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Expression and purification of the mutated form of human GAD65 in different biological systems
No full textRiassunto
Il diabete insulino-dipendente di tipo 1 (T1D), il quale colpisce lo 0.03-0.04% della popolazione, è causato dalla distruzione autoimmune delle cellule beta del pancreas deputate alla produzione di insulina, rendendo necessaria una terapia sostitutiva continua con questo ormone (Gepts, 1965). Questo forma di diabete è considerata una malattia cronica con un forte impatto sociale, a causa della comparsa di complicazioni legate al progredire della malattia ed alla giovane età dei pazienti essendo la più diffusa malattia cronica in bambini e ragazzi al di sotto dei 14 anni di età. Attualmente non esiste una cura della malattia ed il trattamento con insulina rappresenta semplicemente una terapia sostitutiva continua.
L’induzione della tolleranza mediante l’utilizzo di auto antigeni è considerata una strategia utile per la prevenzione o il rallentamento della progressione delle malattie autoimmuni (Harrison, 2005). In particolare, la GAD65 è considerata un possibile vaccino per il diabete di tipo 1 e, di conseguenza, sono stati effettuati diversi studi di immunoterapia antigene-specifica (ASI) utilizzando questo auto antigene per testare la sua efficacia nell’induzione della tolleranza. I risultati ottenuti nel modello animale, rappresentato dai topi NOD (Non-Obese Diabetic mouse), dimostrano la possibilità di fornire un trattamento preventivo contro il diabete mediante la somministrazione di GAD65. Recentemente, un’azienda svedese, “Diamyd Medical”, ha condotto gli studi clinici di Fase II e III in uomo. Nel primo caso, è stato dimostrato che due iniezioni subcutanee da 20 μg ciascuna del farmaco in esame (GAD-alum) può revocare il progredire del T1D in pazienti di età compresa fra 10 e 18 anni in cui la malattia è insorta da breve tempo e fornire protezione contro di essa (Ludvigsson J. et al., 2008), mentre le prove cliniche di Fase III sono fallite.
Sono stati proposti ulteriori studi clinici per testare la possibilità di intervento a tempi diversi rispetto all’insorgenza e sviluppo della malattia, tipi diversi di terapia e nuove modi di somministrazione dell’autoantigene. Le attuali proposte includono terapie preventive in individui ad alto rischio (attualmente in Fase II di sperimentazione clinica), terapie di combinazione che sfruttano l’associazione dell’immunoterapia non antigene-specifica (NASI) mediante un immunosoppressore, con l’immunoterapia antigene-specifica (ASI), utilizzando per esempio la GAD65, e ASI con auto antigeni multipli (Lernmark and Larsson, 2011; Larsson and Lernmark, 2011). Inoltre, la modalità di somministrazione necessita di ulteriori studi, quali, per esempio, l’induzione di tolleranza orale sfruttando la GAD65, come è stato fatto mediante la somministrazione orale dell’insulina.
Viste le prove cliniche in corso o previste in futuro con l’obiettivo di testare diverse formulazioni del vaccino basate su auto antigeni del T1D, attualmente, la produzione di GAD65 umana è un punto cruciale per la pianificazione delle strategie future di prevenzione della malattia. Fino ad ora, la GAD65 umana (hGAD65) è stata ottenuta da diversi sistemi omologi ed eterologi, ma, le attuali piattaforme di produzione sono troppo costose ed incapaci di fornire quantità di auto antigene sufficienti a soddisfare la richiesta per i trattamenti di immunoterapia.
Le due forme della GAD65 umana, hGAD65 e hGAD65mut, sono state, quindi, espresse nel sistema eterologo Escherichia coli per stabilire se entrambe le molecole vengono accumulate come proteine insolubili, come precedentemente già osservato nel caso della hGAD65 (Mauch L. et al., 1993), e se la produzione di hGAD65mut risulta maggiore di quella della forma nativa dell’enzima, come precedentemente descritto in piante di Nicotiana tabacum var. Sr1 (Avesani L. et al., 2010). E stato dimostrato che entrambe le forme della GAD65 umana sono accumulate in corpi di inclusione insolubili e vengono solubilizzate solo in presenza di concentrazioni denaturanti di urea. Inoltre, è stato provato che anche nel sistema batterico la hGAD65mut è prodotta in quantità maggiori rispetto alla hGAD65.
