

Three-dimensional micro computed tomography analysis of the lung vasculature and differential adipose proteomics in the Sugen/hypoxia rat model of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare disease characterized by significant vascular remodeling. The obesity epidemic has produced great interest in the relationship between small visceral adipose tissue depots producing localized inflammatory conditions, which may link metabolism, innate immunity, and vascular remodeling. This study used novel micro computed tomography (microCT) three-dimensional modeling to investigate the degree of remodeling of the lung vasculature and differential proteomics to determine small visceral adipose dysfunction in rats with severe PAH. Sprague-Dawley rats were subjected to a subcutaneous injection of vascular endothelial growth factor receptor blocker (Sugen 5416) with subsequent hypoxia exposure for 3 weeks (SU/hyp). At 12 weeks after hypoxia, microCT analysis showed a decrease in the ratio of vascular to total tissue volume within the SU/hyp group (mean ± standard deviation: 0.27 ± 0.066; P = 0.02) with increased vascular separation (0.37 ± 0.062 mm; P = 0.02) when compared with the control (0.34 ± 0.084 and 0.30 ± 0.072 mm). Differential proteomics detected an up-regulation of complement protein 3 (C3; SU/hyp: control ratio = 2.86) and the adipose tissue–specific fatty acid binding protein-4 (FABP4, 2.66) in the heart adipose of the SU/hyp. Significant remodeling of the lung vasculature validates the efficacy of the SU/hyp rat for modeling human PAH. The upregulation of C3 and FABP4 within the heart adipose implicates small visceral adipose dysfunction. C3 has been associated with vascular stiffness, and FABP4 suppresses peroxisome proliferator–activated receptor, which is a major regulator of adipose function and known to be downregulated in PAH. These findings reveal that small visceral adipose tissue within the SU/hyp model provides mechanistic links for vascular remodeling and adipose dysfunction in the pathophysiology of PAH.

MicroCT analysis of vascular morphometry: a comparison of right lung lobes in the SUGEN/hypoxic rat model of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare disease characterized by significant vascular remodeling within the lung. Clinical computed tomography (CT) scans are routinely used to aid in PAH diagnosis. Animal models, including the Sugen-hypoxic rat model (SU/hyp), of PAH closely mimic human PAH development. We have previously used micro-computed tomography (microCT) to find extensive right lung vascular remodeling in the SU/hyp. We hypothesized that the individual right lung lobes may not contribute equally to overall lung vascular remodeling. Sprague-Dawley rats were subjected to a subcutaneous injection of vascular endothelial growth factor receptor blocker (Sugen 5416) and subsequently exposed to chronic hypoxic conditions (10% O2) for three weeks. Following perfusion of the lung vasculature with an opaque resin (Microfil), the right lung lobes were microCT-imaged with a 10-µm voxel resolution and 3D morphometry analysis was performed separately on each lobe. As expected, we found a significantly lower ratio of vascular volume to total lobe volume in the SU/hyp compared with the control, but only in the distal lobes (inferior: 0.23 [0.21–0.30] versus 0.35 [0.27–0.43], P = 0.02; accessory: 0.27 [0.25–0.33] versus 0.37 [0.29–0.43], P = 0.06). Overall, we observed significantly fewer continuous blood vessels and reduced vascular density while having greater vascular lumen diameters in the distal lobes of both groups (P < 0.05). In addition, the vascular separation within the SU/hyp lobes and the vascular surface area to volume ratio were significantly greater in the SU/hyp lobes compared with controls (P < 0.03). Results for the examined parameters support the overall extensive vascular remodeling in the SU/hyp model and suggest this may be lobe-dependent.

In vivo Endocrine Secretion of Prostacyclin Following Expression of a Cyclooxygenase-1/Prostacyclin Fusion Protein in the Salivary Glands of Rats Via Nonviral Gene Therapy

Pulmonary arterial hypertension (PAH) is a progressive disease that culminates in right heart failure and death. Prostacyclin (PGI2) and its derivatives are effective treatments for PAH when administered as continuous parenteral infusions. This treatment paradigm requires medical sophistication, and patients are at risk for complications from an indewelling catheter; drug interruptions may result in rebound pulmonary hypertension and death. We hypothesized that the salivary gland can be repurposed into an endogenous production site for circulating PGI2 through the expression of a fusion protein embodying cyclooxygenase-1 (Cox1) and prostacyclin synthase (PGIS) domains. We utilized ultrasound-assisted gene transfer, a nonviral gene transfer strategy that achieves robust gene transfer to the salivary gland. We initially found that Cox1-PGIS expression in livers of mice using an adenoviral vector dramatically increased circulating PGI2 relative to untreated rats or rats treated with PGIS alone. We then utilized ultrasound-assisted gene transfer to express Cox1-PGIS in the submandibular glands of rats and showed a significant elevation of circulating PGI2 that corresponded to approximately 30% of that seen in humans undergoing intravenous infusion therapy for PAH. These results suggest the feasibility of gene therapy to drive endogenous biosynthesis of PGI2 as a therapeutic strategy for the treatment of PAH.

In vivo endocrine secretion of prostacyclin following expression of a Cox-1/PGIS fusion protein in the salivary glands of rats via non-viral gene therapy

Pulmonary arterial hypertension (PAH) is a progressive disease that culminates in right heart failure and death. Prostacyclin (PGI2) and its derivatives are effective treatments for PAH when administered as continuous parenteral infusions. This treatment paradigm requires medical sophistication, and patients are at risk for complications from an indewelling catheter; drug interruptions may result in rebound pulmonary hypertension and death. We hypothesized that the salivary gland can be repurposed into an endogenous production site for circulating PGI2 through the expression of a fusion protein embodying cyclooxygenase-1 (Cox1) and prostacyclin synthase (PGIS) domains. We utilized ultrasound-assisted gene transfer, a nonviral gene transfer strategy that achieves robust gene transfer to the salivary gland. We initially found that Cox1-PGIS expression in livers of mice using an adenoviral vector dramatically increased circulating PGI2 relative to untreated rats or rats treated with PGIS alone. We then utilized ultrasound-assisted gene transfer to express Cox1-PGIS in the submandibular glands of rats and showed a significant elevation of circulating PGI2 that corresponded to approximately 30% of that seen in humans undergoing intravenous infusion therapy for PAH. These results suggest the feasibility of gene therapy to drive endogenous biosynthesis of PGI2 as a therapeutic strategy for the treatment of PAH.

Erratum: Ultrasound-assisted non-viral gene transfer to the salivary glands (Gene Therapy (2010) 17 (1318-1324) DOI: 10.1038/gt.2010.86)

Correction to: Gene Therapy (2010) 17, 1318–1324; doi: 10.1038/gt.2010.86 Since the publication of this paper, the authors have noticed that there are several instances in which micrograms were published as milligrams in the Methods section. The correct Methods section is given below.