Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy

Abstract

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating complication associated with drug treatment of cancer for which there are no effective strategies of prevention or treatment. In this study, we examined the effect of intermittent expression of neurotophin-3 (NT-3) or interleukin-10 (IL-10) from replication-defective herpes simplex virus (HSV)-based regulatable vectors delivered by subcutaneous inoculation to the dorsal root ganglion (DRG) on the development of paclitaxel-induced peripheral neuropathy. We constructed two different tetracycline (tet)-on-based regulatable HSV vectors, one expressing NT-3 and the other expressing IL-10, in which the transactivator expression in the tet-on system was under the control of HSV latency-associated promoter 2 (LAP-2), and expression of the transgene was controlled by doxycycline (DOX). We examined the therapeutic effect of intermittent expression of the transgene in animals with paclitaxel-induced peripheral neuropathy modeled by intraperitoneal injection of paclitaxel (16 mg/kg) once a week for 5 weeks. Intermittent expression of either NT-3 or IL-10 3 days before and 1 day after paclitaxel administration protected animals against paclitaxel-induced peripheral neuropathy over the course of 5 weeks. These results suggest the potential of regulatable vectors for prevention of chemotherapy-induced peripheral neuropathy.

Keywords: chemotherapy; intermittent expression; neuroprotective peptides; peripheral neuropathy; therapeutic effect.

Figure 1

Prolonged Regulated HSV Vector Prolonged regulated gene expression was achieved using a modified tet-on system we previously developed in which the expression of the transactivator was under the control of HSV latency-associated promoter 2 (LAP2). (A) The transactivator in the tet-on system is constitutively expressed and binds to the tetracycline response element (TRE)-minimal human cytomegalovirus immediate early promoter (HCMV IEp) of the inducible transgene expression element in the presence of DOX, thus resulting in the expression of the transgene. (B) In the vectors, two copies of the regulatable transgene expression units were inserted into the ICP4 loci of the replication-deficient parental HSV virus. DOX, doxycycline; NT-3, neurotrophin 3; IL-10, interleukin 10; rtTA, reverse tet-controlled transactivator; K, kozak sequence; GOI, gene of interest.

Figure 2

Regulated Expression of NT-3 and IL-10 from the Vectors by DOX In Vitro Complementing 7b cells were infected by vL2rtNT-3 or vL2rtIL-10 with an MOI of 0.5 and exposed to DOX at different concentrations 1 hr after infection. (A and D) NT-3 or IL-10 concentration in the medium (A, NT-3; D, IL-10) was measured by NT-3 or IL-10 ELISA kit after 2 days of DOX treatment. (B, C, E, and F). To test the kinetics of the turn-on and turn-off of NT-3 or IL-10 expression from the vectors, 7b cells were infected with either vL2rtNT-3 or vL2rtIL-10 at an MOI of 0.01 and cultured either in 1 μg/mL DOX-containing medium for 2 days and subsequently in normal medium for 4 days (B, NT-3; E, IL-10) or cultured in normal medium for the first 2 days after vector infection, followed by 4-day culturing with 1 μg/ml /mL DOX-containing medium (C, NT-3; F, IL-10). Under each culture condition, 50 μL of medium was collected every 2 days, and NT-3 or IL-10 concentration in the medium was measured by ELISA.

Figure 3

Induction of NT-3 and IL-10 Expression from the Vector by DOX in Animals Animals were inoculated subcutaneously into the skin of both hindfeet with either vL2rtNT-3 or vL2rtIL-10 and fed DOX-containing chow for 1, 3, and 7 days to examine the induced expression of the transgenes from the vectors, or animals receiving the vectors were fed DOX-containing chow for 3 days, followed by normal food for 4 days, to test the shut-off of the expression of the transgenes from the vectors in the absence of DOX. After each DOX schedule was completed, L4-6 DRGs of both sides were dissected, and NT-3 or IL-10 mRNA and proteins in the DRGs were analyzed by semiquantitative PCR and western blot. β-Actin served as a loading control. (A) Semiquantitative NT-3 PCR and NT-3 mRNA relative amount. 3/4 off, 3 days with DOX treatment/4 days without DOX treatment. (B) NT-3 western blot and NT-3 protein relative amount. (C) Semiquantitative IL-10 PCR and IL-10 mRNA relative amount. (D) IL-10 western blot and IL-10 protein relative amount. *p < 0.05; **p < 0.01; #, 3 days of DOX treatment followed by 4 days of DOX removal.

Figure 4

Impaired Sensory Nerve Electrophysiological Function and Sensorimotor Coordination in Animals with Paclitaxel-Induced Peripheral Neuropathy Paclitaxel-induced peripheral neuropathy was modeled by i.p. injection of paclitaxel (16 mg/kg) once a week for 5 weeks. Sensorimotor co-ordination and sensory nerve electrophysiological function (amplitude and conduction velocity) were evaluated 2 and 5 weeks after the initiation of paclitaxel treatment. (A) Sensorimotor coordination. (B) Amplitude. (C) Conduction velocity. *p < 0.05, **p < 0.01.

Figure 5

Protective Effect of Intermittent Expression of NT-3 or IL-10 from the Vectors on the Development of Paclitaxel-Induced Peripheral Neuropathy Animals were subcutaneously inoculated with either vector vL2rtNT-3 or vL2rtIL-10 or control vector vL2rtGFP and injected i.p. with paclitaxel (16 mg/kg) once a week for 5 weeks to mimic paclitaxel-induced peripheral neuropathy. Naive animals and animals receiving paclitaxel only served as the symptom-negative and -positive controls. Sensorimotor coordination and sensory nerve electrophysiological function (amplitude and conduction velocity) were evaluated 5 weeks after the initiation of paclitaxel treatment. (A) Schematic of the treatment protocol. (B) Effect of intermittent expression of NT-3 (top, sensory nerve amplitude; center, sensory nerve conduction velocity; bottom, sensorimotor coordination). (C) Effect of intermittent expression of IL-10 (top, sensory nerve amplitude; center, sensory nerve conduction velocity; bottom, sensorimotor coordination). *p < 0.05, **p < 0.01.

Figure 6

No Effect of Repeated Paclitaxel Treatments on Induced Expression of the Transgenes from the Vectors To analyze the effect of repeated paclitaxel treatments on the induced expression of the transgenes from the vectors, naive animals and animals receiving paclitaxel alone were fed normal chow for 4 more days, whereas vector-infected animals were fed DOX-containing chow for 4 additional days 1 week after the fifth injection in the repeated paclitaxel treatment schedule. L4-6 DRGs of both hindfeet were dissected for determination of NT-3 or IL-10 mRNA and protein, measured by semiquantitative PCR and western blot. (A) Semiquantitative NT-3 PCR and NT-3 mRNA relative amount. (B) NT-3 western blot and NT-3 protein relative amount. (C) Semiquantitative IL-10 PCR and IL-10 mRNA relative amount. (D) IL-10 western blot and IL-10 protein relative amount. A, naive animals fed normal chow; B, paclitaxel-treated animals fed normal chow; C, vector-inoculated, paclitaxel-treated animals previously fed DOX-containing chow 4 days per week fed DOX-containing chow for 4 additional days; D, vector-inoculated, paclitaxel-treated animals previously fed DOX-containing chow continuously fed DOX-containing chow for 4 additional days.