Protection is not always a good thing: The immune system's impact on gene therapy.

There are many clinical trials underway for the development of gene therapies, and some have resulted in gene therapy products being commercially approved already. Significant progress was made to develop safer and more effective strategies to deliver and regulate genetic products. An unsolved aspect is the immune system, which can affect the efficiency of gene therapy in different ways. Here we present an overview of approved gene therapy products and the immune response elicited by gene delivery systems. These include responses against the vector or its content after delivery and against the product of the corrected gene. Strategies to overcome the hurdles include hiding the vector or/and the transgene product from the immune system and hiding the immune system from the vector/transgene product. Combining different strategies, such as patient screening and intelligent vector design, gene therapy is set to make a difference in the life of patients with severe genetic diseases.

Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of α-l-iduronidase, leading to the storage of dermatan and heparan sulfate. There is a broad phenotypical spectrum with the presence or absence of neurological impairment. The classical form is known as Hurler syndrome, the intermediate form as Hurler-Scheie, and the most attenuated form is known as Scheie syndrome. Phenotype seems to be largely influenced by genotype. Patients usually develop several somatic symptoms such as abdominal hernias, extensive dermal melanocytosis, thoracolumbar kyphosis odontoid dysplasia, arthropathy, coxa valga and genu valgum, coarse facial features, respiratory and cardiac impairment. The diagnosis is based on the quantification of α-l-iduronidase coupled with glycosaminoglycan analysis and gene sequencing. Guidelines for treatment recommend hematopoietic stem cell transplantation for young Hurler patients (usually at less than 30 months of age). Intravenous enzyme replacement is approved and is the standard of care for attenuated-Hurler-Scheie and Scheie-forms (without cognitive impairment) and for the late-diagnosed severe-Hurler-cases. Intrathecal enzyme replacement therapy is under evaluation, but it seems to be safe and effective. Other therapeutic approaches such as gene therapy, gene editing, stop codon read through, and therapy with small molecules are under development. Newborn screening is now allowing the early identification of MPS I patients, who can then be treated within their first days of life, potentially leading to a dramatic change in the disease's progression. Supportive care is very important to improve quality of life and might include several surgeries throughout the life course.

Tolerability and Effects of the Use of Energy-Enriched Infant Formula After Congenital Heart Surgery: A Randomized Controlled Trial.

BACKGROUND: Undernutrition is a common problem among children with congenital heart disease (CHD) and may lead to poorer surgical outcomes. A higher intake of energy during the postoperative period of CHD surgery seems to be associated with better outcomes. This study aimed to investigate the effect of the use of energy-enriched formula (EE-formula) compared with normocaloric formula during 30 days after CHD surgery. METHODS: A randomized controlled trial with patients undergoing heart surgery in a tertiary hospital in southern Brazil from March 2017 to December 2017 was performed. The intervention group received EE-formula (1 kcal/mL), and the control group received normocaloric formula (0.67 kcal/mL). The researcher in charge of anthropometric evaluation was blinded to the randomization. RESULTS: Fifty-nine patients were included; 30 in control group and 29 in intervention group. There were no statistically significant differences between groups regarding age, gender, anthropometry, and surgical risk classification after randomization. A statistically significant difference in z-score of weight for age and in weight gain variation rate between groups after intervention was observed. Antibiotic use was less frequent in the intervention group, and hospital length of stay was shorter. General gastrointestinal side effects were similar between groups, whereas diarrhea was more frequent in the intervention group. However, this side effect was limited and had spontaneous resolution in 4 out of 6 cases. CONCLUSION: This study demonstrates that EE-formula use after heart surgery of patients with CHD is well tolerated and may improve short-term nutrition outcome, decrease hospital stay, and reduce antibiotic use.

Draft genome sequence of a GES-5-producing Serratia marcescens isolated in southern Brazil

Abstract Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides blaGES-5, we were able to identify genes encoding for other β-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines.

Progressive heart disease in mucopolysaccharidosis type I mice may be mediated by increased cathepsin B activity.

