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SUMMARY Mutations in the fukutin-related protein (FKRP) gene result in a broad spectrum of muscular dystrophy (MD) phenotypes, including the severe Walker-Warburg syndrome (WWS). Here, we develop a gene-editing approach that replaces the entire mutant open reading frame with the wild-type sequence to universally correct all FKRP mutations. We apply this approach to correct FKRP mutations in induced pluripotent stem (iPS) cells derived from patients displaying broad clinical severity. Our findings show rescue of functional α-dystroglycan (α-DG) glycosylation in gene-edited WWS iPS cell-derived myotubes. Transplantation of gene-corrected myogenic progenitors in the FKRPP448L-NSG mouse model gives rise to myofiber and satellite cell engraftment and, importantly, restoration of α-DG functional glycosylation in vivo. These findings suggest the potential feasibility of using CRISPR-Cas9 technology in combination with patient-specific iPS cells for the future development of autologous cell transplantation for FKRP-associated MDs., In brief Dhoke et al. develop a universal CRISPR-Cas9 gene-editing strategy for the correction of FKRP mutations. Myogenic progenitors from gene-edited WWS patient-specific iPS cells show rescue of phenotype in vitro and upon transplantation into muscles of FKRP mutant mice., Graphical Abstract

Background Defects in α-dystroglycan (DG) glycosylation characterize a group of muscular dystrophies known as dystroglycanopathies. One of the key effectors in the α-DG glycosylation pathway is the glycosyltransferase fukutin-related protein (FKRP). Mutations in FKRP lead to a large spectrum of muscular dystrophies, including limb girdle muscular dystrophy 2I (LGMD2I). It remains unknown whether stem cell transplantation can promote muscle regeneration and ameliorate the muscle wasting phenotype associated with FKRP mutations. Results Here we transplanted murine and human pluripotent stem cell-derived myogenic progenitors into a novel immunodeficient FKRP-mutant mouse model by intra-muscular injection. Upon both mouse and human cell transplantation, we observe the presence of donor-derived myofibers even in absence of pre-injury, and the rescue of α-DG functional glycosylation, as shown by IIH6 immunoreactivity. The presence of donor-derived cells expressing Pax7 under the basal lamina is indicative of satellite cell engraftment, and therefore, long-term repopulation potential. Functional assays performed in the mouse-to-mouse cohort revealed enhanced specific force in transplanted muscles compared to PBS-injected controls. Conclusions Altogether, our data demonstrate for the first time the suitability of a cell-based therapeutic approach to improve the muscle phenotype of dystrophic FKRP-mutant mice.

Candidemia remains a major cause of morbidity and mortality in the health care environment. The epidemiology of Candida infection is changing, mainly in relation to the number of episodes caused by species C. non-albicans. The overall objective of this study was to evaluate the frequency of yeasts of the genus Candida, in a four-year period, isolated from blood of pediatric patients hospitalized in a public hospital of the city of São Paulo, Brazil. In this period, yeasts from blood of 104 patients were isolated and, the identified species of Candida by phenotypic and genotypic methods were: C. albicans (39/104), C. tropicalis (25/104), C. parapsilosis (23/104), Pichia anomala (6/104), C. guilliermondii (5/104), C. krusei (3/104), C. glabrata (2/104) and C. pararugosa (1/104). During the period of the study, a higher frequency of isolates of C. non-albicans (63.55%) (p = 0.0286) was verified. In this study we verified the increase of the non-albicans species throughout the years (mainly in 2009 and 2010). Thus, considering the peculiarities presented by Candida species, a correct identification of species is recommended to lead to a faster diagnosis and an efficient treatment. Candidemia permance como a maior causa de morbidade e mortalidade em ambiente hospitalar. A epidemiologia de infecções por Candida vem se alterando, principalmente em relação ao número de episódios causados por espécies não-albicans. Este estudo teve como objetivo avaliar a frequência, em um período de quatro anos, de leveduras do gênero Candida isoladas de sangue de pacientes pediátricos internados em hospital público da cidade de São Paulo, Brasil. Neste período foram isoladas leveduras de sangue de 104 pacientes, e as espécies de Candida identificadas, por métodos fenotípicos e genotípicos, foram: C. albicans (39/104), C. tropicalis (25/104), C. parapsilosis (23/104), Pichia anomala (6/104), C. guilliermondii (5/104), C. krusei (3/104), C. glabrata (2/104) e C. pararugosa (1/104). Em todo período do estudo foi observada maior frequência de isolamento de C. não-albicans (63,55%) (p = 0,0286). Neste estudo verificou-se aumento das espécies não-albicans ao longo dos anos (principalmente em 2009 e 2010), assim, ressalta-se que correta identificação em nível de espécie é recomendável, para que isso acarrete diagnóstico rápido e tratamento eficaz.

