• Title
• Abstract
• Introduction
• Content
• Discussion
• References

Future Applications of Intravesical SCL Gene Transfer for the Treatment of Diabetic Neurogenic Bladder

Yan Chen^1*, Jin Sheng Li² and Jian Guo Wen¹

• ¹Department of Urology and urodynamic center, Institute of Clinical Medicine, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
• ²Department of Pediatrics, Children’s Hospital of Zhengzhou 450053, China

---

^*Address for Correspondence: Yan Chen, M.D., Department of Urology and Urodynamic center, Institute of Clinical Medicine, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China, Tel: +86 185 3715 7126; Fax: +86 371 6629 5215; E-mail: chenyan.abby@163.com
 
Citation: Chen Y, Li JS, Wen JG. Future Applications of Intravesical SCL Gene Transfer for the Treatment of Diabetic Neurogenic Bladder. J Urol Nephrol. 2015;2(1): 4.
 
Copyright © 2015 Chen Y, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
 
Journal of Urology & Nephrology | ISSN 2380-0585 | Volume: 2, Issue: 1
 
Submission: 01 June 2015 | Accepted: 09 July 2015 | Published: 13 July 2015
 
Reviewed & Approved by:Dr. Robert Moore, Director of Clinical Urology & Urology Residency Site, Wake Forest University, USA

---

Abstract

Diabetic cystopathy, a complication of diabetes affecting the bladder, is mainly characterized as reduced contractility of the detrusor and increased post-voiding residual volume, which is induced by the reduction of the interstitial cells of Cajal. The c-KIT protein is a specific marker of the interstitial cells of Cajal and the product of a c-KIT proto-oncogene. The interstitial cells of Cajal in high glucose medium express less c-KIT mRNA and protein. SCL gene, serves to assemble SCL complexes on the c-KIT promoter, sustaining c-KIT transcription. Proper transduction of exogenous SCL genetic material, increases c-KIT expression and leads to synthesis of the functional SCL protein. Intravesical, lentiviral vector-mediated gene transfer has been shown efficacious and safe. Therefore, proper intravesical transfer of RNA encoding SCL to the interstitial cells of Cajal may enhance c-KIT transcription and activity in the interstitial cells of Cajal of diabetic bladders, which may improve bladder activity.

---

Keywords

Intravesical; SCL gene; Treatment; Diabetic cystopathy

---

Introduction

Diabetic cystopathy (DCP) is one of the most common complications in diabetes mellitus, affecting over 25% of diabetic patients [1,2]. The main symptoms of DCP are under-activity of the detrusor muscle and increased post-voiding residual volume, which would induce severe urinary tract infection, vesico-ureter reflux, hydronephrosis, and even uremia and renal failure [3,4]. Previous studies have shown that interstitial cells of Cajal (ICCs) in the detrusor have pace-making ability, which is responsible for contraction of the detrusor [5,6]. ICCs are involved in signal transmission between smooth muscle bundles, from efferent nerves to smooth muscles, and from the urothelium to afferent nerves [7,8]. The underactivity of DCP has the disturbance of spontaneous contractility, caused by reduced suburothelial ICCs in DCP patients [9,10]. ICCs in cultured bladder tissue with high glucose medium were found to exhibit poorly self-excited, reduced connections with detrusor cells and nerve terminals, and expressed less c-KIT mRNA and protein than control cells [11]. The c-KIT protein is a specific marker on the cell membrane of ICCs, which is encoded by the c-KIT proto-oncogene [12,13]. Activation of the c-KIT gene can modulate cell growth, differentiation, and phenotype, while mutation of c-KIT leads to ICCs absent [14,15]. The stem cell leukemia gene (SCL) is a tissuespecific transcription factor of the basic helix-loop-helix family, functions in hematopoietic development, is normally expressed n pluripotent hematopoietic precursors, and is downregulated in maturing cells [16,17]. SCL induces c-KIT expression in chromatin. For example, ectopic SCL expression in transgenic mice sustainsc-KIT transcription in developing B-lymphocytes, in which both genes are normally downregulated [18]. Increased SCL expression upregulates c-KIT gene expression in normal bone marrow cells. This c-KIT regulation involves proper activation of the c-KIT promoter by the SCL protein, which coordinates SCL complex binding on the promoter sequence to activate c-KIT transcription [19. Elefanty et al. successfully introduced SCL into mutant mice (SCL lacZ/w) and demonstrated that functional SCL protein was synthesized [20]. For the transfer of genetic material to target tissue, the use of intravesical lentiviral vector-mediated gene delivery to bladder cells has been shown to be efficacious and safe for the treatment of bladder cancers [21]. Taken together, increased SCL expression in DCP patients may initiate a cascade of events, resulting in increased c-KIT activity, thus leading to the rescue of bladder function.