Mucopolysaccharidosis type I (MPS I) is a lysosomal disorder characterized by a deficiency of alpha-L-iduronidase and storage of undegraded glycosaminoglycans (GAGs). Clinical findings of the disease include heart failure, and patients often need valve replacement. It has been shown that, later in life, MPS I mice develop those abnormalities, but to date, there have not been studies on the progression and pathogenesis of the disease. Therefore, in the present study, we evaluated heart function in normal and MPS I male mice from 2 to 8 months of age. Echocardiographic analysis showed left ventricular enlargement with progressive reduction in ejection fraction, fractional area change, and left ventricular fractional shortening in the MPS I hearts at 6 and 8 months of age and a reduction in acceleration time/ejection time ratio of the pulmonary artery starting at 6 months of age, which suggests pulmonary vascular resistance. Histological and biochemical analysis confirmed progressive GAG storage from 2 months of age and onwards in the myocardium and heart valves, which had also increased in thickness. Additionally, macrophages were present in the MPS I heart tissue. Collagen content was reduced in the MPS I mouse valves. Cathepsin B, an enzyme that is known to be able to degrade collagen and is involved in heart dilatation, displayed a marked elevation in activity in the MPS I mice and could be responsible for the heart dilatation and valves alterations observed. Our results suggest that the MPS I mice have progressive heart failure and valve disease, which may be caused by cathepsin B overexpression.

Draft genome sequence of a GES-5-producing Serratia marcescens isolated in southern Brazil.

Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides blaGES-5, we were able to identify genes encoding for other ß-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines.

Long-term restoration of alpha-L-iduronidase activity in fibroblasts from patients with mucopolysaccharidosis type I after non-viral gene transfer

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by deficiency of alpha-L-iduronidase (IDUA). Limitations such as the need for weekly injections, high morbidity and mortality, and high cost of current treatments show that new approaches to treat this disease are required. In this study, we aimed to correct fibroblasts from a patient with MPS I using non-viral gene therapy. Using a plasmid encoding the human IDUA cDNA, we achieved stable high IDUA levels in transfected fibroblasts up to 6 months of treatment. These results serve as proof of concept that a non-viral approach can correct the enzyme deficiency in cells of patients with lysosomal storage disorders, which can be used as a research tool for a series of disease aspects. Future studies should focus on showing if this approach can be useful in small animals and clinical trials (AU)

Neurological outcomes after hematopoietic stem cell transplantation for cerebral X-linked adrenoleukodystrophy, late onset metachromatic leukodystrophy and Hurler syndrome.

OBJECTIVE: To describe survival and neurological outcomes after HSCT for these disorders. METHODS: Seven CALD, 2 MLD and 2 MPS-IH patients underwent HSCT between 2007 and 2014. Neurological examinations, magnetic resonance imaging, molecular and biochemical studies were obtained at baseline and repeated when appropriated. RESULTS: Favorable outcomes were obtained with 4/5 related and 3/6 unrelated donors. Two patients died from procedure-related complications. Nine transplanted patients were alive after a median of 3.7 years: neurological stabilization was obtained in 5/6 CALD, 1/2 MLD, and one MPS-IH patient. Brain lesions of the MPS-IH patient were reduced four years after HSCT. CONCLUSION: Good outcomes were obtained when HSCT was performed before adulthood, early in the clinical course, and/or from a related donor.

Neurological outcomes after hematopoietic stem cell transplantation for cerebral X-linked adrenoleukodystrophy, late onset metachromatic leukodystrophy and Hurler syndrome / Desfechos neurológicos após transplante de células tronco hematopoiéticas na adrenoleucodistrofia ligada ao X, forma cerebral, na leucodistrofia metacromática de início tardio e na síndrome de Hurler

