Adenoviral delivery of Tousled kinase for the protection of salivary glands against ionizing radiation damage

Abstract

Oral complications of salivary hypofunction often afflict cancer patients undergoing radiotherapy for head and neck cancers. Dry mouth or xerostomia is an undesirable consequence of radiotherapy that compromises normal oral functions in addition to causing odynophagia and increasing the patient's risk of oral infections and dental caries. Radiation-induced xerostomia is irreversible, and palliative measures to provide symptomatic relief remain the mainstay of treatment. Previously, we identified a splice variant of a cellular kinase, Tousled-like kinase 1B (TLK1B), which when overexpressed protects normal epithelial cells against ionizing radiation (IR)-induced cell death. To address the need to protect salivary glands in patients undergoing regional radiotherapy, we investigated whether preemptive expression of TLK1B in salivary glands protects against IR. In stably-derived salivary cell lines in vitro, TLK1B expression increased cell survival after IR. Cells expressing exogenous TLK1B were less radiosensitive (A5-TLK1B, a/b¼0.67 Gy; ParC5-TLK1B, a/b¼4.3 Gy) compared to control cells (A5-BK, a/b¼1.7 Gy; ParC5-BK, a/b¼32.7 Gy). Using a recombinant adenovirus serotype 5 viral vector for TLK1B gene transfer into rat submandibular salivary glands in vivo, we demonstrated that TLK1B protects the saliva-secreting acinar cells and better preserves salivary gland function against IR relative to control glands. After a single fraction of 16 Gy, the decline in salivary function at 8 weeks was less pronounced in TLK1B-treated animals (40%) as compared to saline-treated controls (67%). Histopathological analysis demonstrated increase in acinar atrophy, decrease in acinar cell number, and increase in inflammatory infiltrate and fibrosis in irradiated control tissues relative to TLK1B-treated glands. These results show the radioprotective benefits of TLK1B and implicate its usefulness in the management of regional radiotherapy-induced xerostomia.

Recombinant AAV9-TLK1B Administration Ameliorates Fractionated Radiation-Induced Xerostomia

“…We have shown that expression of TLK1B in mouse epithelial cells does not induce oncogenic transformation, but it does increase the cell's ability to overcome radiation injury (Li et al, 2001;SunavalaDossabhoy et al, 2003;Sunavala-Dossabhoy et al, 2005). This holds true for irradiated normal rat salivary acinar and ductal cells transduced with exogenous TLK1B as well (Palaniyandi et al, 2011;Sunavala-Dossabhoy et al, 2012). Although TLK1B facilitates repair of double-strand breaks, its precise role in this process is not completely elucidated.…”

“…Previously, we had shown that a single-dose of 16 Gy compromises rat submandibular gland function, and pretreatment with adenovirus-TLK1B effectively salvaged salivary flow (Palaniyandi et al, 2011). To assess the effect of TLK1B on clinically relevant radiation dosing, animals were irradiated in fractions of 2.5 Gy daily, up to a total of 20 Gy (Fig.…”

“…Using localized adenovirus gene delivery to rat submandibular glands, we recently demonstrated that TLK1B expression effectively attenuates salivary dysfunction after single-dose radiation (Palaniyandi et al, 2011). Nonetheless, short-lived gene expression and immunogenicity of the vector on repeated administration limit the prospect of translating adenovirus-TLK1B to head and neck cancer patient care where radiotherapy is delivered in small doses, daily, for 5-7 weeks.…”

“…Their role in facilitating DNA repair is emphasized by observations that overexpression of the protein protects cells against death by radiation (Li et al, 2001;Sunavala-Dossabhoy et al, 2005), whereas suppression of its kinase activity or knockdown of the protein enhances the cell's susceptibility to DNA damage-induced cell death (SunavalaDossabhoy et al, 2003;Takayama et al, 2010). We have shown before that exogenous TLK1B in rat submandibular salivary glands effectively attenuates xerostomia associated with a single large-dose radiation (Palaniyandi et al, 2011;SunavalaDossabhoy et al, 2012). Now, we demonstrate that sustained expression of TLK1B associated with a single instillation of rAAV9 to rat submandibular glands is sufficient to completely ameliorate salivary dysfunction caused by fractioned radiation.…”

Salivary Glands: A Powerful Experimental System to Study Cell Biology in Live Animals by Intravital Microscopy

“…Notably, for viral-mediated gene therapy in humans, the SGs offer several advantages with respect to other organs: i) the encapsulation of the SG tissue prevents the dissemination of the virus in the rest of the body (Baum et al, 2004;Voutetakis et al, 2004), ii) in case of potential health issues the SGs can be removed since they are not essential for life, iii) the differentiation of the cells provides a relatively stable cell populations for non-integrating vectors, and iv) duacrine (both exocrine and endocrine) protein secretion allows to direct the expressed molecules into either the saliva or the blood stream (Baum et al, 2004). Numerous studies have shown successful gene transfer into both rat and mouse submandibular glands using viral-based approaches, which offer the advantage of a more robust expression of the transgenes (Andresen et al, 2009;Baum and Tran, 2006;Delporte et al, 1996;Honigman et al, 2001;Mastrangeli et al, 1994;Morita et al, 2011;Palaniyandi et al, 2011;Perez et al, 2011;Samuni et al, 2008;Wang et al, 2000;Zheng et al, 2009). However, non viral-mediated approaches have also been utilized, although limited to a small percentage of the cells in the parenchyma (Goldfine et al, 1997;Honigman et al, 2001;Niedzinski et al, 2003a;Niedzinski et al, 2003b;Passineau et al, 2010;Sramkova et al, 2009).…”