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1.
Blood ; 138(18): 1677-1690, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33895800

RESUMO

Adeno-associated virus (AAV)-mediated gene therapy is a novel treatment promising to reduce morbidity associated with hemophilia. Although multiple clinical trials continue to evaluate efficacy and safety, limited cost-effectiveness data have been published. This study compared the potential cost-effectiveness of AAV-mediated factor IX (FIX)-Padua gene therapy for patients with severe hemophilia B in the United States vs on-demand FIX replacement and primary FIX prophylaxis, using either standard or extended half-life FIX products. A microsimulation Markov model was constructed, and transition probabilities between health states and utilities were informed by using published data. Costs were aggregated by using a microcosting approach. A time horizon from 18 years old until death, from the perspective of a third-party payer in the United States, was conducted. Gene therapy was more cost-effective than both alternatives considering a $150 000/quality-adjusted life-year threshold. The price for gene therapy was assumed to be $2 000 000 in the base case scenario; however, one of the 1-way sensitivity analyses was conducted by using observed manufacturing, administration, and 5-year follow-up costs of $87 198 for AAV-mediated gene therapy vector as derived from the manufacturing facility and clinical practice at St Jude Children's Research Hospital. One-way sensitivity analyses revealed 10 of 102 scenarios in which gene therapy was not cost-effective compared with alternative treatments. Notably, gene therapy remained cost-effective in a hypothetical scenario in which we estimated that the discounted factor concentrate price was 20% of the wholesale acquisition cost in the United States. Probabilistic sensitivity analysis estimated gene therapy to be cost-effective at 92% of simulations considering a $150 000/quality-adjusted life-year threshold. In conclusion, based on detailed simulation inputs and assumptions, gene therapy was more cost-effective than on-demand treatment and prophylaxis for patients with severe hemophilia B.


Assuntos
Terapia Genética/economia , Hemofilia B/terapia , Adulto , Simulação por Computador , Análise Custo-Benefício , Hemofilia B/economia , Hemofilia B/epidemiologia , Humanos , Cadeias de Markov , Probabilidade , Estados Unidos/epidemiologia
2.
J Gene Med ; 14(9-10): 609-20, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23065925

RESUMO

BACKGROUND: Adeno-associated virus serotype 9 (AAV9) vectors provide efficient and uniform gene expression to normal myocardium following systemic administration, with kinetics that approach steady-state within 2-3 weeks. However, as a result of the delayed onset of gene expression, AAV vectors have not previously been administered intravenously after reperfusion for post-infarct gene therapy applications. The present study evaluated the therapeutic potential of post-myocardial infarction gene delivery using intravenous AAV9. METHODS: AAV9 vectors expressing firefly luciferase, enhanced green fluorescent protein (eGFP) or extracellular superoxide dismutase genes from the cardiac troponin-T (cTnT) promoter (AcTnTLuc, AcTnTeGFP, AcTnTEcSOD) were employed. AcTnTLuc was administered intravenously at 10 min and at 1, 2 and 3 days post-ischemia/reperfusion (IR), and the kinetics of luciferase expression were assessed with bioluminescence imaging. AcTnTeGFP was used to evaluate the distribution of eGFP expression. High-resolution echocardiography was used to evaluate the effects of AcTnTEcSOD on left ventricular (LV) remodeling when injected 10 min post-IR. RESULTS: Compared to sham animals, luciferase expression at 2 days after vector administration was elevated by four-, 24-, 210- and 213-fold in groups injected at 10 min, 1 day, 2 days and 3 days post-IR, respectively. The expression of cTnT-driven eGFP was strongest in cardiomyocytes bordering the infarct zone. In the efficacy study of EcSOD, post-infarct LV end-systolic and end-diastolic volumes at days 14 and 28 were significantly smaller in the EcSOD group compared to the control. CONCLUSIONS: Systemic administration of AAV9 vectors after IR both elevates and accelerates gene expression that preferentially targets cardiomyocytes in the border zone with pharmacodynamics suitable for the attenuation of LV remodeling.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacocinética , Infarto do Miocárdio/terapia , Reperfusão Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Remodelação Ventricular/genética , Animais , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/terapia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/terapia , Valores de Referência , Transgenes
3.
Magn Reson Med ; 67(1): 201-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21630350

