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Intramuscular vascular endothelial growth factor gene therapy in patients with chronic critical leg ischemia

      Abstract

      Purpose

      We sought to investigate the safety and efficacy of intramuscular gene therapy with vascular endothelial growth factor (VEGF) in patients with chronic critical leg ischemia.

      Methods

      Gene transfer was performed in 24 limbs of 21 patients with rest pain, some of whom also had nonhealing ischemic ulcers (n = 16) due to occlusive peripheral arterial disease. Between 400 μg and 2000 μg of phVEGF165 (400 μg, n = 2; 800 μg, n = 4; 1200 μg, n = 4; 1600 μg, n = 6; and 2000 μg, n = 8) was injected directly into the muscles of the ischemic limb; the same dose was injected 4 weeks later. The ratio of blood pressures at the ankle and brachial artery was measured before and after treatment.

      Results

      Mean (± SD) plasma levels of VEGF increased significantly from 26 ± 31 pg/mL to 63 ± 56 pg/mL (P <0.005), and the ankle-brachial index improved significantly from 0.58 ± 0.24 to 0.72 ± 0.28 (P <0.001). Magnetic resonance angiography showed qualitative evidence of improved distal flow in 19 limbs (79%). Ischemic ulcers healed or improved markedly in 12 limbs (75%). Rest pain was relieved or improved markedly in 20 limbs (83%). Amputation was performed in two limbs because of wound infection. Complications were limited to transient leg edema in six limbs.

      Conclusion

      Intramuscular gene therapy with VEGF165 for patients with chronic critical leg ischemia is safe, feasible, and effective.
      Despite advances in the treatment of occlusive peripheral arterial disease, many patients cannot be managed adequately with either medical therapy (
      • Hiatt W.R.
      Medical treatment of peripheral arterial disease and claudication.
      ) or revascularization procedures (
      • Beard J.D.
      Chronic lower limb ischemia.
      ). In some patients, the anatomic extent and distribution of arterial occlusion are too severe to permit relief of pain or healing of ischemic ulcers. Therapeutic angiogenesis, by promoting development of collateral vessels, may be useful in these patients (
      • Pu L.Q.
      • Sniderman A.D.
      • Arekat Z.
      • et al.
      Angiogenic growth factor and revascularization of the ischemic limb evaluation in a rabbit model.
      ,
      • Takeshita S.
      • Zheng L.P.
      • Brogi E.
      • et al.
      Therapeutic angiogenesis a single intra-arterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hindlimb model.
      ).
      Several growth factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor, can stimulate the development of collateral vessels in animal models of hind limb ischemia (
      • Baffour R.
      • Berman J.
      • Garb J.L.
      • et al.
      Enhanced angiogenesis and growth of collateral by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia dose-response effect of basic fibroblast growth factor.
      ,
      • Takeshita S.
      • Pu L.Q.
      • Zheng L.
      • et al.
      Vascular endothelial growth factor induces dose-dependent revascularization in a rabbit model of persistent limb ischemia.
      ). Among the growth factors, VEGF is specifically mitogenic for endothelial cells (
      • Leung D.W.
      • Cachianes G.
      • Kuang W.J.
      • et al.
      Vascular endothelial growth factor is a secreted angiogenic mitogen.
      ,
      • Keck P.J.
      • Hauser S.D.
      • Krivi G.
      • et al.
      Vascular permeability factor, an endothelial cell mitogen related to PDGF.
      ,
      • Plouet J.
      • Schilling J.
      • Gospodarowicz D.
      Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells.
      ), an important advantage of VEGF for gene therapy because endothelial cells are responsible for neovascularization. Intramuscular gene therapy with VEGF promotes collateral vessel development, relieves ischemic symptoms, and improves endothelial function in patients with critical limb ischemia (
      • Baumgartner I.
      • Pieczek A.
      • Manor O.
      • et al.
      Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.
      ,
      • Rajagopalan S.
      • Shah M.
      • Luciano A.
      • et al.
      Adenovirus-mediated gene transfer of VEGF121 improves lower-extremity endothelial function and flow reserve.
      ). However, the minimal effective dose of VEGF plasmid is uncertain, and it is also not known whether racial differences affect therapeutic results. Therefore, we studied several doses of intramuscular VEGF plasmid for the treatment of chronic critical leg ischemia in Chinese patients.

