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【摘要】
Objective— We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library. UCHL1 is expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson?s disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor- B (NF- B) inactivation in ECs and vascular smooth muscle cells (VSMCs).
Methods and Results— From Northern blot and immunohistochemical analysis, the UCHL1 gene was endogenously expressed in vascular ECs, VSMCs, and brain tissue. Expression of UCHL1 was markedly increased in the neointima of the balloon-injured carotid artery and was also present in atherosclerotic lesions from human carotid arteries. Overexpression of the UCHL1 gene significantly attenuated tumor necrosis factor (TNF)- –induced NF- B activity in vascular cells and increased inhibitor of kappa B- (I B- ), possibly through the attenuation of I B- ubiquitination, leading to decreased neointima in the balloon-injured artery. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF- B activity in VSMCs.
Conclusions— These data suggest that UCHL1 may partially attenuate vascular remodeling through inhibition of NF- B activity.
The ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene encodes for a deubiquitinating enzyme and is associated with Parkinson disease. UCHL1 gene expression was characterized in the neointima of the balloon-injured artery and in human atherosclerotic lesions. In vascular cells, UCHL1 attenuated TNF- –induced NF- B activity, possibly through the attenuation of I B- ubiquitination.
【关键词】 UCHL NF B deubiquitinating enzyme atherosclerosis
Introduction
The vasculature is capable of sensing changes within its milieu, integrating these signals by intercellular communication, and changing itself through the local production of mediators that influence structure as well as function (eg, "vascular remodeling"). 1 In the process of vascular remodeling, nuclear factor- B (NF- B) plays a pivotal role in the regulation of coordinated transactivation of genes involved in the expression of interleukins, intercellular adhesion molecules, vascular cell adhesion molecules, and endothelial leukocyte adhesion molecules. 2 NF- B activity is regulated in the cytoplasm through its association with I B- which is regulated by ubiquitin-proteasome system. Because phosphorylation of I B- is forwarded to be ubiquitinated by ubiquitinating enzymes and degraded by proteasomes, 3 the ubiquitin-proteasome system is a critical mediator of NF- B activity.
Ubiquitin-mediated protein degradation plays a crucial role in various cellular processes, including signal transduction, cell differentiation, and stress response. 4 By contrast, the deubiquitination system prevents protein degradation by reversing the ubiquitination process via the disassembly of the poly-ubiquitin chain, recycling active ubiquitin by the removal of ubiquitin from its covalently linked protein, and generating monomeric ubiquitin from its precursor fusion protein. 5 Ubiquitinated I B- is also able to escape from degradation by deubiquitination. Regardless, regulation of transcription factor NF- B remains the most important link between the ubiquitin-proteasome system and inflammation. Even though the proteasome inhibitor MG132 effectively reduces neointima formation, which corresponds to strong antiproliferative, antiinflammatory, and proapoptotic effects, 6 it remains unclear whether deubiquitinating enzyme is involved in vascular remodeling.
We recently used a functional gene screening system with the HVJ-E vector 7 to identify ubiquitin C-terminal hydroxylase L1 (UCHL1) as an antiremodeling factor that is expressed in human endothelial cells. UCHL1 is a causative gene in Parkinson disease (PD), which is a neurodegenerative disorder associated with dysfunction of the ubiquitin-proteasome system. 8 The goal of the present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor- B (NF- B) inactivation in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs).
Methods
Functional Screening Using Hemagglutinating Virus of Japan Envelope (HVJ-E)
Several kinds of cells were maintained as previously described. 9 Functional screening of a cDNA library using HVJ-E vector was performed as previously described 7,10 using human umbilical venous endothelial cell (HUVEC) cDNA Library 11 (a kind gift from Prof Hiroshi Nojima, Research Institute for Microbial Diseases, Osaka University, Japan).
Plasmid DNA Construction
Gateway cloning technology (Invitrogen) was used to construct expression vectors in accordance with the manufacturer?s instructions. Briefly, human UCHL1 and bovine eNOS were subcloned into D-TOPO to construct entry vectors and were then transferred into the mammalian expression vector, pcDNA3.1 or pCAGGS, 12 through a LR-recombination reaction, creating pcDNA3.1-UCHL1, pCAGGS-FLAG-UCHL1, and pCAGGS-eNOS.
