Michael J. Passineau

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.

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.

Harvest of pulmonary artery endothelial cells from patients undergoing right heart catheterization.

Endothelial dysfunction is a hallmark of pulmonaryarterial hypertension (PAH). Although the exact roleendothelial dysfunction plays in the initiation or progressionof PAH is not yet clear, impaired apoptosis and aberrantsignaling in endothelial cells is observed in most forms ofPAH. A variety of approaches have been utilized to studyendothelial cell biology in the context of PAH. Most studieshave focused on in situ visualization or purification ofpulmonary artery endothelial cells (PAECs) from explantedlungs of PAH patients, using untransplanted human donorlungs as controls. Although very useful for comparingendothelial cell biology in PAH versus normal pulmonaryvasculature, this approach is unable to provide informationon dynamic changes in PAEC biology over time within aliving patient.The definitive diagnosis of PAH requires measurement ofmean pulmonary artery (PA) pressure via right heartcatheterization (RHC), and thus RHC serves as a “goldstandard” for diagnosis and as a gateway for the manage-ment of PAH patients. We hypothesized that PAECs mightbe dislodged from the PA vessel wall by the balloon tip ofthe flow-directed pulmonary artery (Swan–Ganz) catheterduring RHC and would remain adherent to the balloon whenit is collapsed and the catheter extracted. Herein we reportour successful efforts to isolate, characterize and expandPAECs recovered from Swan–Ganz catheter balloons afterroutine RHC for the diagnosis and management of PAH.After obtaining institutional review board approval, 24patients undergoing RHC consented to participate in ourstudy. After the RHC procedure, the last 2 inches of theSwan–Ganz catheter, including the inflatable balloon, wascut and placed in a tube containing endothelial cell media(Cell Applications, Inc., San Diego, CA) and placed on iceuntil the samples were further processed. In the laboratory,catheter tips were washed vigorously 5 with phosphate-buffered saline (PBS)/trypsin and centrifuged at 1,500 rpmfor 10 minutes. The resulting pellets were re-suspended inammonium–chloride–potassium (ACK)/PBS solution tolyse the erythrocytes. These suspensions were centrifugedat 2,500 rpm for 10 minutes to pellet the cells. An aliquot ofthis final cell harvest was analyzed by fluorescence-activated cell sorting (FACS) with antibodies for endothelialantigens and the balance transferred to primary culture.In these studies, we relied on the classic marker profile forendothelial cells as reported in 2001 by Mancuso et al,

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.

In situ expression of Bcl-2 in pulmonary artery endothelial cells associates with pulmonary arterial hypertension relative to heart failure with preserved ejection fraction

We have previously reported that pulmonary artery endothelial cells (PAECs) can be harvested from the tips of discarded Swan-Ganz catheters after right heart catheterization (RHC). In this study, we tested the hypothesis that the existence of an antiapoptotic phenotype in PAECs obtained during RHC is a distinctive feature of pulmonary arterial hypertension (PAH; World Health Organization group 1) and might be used to differentiate PAH from other etiologies of pulmonary hypertension. Specifically, we developed a flow cytometry-based measure of Bcl-2 activity, referred to as the normalized endothelial Bcl-2 index (NEBI). We report that higher NEBI values are associated with PAH to the exclusion of heart failure with preserved ejection fraction (HFpEF) and that this simple diagnostic measurement is capable of differentiating PAH from HFpEF without presenting addition risk to the patient. If validated in a larger, multicenter study, the NEBI has the potential to assist physicians in the selection of appropriate therapeutic interventions in the common and dangerous scenario wherein patients present a clinical and hemodynamic phenotype that makes it difficult to confidently differentiate between PAH and HFpEF.

A Novel Compound, “FA-1” Isolated from Prunus mume , Protects Human Bronchial Epithelial Cells and Keratinocytes from Cigarette Smoke Extract-Induced Damage

Extract of the Japanese apricot (JAE) has biological properties as an antioxidant and anti-inflammatory agent. We hypothesized that JAE might exert therapeutic effects on cigarette smoke (CS)-induced DNA damage and cytotoxicity. In this study, we found that concentrated JAE protects against cigarette smoke extract (CSE)-induced cytotoxicity and DNA damage accompanied by increased levels of aldehyde dehydrogenase (ALDH)2, 3A1, and Werner’s syndrome protein (WRN) in immortalized human bronchial epithelial cells (HBEC2) and normal human epidermal keratinocytes (NHEK). Using the centrifugal partition chromatography (CPC) method, we identified an undescribed compound, 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal (which we named FA-1), responsible for the protective effects against CSE. This chemical structure has not been reported from a natural source to date. Protective effects of isolated FA-1 against CSE were observed in both HBEC2 and NHEK cells. The studies described herein suggest that FA-1 isolated from JAE protects against CSE-induced DNA damage and apoptosis by augmenting multiple isozymes of ALDH and DNA repair and reducing oxidative stress.

Beating-heart registration for organ-mounted robots

Organ‐mounted robots address the problem of beating‐heart surgery by adhering to the heart, passively providing a platform that approaches zero relative motion. Because of the quasi‐periodic deformation of the heart due to heartbeat and respiration, registration must address not only spatial registration but also temporal registration.

CRISPR-Cas9 HDR system enhances AQP1 gene expression

Ionizing radiation (IR) isthe primarytherapeutic tool to treat patients with cancerous lesions located in the head and neck. In many patients, IR results in irreversible and severe salivary gland dysfunction or xerostomia. Currently there are no effective treatment options to reduce the effects of xerostomia. More recently, salivary gland gene therapy utilizing the water-specific protein aquaporin 1 (AQP1) has been of great interest to potentially correct salivary dysfunction. In this study, we used CRISPR-Cas9 gene editing along with the endogenous promoter of AQP1 within theHEK293 and MDCK cell lines. The successful integration of the cytomegalovirus (CMV) promoterresultedin a significant increase of AQP1 gene transcription and translation. Additionalfunctional experiments involvingthe MDCK cell line confirmedthat over-expressed AQP1increasedtransmembrane fluid flux indicative of increased intracellular fluid flux. The off-target effect of designed guided RNA sequence was analyzed and demonstrateda high specificity for the Cas9 cleavage. Considering the development of new methods for robust DNA knock-in, our results suggest that endogenous promoter replacement may be a potential treatment forsalivary gland dysfunction.

Salivary Gland Gene Therapy in Experimental and Clinical Trials

Salivary gland gene therapy presents an opportunity to reprogram the organ on the molecular level and achieve unprecedented therapeutic advancements. This chapter will review the basic biology of gene transfer, with emphasis on those vector systems that have performed well in the salivary gland in animal models. Various therapeutic applications of salivary gland gene therapy will be discussed, including radiation-induced xerostomia and Sjogren’s syndrome. The concept of salivary glands as endogenous bioreactors for systemic gene therapeutics in monogenetic and acquired diseases will also be reviewed.