      Methods

      Patient selection

      Patients qualified for intramuscular gene therapy if they had chronic critical limb ischemia (
      European Working Group on Critical Leg Ischemia
      Second European consensus document on chronic critical leg ischemia.
      ), including rest pain or nonhealing ischemic ulcers for a minimum of 4 weeks without evidence of improvement in response to conventional therapies, and were not optimal candidates for surgical or percutaneous revascularization (
      Standards of Practice Committee of the Society of Cardiovascular and Interventional Radiology
      Guidelines for percutaneous transluminal angioplasty.
      ). The ratio of systolic blood pressure at the ankle and the brachial artery (the ankle-brachial index) was <0.6, or the great toe pressure was <30 mm Hg in patients with noncompressible ankle arteries. All patients had angiographic evidence of superficial femoral artery or infrapopliteal disease in the index limb. All patients had no new medications started and did not stop any medications that would have reduced flow. We excluded patients with radiographic and radioisotopic evidence of osteomyelitis in the ischemic extremity; a history of alcohol or drug abuse within the past 3 months; previous or current history of neoplasm; hepatic dysfunction; or evidence of possible malignancies following evaluation with carcinoembryonic antigen levels, chest radiographs, computerized tomographic scan, and mammography in women or prostate examination and prostate-specific antigen levels in men. The protocol was approved by the Human Research Committee of our institution and the Department of Health, Executive Yuan, Taiwan. All patients gave written informed consent to participate.
      Patients were followed on a biweekly basis for the first 10 weeks after gene therapy, and at monthly intervals thereafter. Ischemic ulcers were documented by color photography. Shrinkage of the ulcer area to less than half of the baseline area was defined as improvement. Improvement in rest pain was evaluated by questionnaire and the amounts of analgesic medications used before and after therapy. Visual acuity and fundoscopic examination were checked before and 4 weeks after the second injection of plasmid in patients with diabetes mellitus.

      Plasmid deoxyribonucleic acid (phVEGF165) preparation and administration

      All patients received a eukaryotic expression vector encoding the VEGF165 gene (
      • Tischer E.
      • Mitchell R.
      • Hartmann T.
      • et al.
      The human gene for vascular endothelial growth factor multiple protein forms are encoded through alternative exon splicing.
      ,
      • Isner J.M.
      • Pieczek A.
      • Schainfield R.
      • et al.
      Clinical evidence of angiogenesis following arterial gene transfer of phVEGF165.
      ). Preparation and purification of the plasmid from cultures of phVEGF165-transformed Escherichia coli were performed in the central laboratory at our hospital with the endotoxin-free column method (Qiagen Mega Kit, Qiagen Inc., Valencia, California). The purified plasmid was stored in vials and pooled for quality-control analysis.
      Aliquots of 400 μg of phVEGF165 were diluted in sterile saline, and one to five 2-mL aliquots (total, 400 μg to 2000 μg) were injected directly into the calf muscles of the ischemic limb. The injection sites were selected arbitrarily according to available muscle mass in the calf. A second injection of the same dose was administered 4 weeks later. Patients were not aware of the dose they received.

      Plasma levels of vascular endothelial growth factor

      Plasma VEGF levels were measured at baseline and 2 weeks after the first dose of VEGF to detect evidence of gene expression. Samples were immediately centrifuged for 20 minutes at 4000 rpm at 4°C and stored at –70°C until analysis. Plasma VEGF levels were measured with an enzyme immunoassay technique according to the manufacturer’s instruction (R & D Systems, Inc., Minneapolis, Minnesota). Results were compared with a standard curve of human VEGF with a lower detection limit of 5 pg/mL. Samples were checked by serial dilution and were performed at least in duplicate. The intra-assay coefficient of variation was 4.1%, and the interassay coefficient of variation was 5.6%.