Northern and Western Blotting and Immunoprecipitation
Northern and Western blotting was performed as previously described. 13,14 Cells were subjected to immunoprecipitation and immunoblot analysis as previously described. 15 Of the 200 µL of cell lysates (50 µg), 10 µL was used for analysis of the relative expression levels of I B- and internal control proteins, and the remainder of the cell lysates was used for immunoprecipitation experiments with 0.5 mg anti-I B- (1:1000, sc-371, Santa Cruz Biotechnology Inc) and after immunoblotting with anti-ubiquitin (Ub) (1:2500, sc-8017, Santa Cruz Biotechnology Inc).
Immunochemical Staining
Rat balloon-injured carotid artery was prepared as previously described. 16 At least 3 individual sections (4 µm) from the middle of the injured arterial segments were fixed in 4% PFA for 15 minutes and then immunostained as previously described. 17 All experimental protocols were approved by the Osaka University Graduate School of Medicine Standing Committee on Animals. The tissue specimens were obtained from symptomatic patients who underwent carotid endartectomy for high-grade carotid stenosis. Written informed consent was obtained from all patients, and all protocols were approved by the Ethics Committee of Osaka University.
In Vivo Gene Transfer Into Rat Balloon-Injured Carotid Artery
The HVJ-liposome method was used for transfection of plasmid DNA in rat carotid artery as previously described. 16,18 Briefly, a cannula was introduced into the common carotid artery through the external carotid artery. Then, 200 µL of HVJ-liposome complex was infused into the segment and incubated for 10 minutes at room temperature.
Statistical Analysis
All values are expressed as mean±SD. Data were compared using the ANOVA followed by Dunnett test for pair-wise comparisons against "control" and by Tukey test for multiple comparisons. All statistical analysis was performed using Stat-View 5.0 software (SAS. Institute Inc). Values of P <0.05 were considered to represent statistical significance.
Detailed information is contained in the supplemental methods.
Results
Functional Screening of Antivascular Remodeling Factor
Screening of a HUVEC cDNA library for genes that inhibit PDGF-stimulated cell growth and that inhibit NF- B activity resulted in isolation of several candidate genes, including UCHL1. As we have previously demonstrated the importance of NF- B in vascular cells to attenuate vascular remodeling, 19,20 we further examined the effect of UCHL1 on NF- B activity in vascular cells. Overexpression of UCHL1 significantly attenuated NF- B activity in endothelial cells and smooth muscle cells (supplemental Figure IA through ID). Gel shift mobility assay demonstrated that overexpression of the UCHL1 also attenuated binding of NF- B to the consensus sequence in endothelial cells (supplemental Figure IE).
Expression of UCHL1 in Vascular Cells
Immunostaining demonstrated UCHL1 protein in the cytoplasm and nucleus in human aortic endothelial cells (HAECs) and human aortic smooth muscle cells (HASMCs) in 5% FBS (supplemental Figure IIA). Northern blot analysis demonstrated mRNA expression of UCHL1 in HAECs ( Figure 1 A) and HASMCs (supplemental Figure IIB). Treatment of HAECs and HASMCs with several growth factors or cytokines (ie, human recombinant epidermal growth factor [EGF: 10 ng/mL], fibroblast growth factor-2 [FGF2: 10 ng/mL], vascular endothelial growth factor [VEGF: 10 ng/mL], TNF- [TNF- : 10 ng/mL], and lipopolysaccharide [LPS: 30 ng/mL], angiotensin II [AngII: 1 µmol/L], hydrogen peroxide [H 2 O 2; 100 µmol/L]) for 24 hours did not significantly alter UCHL1 mRNA levels ( Figure 1 A and supplemental Figure IIB). Treatment of cultured VSMCs with TNF- (10 ng/mL) for 24 and 48 hours did not significantly alter UCHL1 mRNA levels, whereas long term treatment (96 hours) with TNF- (10 ng/mL) significantly increased UCHL1 mRNA levels (3.63±0.25-fold; P <0.01; Figure 1 B).