ABSTRACT Hematopoietic stem cell transplantation (HSCT) is the only available treatment for the neurological involvement of disorders such as late-onset metachromatic leukodystrophy (MLD), mucopolysaccharidosis type I-Hurler (MPS-IH), and X-linked cerebral adrenoleukodystrophy (CALD). Objective To describe survival and neurological outcomes after HSCT for these disorders. Methods Seven CALD, 2 MLD and 2 MPS-IH patients underwent HSCT between 2007 and 2014. Neurological examinations, magnetic resonance imaging, molecular and biochemical studies were obtained at baseline and repeated when appropriated. Results Favorable outcomes were obtained with 4/5 related and 3/6 unrelated donors. Two patients died from procedure-related complications. Nine transplanted patients were alive after a median of 3.7 years: neurological stabilization was obtained in 5/6 CALD, 1/2 MLD, and one MPS-IH patient. Brain lesions of the MPS-IH patient were reduced four years after HSCT. Conclusion Good outcomes were obtained when HSCT was performed before adulthood, early in the clinical course, and/or from a related donor.

RESUMO O transplante de células tronco hematopoiéticas (TCTH) é o único tratamento disponível para o envolvimento neurológico de doenças como a leucodistrofia metacromática (MLD), a mucopolissacaridose tipo I-Hurler (MPS-IH) e a adrenoleucodistrofia (CALD). Objetivos Descrever a sobrevida e os desfechos neurológicos após o TCTH nessas doenças. Métodos Sete pacientes CALD, 2 MLD e 2 MPS-IH realizaram TCTH entre 2007 e 2014. Avaliações neurológicas, ressonância nuclear magnética e estudos bioquímicos e moleculares foram feitos no baseline e repetidos quando apropriado. Resultados Desfechos favoráveis foram obtidos em 4/5 TCTH de doadores relacionados e em 3/6 não relacionados. Dois pacientes faleceram de complicações do procedimento. Nove transplantados sobreviveram após uma mediana de 3,7 anos: estabilização neurológica foi obtida em 5/6 CALD, ½ MLD e em um caso MPS-IH. As lesões encefálicas de um caso MPS-IH reduziram-se quatro anos após o TCTH. Conclusão Bons desfechos foram obtidos quando o TCTH foi feito antes da vida adulta, cedo no curso clínico e/ou a partir de um doador relacionado.

Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53.

Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect. Several genotyping methods are used for the molecular diagnosis of LFS/LFL, however Sanger sequencing is still considered the gold standard. We compared performance, cost and turnaround time of Sanger sequencing, PCR-RFLP, TaqMan-PCR and HRM in the p.R337H genotyping. The performance was determined by analysis of 95 genomic DNA samples and results were 100% concordant for all methods. Sequencing was the most expensive method followed by TaqMan-PCR, PCR-RFLP and HRM. The overall cost of HRM increased with the prevalence of positive samples, since confirmatory sequencing must be performed when a sample shows an abnormal melting profile, but remained lower than all other methods when the mutation prevalence was less than 2.5%. Sequencing had the highest throughput and the longest turnaround time, while TaqMan-PCR showed the lowest turnaround and hands-on times. All methodologies studied are suitable for the detection of p.R337H and the choice will depend on the application and clinical scenario.

The effects of anesthetic regimen in 90% hepatectomy in rats.

PURPOSE: To evaluate the influence of the anesthetic regimen on anesthetic recovery, survival, and blood glucose levels following a 90% partial hepatectomy in rats. METHODS: Thirty adult male Wistar rats were divided into two groups according to their anesthetic regimens: intraperitoneal ketamine and xylazine or inhaled isoflurane. In order to prevent hypoglycemia, glucose was administered intraperitoneally and glucose (20%) was added to the drinking water. RESULTS: Anesthetic recovery time was longer in the ketamine and xylazine group. The survival rate after 72 hours was lower (log rank=0.0001) in the ketamine and xylazine group (0.0%) than in the isoflurane group (26.7%). The blood glucose after six hours was lower (p=0.017) in the ketamine and xylazine group (63 ± 31.7 mg/dL) than in the isoflurane group (98 ± 21.2 mg/dL). The prolonged anesthesia recovery time associated with ketamine and xylazine decreased the survival rate and blood glucose levels after 90% hepatectomy. CONCLUSION: Isoflurane anesthesia reduced the recovery time and incidence of hypoglycemia and increased the survival rate in the early hours, providing a therapeutic window that is suitable for experimental studies.