      Ankle-brachial index

      Resting ankle-brachial indices were calculated as the ratio of the lowest pressure from either the posterior or anterior tibial arteries divided by the greatest brachial systolic pressure, which were obtained 1 week before and 4 weeks after completing the two injections. A technician who was unaware of the treatment status of the patients performed this examination.

      Magnetic resonance angiography

      Moving-bed infusion-tracking magnetic resonance (MR) angiography (
      • Ho K.Y.
      • Leiner T.
      • de Haan M.W.
      • et al.
      Peripheral vascular tree stenosis evaluation with moving-bed infusion-tracking MR angiography.
      ) was performed 1 week before and 4 weeks after completing the two injections. Angiograms were obtained with a 1.5-T MR system (Impact; Siemens, Erlangen, Germany). A body coil was used for signal transmission and reception. The dynamic study was acquired twice; before infusion of contrast material and during infusion. To obtain accurate and reproducible table movement, a wooden stick with three premeasured stops was used. After acquisition of the unenhanced study, 0.4 mL/kg of preheated gadopentetate dimeglumine was injected. The two dynamic studies were reconstructed; nonenhanced volumes were subtracted from corresponding gadolinium-enhanced volumes in all regions. An increase in the number of visible vessels, or an increase in the intensity or apparent size of a previously visible vessel, was considered improvement. A radiologist who was not aware of the treatment status of the patients interpreted the MR angiograms. Quantitative MR angiographic analysis of collateral vessel development was used to derive an angiographic score for each film, defined as the ratio of grid intersections crossed by opacified arteries divided by the total number of grid intersections from knee to ankle.

      Statistical analysis

      Paired t tests were used to compare continuous variables before and after therapy; analysis of variance followed by Scheffé’s procedure was used to compare three or more means. A value of P <0.05 was considered significant.

      Results

      A total of 21 patients with 24 limbs met all eligibility criteria and were treated with intramuscular injections of phVEGF165. Their mean (± SD) age was 65 ± 16 years (range, 21 to 84 years), and 6 of the patients were women (Table). Patients had rest pain in all 24 limbs; there were nonhealing ischemic ulcers in 16 limbs and gangrene in 4 limbs. Five patients had a prior surgical bypass graft, and 4 had undergone previous toe amputations. Six patients were current smokers, and 6 were ex-smokers; 10 had diabetes mellitus, 7 had hypertension, and 6 had hyperlipidemia.
      TableClinical Characteristics of Patients, Dose Protocol for phVEGF165, and Leg Edema after Therapy
      Patient No.SexAge (years)SmokingDiabetes MellitusSigns and SymptomsAffected Limb
      Indicates the first leg that was treated. Patients 3, 6, and 10 were also treated in the contralateral leg.
      Underlying Vascular DiseaseVEGF DoseBooster (2000 μg)Edema after Therapy
      1M72++Rest painLeftAtherosclerosis400 μg × 2+0
      2F670+Rest painLeftAtherosclerosis400 μg × 2+0
      3M84+0Rest painLeftAtherosclerosis800 μg × 200
      Rest painRightAtherosclerosis1200 μg × 200
      4M75++Rest painLeftAtherosclerosis800 μg × 2+0
      5F750+Rest pain, ulcerLeftAtherosclerosis800 μg × 200
      6M39+0Rest painRightBurger’s disease800 μg × 200
      Rest painLeftBurger’s disease2000 μg × 200
      7M700+Rest painLeftAtherosclerosis1200 μg × 200
      8M740+Rest pain, ulcerLeftAtherosclerosis1200 μg × 200
      9M41+0Rest pain, ulcerLeftBurger’s disease1200 μg × 2+0
      10M73++Rest pain, ulcerLeftAtherosclerosis1600 μg × 200
      Rest pain, ulcerRightAtherosclerosis2000 μg × 20+
      11
      Amputation below the knee joint was performed in patients 17 and 18. Patient 11 also failed to respond to treatment.
      M610+Rest pain, ulcerRightAthersclerosis1600 μg × 200
      12M5300Rest pain, ulcerLeftAtherosclerosis1600 μg × 2++
      13M21+0Rest pain, ulcerRightBurger’s disease1600 μg × 20+
      14M76+0Rest pain, ulcer, toe gangreneRightAtherosclerosis1600 μg × 20+
      15F590+Rest pain, ulcerRightAtherosclerosis1600 μg × 20+
      16F71+0Rest pain, ulcerLeftAtherosclerosis2000 μg × 200
      17
      Amputation below the knee joint was performed in patients 17 and 18. Patient 11 also failed to respond to treatment.
      M780+Rest pain, ulcer, toe gangreneLeftAtherosclerosis2000 μg × 200
      18
      Amputation below the knee joint was performed in patients 17 and 18. Patient 11 also failed to respond to treatment.
      F7800Rest pain, ulcerRightAtherosclerosis2000 μg × 20+
      19M73+0Rest pain, ulcer, toe gangreneRightAtherosclerosis2000 μg × 200
      20M42+0Rest pain, ulcerLeftBurger’s disease2000 μg × 200
      21F79++Rest pain, ulcer, toe gangreneLeftAthersclerosis2000 μg × 200
      + = present; 0 = not present; F = female; M = male; VEGF = vascular endothelial growth factor.
      * Indicates the first leg that was treated. Patients 3, 6, and 10 were also treated in the contralateral leg.
      Amputation below the knee joint was performed in patients 17 and 18. Patient 11 also failed to respond to treatment.