Figure 1. Expression of UCHL1 in vascular cells. A, Representative Northern blot of human UCHL1 in HAECs. Corresponding β-actin or GAPDH expression was used to standardize loading. "control" indicates no stimulation. Cultured cells were treated with human recombinant EGF (10 ng/mL), FGF2 (10 ng/mL), VEGF (10 ng/mL), TNF- (10 ng/mL), or hydrogen peroxide (H 2 O 2; 100 µmol/L) for 24 hours. Experiments were performed in duplicate. B, Quantification of UCHL1 expression by quantitative real time RT-PCR after TNF- (10 ng/mL) treatment for 0, 24, 48, and 96 hours in HASMCs (n=4 per group in triplicate). Results are expressed as ratio of relative gene expression of UCHL1 in HASMCs with TNF- treatment to that without TNF- treatment.
Role of UCHL1 in Cultured Vascular Endothelial Cells
Treatment of BAECs or HAECs with TNF- (10 ng/mL) resulted in a significant increase in NF- B activity, whereas overexpression of UCHL1 significantly attenuated both basal and TNF- –induced NF- B activity (56% inhibition in BAECs and 85% inhibition in HAECs; P <0.05, compared with control under TNF- treatment). In fact, the inhibitory effect of UCHL1 was similar to that of eNOS ( Figure 2 A and supplemental Figure IIIA). In HAECs, immunofluorescent staining with anti-p65 antibody showed that p65, NF- B subunit, was rapidly translocated into nucleus after the treatment of TNF- for 30 minutes, whereas in UCHL1-trasnfected ECs, p65 remained to be located in the cytoplasm after the same treatment (supplemental Figure IVA). Although the treatment of TNF- is known to decrease eNOS expression through NF- B activation, 21 overexpression of UCHL1 attenuated TNF- –induced decreases in eNOS expression (supplemental Figure VA). Similarly, treatment of ECs with 2 different type of proteasome inhibitors, aspirin or MG132 attenuated TNF- –induced eNOS downregulation (supplemental Figure VA).
Figure 2. Role of UCHL1 in cultured endothelial cells. A, Effect of overexpressed UCHL1 on NF- B activity evaluated by the luciferase gene driven by the NF- B binding site in BAECs. "control" indicates overexpressed GFP gene, "UCHL1" indicates overexpressed human UCHL1 gene, and "eNOS" indicates over-expressed eNOS gene. "TNF- " indicates treatment with human recombinant TNF- (10 ng/mL) for 12 hours. * P <0.05 vs control. # P <0.05 vs TNF- (–). n=6 per group in triplicate. B, Representative Western blot of phospho-I B- (P-I B- ), I B-, and UCHL1 protein in BAECs. "control" indicates overexpressed GFP gene, and "UCHL1" indicates overexpressed human UCHL1 gene. Corresponding β-actin expression was used to standardize loading. Experiments were performed in triplicate. C, Analysis of ubiquitinated I B- by immunoprecipitation in BAECs. "IP:I B- " indicates immunoprecipitation with I B- and "IB:Ub" indicates immunoblot with anti-ubiquitin antibody. "control" indicates overexpressed GFP gene, "UCHL1" indicates overexpressed human UCHL1 gene. Corresponding GAPDH expression was used to standardize loading. Experiments were performed in triplicate.
As for the expression of NF- B–driven gene, overexpression of UCHL1 in ECs attenuated TNF- –induced expression of inducible NOS (iNOS; 54% inhibition), Manganese superoxide dismutase (Mn-SOD; 35% inhibition), heme oxygenase-1 (HO-1; 46% inhibition), vascular cell adhesion molecule-1 (VCAM-1; 74% inhibition), intercellular adhesion molecule-1 (ICAM-1; 57% inhibition), and E-selectin (37% inhibition), respectively ( P <0.05 compared with control), as assessed by quantitative real-time–polymerase chain reaction (PCR; supplemental Figure VB).
We further examined how UCHL1 induced the NF- B inactivation. I B- was phosphorylated and downregulated at 5 minutes after treatment with human recombinant TNF- (10 ng/mL) in BAECs (data not shown). Five minutes after treatment with TNF-, I B- expression was relatively high in UCHL1-overexpressed BAECs despite the fact that phospho–I B- expression was similar with or without overexpressed UCHL1 ( Figure 2 B and supplemental Figure IVB). Overexpression of UCHL1 resulted in decreased basal ubiquitination of I B- and higher protein levels of I B- ( Figure 2 C and supplemental Figure IVB).