Non-viral transfer of the growth hormone gene to different muscles of immunodeficient dwarf (lit/scid) mice is under study with the objective of improving phenotypic correction via this particular gene therapy approach. Plasmid DNA was administered into the exposed quadriceps or non-exposed tibialis cranialis muscle of lit/scid mice followed by electroporation, monitoring several growth parameters. In a 6-month bioassay, 50μg DNA were injected three times into the quadriceps muscle of 80-day old mice. A 50% weight increase, with a catch-up growth of 21%, together with a 16% increase for nose-to-tail and tail lengths (catch-up=19-21%) and a 24-28% increase for femur length (catch-up=53-60%), were obtained. mIGF1 serum levels were ~7-fold higher than the basal levels for untreated mice, but still ~2-fold lower than in non-dwarf scid mice. Since treatment age was found to be particularly important in a second bioassay utilizing 40-day old mice, these pubertal mice were compared in a third bioassay with adult (80-day old) mice, all treated twice with 50μg DNA injected into each tibialis cranialis muscle, via a less invasive approach. mIGF1 concentrations at the same level as co-aged scid mice were obtained 15days after administration in pubertal mice. Catch-up growth, based on femur length (77%), nose-to-tail (36%) and tail length (39%) increases was 40 to 95% higher than those obtained upon treating adult mice. These data pave the way for the development of more effective pre-clinical assays in pubertal dwarf mice for the treatment of GH deficiency via plasmid-DNA muscular administration.

The cDNAs of the α-subunit of the pituitary gonadotrophic hormones (GTHα) of fish of the order Osteoglossiformes or the superorder Osteoglossomorpha have never been sequenced. For a better understanding the phylogenetic diversity and evolution of PGHα in fish and for future biotechnological synthesis of the gonadotrophic hormones (ag-FSH and ag-LH), of Arapaima gigas, one of the largest freshwater fishes of the world, its GTHα cDNA was synthesized by reverse transcriptase and the polymerase chain reaction starting from total pituitary RNA. The ag-GTHα-subunit was found to be encoded by 348 bp, corresponding to a protein of 115 amino acids, with a putative signal peptide of 24 amino acids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for forming 5 disulfide linkages, 2 putative N-linked glycosylation sites and 3 proline residues, were found to be conserved on the basis of the known sequences of vertebrate gonadotrophic hormones. Phylogenetic analysis, based on the amino acid sequences of 38 GTHα-subunits, revealed the highest identity of A. gigas with members of the Acipenseriformes, Anguilliformes, Siluriformes and Cypriniformes (87.1-89.5 %) and the lowest with Gadiformes and Cyprinodontiformes (55.0 %). The obtained phylogenetic tree agrees with previous analysis of teleostei, since A. gigas, of the order of Osteoglossiformes, appears as the sister group of Clupeocephala, while Elopomorpha forms the most basal group of all other teleosts.