      PhVEGF165 dose

      The dose of VEGF ranged from 400 μg to 2000 μg (Table). Several patients received a 2000-μg booster dose 2 to 3 months after the second injection (2 patients treated 400 μg, 1 patient treated with 800 μg, 1 patient treated with 1200 μg, and 1 patient treated with 1600 μg). Three patients had bilateral critical limb ischemia; the second limb was treated 1 month after treatment of the first limb. The maximal treatment dose for one limb was 5200 μg (patient 12); for bilateral treatment, the maximal dose was 7200 μg (patient 10).

      Transgene expression

      Mean blood levels of VEGF increased significantly from 26 ± 31 pg/mL before treatment to 63 ± 56 pg/mL (P <0.005) 2 weeks after the first dose of gene therapy (Figure 1A). The increase in VEGF levels, however, was highly variable; although there appeared to be a dose-related response (Figure 1B), it was not statistically significant (P = 0.46).
      Figure thumbnail GR1
      Figure 1A: Plasma vascular endothelial growth factor (VEGF) levels before treatment and 2 weeks after intramuscular injection of phVEGF165. The dot and bar indicate mean ± SD; the asterisk indicates P = 0.001. B: Change in plasma VEGF levels by doses of phVEGF165. The bar indicates the mean; the error bar indicates the SD.

      Clinical follow-up and safety assessment

      Intramuscular gene transfer induced no or mild local discomfort for up to 48 hours after injection. Mild (limited to below the ankle) and transient leg edema occurred in 6 (25%) of the 24 treated limbs, and only in patients with nonhealing ischemic ulcers who had been treated with 1600 or 2000 μg of VEGF (Table). Edema subsided spontaneously without use of diuretics.
      All patients were followed for at least 6 months. Amputation of the affected limb because of a large ulcer with a severe wound infection was performed in 2 patients who had been treated with 2000 μg of VEGF. Rest pain or the leg ulcer improved markedly within 1 month in 4 of the 5 patients treated with a booster injection for persistent rest pain.
      Overall, rest pain resolved completely in 12 limbs and improved markedly in eight limbs. The ischemic ulcer healed in six limbs (Figure 2) and improved markedly in 6 limbs. Thus, therapeutic benefit was demonstrated by regression of rest pain in 20 (83%) of 24 ischemic limbs and improved tissue integrity in 12 (75%) of 16 ischemic limbs with ulceration. Treatment failure occurred in 2 patients treated with 400 μg, 1 patient treated with 800 μg, 1 patient treated with 1200 μg, and 1 patient treated with 1600 μg. No diabetic patient had a decline in visual acuity or clinically apparent fundoscopic changes.
      Figure thumbnail GR2
      Figure 2Limb salvage after gene therapy. A 76-year-old man presented with nonhealing wounds for 1 month on the right ankle and foot (A). After gene therapy (B), the wound healed completely. His ankle-brachial index increased by 0.37 in association with increased flow on magnetic resonance angiography. A 39-year-old man presented with nonhealing ulcers on the right foot for 1 year (C). After gene therapy (D), the wounds improved markedly. The ankle-brachial index increased by 0.2 after gene therapy.

