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Self-inactivating gammaretroviral vectors for the gene therapy of chronic granulomatous disease

By Bibiana Moreno Carranza

Abstract

Chronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency characterized by defective intracellular oxidative killing of ingested invading microbes by PMN and monocytes. It is caused by mutations in one of the four genes coding for the essential subunits of the NADPH oxidase (gp91phox, p47phox, p67phox and p22phox). Approximately 75% of the CGD cases are due to mutations in the gp91phox gene. If regular care and conventional therapy fail, the recommended therapy is allogeneic bone marrow transplantation (BMT), but only if a matched donor is available. A therapeutic option for patients lacking suitable donors is the genetic modification of autologous hematopoietic stem cells. The gene therapy offers an interesting alternative to BMT since it implies a less invasive treatment and represents a possibly unique curative option for patients with no suitable donor. Gammaretroviral vectors were already used in some gene therapy trials for CGD and other immunodeficiencies showing relevant clinical benefit. However, these trials uncovered an unexpected mutagenic side effect. If the retrovial integration ocurrs near to, or into proto-oncogenes this might lead to clonal dominance or even malignant transformation (Hacein-Bey-Abina et al., 2003a; Ott et al., 2006). Therefore, there was a need to further improve the safety of these vectors and to this end the self-inactivating gammaretroviral vectors were engineered. Non essential sequences for virus infectivity and integration, which might influence the surrounding gene expression, were deleted in these vectors. In the first set of experiments, a series of SIN gamma retroviral vectors was cloned driving the expression of the wild-type gp91phox cDNA under the control of a viral constitutive SFFV promoter. However initial studies with these vectors failed because the titers of the virus produced by transient transfection protocols were extremely low (<5x105 TU/ml). Therefore, a codon optimization of the gp91phox cDNA was considered as an alternative. The codon optimized synthetic gp91phox gene was used to construct a SIN gammaretroviral vector, again under the control of the SFFV promoter (Schambach et al., 2006c). With this vector an increase in titer was observed compared to the native gp91phox sequence, which was due to the improved transcription in 293T transfected cells. The enhancement of the synthetic gp91phox transcription led to a higher internal transcript production and protein expression. An enhanced superoxide production in transduced myelomonocytic X-CGD PLB-985 populations was also detected. All these data indicate that the synthetic gp91phox might represent an excellent alternative to those former constructs expressing the native gp91phox transgene. Since it was postulated that the SFFV promoter could still cause transactivation of neighboring genes due to its strength (Modlich et al., 2006), three different non-viral promoters were tested, one constitutive (the EFs promoter) and two myeloid-specific promoters (the c-fes and MRP8 promoter). The three SIN gammaretroviral vectors were able to generate high titers after transient transfection of 293T packaging cells, to efficiently transduce the X-CGD PLB-985 cell line and to reconstitute the NADPH oxidase activity to a high degree. In mouse transplantation experiments, the EFs promoter showed a high variable transgene expression in the different lineages analyzed, and the c-fes promoter showed also a ubiquitinous expression. In contrast, the MRP8 promoter showed a high myeloid specificity since gp91phox expression in mSca-1+ cells and lymphoid B cells from transplanted mice was extremly low and even absent. However, the lowest levels of transgene expression were observed in the myeloid populations both in bone marrow and peripheral blood with this vector. When the oxidase reconstitution ability of these promoters was tested, the numbers of superoxide producing cells obtained were similar than those observed in the clinical X-CGD trial conducted by the groups of Dr. M. Grez and Prof. R. A. Seger (over 35% in one patient and ~15% in the second), which led to the eradication of therapy refractory infections (Ott et al., 2006). Between the three constructs, the MRP8 promoter was less effective in restoring the NADPH oxidase activity than the EFs and c-fes promoters. The c-fes promoter reached the highest levels of DHR reactive cells in the highest number of mice. Overall, these data showed that between all constructs tested, the c-fes containing construct in combination with the codon optimized gp91phox sequence showed the best performance within the SIN gammaretroviral backbone. It generated the highest titers in combination with a better NADPH oxidase reconstituting ability. One main goal in the development of SIN gammaretroviral vectors is reducing the genotoxic effect due to random vector integration. An improved gene transfer and expression, and a constant performance are also highly desirable. The present study shows that the c-fes SIN vector in combination with the synthetic gp91phox may be considered as an effective gene therapy strategy for the restoration of the NADPH oxidase activity in CGD. It allows the use of a cellular promoter generating adequate physiological levels of the therapeutic protein and reduces the number of vector copies required for a therapeutic effect.Bei der Septischen Granulomatose (engl.: CGD chronic granulomatous disease) handelt es sich um eine seltene, vererbbare primäre Immunschwäche, welche sich durch einen Defekt im