RESUMO

T(2) -weighted, cardiac magnetic resonance imaging (T(2) w CMR) can be used to noninvasively detect and quantify the edematous region that corresponds to the area at risk (AAR) following myocardial infarction (MI). Previously, CMR has been used to examine structure and function in mice, expediting the study of genetic manipulations. To date, CMR has not been applied to imaging of post-MI AAR in mice. We developed a whole-heart, T(2) w CMR sequence to quantify the AAR in mouse models of ischemia and infarction. The ΔB(0) and ΔB(1) environment around the mouse heart at 7 T were measured, and a T(2) -preparation sequence suitable for these conditions was developed. Both in vivo T(2) w and late gadolinium enhanced CMR were performed in mice after 20-min coronary occlusions, resulting in measurements of AAR size of 32.5 ± 3.1 (mean ± SEM)% left ventricular mass, and MI size of 50.1 ± 6.4% AAR size. Excellent interobserver agreement and agreement with histology were also found. This T(2) w imaging method for mice may allow for future investigations of genetic manipulations and novel therapies affecting the AAR and salvaged myocardium following reperfused MI.


Assuntos
Edema Cardíaco/etiologia , Edema Cardíaco/patologia , Imageamento por Ressonância Magnética/métodos , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
4.
Mol Ther Methods Clin Dev ; 19: 275-284, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33102619

RESUMO

With many ongoing clinical trials utilizing adeno-associated virus (AAV) gene therapy, it is necessary to find scalable and serotype-independent primary capture and recovery methods to allow for efficient and robust manufacturing processes. Here, we demonstrate the ability of a hydrophobic interaction chromatography membrane to capture and recover AAV1, AAV5, AAV8, and AAV "Mutant C" (a novel serotype incorporating elements of AAV3B and AAV8) particles from cell culture media and cell lysate with recoveries of 76%-100% of loaded material, depending on serotype. A simple, novel technique that integrates release and recovery of cell-associated AAV capsids is demonstrated. We show that by the addition of lyotropic salts to AAV-containing cell suspensions, AAV is released at an equivalent efficiency to mechanical lysis. The addition of the lyotropic salt also promotes a phase separation, which allows physical removal of large amounts of DNA and insoluble cellular debris from the AAV-containing aqueous fraction. The AAV is then captured and eluted from a hydrophobic interaction chromatography membrane. This integrated lysis and primary capture and recovery technique facilitates substantial removal of host-cell DNA and host-cell protein impurities.

5.
Hum Gene Ther Methods ; 27(3): 112-21, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27229773

RESUMO

Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.


Assuntos
Fator IX/biossíntese , Terapia Genética , Vetores Genéticos/uso terapêutico , Reatores Biológicos , Dependovirus/genética , Fator IX/genética , Células HEK293 , Humanos , Transfecção
6.
J Am Heart Assoc ; 5(4)2016 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-27107133

RESUMO

BACKGROUND: Ivabradine selectively inhibits the pacemaker current of the sinoatrial node, slowing heart rate. Few studies have examined the effects of ivabradine on the mechanical properties of the heart after reperfused myocardial infarction (MI). Advances in ultrasound speckle-tracking allow strain analyses to be performed in small-animal models, enabling the assessment of regional mechanical function. METHODS AND RESULTS: After 1 hour of coronary occlusion followed by reperfusion, mice received 10 mg/kg per day of ivabradine dissolved in drinking water (n=10), or were treated as infarcted controls (n=9). Three-dimensional high-frequency echocardiography was performed at baseline and at days 2, 7, 14, and 28 post-MI. Speckle-tracking software was used to calculate intramural longitudinal myocardial strain (Ell) and strain rate. Standard deviation time to peak radial strain (SD Tpeak Err) and temporal uniformity of strain were calculated from short-axis cines acquired in the left ventricular remote zone. Ivabradine reduced heart rate by 8% to 16% over the course of 28 days compared to controls (P<0.001). On day 28 post-MI, the ivabradine group was found to have significantly smaller end-systolic volumes, greater ejection fraction, reduced wall thinning, and greater peak Ell and Ell rate in the remote zone, as well as globally. Temporal uniformity of strain and SD Tpeak Err were significantly smaller in the ivabradine-treated group by day 28 (P<0.05). CONCLUSIONS: High-frequency ultrasound speckle-tracking demonstrated decreased left ventricular remodeling and dyssynchrony, as well as improved mechanical performance in remote myocardium after heart rate reduction with ivabradine.