I sistemi di produzione vegetali possono offrire vantaggi dal punto di vista economico e della produzione su larga scala di proteine ampiamente utilizzate in ambito terapeutico (Ma J.C.K. et al., 2005 a, b; Barasan and Rodriguez-Cerezo, 2008). Di conseguenza, il principale obiettivo del progetto di ricerca è la valutazione dell’idoneità del sistema vegetale per la produzione della GAD65 umana.
La GAD65 umana è stata precedentemente espressa in piante di tabacco transgeniche, ma i livelli di produzione si sono rivelati deludenti raggiungendo valori massimi pari allo 0.25% delle proteine solubili totali (Total Soluble Proteins, TSP) (Porceddu A. et al., 1999; Ma S. et al., 2004; Wang et al., 2008; Avesani L. et al., 2003). Recentemente, una forma mutata e cataliticamente inattiva della GAD65 umana, hGAD65mut, è stata espressa in piante transgeniche di Nicotiana tabacum var. Sr1. Le piante più alte esprimenti hanno accumulato livelli di proteina ricombinante dieci volte maggiori (2.2% TSP) rispetto a quelli ottenuti nelle piante più alte esprimenti trasformate con la hGAD65 (Avesani L. et al., 2010). Tale sistema di produzione vegetale necessita di essere ulteriormente caratterizzato in modo da poter verificare l’ipotesi secondo cui le proprietà catalitiche della forma nativa della GAD65 umana possono contribuire ai bassi livelli di produzione. In studi precedenti, infatti, è stato dimostrato l’assenza di attività enzimatica in vitro per la forma mutata della GAD65. Di conseguenza, si è effettuato un saggio enzimatico in vivo con il quale è stata confermata l’assenza di attività enzimatica della stessa molecola anche nel sistema eterologo vegetale.
Un secondo sistema vegetale è stato testato per la produzione di entrambe le forme della GAD65 umana. Nicotiana tabacum var. Maryland mammoth è una varietà di tabacco che produce una maggiore quantità di biomassa della più diffusa Sr1 e, quindi, potrebbe essere un eccellente candidato per la produzione di proteine ricombinanti ampiamente utilizzate in ambito terapeutico. Per questo, entrambe le forme della GAD65 umana, hGAD65 e hGAD65mut, sono state espresse in piante di tabacco transgeniche della varietà Maryland mammoth, dimostrando che, anche in tale sistema vegetale, la produzione di hGAD65mut è maggiore di quella della forma nativa dell’enzima, come precedentemente descritto in piante di tabacco transgeniche della varietà Sr1 (Avesani L. et al., 2010).
L’1% delle proteine solubili totali è generalmente il livello di espressione minimo richiesto per considerare l’estrazione di proteine ad uso farmaceutico derivate da pianta economicamente vantaggiosa (Ma J.C.K. et al., 2003). Visto che tale valore è stato raggiunto e superato già nella prima generazione di piante di Nicotiana tabacum var. Sr1, si è proceduto alla messa a punto del downstream processing della proteina ricombinante dal sistema vegetale. I risultati ottenuti durante il processo di selezione della popolazione di piante esprimenti la hGAD65mut in modo stabile ed omogeneo e di studio e valutazione del protocollo di estrazione e purificazione della proteina dal tessuto fogliare sono riportati e discussi.Abstract
Type 1 insulin-dependent diabetes (T1D) which afflicts 0.03-0.04% of population is caused by autoimmune destruction of insulin-secreting beta cells, leading to an insulin deficiency (Gepts, 1965). It is considered a chronic disease with a strong social impact because of high prevalence of late-onset complications and the young age of affected patients being the most frequent chronic disease in children younger than 14-years old. Until now there are no possibilities to cure it and insulin treatment is only a life-long replacement therapy.
Tolerance induction through autoantigen administration is one of the strategies useful to prevent or to slow down autoimmune diseases (Harrison, 2005). In particular, for T1D GAD65 has been evaluated as a good candidate vaccine and different Antigen-Specific Immunotherapy (ASI) studies using this autoantigen have been done to test its efficacy in tolerance induction. Results obtained in the non-obese diabetic (NOD) mouse models indicate the potential of GAD65 administration to provide a preventive treatment for diabetes. Recently, Diamyd Medical, a Swedish company, has conducted phase II and phase III clinical trials in humans: in the first case it was demonstrated that two subcutaneous injections of 20μg of alum-formulated GAD65 can reverse the progress of recent-onset T1D in 10 to 18-years-old patients and give protection against it (Ludvigsson et al., 2008), while phase III trials failed.