The effects of anesthetic regimen in 90% hepatectomy in rats / Os efeitos do regime anestésico na hepatectomia de 90% em ratos

PURPOSE: To evaluate the influence of the anesthetic regimen on anesthetic recovery, survival, and blood glucose levels following a 90% partial hepatectomy in rats. METHODS: Thirty adult male Wistar rats were divided into two groups according to their anesthetic regimens: intraperitoneal ketamine and xylazine or inhaled isoflurane. In order to prevent hypoglycemia, glucose was administered intraperitoneally and glucose (20%) was added to the drinking water. RESULTS: Anesthetic recovery time was longer in the ketamine and xylazine group. The survival rate after 72 hours was lower (log rank=0.0001) in the ketamine and xylazine group (0.0%) than in the isoflurane group (26.7%). The blood glucose after six hours was lower (p=0.017) in the ketamine and xylazine group (63±31.7 mg/dL) than in the isoflurane group (98±21.2 mg/dL). The prolonged anesthesia recovery time associated with ketamine and xylazine decreased the survival rate and blood glucose levels after 90% hepatectomy. CONCLUSION: Isoflurane anesthesia reduced the recovery time and incidence of hypoglycemia and increased the survival rate in the early hours, providing a therapeutic window that is suitable for experimental studies.

OBJETIVO: Avaliar a influência do regime anestésico sobre a recuperação anestésica, a sobrevida em 72 horas e a glicemia após hepatectomia parcial de 90% em ratos. MÉTODOS: Trinta ratos Wistar machos adultos foram distribuídos em dois grupos conforme o regime anestésico: combinação de ketamina e xilazina intraperitoneal ou isoflurano inalatório. Para prevenção de hipoglicemia foi administrada glicose intraperitoneal e adicionado glicose (20%) na água de beber. RESULTADOS: A recuperação anestésica no grupo ketamina e xilazina foi mais prolongada. Durante primeira hora após hepatectomia, nenhum rato anestesiado com ketamina e xilazina despertou. Todos do grupo isoflurano estavam ativos minutos após final da cirurgia. A sobrevida em 72 horas foi menor (Log rank=0,0001) no grupo ketamina e xilazina (0,0%) que no grupo isoflurano (26,7%). Glicemia em 6 horas do grupo ketamina e xilazina (63±31,7 mg/dL) foi menor (p=0,017) que no grupo isoflurano (98 ±21,2 mg/dL). Prolongado tempo de recuperação anestésica com ketamina e xilazina diminuiu sobrevida e glicemia após hepatectomia 90%. CONCLUSÃO: Anestesia com isoflurano reduziu tempo de recuperação e hipoglicemia, além de aumentar a sobrevida nas primeiras horas, possibilitando uma janela terapêutica adequada para estudos experimentais.

Peritonite bacteriana espontânea: novas perspectivas / Spontaneuous bacterial peritonitis: new perspectives

A peritonite bacteriana espontânea (PBE) é uma das infecções mais frequentes no paciente cirrótico, estando associada à alta morbi-mortalidade. Descrita desde a metade dos anos de 1960, diretrizes recentemente publicadas apontam para o surgimento de novos conceitos relacionados à patogênese, ao diagnóstico e ao tratamento da PBE, motivo desta revisão. Dentre os principais aspectos discutidos destacam-se o conceito de translocação bacteriana patológica e a identificação de polimorfismos genéticos associados ao desenvolvimento da PBE, este último sugerindo a existência de um fator de risco adicional para a infecção. Os critérios diagnósticos e terapêuticos são revisados, sendo enfatizada a crescente ocorrência de resistência bacteriana naqueles pacientes que desenvolvem PBE nosocomial, situação na qual é sugerida uma abordagem terapêutica específica. Discute-se finalmente, as atuais indicações de profilaxia primária e secundária. Quando pertinente, serão também apresentados os aspectos relacionados à PBE que acomete o paciente pediátrico (AU)