      Change in ankle-brachial index

      The mean ankle-brachial index increased significantly from 0.58 ± 0.24 before treatment to 0.72 ± 0.28 (P <0.001) 4 weeks after completing two injections (Figure 3A). The increase in ankle-brachial index in patients receiving 1600 μg was significantly higher than that in patients receiving 400 μg or 800 μg (Figure 3B), although there was no further improvement in patients who received the 2000-μg dose. The mean ankle-brachial index in the 5 patients who received booster doses changed from 0.47 ± 0.22 before therapy to 0.56 ± 0.21 after two injections to 0.66 ± 0.18 after the booster dose.
      Figure thumbnail GR3
      Figure 3A: Ankle-brachial index before and 4 weeks after gene therapy. The dot and bar indicate mean ± SD; the asterisk indicates P <0.001. B: Increase (mean ± SD) in ankle-brachial index by doses of phVEGF165. VEGF = vascular endothelial growth factor.

      Change in magnetic resonance angiography

      Magnetic resonance angiography showed superficial femoral artery or infrapopliteal disease in all 24 limbs, 19 of which showed qualitative evidence of improved distal flow after gene therapy (Figure 4). The mean MR angiographic score increased significantly from 0.37 ± 0.10 before treatment to 0.47 ± 0.11 after gene therapy (P <0.01; (Figure 5).
      Figure thumbnail GR4
      Figure 4Magnetic resonance angiogram before (A) and 4 weeks after (B) gene therapy. After gene therapy, signal enhancement and collaterals are evident (arrow), consistent with improved flow in the ischemic limb.
      Figure thumbnail GR5
      Figure 5Magnetic resonance (MR) angiographic score before and 4 weeks after gene therapy. The dot and bar indicate mean ± SD; the asterisk indicates P = 0.00.

      Discussion

      The prognosis and quality of life for patients with chronic critical leg ischemia, as manifested by rest pain or ischemic ulcers, are poor (
      • Albers M.
      • Fratezi A.C.
      • DeLuccia N.
      Assessment of quality of life of patients with severe ischemia as a result of infrainguinal arterial occlusive disease.
      ), and no pharmacologic treatment has been shown to be effective in these patients (
      • Isner J.M.
      • Rosenfield K.
      Redefining the treatment of peripheral artery disease.
      ). Indeed, amputation, despite the associated morbidity, mortality, and functional implications (
      • Most R.S.
      • Sinnock P.
      The epidemiology of lower extremity amputations in diabetic individuals.
      ,

      Taylor LM Jr, Porter JM. Natural history and non-operative treatment of chronic lower extremity ischemia. In: Rutherford BB, ed. Vascular Surgery. Philadelphia, Pennsylvania: W. B. Saunders;1989:656