New clinical studies have been proposed including new challenges in timing, different types of therapies and new administration routes. Current proposals include preventive therapies in high-risk individuals (current Phase II trial), combination therapies exploiting the combination of Non-Antigen Specific Immunotherapy (NASI) through an immunosoppressor, together with Antigen-Specific Immunotherapy (ASI), using for example GAD65, and ASI using multiple autoantigens (Lernmark and Larsson, 2011; Larsson and Lernmark, 2011). In addition the route of administration needs further studies, such as oral tolerance induction through GAD65, as it has been done using oral insulin.
At present, human GAD65 production is a central point for planning future T1D prevention strategies because of the undergoing and future trials using different vaccine preparations based on T1D autoantigens. Until now human GAD65 (hGAD65) has been obtained from different homologous and heterologous platforms. However, actual production platforms are too expensive and unable to provide sufficient quantity of this autoantigen to meet demand for immunotherapy treatments.
Both forms of human GAD65, hGAD65 and hGAD65mut, are expressed in E.coli heterologous system to sort out if they are both accumulated as insoluble proteins, as previously described for human GAD65 (Mauch L. et al., 1993), and if hGAD65mut yield is higher than that of the wild type form of the enzyme, as previously reported in Nicotiana tabacum var. Sr1 plants (Avesani L. et al., 2010). They demonstrated to be both accumulated as insoluble inclusion bodies and were solubilized by the use of denaturing concentration of urea. Western and radioimmunoassay analyses demonstrated that hGAD65mut accumulated at higher levels than hGAD65.
Plant-based systems may offer advantages in terms of economy and scalability for the large-scale production of therapeutic proteins in high demand (Ma J.C.K. et al., 2005 a, b; Barasan and Rodriguez-Cerezo, 2008). Thus, the principal aim of the PhD project is the evaluation of plant-based platform feasibility for human GAD65 production.
Human GAD65 has previously been expressed in transgenic tobacco plants but yields were disappointing (maximum 0.25% of total soluble protein, TSP) (Porceddu et al., 1999; Ma S. et al., 2004; Wang et al., 2008; Avesani L. et al., 2003). In a recent study, a mutated catalytically-inactive form of human GAD65 (hGAD65mut) was expressed in transgenic Nicotiana tabacum var. Sr1 plants. hGAD65mut-highest expressing plants accumulated 10-fold (2.2% TSP) higher levels of recombinant protein than hGAD65-highest expressing plants (Avesani L. et al., 2010). This plant platform production system needs to be characterized thoroughly in order to verify the hypothesis by which the catalytic properties of native hGAD65 could contribute to its poor yields.
Since in previous studies it has been demonstrated in vitro the lack of the enzymatic activity for hGAD65mut, an enzymatic assay in vivo is performed in order to demonstrated the absence of enzymatic activity of the mutated form of GAD65 also in the heterologous plant-based system. Results of the assay are discussed.
An additional plant-based platform is tested for the production of both forms of human GAD65. In fact, being Nicotiana tabacum var. Maryland mammoth a higher leaf biomass producing variety than the most widely used one, Sr1, it can be, potentially, an excellent candidate for the production of recombinant pharmaceutical proteins with a large demand. Both forms of human GAD65, hGAD65 and hGAD65mut, are expressed in this plant-based platform, demonstrating that also in this system hGAD65mut yield is higher than that of the wild type form of the enzyme, as previously already described in Nicotiana tabacum var. Sr1 plants (Avesani L. et al., 2010).