Role of UCHL1 in Cultured Smooth Muscle Cells
Treatment of A7r5 (embryonic rat aortic smooth muscle cells) or HASMCs with TNF- (10 ng/mL) for 12 hours significantly upregulated NF- B activity, whereas overexpression of the UCHL1 gene significantly attenuated NF- B activity, particularly in TNF- –stimulated samples (46% inhibition in A7r5 and 63% inhibition in HASMCs; P <0.05, compared with control under TNF- treatment) as shown in Figure 3 A and supplemental Figure IIIB. In fact, the inhibitory effect of UCHL1 was almost similar to that of eNOS.
Figure 3. Role of UCHL1 in cultured smooth muscle cells. A, Effect of overexpressed human UCHL1 on NF- B activity, as evaluated by the luciferase gene driven by the NF- B binding site in A7r5. "Control" indicates overexpressed GFP gene, "UCHL1" indicates overexpressed human UCHL1 gene, and "eNOS" indicates overexpressed eNOS gene. "TNF- " indicates treatment with human recombinant TNF- (10 ng/mL) for 12 hours. * P <0.05 vs control. # P <0.05 vs TNF- (–). n=6 per group in triplicate. B, Representative EMSA for NF- B activity in A7r5. "control" indicates overexpressed GFP gene, "UCHL1" indicates overexpressed human UCHL1 gene, "scramble" indicates transfection of scramble siRNA, "siRNA for UCHL1" indicates suppression of UCHL1 gene by transient transfection of siRNA for UCHL1. "competitor" indicates addition of non–RI- labeled consensus oligodeoxynucleotide of NF- B binding sequence in "control" or "scramble" sample. "FP" indicates free probe (nonbinding RI labeled oligodeoxynucleotide). "TNF- " indicates treatment with human recombinant TNF- (10 ng/mL) for 12 hours.
Suppression of UCHL1 expression in A7r5 using siRNA was confirmed by real-time RT-PCR (73% inhibition, relative expression to GAPDH: control, 1.00±0.63; siRNA, 0.27±0.12). Indeed, the transfection of siRNA in A7r5 cells resulted in an increase in NF- B activity regardless of TNF- (10 ng/mL) stimulation for 12 hours (supplemental Figure VIA). In the gel shift mobility assay, overexpression of UCHL1 gene also attenuated the binding of NF- B to DNA consensus sequence, and suppression of UCHL1 increased the binding of NF- B ( Figure 3 B). They were consistent with the results obtained in experiments with the luciferase gene driven by the NF- B binding site. Overexpressed UCHL1 attenuated TNF- –induced cytokine expression (66% inhibition in TNF-, 54% inhibition in interleukin -6, and 57% inhibition in matrix metalloprotease -9); P <0.05 compared with control), as quantified by real-time PCR (supplemental Figure VIB).
Expression of UCHL1 in Intact and Injured Arteries
Northern blot analysis demonstrated mRNA expression of human UCHL1 in aorta (supplemental Figure VIIA). Immunostaining analysis showed that UCHL1 was expressed in whole brain, brain microvessels, rat common carotid artery, and rat abdominal artery (supplemental Figures VIIB, VIIIA, and VIIIB).
UCHL1 expression was high in the neointimal smooth muscle cells of balloon-injured carotid artery, as confirmed by coimmunostaining with the VSMC specific marker, anti– -smooth muscle (SM) actin ( Figure 4 A). The mRNA levels of UCHL1 were increased 25-fold compared with sham-operated common carotid artery quantified by real-time PCR ( P <0.05). UCHL1 was also expressed in ECs and VSMCs in atherosclerotic lesions from human carotid arteries ( Figure 4 B).