Assuntos
Benzazepinas/uso terapêutico , Frequência Cardíaca/efeitos dos fármacos , Contração Miocárdica/fisiologia , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/complicações , Função Ventricular Esquerda/fisiologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Fármacos Cardiovasculares/uso terapêutico , Modelos Animais de Doenças , Ecocardiografia , Frequência Cardíaca/fisiologia , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Ivabradina , Imagem Cinética por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/diagnóstico , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Função Ventricular Esquerda/efeitos dos fármacos
7.
Mol Ther Methods Clin Dev ; 3: 16015, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27069949

RESUMO

With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.

8.
Oxid Med Cell Longev ; 2015: 585297, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26161239

RESUMO

BACKGROUND: Activation of the adenosine A2B receptor (A2BR) can reduce myocardial ischemia/reperfusion (IR) injury. However, the mechanism underlying the A2BR-mediated cardioprotection is less clear. The present study was designed to investigate the potential mechanisms of cardioprotection mediated by A2BR. METHODS AND RESULTS: C57BL/6 mice underwent 40-minute ischemia and 60-minute reperfusion. ATL-801, a potent selective A2BR antagonist, could not block ischemic preconditioning induced protection. BAY 60-6583, a highly selective A2BR agonist, significantly reduced myocardial infarct size, and its protective effect could be blocked by either ATL-801 or wortmannin. BAY 60-6583 increased phosphorylated Akt (p-Akt) levels in the heart at 10 min of reperfusion, and this phosphorylation could also be blocked by ATL-801 or wortmannin. Furthermore, BAY 60-6583 significantly increased M2 macrophages and decreased M1 macrophage and neutrophils infiltration in reperfused hearts, which also could be blocked by wortmannin. Meanwhile, confocal imaging studies showed that the majority of Akt phosphorylation in the heart was colocalized to CD206+ cells in both control and BAY 60-6583 pretreated hearts. CONCLUSION: Our results indicated that pretreatment with BAY 60-6583 protects the heart against myocardial IR injury by its anti-inflammatory effects, probably by modulating macrophages phenotype switching via a PI3K/Akt pathway.


Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Macrófagos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor A2B de Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Aminopiridinas/farmacologia , Androstadienos/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Precondicionamento Isquêmico Miocárdico , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptor A2B de Adenosina/química , Transdução de Sinais , Wortmanina
9.
Physiol Rep ; 3(3)2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25825543

RESUMO

Extracellular matrix (ECM) degradation after myocardial infarction (MI) leaves the myocardium structurally weakened and, as a result, susceptible to early infarct zone dyskinesia and left ventricular (LV) remodeling. While various cellular and biomaterial preparations have been transplanted into the infarct zone in hopes of improving post-MI LV remodeling, an allogeneic cardiac muscle-derived ECM extract has yet to be developed and tested in the setting of reperfused MI. We sought to determine the effects of injecting a novel cardiac muscle-derived ECM into the infarct zone on early dyskinesia and LV remodeling in a mouse model of MI. Cardiac muscle ECM was extracted from frozen mouse heart tissue by a protocol that enriches for basement membrane constituents. The extract was injected into the infarct zone immediately after ischemia/reperfusion injury (n = 6). Echocardiography was performed at baseline and at days 2, 7, 14, and 28 post-MI to assess 3D LV volumes and cardiac function, as compared to infarcted controls (n = 9). Early infarct zone dyskinesia was measured on day 2 post-MI using a novel metric, the dyskinesia index. End-systolic volume was significantly reduced in the ECM-treated group compared to controls by day 14. Ejection fraction and stroke volume were also significantly improved in the ECM-treated group. ECM-treated hearts showed a significant (P < 0.005) reduction in dyskinetic motion on day 2. Thus, using high-frequency ultrasound, it was shown that treatment with a cardiac-derived ECM preparation reduced early infarct zone dyskinesia and post-MI LV remodeling in a mouse model of reperfused MI.

10.
PLoS One ; 8(9): e75894, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086659

RESUMO

AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×10(10) to 1.8×10(11) viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×10(11) viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.


