Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction

Abstract

Head and neck cancer patients treated with irradiation often present irreversible salivary gland hypofunction for which no conventional treatment exists. We recently showed that recombinant neurturin, a neurotrophic factor, improves epithelial regeneration of mouse salivary glands in ex vivo culture after irradiation by reducing apoptosis of parasympathetic neurons. Parasympathetic innervation is essential to maintain progenitor cells during gland development and for regeneration of adult glands. Here, we investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. First, ex vivo fetal salivary gland culture was used to compare the neurturin adenovirus with recombinant neurturin, showing they both improve growth after irradiation by reducing neuronal apoptosis and increasing innervation. Then, the neurturin adenovirus was delivered to mouse salivary glands in vivo, 24 hr before irradiation, and compared with a control adenovirus. The control-treated glands have ∼50% reduction in salivary flow 60 days post-irradiation, whereas neurturin-treated glands have similar flow to nonirradiated glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with irradiation, but not with neurturin treatment. Our findings suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

CERE-120 Prevents Irradiation-Induced Hypofunction and Restores Immune Homeostasis in Porcine Salivary Glands

“…Interestingly, at this late stage (300 days) after IR and CERE-120 treatment, there were no DEGs directly related to parasympathetic or sympathetic function in the RNA-seq analysis; therefore, we screened genes that were DEGs after adenovirus-NRTN treatment at 60 days in our previous work. 8 While there were no significant changes in gene expression, there was variability in gene expression among SMGs. Genes such as Vip , Adra2b , Vacht , and Tubb3 increased to a level similar to control levels with IR-CERE treatment ( Figure S2 A).…”

“… 23 The CERE-120 (10 6 , 10 8 , or 10 10 viral particles/gland [vp/g]) or AAV2-GFP (10 10 vp/g) vectors were administered by retro-ductal infusion into SMGs at 60 days post-IR. A fractionated IR dose (6 × 5 Gy) was used to induce hyposalivation, 8 which was measured by pilocarpine stimulation of whole saliva production. Saliva was collected 90 days post-IR, and the IR control group produced ∼65% less saliva compared to the non-IR group (baseline), irrespective of whether the AAV2-GFP was delivered pre- or post-IR ( Figures 1 C–1E).…”

“…Previously, we showed NRTN treatment protected neuronal cells from apoptosis after IR-induced damage, and there was improved innervation, epithelial morphogenesis, and gene expression. 8 , 9 , 12 However, we had used recombinant NRTN protein or adenovirus-NRTN treatment for these studies, and their use in clinical settings is unlikely. Recombinant NRTN has a low solubility and short half-life, 33 and adenoviruses may elicit immune responses, 7 whereas AAV2 is considered safe and causes minimal inflammation.…”

“…The NRTN-treated glands had a similar flow to non-irradiated (non-IR) glands, and expression of neuronal markers, such as Ret , Tubb3 , Vacht , and Th , increased, suggesting that NRTN gene therapy may prevent IR-induced hypofunction. 8 To advance this therapy toward the clinic, we needed to change the viral vector to an adeno-associated virus serotype 2 (AAV2), which has longer expression and fewer immune side effects and is now the vector of choice for the current AAV2-AQP1 gene therapy clinical trial 7 (ClinicalTrials.gov: NCT02446249 ). Here, we used an AAV2-based vector encoding human NRTN (CERE-120), which was made by Ceregene, a part of Sangamo BioSciences.…”

Gene therapy and its applicability in Dentistry

“…Researches and treatments through genetic transcription showed beneficial modulatory effects, treating inflammatory processes and regulating the fluids of the secretory glands. Thus, high expectations guide the reestablishment of the oral homeostasis resulting from use of these new technologies [9,10,11].…”