As craniossinostoses, resultantes da fusão precoce de uma ou mais suturas cranianas, representam um grupo heterogêneo de doenças que ocorre aproximadamente em 1:2000 nascidos vivos. Caracterizam-se por grande variabilidade clínica e heterogeneidade genética, com raras exceções como a Síndrome de Apert. As craniossinostoses podem ser classificadas em não sindrômicas, quando o único problema do paciente é a fusão precoce da sutura, e sindrômica quando outros problemas estão presentes. Dentre as craniossinostoses sindrômicas, as mais comuns são a Síndrome de Crouzon, Pfeiffer e Apert. O diagnóstico diferencial entre estas 3 síndromes é dado principalmente pelo grau de comprometimento dos membros, sendo que na Síndrome de Crouzon, a mais leve, não há comprometimento de membros, enquanto que na Síndrome de Apert, a mais grave, os membros estão sempre comprometidos. O mecanismo mutacional é identificado em aproximadamente 58% dos pacientes Síndrome de Crouzon, sendo que o gene FGFR2 corresponde à 48% desse total e mutações nos genes TWIST1 e FGFR3 correspondem aos 10% dos casos restantes. Assim sendo, cerca de 42% dos casos com características clínicas compatíveis com a Síndrome de Crouzon, não apresentam mutações em nenhum destes três genes, e, portanto, mutações em outros genes podem ser responsáveis por este quadro clínico.Aproximadamente 45% dos casos de Pfeiffer não estão relacionadas a mutações nos genes FGFR1 e FGFR2, indicando que outros genes também podem estar envolvidos. Por outro lado, todos os casos de Síndrome de Apert são causados por mutações no gene FGFR2. As mutações em FGFR2 apresentam uma correlação parcial entre genótipo e fenótipo, contudo, casos graves, como aqueles com Síndrome de Pfeiffer tipo II ou III são ainda muito pouco caracterizados. Experimentos utilizando-se inserção retroviral em camundongos levaram a identificação de um fenótipo semelhante à síndrome de Crouzon, o qual parece ter sido causado por um aumento da expressão do produto dos genes FGF3 e FGF4. Além disso, a implantação de beads contendo FGF4 em crânios de camundongos neonatais acelerou o fechamento das suturas, em decorrência do aumento da proliferação e diferenciação dos osteoblastos. Assim sendo, é possível que mutações nos genes FGF3 e FGF4 possam causar um fenótipo semelhante à Síndrome de Crouzon ou craniossinostoses sindrômicas.A identificação de mutações patogênicas do gene TWIST1 em pacientes com síndrome de Saethre-Chotzen, tem colaborado para a caracterização de domínios funcionais da proteína codificada por este gene. Até o momento, estas mutações, que podem ser do tipo substituição de aminoácido ou que criam códigos de parada prematura, levam a perda da função da proteína por diferentes processos, e, portanto, o mecanismo responsável pela síndrome é o da haploinsuficiência. Até o momento, apenas mutações em regiões codificadoras têm sido associadas à doença e há muita pouca informação a respeito do efeito de mutações na região reguladora do gene TWIST1.Em vista do exposto acima, julgamos importante testarmos os genes FGF3 e FGF4 em pacientes com Síndrome de Crouzon e craniossinostose sindrômica sem mutações nos genes candidatos, com o objetivo de verificar se mutações nesses possam ser responsáveis pelo fenótipo dos pacientes. Julgamos também importante verificar se uma paciente com Pfeiffer tipo 3 era portadora de mutação patogênica no gene FGFR2 bem como identificar mutações em 7 pacientes com a síndrome de Saethre-Chotzen. Ainda, realizamos estudos funcionais para verificar se uma mutação nova localizada em região reguladora do gene TWIST1 é responsável pelo fenótipo da paciente. Craniosynostosis, defined as the premature fusion of one or more cranial suture, are a very heterogeneous group of disorders, with an occurrence of about 1: 2000 live births. Except for rare syndromes, such as Apert, most of the cranioysnostotic conditions are characterized by wide clinical variability and genetic heterogeneity. Craniosynostosis can be classified into non syndromic forms - premature suture fusion is the only clinical alteration, and syndromic ones - premature cranial suture fusion associated with other anomalies. Apert, Pfeiffer, Crouzon and Saethre-Chotzen are the most common syndromic forms of craniosynostosis. The mutational mechanism is identified in approximately 58% of patients with Crouzon syndrome: FGFR2 mutations account for most of the cases (more than 80%) while mutations in TWIST1 and FGFR3 causes the remaining of them. Therefore, about 42% of the cases with clinical characteristics of Crouzon syndrome do not harbor mutations in any of these 3 genes and other mutational mechanisms might be responsible for the phenotype. Approximately 45% of Pfeiffer syndrome cases is not caused by mutations in the FGFR1 or FGFR2 genes, thus also suggesting genetic heterogeneity for this phenotype. On the other hand, all cases with Apert syndrome are caused by specific mutations in FGFR2. FGFR2 mutations show a partial genotype-phenotype correlation; however, severe cases, such as Pfeiffer types II and III are still very poorly characterized. Retroviral insertion experiments in mouse lead to the identification of animals with a phenotype that resembles Crouzon syndrome. It was suggested that this phenotype was caused by upregulation of the FGF3 and FGF4 genes as a consequence of the insertion of the retrovirus between these 2 genes . In addition, implantation of beads with FGF4 in the cranium of neonatal mice has increased the fusion of the cranium sutures . Therefore, it is possible that gain of function mutations in the FGF3 e FGF4 genes might lead to Crouzon or syndromic craniosynostosis.Characterization of pathogenic mutations in the TWIST1 gene among patients with Saethre-Chotzen syndrome has contributed for a better understanding of the protein domains of the corresponding protein. These mutations, which are missense or null, cause loss of function of the protein and haploinsufficiency of the TWIST1 gene has been postulated as the mechanism model for Saethre-Chotzen syndrome.Up to now, only pathogenic mutations in coding regions have been associated with the disease and there is very little information about the effect of mutations on the regulatory regions of the TWIST1 gene on the phenotype.Considering the above information, the aims of the present work were: a) verify if mutations in FGF3 and FGF4 can cause craniosynostosis; b) verify if the severe phenotype in a patient Pfeiffer type 3 is due to a specific mutation in FGFR2; c) identify mutations in 7 patients with Saethre-Chotzen syndrome; d) verify if a mutation in the regulatory region of the TWIST1 gene can cause craniosynostosis.