oxidativen Abtöten intrazellulär aufgenommener Mikroben durch Phagozyten auszeichnet. In Folge dessen erleiden Patienten mit CGD immer wieder schwerste Infektionen durch Bakterien und Pilze. Ursache für die Septische Granulomatose ist eine Mutation in einem der vier Gene (gp91phox, p47phox, p67phox und p22phox), die für die essentiellen Untereinheiten der NADPH Oxidase kodieren. In zirka 75% aller CGD-Fälle treten die Mutationen im gp91phox Gen auf. Weil dieses Gen auf dem X-Chromosom liegt (Xp21.1 Locus), handelt es sich somit um eine X-chromosomal gekoppelte Vererbung. In den übrigen Fällen der CGD erfolgt die Vererbung autosomal-rezessiv. Bei dieser Form der Krankheit sind Frauen und Männer gleichermaßen betroffen. Führen Standardbehandlungen und konventionelle Therapien zu keinem Erfolg, verbleibt als einziger Therapieansatz eine allogene Knochenmarktransplantation. Diese kann jedoch nur durchgeführt werden, wenn ein passender Spender vorhanden ist. Die begrenzte Verfügbarkeit adäquater Spender, die methodisch bedingte Toxizität der Transplantationsprozedur sowie posttransplantationale Komplikationen, wie beispielsweise die Transplantat-Wirt Krankheit (engl. graft versus host disease), erschweren eine breite Anwendung der allogenen Knochenmarktransplantation zur Behandlung von CGD. Ein alternativer therapeutischer Ansatz ist die genetische Modifikation der eigenen (=autologen) hämatopoetischen Stammzellen. Zwei der wichtigsten Vorteile einer Gentherapie im Vergleich zur allogenen Knochenmarktransplantation sind die geringere Belastung für die Patienten sowie die Möglichkeit der Therapie von Patienten ohne passenden Spender. In einigen gentherapeutischen Studien zur CGD und anderen Immundefizienzkrankheiten konnten Gammaretrovirale Vektoren einen deutlichen klinischen Nutzen für die Patienten aufzeigen. Die Studien zeigten jedoch teils erhebliche Nebenwirkungen, wie die Entwicklung maligner Zellpopulationen oder einen selektiven Wachstumsvorteil der genkorrigierten Zellen, der zu einer oligo- oder gar monoklonalen Expansion der Zellen führte. Diese Phänomene entstehen in Folge der Integration des Vektors in das Wirtsgenom, welche jedoch ein unumgänglicher Schritt im Replikationszyklus der Retroviren darstellt. Aus diesem Grund bestand der Bedarf die Sicherheit dieser Vektoren weiter zu verbessern, was in der Weiterentwicklung der sog. Selbst-Inaktivierenden Gammaretroviren (SIN Retrovektoren) resultierte. Bei diesen Genvektoren sind Promotor / Enhancer-Bereiche im 3´LTR des Vektors deletiert, wodurch der Einfluss auf die Genexpression in der Nachbarschaft der Integrationsstelle minimiert werden soll. Wenngleich diese Vektoren häufig eine geringere Effizienz in der Virusproduktion zeigen, stellt die Verwendung eines internen Promotors einen entscheidenden Vorteil dar. So ermöglichen es diese Vektoren z. B. die Expression eines Transgens unter die Kontrolle geeigneter, gewebespezifischer Promotoren in unterschiedlichen Zellen zu stellen. Die vorliegende Studie begann mit der Klonierung SIN Gammaretroviraler Vektoren. Die ersten getesteten Vektoren exprimieren die gp91phox Wildtyp cDNA, wobei der konstitutive SFFV LTR als interner Promotor fungiert. Anfängliche Studien mit diesen Vektoren scheiterten, da der Virustiter nach transienter Transfektion zu niedrig war (<5x105 TU/ml). Da der Spleißvorgang im Zellkern mit dem Kernexportmechanismus der endogenen mRNA gekoppelt ist, wurde vermutet, dass das Einbringen eines Introns in das virale Genom eine Steigerung des mRNA Exports zur Folge hätte. Der Einbau eines Spleißakzeptors und einer Spleißdonorstelle in die Leader Sequenz des Vektors brachte jedoch keine Erhöhung des Titers. Genauso wenig zeigte die gerichtete Mutation (site-directed mutagenesis) eines kryptischen Spleißakzeptors (cSA) in der gp91phox Sequenz (gp91m) einen Effekt auf den Virustiter. Als Alternative kam daher eine vollständige Codonoptimierung der gp91phox cDNA in Betracht. Die Strategie der Codonoptimierung wird benutzt, um die heterologe Genexpression von Genen, die in fremde Genome eingeführt werden, zu verbessern. Alle Codons einer proteinkodierenden DNA werden dabei auf den bevorzugten Codongebrauch im Zielorganismus abgestimmt. Diese Methode wurde bereits für Reporterproteine wie GFP und Luziferase angewandt. In verschiedenen Organismen konnte gezeigt werden, dass eine vorher niedrige Genexpression durch diese Methode erhöht werden kann. Die Firma GeneArt (Regensburg, Deutschland) analysierte und verbesserte die Sequenz der gp91phox mRNA des Wildtypes. Dabei wurde die Sequenz zunächst so modifiziert, dass wenig verwendete Codons durch häufiger benutzte ersetzt wurden. Der Codon Adaption Index (CAI) ist ein relatives Maß dafür wie nahe - verglichen mit einer Reihe von Referenzgenen - der Codongebrauch eines Genes am Optimum liegt. Der CAI konnte beim gp91phox von 0,77 auf 0,99 optimiert werden. Zusätzlich wurden einige Motive identifiziert, die entweder die Genexpression oder die Virusproduktion beeinträchtigen könnten. Aufgrund dessen wurden interne Polyadenylierungsstellen, zusätzliche kryptische Spleißstellen und RNA instabilisierende Motive, wie z. B. AU-reiche Elemente, gegen weniger kritische Sequenzen ausgetauscht. Wichtig ist dabei anzumerken, dass die Aminosäuresequenz des Gp91phox Proteins bei all diesen Modifikationen nicht verändert wurde. Die so veränderte synthetische gp91phox cDNA (gp91s) wurde im Folgenden für den SIN Gammaretroviralen Vektor SERS11.SF.gp91s.Wm verwendet. Dieses Plasmid beinhaltet den internen SFFV Promotor und die gleiche Promotor/Enhancer Anordnung ..

Topics: ddc:610
Year: 2008
OAI identifier: oai:publikationen.ub.uni-frankfurt.de:5991

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