---

The Hypotheses

Given the results of previous studies, we hypothesize that intravesical SCL gene transfer by viral vectors, such as the lentiviral vector, has high potential to promote c-KIT gene expression in ICCs and relieve bladder under-activity in DCP patients. During infection of ICCs, the viral vector carrying the SCL RNA is transduced into the ICCs, reverse-transcribed into double-stranded DNA (dsDNA) in the cytoplasm, and transported into the nucleus to stably integrate into the ICC genome. In DCP patients, the exogenous SCL may be expressed, and SCL protein may be synthesized in ICCs. Because DCP causes downregulation of the c-KIT gene, newly produced SCL may activate and sustain c-KIT transcription by binding the promoter. These reactions may enhance/sustain c-KIT gene transcription and protein synthesis in DCP patients; therefore, bladder under-activity may be relieved.

---

Discussion

DCP-related alterations of the detrusor are attributed to several mechanisms: changes in cellular excitability or intercellular communication; changes in receptor density, distribution, and function; alterations to intracellular signal transduction; and molecular or genetic changes [7,8]. All of these mechanisms are induced by abnormalities in ICCs in the bladder [22,23]. At the molecular level, c-KIT, which is a transmembrane receptor on the ICC cell membrane, is responsible for these abnormalities. Under normal conditions, the stem cell factor (SCF) activates its receptor (c-KIT), and this triggers the activation of MAP kinase and conducts the signal to the cell nucleus to modulate cell growth, differentiation, and phenotype [24]. ICCs and detrusor smooth muscle cells originate from the same cell type, which are embryonic, c-KITpositive, mesenchymal precursor cells. At the late embryo stage, the mesenchymal precursor cells receiving the c-KIT signal differentiate into ICCs, while precursor cells receiving no c-KIT signal become smooth muscle cells[15]. Mutation of the dominant white spotting (W) locus in chromatin, where c-KIT has been mapped in embryonic precursor cells, leads to ICCs absent in the mouse [25]. In fact, the c-KIT protein is a detection marker, and it plays a crucial role in the control of bladder function. The inhibition of c-KIT receptor induces ATP-K+ channel opening and cell membrane hyper-polarization in ICCs; concomitantly, decreased excitability and contractility of the bladder have been detected, which implies that c-KIT protein inhibition in ICCs decreases bladder activity [26,7].


SCL is a crucial regulator of diverse developmental processes, such as hematopoiesis, neurogenesis, and myogenesis [28]. SCL is required for c-KIT expression and function in the hemopoietic cell line and has been shown to induce c-KIT transcription in chromatin. In a functional screen of TF-1 cells expressing antisense SCL 20, c-KIT receptor function and expression were defective. However, codelivery of SCL in the sense orientation rescued c-KIT gene expression, which suggests that the latter is a potential downstream target of SCL [19]. Ectopic SCL expression in transgenic mice induces sustained c-KIT transcription in developing B cells. SCL levels determine c-KIT gene expression in hematopoietic cells, and c-KIT expression is directly controlled by SCL. In fact, SCL serves to nucleate the assembly of a multi-protein complex (SCL complex) formed on the c-KIT promoter, which contains SCL, Lim-only 2, GATA-1/GATA-2, E2A, LIM domain binding protein 1, and specificity protein 1 (Sp1) zinc finger protein. The SCL complex activates the c-KIT promoter; specifically, SCL, E2A, and Sp1 have been shown to occupy the c-KIT promoter in vivo in TF-1 cells [16-18]. Therefore, c-KIT is a direct target of the transcription factor SCL and its partners, and activation of the c-KIT promoter depends on SCL. Therefore, exogenously introduced SCL genetic material may help cells sustain or enhance c-KIT function.