BACKGROUND: Pfeiffer syndrome (PS; OMIM #101600) is an autosomal dominant disorder characterized by craniosynostosis, midface hypoplasia, broad thumbs, brachydactyly, broad great toes, and variable syndactyly. CASE: We report a case of PS (type 3) with tracheal and visceral involvement and sacrococcygeal eversion. The patient shows facial dysmorphism with macrocephaly, dolichocephaly, and trigonocephaly, and an asymmetric skull, bilateral and severe exophthalmia with shallow orbits and ocular hypertelorism, downslanting palpebral fissures, constant strabismus, short anterior cranial base, and midface hypoplasia. CONCLUSIONS: Molecular analysis of the FGFR2 gene in this patient revealed a point mutation (c.890G>C NM_000141). This mutation leads to the substitution of the residue tryptophan at position 290 to cysteine in the protein (p.Try290Cys). These data reinforce the hypothesis that the p.Trp290Cys mutation is more often associated with a severe and poor prognosis of PS. Furthermore they suggest that the presence of sacrococcygeal defects is not associated with any specific FGFR2 mutation. Birth Defects Research (Part A), 2006. © 2006 Wiley-Liss, Inc.

The common gonadotrophic hormone α-subunit (GTHα) has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHβ and LHβ subunits have also been isolated from the same species of fish. The FSH β-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LH β-subunit consists of 141 amino acids with a putative 24 amino acid amino acid signal peptide and a 117 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the order of Anguilliformes (61%) for FSHβ and of Cypriniformes (76%) for LHβ, followed by Siluriformes, 53% for FSHβ and 75% for LHβ. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHβ and 51.4% for LHβ. Three dimensional models of ag-FSH and ag-LH, generated by using the crystal structures of h-FSH and h-LH as the respective templates and carried out via comparative modeling and molecular dynamics simulations, suggested the presence of the so-called "seat-belt", favored by a disulfide bond formed between the 3rd and 12th cysteine in both β-subunits. The sequences found will be used for the biotechnological synthesis of A. gigas gonadotrophic hormones (ag-FSH and ag-LH). In a first approach, to ascertain that the cloned transcripts allow the expression of the heterodimeric hormones, ag-FSH has been synthesized in human embryonic kidney 293 (HEK293) cells, preliminarily purified and characterized.