      ,
      • Wolfe J.H.N.
      Defining the outcome of critical ischemia a one year prospective study.
      ), is often recommended as a solution for disabling symptoms (
      • Eneorth M.
      • Person B.M.
      Amputation for occlusive arterial disease a multicenter study of 177 amputees.
      ,
      • Campbell W.B.
      • Johnston J.A.
      • Kernick V.F.
      • Rutter E.A.
      Lower limb amputation striking the balance.
      ,
      • Dawson I.
      • Keller B.P.
      • Brand R.
      • et al.
      Late outcomes of limb loss after failed infrainguinal bypass.
      ,
      • Skinner J.A.
      • Cohen A.T.
      Amputation for premature peripheral atherosclerosis do young patients do better?.
      ). Thus, the need for alternative treatment strategies is compelling. Intramuscular injection of naked plasmid deoxyribonucleic acid has a wide range of applications, because striated muscle can take up and express foreign genes (
      • Wolff J.A.
      • Malone R.W.
      • Williams P.
      • et al.
      Direct gene transfer into mouse muscle in vivo.
      ,
      • Wolff J.A.
      • Williams P.
      • Acsadi G.
      • et al.
      Conditions affecting direct gene transfer into rodent muscle in vivo.
      ,
      • Wolff J.A.
      • Ludtke J.J.
      • Acsadi G.
      • et al.
      Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle.
      ). Gene therapy with VEGF has been used successfully in a patient with critical leg ischemia who was treated with arterial infusion of VEGF165 plasmid (
      • Isner J.M.
      • Pieczek A.
      • Schainfield R.
      • et al.
      Clinical evidence of angiogenesis following arterial gene transfer of phVEGF165.
      ). A previous study showed that a fixed dose of intramuscular gene therapy with phVEGF165 was safe and effective in the treatment of patients with critical limb ischemia (
      • Baumgartner I.
      • Pieczek A.
      • Manor O.
      • et al.
      Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.
      ). However, high-dose VEGF induces angioma formation in the rat heart (
      • Schwarz E.R.
      • Speakman M.T.
      • Patterson M.
      • et al.
      Evaluation of the effects of intramuscular injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat—angiogenesis and angioma formation.
      ), and the efficacy and safety of this kind of gene therapy needs clarification (
      • Isner J.M.
      • Vale P.R.
      • Symes J.F.
      • Losordo D.W.
      Assessment of risks associated with cardiovascular gene therapy in human subjects.
      ,
      • Manninen H.I.
      • Makinen K.
      Gene therapy for peripheral arterial disease.
      ). Compared with that previous study (
      • Baumgartner I.
      • Pieczek A.
      • Manor O.
      • et al.
      Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.
      ), we observed a lower complication rate (25% vs. 67%) and a higher failure rate (25% vs. 10%), perhaps because we used a lower dose of phVEGF165.
      Although the ankle-brachial index, which is subject to measurement error, does not parallel local perfusion changes, an increase of >0.1 in the index indicates a successful surgical or percutaneous intervention (
      • Rutherford R.B.
      • Becker G.J.
      Standards for evaluating and reporting the results of surgical and percutaneous therapy for peripheral arterial disease.
      ). We observed increases in the mean ankle-brachial index after gene therapy in patients treated with at least 1200-μg injections of phVEGF165, suggesting that 2400 μg of phVEGF165 (all patients received two doses) is the minimal effective dose for the treatment of chronic critical leg edema in Chinese patients.
      Traditionally, conventional arteriography has served as the gold standard for the diagnostic evaluation of lower extremity vascular disease. However, conventional arteriography is invasive and potentially hazardous. Magnetic resonance angiography has been shown to provide similar information as conventional angiography (
      • Ho K.Y.
      • Leiner T.
      • de Haan M.W.
      • et al.
      Peripheral vascular tree stenosis evaluation with moving-bed infusion-tracking MR angiography.
      ,
      • Glikerman D.J.
      • Obregon R.G.
      • Schmiedl U.P.
      • et al.
      Cardiac-gated MR angiography of the entire lower extremity a prospective comparison with conventional angiography.
      ); it is noninvasive and can be performed in outpatients, as in our study.
      Our study has several limitations. It was not randomized, placebo-controlled, or double-blind. However, we believe that the improvement in clinical symptoms and signs in our study cannot be attributed to a placebo effect, as patients were unaware of their phVEGF165 dose, and there was no therapeutic benefit from low doses of phVEGF165. Although patients who received higher doses appeared to have greater improvement, this finding cannot substitute for a true control group. We did perform the ankle/brachial index and MR angiographic examination in a blinded manner. Finally, we did not perform exercise testing to determine walking capacity.
      In conclusion, we found that intramuscular VEGF gene transfer was safe and effective in Chinese patients with chronic critical leg ischemia. The benefits of treatment on the ankle-brachial index appeared to require at least a 1200-μg dose. Edema after intramuscular gene therapy with VEGF was dose related, perhaps because more patients with ulcers, who had a greater risk of edema, were treated with higher doses. Clinical efficacy, including resolution of rest pain, limb salvage, and healing of ischemic ulcers, was associated with objective findings of improved ankle-brachial indices and blood flow on MR angiography. However, two limbs were amputated because of severe infection of an ulcer, emphasizing that meticulous wound care is necessary.

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