Figure 4. Expression of UCHL1 in intact and injured arteries. A, Representative pictures of immunofluorescent staining in rat common carotid artery 14 days after balloon injury ( x 200 magnification). "DAPI" indicates nuclear staining (blue), " -SM actin" indicates the staining with anti– -SM actin antibody (green), "UCHL1" indicates the staining with anti-UCHL1 antibody (red), and "Merge" indicates staining with all three. Arrows (yellow) indicate the neointimal formation. B, Representative picture of immunofluorescent staining in atherosclerotic lesion of human carotid artery obtained from symptomatic patients ( x 200 magnification). Arrows (white) indicate endothelium. "DAPI" indicates nuclear staining (blue), " -SM actin" indicates the staining with anti- -SM actin antibody (green), "UCHL1" indicates the staining with anti-UCHL1 antibody (red), and "Merge" indicates staining with all three.
Functional Analysis of UCHL1 In Vivo
Expression of transfected UCHL1 gene was confirmed by RT-PCR and immunohistochemistry in carotid arteries 3 days after balloon injury (data not shown). As shown in Figure 5 A, overexpression of UCHL1 plasmid resulted in significant inhibition of neointimal formation, as quantified by measurement of neointimal and medial area and by calculation of the ratio of neointimal-to-medial area at 2 weeks after balloon injury (supplemental Figure IX, P <0.01). Accompanied with an inhibition of neointimal formation by transfection of UCHL1 plasmid DNA, the inhibition of NF- B activation was examined assessed by immunostaining at 1 week after transfection. These anti-p50 and anti-p65 (NF- B subunits) antibodies recognize the epitopes overlapping the nuclear location signal of the subunit of NF- B heterodimer, and fairly selectively bind to the activated form of NF- B. Thus, these results suggest that overexpressed UCHL1 attenuates NF- B activation in vivo ( Figure 5 B and supplemental Figure X).
Figure 5. Effects of UCHL1 on balloon-injured arteries. A, Effect of overexpressed UCHL1 on neointimal formation in rat balloon injury model at 2 weeks after transfection. The panel shows the representative cross sections of balloon-injured vessels by hematoxylin-and-eosin staining ( x 40 magnification). "Control" indicates the transfection of LacZ plasmid. "UCHL1" indicates the transfection of UCHL1 plasmid. B, Effect of overexpressed UCHL1 on NF- B inactivation in rat balloon injury model at 1 week after transfection, as assessed by immunostaining with anti-p65 antibody (green) and anti–ICAM-1 antibody (red). Left panel shows representative cross sections stained with anti-p65 antibody (green: x 500 magnification) and right panel stained with anti–ICAM-1 antibody (red: x 500 magnification). "Control" indicates the transfection of LacZ plasmid. "UCHL1" indicates the transfection of UCHL1 plasmid. "DAPI" indicates nuclear staining (blue). "Merge" indicates staining with both. Arrows (white) indicate neointimal formation.
Furthermore, the expression of NF- B regulatory proteins, ICAM-1, and MMP-9, were suppressed in the neointima of the overexpressed UCHL1 group, as assessed by immunostaining at 1 week after transfection ( Figure 5 B and supplemental Figure X).
These results in vitro and in vivo suggest that UCHL1 may deubiquitinate I B-, thereby resulting in NF- B inactivation ( Figure 6 ).
Figure 6. Summary scheme of UCHL1 function in vascular cells. TNF- induces the phosphorylation (P) of I B-, which leads to ubiquitination (Ub) and degradation of I B- by proteasome. In vascular cells, UCHL1 attenuates TNF- –induced NF- B activation via stabilization of I B-, possibly through the deubiquitination of I B-. Thus, UCHL1 may reduce NF- B–driven cytokines, such as inflammatory cytokines or adhesion molecules, and increase eNOS protein.
Discussion
The present study revealed that UCHL1 was expressed in atherosclerotic lesion and that UCHL1 may participate in vascular remodeling via inhibition of NF- B activity. Specifically, UCHL1 attenuated TNF- –induced NF- B activity and increased eNOS expression, which may directly attenuate atherosclerosis leading to the reduction of ischemic vascular disease. 23 Indeed, overexpressed UCHL1 reduced neointima formation in balloon-injured artery. The present study provides the first evidence that a deubiquitinating enzyme can modulate vascular remodeling.