The possibilities for non-viral GH gene therapy are studied in immunocompetent dwarf mice (lit/lit). As expression vector we used a plasmid previously employed in immunodeficient dwarf mice (pUBI-hGH-gDNA) by replacing the human GH gene with the genomic sequence of mouse-GH DNA (pUBI-mGH-gDNA). HEK-293 human cells transfected with pUBI-mGH-gDNA produced 3.0 µg mGH/10(6) cells/day compared to 3.7 µg hGH/106 cells/day for pUBIhGH- gDNA transfected cells. The weight of lit/lit mice treated with the same two plasmids (50 µg DNA/mouse) by electrotransfer into the quadriceps muscle was followed for 3 months. The weight increase up to 15 days for mGH, hGH and saline treated mice were 0.130, 0.112 and 0.027 g/mouse/day. Most sera from hGH-treated mice contained anti-hGH antibodies already on day 15, with the highest titers on day 45, while no significant anti-mGH antibodies were observed in mGH-treated mice. At the end of 3 months, the weight increase for mGH-treated mice was 34.3%, while the nose-to-tail and femur lengths increased 9.5% and 24.3%. Mouse-GH and hGH circulating levels were 4-5 ng/mL 15 days after treatment, versus control levels of ~0.7 ng GH/mL (P

In previous work, sustained levels of circulating human growth hormone (hGH) and a highly significant weight increase were observed after electrotransfer of naked plasmid DNA (hGH-DNA) into the muscle of immunodeficient dwarf mice (lit/scid). In the present study, the efficacy of this in vivo gene therapy strategy is compared to daily injections (5μg/twice a day) of recombinant hGH (r-hGH) protein, as assessed on the basis of several growth parameters. The slopes of the two growth curves were found to be similar (P > 0.05): 0.095 g/mouse/d for protein and 0.094 g/mouse/d for DNA injection. In contrast, the weight increases averaged 35.5% (P

Prolactin (PRL) is a pleiotropic hormone produced by lactotroph cells of the anterior pituitary gland and is mainly related to lactation control and reproduction. Recombinant mouse prolactin (r-mPRL), never obtained in its authentic form, can be very useful for research and tests in animal models, in which human prolactin (hPRL) is usually employed in a heterologous mode. Synthesis of r-mPRL was carried out here via secretion in Escherichia coli periplasmic space using a plasmid containing mPRL cDNA joined to the DsbA signal peptide sequence under the control of a constitutive major leftward promoter of the bacteriophage λ (λPL). Fermentation in a pilot bioreactor was carried out at 30°C, with 6 H of induction at 37°C, reaching an optical density of 23 A600 units, a specific yield of 0.06–0.1 µg mPRL/(mL A600), and a concentration of up to 2.2 µg/mL. Even with such a low yield and a poor mass fraction, r-mPRL was purified via a three-step laboratory process based on hydrophobic chromatography, reversed-phase high-performance liquid chromatography, and high-performance size-exclusion chromatography (HPSEC). The purified hormone was then characterized using SDS-PAGE, Western blotting, and HPSEC and showed, by Nb2 rat lymphoma cell proliferation assay, a bioactivity of 39.5 IU/mg, determined against the International Standard of recombinant hPRL [World Health Organization (WHO)-97/714].

Recombinant growth hormone (GH) was one of the first proteins to be synthesized via DNA recombinant techniques in the late seventies. It was also one of the first proteins to be used in studies of animal models for gene therapy, already in the eighties. This was due to the real therapeutic need for GH, combined with the fact that its detection by well-known immunoassay methods is facile and sensitive. Moreover, evident phenotypic effects can be observed and measured in several animal models (e.g., dwarf mice), some of which have GH deficiencies that closely resemble their human counterparts. GH gene therapy has the potential advantage of circumventing laborious and expensive purification processes, quality control procedures and the repetitive injections that are required in the conventional treatment. The ideal situation would, of course, be to introduce the deficient protein into the circulation via a mechanism that resembles the natural process. These treatments have not yet reached the clinical stage for humans, the major challenge being to achieve a sustainable and regulated in vivo GH secretion. However, several interesting and promising ex vivo and direct in vivo DNA administration strategies for GH gene therapy have been developed and studied using animal models. These studies obviously open the way for the systemic delivery of other therapeutic proteins in addition to GH. The various ex vivo models for GH gene therapy are based on the use of target cells, such as keratinocytes, fibroblasts, endothelial cells, peritoneal mesothelial cells, or skeletal myoblasts. These cells can also be encapsulated to prevent rejection when implanted in allogeneic hosts. The majority of the methodologies are carried out via cell isolation and in vitro cultivation, genetic modification by viral or non-viral vectors containing the GH gene and re-implantation of the secreting cells onto the animal. Primary keratinocytes are one of the most attractive vehicles for gene transfer and gene therapy. They are among the most accessible cells in the body and can be serially propagated in culture; the procedure for their transplantation is already well established, e.g., for burn patients, and the therapy can be reversed by excision of the genetically modified tissue. Cutaneous gene therapy has already been demonstrated to be a powerful

