Gene therapy with replication-deficient herpes simplex virus vectors encoding poreless TRPV1 or protein phosphatase 1α (PP1α) reduces detrusor overactivity in mice with spinal cord injury

Shimizu N1, Saito T2, Igarashi N2, Gotoh D2, Majima T2, Takai S2, Hirayama A3, William F G4, Joseph C G4, Uemura H1, Yoshimura N2

Research Type

Basic Science / Translational

Abstract Category

Neurourology

Abstract 203
Neurourology and Interventions
Scientific Podium Short Oral Session 10
Wednesday 4th September 2019
14:30 - 14:37
Hall G3
Spinal Cord Injury Animal Study Detrusor Overactivity Basic Science Infection, Urinary Tract
1.Dept of Urology-Kindai University, Faculty of Medicine, 2.Dept of Urology- University of Pittsburgh, 3.Dept of Urology- Kindai University Nara Hospital, Faculty of Medicine, 4.Dept of Microbiology & Molecular Genetics, University of Pittsburgh
Presenter
N

Nobutaka Shimizu

Links

Abstract

Hypothesis / aims of study
Chronic spinal cord injury (SCI) rostral to the lumbosacral level induces detrusor overactivity (DO) during the storage phase, which is mediated by spinal reflexes triggered by hyperexcitable C-fiber afferent pathways. During the voiding phase, inefficient voiding is commonly observed due to detrusor-sphincter dyssynergia after SCI.  It has also been reported that transient receptor potential vanilloid-1 (TRPV1) receptors predominantly expressed in C-fibre afferent pathways greatly contribute to DO in SCI.
However, the clinical application of TRPV1 antagonists for chronic pain has been hampered partly due to their adverse events (AEs) such as hyperthermia and impaired noxious heat sensation [1]. Hence, the development of local therapies that can target TRPV1 receptors expressed in the affected organs and their afferent pathways without inducing systemic AEs would be useful for the treatment of DO in SCI.
Herpes simplex virus (HSV) has a natural property that it is transported from primary infection sites to their afferent pathways, which could offer an organ-specific treatment for sensory nerve-related diseases.  A previous study also indicated that deletion of the channel pore-forming domain of TRPV1 receptor inhibits the assembly including an aqueous pore, which blocked the channel function [2]. It has also been previously reported that HSV vector-mediated gene delivery of poreless TRPV1, in which the segment in C terminus of TRPV1 receptor is deleted to suppress TRPV1 activation, or protein phosphate 1α (PP1α), which negatively modulates TRPV1 activation, had a therapeutic effect on TRPV1-mediated bladder overactivity and pain behavior in rats with acute chemical irritation [3].
Therefore, this study investigated the effect of HSV vectors-mediated gene delivery of non-functional, poreless TRPV1 or PP1α in storage and voiding dysfunction in SCI mice.
Study design, materials and methods
Female C57BL/6N (8-9 weeks old) mice weighing 18-20g were used, and SCI was induced by complete transection of the Th8/9 spinal cord under isoflurane anesthesia. Two weeks after SCI, replication-deficient HSV vectors encoding porelss TRPV1 or PP1α were injected into the bladder wall. Green fluorescent protein (GFP) vectors, which have the same mutant background as poreless TRPV1 or PP1α vectors, respectively, except that ICP4 genes were replaced with GFP transgene, were used as control vectors. 
SCI mice were divided into 3 groups; (1) SCI mice inoculated with GFP vectors (n=6), (2) SCI mice inoculated with poreless TRPV1 vectors (n=3), (3) SCI mice inoculated with PP1α vectors (n=4).  After spinal cord transection, their bladders were manually squeezed to eliminate urine once daily for 4 weeks until cystometric evaluation. SCI mice were evaluated using single-filling cystometry (CMG) under an awake condition. In single CMG recordings, the number of non-voiding contractions (NVCs), micturition pressure (MP), post-void residual volume (PVR) and voiding efficiency (VE) were evaluated in each SCI mouse (Figure 1, 2).
Bladder were then removed from GFP, poreless TRPV1 and PP1α vector- inoculated SCI mice as well as GFP vector-inoculated normal (spinal intact) mice to evaluate the TRPV1 phosphorylation level by Western blot assay.
Results
GFP expression was seen in L6 dorsal root ganglion (DRG) sections after HSV-GFP vector inoculation into the bladder wall.  Compared to GFP vector-inoculated SCI mice, NVCs during bladder filling were significantly reduced (Figure 1), and MP, PVR and VE were not significantly different between GFP vector-inoculated, poreless TRPV1 and PP1α vector-inoculated SCI mice (Figure 2). Western blot assay revealed the lower levels of phosphorylated TRPV1 in the porelss TRPV1 and PP1α vector-inoculated bladders compared with GFP vector-inoculated and normal mouse bladders.
Interpretation of results
These results indicate that; (1) HSV vectors injected into the bladder wall are transported to L6 DRG neurons through bladder afferent pathways, (2) HSV vectors-mediated gene delivery of poreless TRPV1 or PP1α significantly reduced DO after SCI and (3) HSV vectors-mediated gene delivery of poreless TRPV1 or PP1α also reduced the phosphorylated level of TRPV1 in the bladder.
Concluding message
Gene therapy with replication-deficient herpes simplex virus vectors encoding poreless TRPV1 or PP1α, which can suppress TRPV1 receptor activation in the bladder and bladder afferent pathways, could be a novel treatment that can avoid systemic adverse events for neurogenic lower urinary tract dysfunction such as SCI.
Figure 1
Figure 2
References
  1. Expert Opin Investig Drugs 21, 1351-69, 2012
  2. Nat Methods 4, 733-9, 2007
  3. Neurourology and Urodynamics.;38:582-590 2019
Disclosures
<span class="text-strong">Funding</span> KAKENHI for Early-Career Scientists (18K16751) , DOD W81XWH-17-1-0403, <span class="text-strong">Clinical Trial</span> No <span class="text-strong">Subjects</span> Animal <span class="text-strong">Species</span> mouse <span class="text-strong">Ethics Committee</span> University of Pittsburgh Institutional Animal Care and Use Committee