1% TSP yield is usually regarded as the minimum required to make the extraction of a plant-derived pharmaceutical protein economically feasible (Ma J.K.C. et al., 2003). Since this threshold was exceeded in the first generation of hGAD65mut-transgenic Nicotiana tabacum var. Sr1 plants, a protocol for downstream processing of the recombinant protein from plant systems was investigated. Results obtained during the set up of the extraction and purification protocol are discussed
Perspectives for autoimmune diabetes prevention using plants
No full textAutoimmune diabetes is the most frequent chronic disease of childhood and it is characterised by long-term and severe complications; different therapeutic approaches have been used in order to prevent and to suppress the disease at an experimental level but no one of them reached so far positive results in human trials. However, the results of clinical trials using autoantigen-based therapies suggested new insight for the research of a vaccine.
The 65 kDa isoform of human glutamic acid decarboxylase, one of the major autoantigen associated with the disease, is regarded as crucial for future studies on diabetes prevention and suppression.
GAD65 is actually produced using conventional expression systems (mainly based on yeast and baculovirus/insect cell) and the final product is characterised by high costs.
We exploited plants as a platform for the production of human GAD65 by using stable and transient expression and by engineering the protein at different degrees. Our results indicate that plants are a feasible and versatile system for the production of this recombinant protein. The results obtained in the perspective of new therapeutic approaches are described and discussed
Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective
No full textIn recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms
Plant-Based Systems for Vaccine Production
No full text: Plant systems have been used as biofactories to produce recombinant proteins since 1983. The huge amount of data, collected so far in this framework, suggests that plants display several key advantages over existing traditional platforms when they are intended for therapeutic uses, including safety, scalability, and the speed in obtaining the final product.Here, we describe a method that could be applied for the expression and production of a candidate subunit vaccine in Nicotiana benthamiana plants by transient expression, defining all the protocols starting from plant cultivation to target recombinant protein purification
A downstream process allowing the efficient isolation of a recombinant amphiphilic protein from tobacco leaves
No full textThe 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major autoantigen in autoimmune diabetes. The heterologous production of hGAD65 for diagnostic and therapeutic applications is hampered by low upstream productivity and the absence of a robust and efficient downstream process for product isolation. A tobacco-based platform has been developed for the production of an enzymatically-inactive form of the protein (hGAD65mut), but standard downstream processing strategies for plant-derived recombinant proteins cannot be used in this case because the product is amphiphilic. We therefore evaluated different extraction buffers and an aqueous micellar two-phase system (AMTPS) to optimize the isolation and purification of hGAD65mut from plants. We identified the extraction conditions offering the greatest selectivity for hGAD65mut over native tobacco proteins using a complex experimental design approach. Under our optimized conditions, the most efficient initial extraction and partial purification strategy achieved an overall hGAD65mut yield of 92.5% with a purification factor of 12.3 and a concentration factor of 23.8. The process also removed a significant quantity of phenols, which are major contaminants present in tobacco tissue. This is the first report describing the use of AMTPS for the partial purification of an amphiphilic recombinant protein from plant tissues and our findings could also provide a working model for the initial recovery and partial purification of hydrophobic recombinant proteins from transgenic tobacco plants
Expression of a mutated form of GAD65 in heterologous systems
No full textType 1 insulin-dependent diabetes mellitus (T1DM) which afflicts 0.2-0.3% of population is caused by autoimmune destruction of insulin-secreting beta cells. The young age of affected patients, the need for life-long insulin therapy and the high prevalence of late-onset complications make T1DM a major health problem. The smaller isoform of glutamic acid decarboxylase of 65 kDa (GAD65) is the major autoantigen in human T1DM and it has recently demonstarated that two injections of the molecule can give protection against this autoimmune disease. T1DM requires a primary prevention because the disease has a complex genetic basis, making difficult to identify in the population people at risk of developing it. Vaccination studies and subsequent vaccination treatment of a lot of people need large quantity of purified protein, but the current production systems are too much expensive and unable to provide enough GAD65 to meet global demand. We have previously shown that GAD65 can be expressed in transgenic tobacco plants but yields are disappointing. In order to improve its expression level we use different heterologous systems such as Nicotiana tabacum plants, E.coli inducible system and insect cells/Baculovirus to express two different forms of the recombinant human GAD65: the wild type form of the enzyme (hGAD65) and the mutated form with no catalytic activity (hGAD65mut), hypothesising that the enzymatic activity might interfere with its accumulation in heterologous systems. In previous studies it has been demonstrated in vitro the lack of the enzymatic activity for the hGAD65mut and we show that GAD65mut accumulates to higher levels in transgenic plants and in E.coli inducible system than its enzymatically active counterpart, indicating that the catalytic properties of GAD65 contribute to its poor yields. To demonstrated the absence of enzymatic activity of the mutated form of GAD65 (GAD65mut) also in the heterologous systems we perform an enzymatic assay in vivo,. The results of the assay and the difference among the expression levels obtained in the heterologous systems are discussed
Comparative analysis of different biofactories for the production of a major diabetes autoantigen.