UCHL1 (also known as pGp9.5) is predominantly expressed in central and peripheral neurons. Mutations in the UCHL1 gene have been identified in a single German PD family with a reduced penetrance inheritance pattern. 24 Further, a polymorphism in exon 3 of the UCHL1 gene (G18 years) is associated with a reduced susceptibility to PD in some populations 25,26 and may be related to onset of Huntington?s disease. 27 UCHL1 hydrolyzes small C-terminal adducts of ubiquitin to generate the ubiquitin monomer, making it an important component of the ubiquitin-proteasome system. Further, the ubiquitin-proteasome pathway has an important role in vascular remodeling 6 and cardiac fibrosis, 28 as demonstrated by the observation that treatment of cardiovascular cells with proteasome inhibitors results in strong antiproliferative, antiinflammatory, and proapoptotic effects. 6 In this study, although overexpressed UCHL1 attenuated the TNF- –induced inflammatory action, endogenous UCHL1 may not affect the first action of TNF- –induced inflammation because the induction of UCHL1 expression by TNF- was delayed. This suggests that UCHL1 cannot be directly targeted, but is chronically upregulated by several stimulants in vascular remodeling. This is consistent with recent studies that have demonstrated that full-length UCHL1 is a major target of oxidative damage and that UCHL1 is downregulated in the brain of patients with Alzheimer or Parkinson disease. 29 Thus, deubiquitinating enzymes, such as UCHL1, may act as suppressors during the activation of ubiquitin-proteasome system in atherosclerosis lesions.
Recent evidence has shown that UCHL1 is also highly expressed in carcinomas of various tissue origins, including those from brain, lung, breast, kidney, colon, prostate, pancreas, and mesenchymal tissues. 30 Indeed, in 1 lung cancer cell line, UCHL1 interacts with the Jun activation domain binding protein, JAB1, and a cyclin dependent kinase inhibitor, p27 (Kip1). 31 Further, UCHL1 exerts an antiproliferative response, and its expression may be induced as a response to tumor growth. 32 This is consistent with observations from the present study that overexpressed UCHL1 attenuated neointimal formation in balloon-injured artery. Interestingly, aspirin and statins, 2 of the most successful drugs in the attenuation of cardiovascular events, both exert an inhibitory effect on the proteosome. 22,33 From the perspective of the ubiquitin-proteasome system, the pleiotropic effect of aspirin was reported in the upregulation of eNOS expression. 22 In combination with the observation that aspirin upregulated I B- protein through inhibition of ubiquitin-proteasome system, 22 the present study may provide a new understanding of the mechanisms by which pharmacological agents may prevent atherosclerosis. Further study to determine the mechanisms by which UCHL1 or another deubiquitinating enzyme contributes to the process of cardiovascular diseases would be of benefit.
In conclusion, this study demonstrated expression of UCHL1 in vascular endothelial cells and smooth muscle cells and the antiinflammatory action of UCHL1 in the process of vascular remodeling. The UCHL1 gene may represent a novel therapeutic target for the attenuation of atherosclerosis and the prevention of cardiovascular events.
Acknowledgments
We thank all members of the Gene Therapy Science laboratory for critical discussion during the course of this work. We also thank Prof Nojima in Osaka University for providing the cDNA library.
Sources of Funding
This work was supported by the Northern Osaka (Saito) Biomedical Knowledge-Based Cluster Creation Project, Mitsubishi Pharma Research Foundation (to H.H), American Diabetes Association (to T.C, JFA 7-05-JF-12), the Salt Science Research Foundation (to T.O.), Takeda Science Foundation (to T.O.), and the Japan Heart Foundation (to T.K.), Grants-in-Aid for Scientific Research (15220401, 17090101) from the Japanese Ministry of Health, Labor, and Welfare, and Grants-in-Aid for Scientific Research (17650203, 16659224) from the Ministry of Education, Science, Sports and Culture of Japan.
Disclosures
None.
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作者单位：Department of Geriatric Medicine (Y.T., T.K., Y.B., O.Y., H.R., T.O.), Osaka University Graduate School of Medicine, Japan; the Division of Gene Therapy Science (Y.T., H.N., Y.S., H.H., Y.K., T.N., Y.K.), Osaka University Graduate School of Medicine, Japan; the Division of Clinical Gene Therapy (R.M