Spontaneous bacterial peritonitis (SBP) is one of the most common infections in cirrhotic patients and is associated with high morbidity and mortality. Described since the mid-1960s, recently published guidelines point to the emergence of new concepts related to the pathogenesis, diagnosis and treatment of SBP, subject of this review. The main aspects discussed include the concept of pathological bacterial translocation and identification of genetic polymorphisms associated with the development of SBP, the latter suggesting the existence of an additional risk factor for infection. The diagnostic and therapeutic criteria are reviewed, with an emphasis on the increasing occurrence of bacterial resistance in patients who develop nosocomial SBP, in which case a specific therapeutic approach is suggested. Finally we discuss the current indications for primary and secondary prophylaxis. Where relevant, SBP-related aspects affecting the pediatric patient will also be presented (AU)

Common N-acetylgalactosamine-6-sulfate sulfatase (GALNS) exon mutations in Brazilian patients with mucopolysaccharidosis IVA (MPS IVA)

Morquio A Syndrome (mucopolysaccharidosis IVA - MPS IVA, OMIM# 253000) is an autosomal recessive inborn error of metabolism caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). We investigated five unrelated Brazilian MPS IVA families for mutations in exons 4, 5, 9 and 10 of the GALNS gene. Six out of the 10 mutant alleles were identified. Taken together with a previous study, which included six unrelated families, common mutations among Brazilian patients were p.N164T, p.G116S and p.G301C. Among one hundred control subjects three novel silent mutations were found (p.A107A; GCC -> GCT, p.Y108Y; TAC -> TAT, p.P357P; CCG -> CCA). Screening starting with exons 4, 5, 9, 10 and 11 may be a good strategy for genotyping of Brazilian patients since these exons include 73 percent of all mutations identified in the current and previous studies.

A perspectiva estratégica na gestão de um laboratório de pesquisa da área da saúde / The strategic perspective in the administration of a research laboratory in healthcare

O Centro de Terapia Gênica é um laboratório compartilhado, implantado em 2002, que atuana área de genética e localiza-se no Centro de Pesquisas do Hospital de Clínicas de Porto Alegre.Tem como característica principal ser um prestador de serviços de biologia molecular e celular para os projetos de pesquisa que se desenvolvem no hospital. Os resultados financeiros negativos ao final de cada período motivaram este estudo, que teve como objetivo desenvolver e aplicar o sistema de gestão balanced scorecard. Realizou-se a revisão da literatura, o levantamento de dados do laboratório e o desenvolvimento do planejamento estratégico. O trabalho foi implementado em novembro de 2004. Os resultados foram a construção do mapa estratégico, definição de metas para o período 2005-2008, crescimento da receita de faturamento em 21%, redução dos custos operacionais em 43%, estabelecimento dos custos de 39% das técnicas básicas do laboratório e aumento da captação de projetos em 47%. Este estudo mostra que a gestão das atividades de pesquisa, vista sob uma perspectiva estratégica, poderá agregar valor tanto científico como econômico à instituição, expresso sob a forma dos novos conhecimentos gerados,publicações realizadas, captação de recursos para a pesquisa e redução dos custos operacionais.

The Centro de Terapia Gênica (Gene Therapy Center), which was established in 2002, is a shared laboratory specialized in genetics. It is located at Research Center, Hospital de Clínicasde Porto Alegre. Its main characteristic is to provide services of molecular and cellular biologyfor research projects that are being developed at the hospital. This study was motivated by thenegative financial results at the end of each year. Therefore, it aims to develop and apply thebalanced scorecard, a strategic management system. We performed a literature review, laboratory data collection, and development of the strategic planning. This study was implemented on November 2004. Results were: elaboration of a strategic map, definition of goals for 2005-2008, increase in revenue by 21%, reduction in operational costs by 43%, establishment of the costs concerning 39% of basic laboratory techniques, and increase in submission of projects by 47%.This study shows that the strategic administration of research activities may add scientific and economic value to the institution. This value is expressed as increased knowledge, publications, generation of resources for research, and reduction in operational costs.