Kurita et al. showed that transduction of exogenous TAL1/SCL cDNA into embryonic stem cells using lentiviral vectors is efficient and safe [29]. The viral vectors can deliver significant amounts of genetic information into host cells and integrate the newly synthesized dsDNA into the cellular genome successfully and safely [30,31]. Accordingly, genetically engineered viral vectors, such as lentivirus from the retrovirus family, are currently the most efficient tools of gene delivery. Viral vectors contain a viral promoter, which is used to control the expression of the transgene, and virulence genes are removed. Viral vectors are safe to use in the laboratory with security modifications to eliminate pathogenicity [32]. Viral vectors, e.g., the lentiviral vector, can transduce a wide range of dividing and non-dividing mammalian cell types [33,34]. Thus, it is possible that a lentiviral vector harboring SCL RNA can transduce ICCs into the mammalian bladder. Upon infection, the single-stranded RNA is transduced into the ICCs and reverse-transcribed to dsDNA in the cytoplasm; then the resulting dsDNA would integrate into the genome of the ICCs (figure 1A). In DCP patients, exogenously introduced SCL may be expressed, SCL protein may be synthesized, and the SCL complex would assemble in the ICCs. In DCP patients with downregulated c-KIT transcription, the assembled SCL complex may bind the promoter to enhance or sustain c-KIT transcription. Thus, ICC function may be restored, and bladder under-activity may be relieved (figure 1B).


In fact, intravesical viral gene transfer has been applied to treat bladder cancer, and the procedure was well tolerated; the bladder urothelium appears to prevent systemic dissemination of the viral particles [21]. Lentivirus is the commonly used vector for these procedures. Lu et al. successfully applied lentivirus-mediated RNA interference to knockdown clusterin in bladder 


Some urologists may suspect the intravesical application of lentiviral particles might cause some concern for local/systemic immunogenic responses, and there is a possibility of poor viral vector permeability through the watertight barrier formed by the urothelium. A previous study in Taiwan developed an intravesical instillation of an in situ biodegradable hydrogel system (15% hydrogel) containing a lentiviral vector harboring WWOX, a rat bladder tumor suppressor gene, to treat bladder cancer. The hydrogel system showed promise for in situ delivery of lentiviral vectors to bladders and demonstrated gene expression for bladder disease therapy without local/systemic immunogenic responses 36]. Another research using lentiviral vector-mediated antisense oligonucleotides targeting heat shock protein-27 with the tubulin inhibitor, taltobulin (HTI-286), discovered that strong preclinical proof-of-principle for intravesical administration of oligonucleotides in combination with HTI-286 for the treatment of high-grade bladder cancer 37].



Figure 1: SCL transfer by the lentiviral vector into an interstitial cell of Cajal (ICC). (A) Lentiviral vector carrying SCL RNA enters an ICC, and the SCL gene is integrated into the host DNA in the nucleus. (B) The SCL gene specifically enhances c-KIT promoter activity, and c-KIT gene transcription increases




 Taken together, lentivirus-mediated gene delivery to bladder cells is a safe and promising way to treat underactivity of DCP by SCL gene enhancing c-KIT gene expression to rescue the bladder function, so transfection of DNA via an intravesical route using a viral vector is a new strategy of transgene expression for bladder disease therapy. Therefore, we proposed other researchers to explore this treatment strategy in detail, not only for diabetic cystopathy but also for other clinical problems, which can ameliorate the quality of life of our patients, and seems therefore valuable.