Assuntos
Dependovirus/metabolismo , Técnicas de Silenciamento de Genes/métodos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Miocárdio/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Animais , Western Blotting , Primers do DNA/genética , Dependovirus/genética , Proteínas de Fluorescência Verde/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência
11.
PLoS One ; 7(7): e41736, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22848585

RESUMO

Skeletal myoblast transplantation has therapeutic potential for repairing damaged heart. However, the optimal conditions for this transplantation are still unclear. Recently, we demonstrated that satellite cell-derived myoblasts lacking the MyoD gene (MyoD(-/-)), a master transcription factor for skeletal muscle myogenesis, display increased survival and engraftment compared to wild-type controls following transplantation into murine skeletal muscle. In this study, we compare cell survival between wild-type and MyoD(-/-) myoblasts after transplantation into infarcted heart. We demonstrate that MyoD(-/-) myoblasts display greater resistance to hypoxia, engraft with higher efficacy, and show a larger improvement in ejection fraction than wild-type controls. Following transplantation, the majority of MyoD(-/-) and wild-type myoblasts form skeletal muscle fibers while cardiomyocytes do not. Importantly, the transplantation of MyoD(-/-) myoblasts induces a high degree of angiogenesis in the area of injury. DNA microarray data demonstrate that paracrine angiogenic factors, such as stromal cell-derived factor-1 (SDF-1) and placental growth factor (PlGF), are up-regulated in MyoD(-/-) myoblasts. In addition, over-expression and gene knockdown experiments demonstrate that MyoD negatively regulates gene expression of these angiogenic factors. These results indicate that MyoD(-/-) myoblasts impart beneficial effects after transplantation into an infarcted heart, potentially due to the secretion of paracrine angiogenic factors and enhanced angiogenesis in the area of injury. Therefore, our data provide evidence that a genetically engineered myoblast cell type with suppressed MyoD function is useful for therapeutic stem cell transplantation.


Assuntos
Proteína MyoD/genética , Mioblastos/transplante , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Neovascularização Fisiológica , Transplante de Células-Tronco , Disfunção Ventricular Esquerda/cirurgia , Animais , Diferenciação Celular , Hipóxia Celular , Proliferação de Células , Sobrevivência Celular , Quimiocina CXCL12/genética , Técnicas de Cocultura , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/patologia , Mioblastos/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/cirurgia , Miocárdio/metabolismo , Fator de Crescimento Placentário , Proteínas da Gravidez/genética
12.
Proc Natl Acad Sci U S A ; 104(42): 16552-7, 2007 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-17940048

RESUMO

MyoD is a myogenic master transcription factor that plays an essential role in muscle satellite cell (muscle stem cell) differentiation. To further investigate the function of MyoD in satellite cells, we examined the transplantation of satellite cell-derived myoblasts lacking the MyoD gene into regenerating skeletal muscle. After injection into injured muscle, MyoD(-/-) myoblasts engrafted with significantly higher efficiency compared with wild-type myoblasts. In addition, MyoD(-/-) myoblast-derived satellite cells were detected underneath the basal lamina of muscle fibers, indicating the self-renewal property of MyoD(-/-) myoblasts. To gain insights into MyoD gene deficiency in muscle stem cells, we investigated the pathways regulated by MyoD by GeneChip microarray analysis of gene expression in wild-type and MyoD(-/-) myoblasts. MyoD deficiency led to down-regulation of many muscle-specific genes and up-regulation of some stem cell markers. Importantly, in MyoD(-/-) myoblasts, many antiapoptotic genes were up-regulated, whereas genes known to execute apoptosis were down-regulated. Consistent with these gene expression profiles, MyoD(-/-) myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival compared with wild-type myoblasts. Forced expression of MyoD or the proapoptotic protein Puma increased cell death in MyoD(-/-) myoblasts. Therefore, MyoD(-/-) myoblasts may preserve stem cell characteristics, including their resistance to apoptosis, expression of stem cell markers, and efficient engraftment and contribution to satellite cells after transplantation. Furthermore, our data offer evidence for improved therapeutic stem cell transplantation for muscular dystrophy, in which suppression of MyoD in myogenic progenitors would be beneficial to therapy by providing a selective advantage for the expansion of stem cells.


Assuntos
Músculo Esquelético/fisiologia , Proteína MyoD/genética , Regeneração/genética , Células Satélites de Músculo Esquelético/fisiologia , Transplante de Células-Tronco , Animais , Apoptose/genética , Expressão Gênica , Camundongos , Camundongos Mutantes , Mitocôndrias/genética , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Células Satélites de Músculo Esquelético/transplante
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