Induced pluripotent stem cells (iPSCs) can be created by forcing expression of certain genes in fibroblasts or other somatic cell types, reversing them to a pluripotent state similar to that of embryonic stem cells (ESC). Here, we used human immature dental pulp stem cells (hIDPSCs) as an alternative source for creating iPSC. hIDPSCs can be easily isolated from accessible tissue of young and adult patients. hIDPSCs possess a fibroblast-like morphology, retaining characteristics of adult multipotent stem cells. Reprogramming of hIDPSCs was fast, producing primary hIDPSC-iPSC colonies even under feeder-free conditions. hIDPSCs acquired ESC-like morphology, expressed pluripotent markers, possessed stable, normal karyotypes, and demonstrated the ability to differentiated in vitro and in vivo. Our data demonstrate that hIDPSCs-iPSCs offer an advantageous cell system for future cell therapy and basic studies, particularly as a model for pediatric developmental disorders.

In this report, we present a boy with lower lip pits, distinct craniofacial dysmorphism with cleft lip and palate, central nervous system malformation, and severe mental retardation. Similar but less pronounced facial findings were present in his mentally normal mother and maternal grandfather, both presenting with lower lip pits. Cleft lip was present in patient's father. Analysis of the VWS1 and VWS2 regions were performed to elucidate the molecular basis of the phenotype of the propositus. Screening for mutations at the IRF6 gene detected a pathogenic mutation (c.960G>C) in the propositus and in his mother; and a single nucleotide polymorphism (c.175-5C>G) in the propositus and in his father. Clinical and genetic aspects of this case are discussed.

Apert syndrome (AS), a severe form of craniosynostosis, is caused by dominant gain-of-function mutations in FGFR2. Because the periosteum contribution to AS cranial pathophysiology is unknown, we tested the osteogenic potential of AS periosteal cells (p.Ser252Trp mutation) and observed that these cells are more committed toward the osteoblast lineage. To delineate the gene expression profile involved in this abnormal behavior, we performed a global gene expression analysis of coronal suture periosteal cells from seven AS patients (p.Ser252Trp), and matched controls. We identified 263 genes with significantly altered expression in AS samples (118 upregulated, 145 downregulated; SNRor= |0.4|, Por= 0.05). Several upregulated genes are involved in positive regulation of cell proliferation and nucleotide metabolism, whereas several downregulated genes are involved in inhibition of cell proliferation, gene expression regulation, cell adhesion, and extracellular matrix organization, and in PIK3-MAPK cascades. AS expression profile was confirmed through real-time PCR of a selected set of genes using RNAs from AS and control cells as well as from control cells treated with high FGF2 concentration, and through the analysis of genes involved in FGF-FGFR signaling. Our results allowed us to: (a) suggest that AS periosteal cells present enhanced osteogenic potential, (b) unravel a specific gene expression signature characteristic of AS periosteal cells which may be associated with their osteogenic commitment, (c) identify a set of novel genes involved in the pathophysiology of AS or other craniosynostotic conditions, and (d) suggest for the first time that the periosteum might be involved in the pathophysiology of AS.

Background The Hormone Group of IPEN develops researches on recombinant pituitary hormones produced in Escherichia coli at laboratory scale, including human growth hormone (hGH) and prolactin (hPRL). The best results were obtained using the lPL promoter and the signal peptide DsbA, and so the protein of interest is secreted into the bacterial periplasmic space, in its authentic form without an initial methionine [1,2]. The expression, under control of the lPL promoter, regulated by the thermosensitive repressor, is used for large scale production in E. coli. Thermal induction presents a great advantage over chemical inducers like IPTG or nalidixic acid, that are expensive and dangerous for manipulators and environment, but it presents the disadvantage that proteins particularly thermolabile like hPRL could suffer proteolysis and aggregation, influencing negatively the production. Considering the limitations on the use of IPTG and the periplasmic secretion, successfully obtained with our hGH expression vector using lPL promoter and W3110 strain, we decided to use this system for IFN-a2a production. The goal of this work was the construction of the IFN-a2a vector, obtaining an the IFN-a2a periplasmic expression.