---

References

1. Daneshgari F, Moore C (2006) Diabetic uropathy. Semin Nephrol 26: 182-185.
2. Goldstraw MA, Kirby MG, Bhardwa J, Kirby RS (2007) Diabetes and the urologist: a growing problem. BJU Int 99: 513-517.
3. Bansal R, Agarwal MM, Modi M, Mandal AK, Singh SK (2011) Urodynamic profile of diabetic patients with lower urinary tract symptoms: association of diabetic cystopathy with autonomic and peripheral neuropathy. Urology 77: 699-705.
4. Andersson KE, Arner A (2004) Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev 84: 935-986.
5. Shafik A, El-Sibai O, Shafik AA, Shafik I (2004) Identification of interstitial cells of Cajal in human urinary bladder: concept of vesical pacemaker. Urology 64: 809-813.
6. Piaseczna-Piotrowska AM, Dzieniecka M, Kulig A, Danilewicz M, Chilarski A (2011) Different distribution of c-kit positive interstitial cells of Cajal-like in children's urinary bladders. Folia Histochem Cytobiol 49: 431-435.
7. Davidson RA, McCloskey KD (2005) Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth muscle and neurons. J Urol 173: 1385-1390.
8. Johnston L, Carson C, Lyons AD, Davidson RA, McCloskey KD (2008) Cholinergic- induced Ca2+ signaling in interstitial cells of Cajal from the guinea pig bladder. Am J Physiol Renal Physiol 294: F645-55.
9. Li YF, Wang QZ, Ding GF (2009) Changes and significances of urodynamics and cajal-like cells in diabetic cystopathy of the early stage. J Clin Urol 24: 694-697.
10. Wang Y, Fang Q, Lu Y, Song B, Li W, et al. (2010) Effects of mechanical stretch on interstitial cells of Cajal in guinea pig bladder. J Surg Res 164: e213-e219.
11. Wang QZ, Ji SQ, Zhu GD, Li YF, Cai ZQ, et al. (2009) The morphologic changes and significance of interstitial cells of Cajal-like cells in the bladder of diabetic guinea pig. J Clin Exp Pathol 25: 631-634.
12. Liu YG, Wang QZ, Ding GF, Liu XJ, Zhou JM, et al. (2011) Effects of SCF on the expressions of c-kit mRNA and protein of interstitial cells of Cajal- like cells in the bladder of guinea pig in the high glucose environment. J Clin Exp Pathol 27: 282-285.
13. Rasmussen H, Rumessen JJ, Hansen A, Smedts F, Horn T (2009) Ultrastructure of Cajal-like interstitial cells in the human detrusor. Cell Tissue Res 335: 517-527.
14. Chen W, Jiang C, Jin X, Shen W, Song B, et al. (2011) Roles of stem cell factor on loss of interstitial cells of Cajal in bladder of diabetic rats. Urology 78: 1443 e1-e6.
15. Torihashi S, Nishi K, Tokutomi Y, Nishi T, Ward S, et al. (1999) Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype. Gastroenterology 117: 140-148.
16. Lacombe J, Krosl G, Tremblay M, Gerby B, Martin R, et al. (2013) Genetic interaction between Kit and Scl. Blood 122: 1150-1161.
17. Curtis DJ, Salmon JM, Pimanda JE (2012) Concise review: Blood relatives: formation and regulation of hematopoietic stem cells by the basic helix-loop-helix transcription factors stem cell leukemia and lymphoblastic leukemia-derived sequence 1. Stem Cells 30: 1053-1058.
18. Lécuyer E, Herblot S, Saint-Denis M, Martin R, Begley CG, et al. (2002) The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1. Blood 100: 2430-2440.
19. Krosl G, He G, Lefrancois M, Charron F, Roméo PH, et al. (1998) Transcription factor SCL is required for c-kit expression and c-Kit function in hemopoietic cells. J Exp Med 188: 439-450.