No full textThe 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major diabetes autoantigen that can be used for the diagnosis and (more recently) the treatment of autoimmune diabetes. We previously reported that a catalytically-inactive version (hGAD65mut) accumulated to tenfold higher levels than its active counterpart in transgenic tobacco plants, providing a safe and less expensive source of the protein compared to mammalian production platforms. Here we show that hGAD65mut is also produced at higher levels than hGAD65 by transient expression in Nicotiana benthamiana (using either the pK7WG2 or MagnICON vectors), in insect cells using baculovirus vectors, and in bacterial cells using an inducible-expression system, although the latter system is unsuitable because hGAD65mut accumulates within inclusion bodies. The most productive of these platforms was the MagnICON system, which achieved yields of 78.8 μg/g fresh leaf weight (FLW) but this was substantially less than the best-performing elite transgenic tobacco plants, which reached 114.3 μg/g FLW after six generations of self-crossing. The transgenic system was found to be the most productive and cost-effective although the breeding process took 3 years to complete. The MagnICON system was less productive overall, but generated large amounts of protein in a few days. Both plant-based systems were therefore advantageous over the baculovirus-based production platform in our hand
Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes
Full text linkPlant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required
PVX a powerful tool for autoimmune disease diagnosis
No full textWe explored the use of Potato Virus X as a scaffold for the expression of an immunodominant peptide associated to Sjogren’s syndrome.
Sjögren's syndrome (SjS) is a chronic autoimmune disease in which the body's white cells destroy the exocrine glands, specifically the salivary and lacrimal glands, that produce saliva and tears, respectively.
Human lipocalin was recently identified as a primary autoantigen associated to the disease and the immunodominant peptide associated to the protein was discovered using the sera of SjS patients. Autoantibodies directed to the peptide allow to identify in the population patients whose sera was negative to anti nuclear antibodies and to the rheumatoid factor.
The lipocalin peptide was cloned into a PVX-based vector which mediates the peptide display on the virus coat protein, yielding Chimeric Virus Particles (CVPs). CVPs allow a high-density exposure of the peptide on the viral surface.
The CVPs were used for coating an ELISA plate giving an assay which reproducibility, stability and sensitive was compared to the use of the peptide alone
GFP-based expression of truncated GSD65mut forms in high-throughput platforms
No full textType 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the destruction of insulin-secreting pancreatic beta cells, causing the need of life-long insulin therapy. The 65 kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen involved in the disease development. Recent phase II clinical trials have shown that administration of alum-formulated GAD65 lead to a significant preservation of residual insulin secretion without serious adverse effects (Ludvigsson et al., 2008); large-scale confirmatory studies are underway in Europe and in the USA. Current systems for the production of large quantity of purified recombinant protein are very expensive and inadequate to provide enough GAD65 to meet the global demand. We have previously shown that GAD65 and a mutated catalytically-inactive form of the protein (GAD65mut) can be expressed in transgenic tobacco plants (Avesani et al., 2003; Avesani et al., 2010). GAD65mut accumulates 10-fold higher than GAD65 and retains the immunogenic properties required to treat T1DM (Avesani et al., 2010). In order to develop a system for the highefficient production and purification of GAD65, we engineered GAD65mut to various extents to obtain soluble forms of the molecule. It is well documented that GAD65 undergoes some posttranslational modifications in the N-terminal domain that result in a firmly membrane-anchored protein, which is only released by the use of detergents, thus complicating downstream processing of the protein. In the present work we described and discussed the solubility and accumulation levels of three N-truncated forms of GAD65mut in comparison with full-length GAD65mut and GAD65 in a plant-based and a yeast-based platform. The two platforms have been chosen for the high-throughput and fast expression of the molecules. Furthermore, in both the systems, GAD65- based forms have been C-terminal fused with GFP in order to easily determine the recombinant proteins sub-cellular localization
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