20. Elefanty AG, Begley CG, Metcalf D, Barnett L, Köntgen F, et al. (1998) Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy. Proc Natl Acad Sci U S A 95: 11897-11902.
21. Perrotte P, Wood M, Slaton JW, Wilson DR, Pagliaro L, et al. (2000) Biosafety of in vivo adenovirus-p53 intravesical administration in mice. Urology 56: 155-159.
22. Yoshimura N, Chancellor MB, Andersson KE, Christ GJ (2005) Recent advances in understanding the biology of diabetes-associated bladder complications and novel therapy. BJU Int 95: 733-738.
23. McCloskey KD (2013) Bladder interstitial cells: an updated review of current knowledge. Acta Physiol (Oxf) 207: 7-15.
24. Kimura S, Kawakami T, Kawa Y, Soma Y, Kushimoto T, et al. (2005) Bcl-2 reduced and fas activated by the inhibition of stem cell factor/KIT signaling in murine melanocyte precursors. J Invest Dermatol 124: 229-234.
25. Iino S, Horiguchi S, Horiguchi K (2011) Interstitial cells of Cajal in the gastrointestinal musculature of W (jic) c-kit mutant mice. J Smooth Muscle Res 47: 111-121.
26. Kojima Y, Shibata Y, Imura M, Sasaki S, Kohri K (2011) Role of KIT-positive interstitial cells of cajal in the urinary bladder and possible therapeutic target for overactive bladder. Adv Urol 2011: 816342.
27. Kim BJ, Chae H, Kwon YK, Choi S, Jun JY, et al. (2010) Effects of imatinib mesylate in interstitial cells of Cajal from murine small intestine. Biol Pharm Bull 33: 993-997.
28. Begley CG, Green AR (1999) The SCL gene: from case report to critical hematopoietic regulator. Blood 93: 2760-2770.
29. Kurita R, Sasaki E, Yokoo T, Hiroyama T, Takasugi K, et al. (2006) Tal1/Scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset (Callithrix jacchus) embryonic stem cells. Stem Cells 24: 2014-2022.
30. Ni S, Zhao J, Fu Z, Liu H (2014) Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 overexpression induces beta-2 adrenergic receptor desensitization in airway smooth muscle cells. J Thorac Dis 6: 118-125.
31. Cribbs AP, Kennedy A, Gregory B, Brennan FM (2013) Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells. BMC Biotechnol 13: 98.
32. Dismuke DJ, Tenenbaum L, Samulski RJ (2013) Biosafety of recombinant adeno-associated virus vectors. Curr Gene Ther 13: 434-452.
33. D'Costa J, Mansfield SG, Humeau LM (2009) Lentiviral vectors in clinical trials: Current status. Curr Opin Mol Ther 11: 554-564.
34. Schambach A, Baum C (2008) Clinical application of lentiviral vectors - concepts and practice. Curr Gene Ther 8: 474-482.
35. Lu J, Luo JH, Pang J, Cao JZ, Wu RH, et al. (2012) Lentivirus-mediated RNA interference of clusterin enhances the chemosensitivity of EJ bladder cancer cells to epirubicin in vitro. Mol Med Rep 6: 1133-1139.
36. Liu C, Tsai T, Fan S, Nien C, Chang L (2013) Hydrogel for enhanced intravesical delivery of lentiviral vector expressing WWOX in vivo. The 39th Annual Meeting & Exposition of the Controlled Release Society, Québec, Canada.
37. Matsui Y, Hadaschik BA, Fazli L, Andersen RJ, Gleave ME, et al. (2011) Intravesical combination treatment with antisense oligonucleotides targeting heat shock protein-27 and HTI-286 as a novel strategy for high-grade bladder cancer. Mol Cancer Ther 8: 2402-2011.