title: Transgene optimization significantly improves SIN vector titers, gp91phox expression and reconstitution of superoxide production in X-CGD cells.
abstract: Gene therapy has proven to be of potential value for the correction of inherited hematopoietic disorders. However, the occurrence of severe side effects in some of the clinical trials has questioned the safety of this approach and has hampered the use of long terminal repeat-driven vectors for the treatment of a large number of patients. The development of self-inactivating (SIN) vectors with reduced genotoxicity provides an alternative to the currently used vectors. Our initial attempts to use SIN vectors for the correction of a myeloid disorder, chronic granulomatous disease, failed due to low vector titers and poor transgene expression. The optimization of the transgene cDNA (gp91(phox)) resulted in substantially increased titers and transgene expression. Most notably, transgene optimization significantly improved expression of a second cistron located downstream of gp91(phox). Thus, optimization of the transgene sequence results in higher expression levels and increased therapeutic index allowing the use of low vector copy numbers per transduced cell and weaker internal promoters.
authors.affiliation: Division of Applied Virology and Gene Therapy, Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Moreno-Carranza
authors.forename: M
authors.initials: M
authors.lastname: Gentsch
authors.forename: S
authors.initials: S
authors.lastname: Stein
authors.forename: A
authors.initials: A
authors.lastname: Schambach
authors.forename: G
authors.initials: G
authors.lastname: Santilli
authors.forename: E
authors.initials: E
authors.lastname: Rudolf
authors.forename: M F
authors.initials: MF
authors.lastname: Ryser
authors.forename: S
authors.initials: S
authors.lastname: Haria
authors.forename: A J
authors.initials: AJ
authors.lastname: Thrasher
authors.forename: C
authors.initials: C
authors.lastname: Baum
authors.forename: S
authors.initials: S
authors.lastname: Brenner
authors.forename: M
authors.initials: M
authors.lastname: Grez
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D006105:Granulomatous Disease, Chronic / Q000378:metabolism / Q000628:therapy*; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000821:virology; D006801:Humans; D018189:Immunomagnetic Separation; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013481:Superoxides / Q000032:analysis; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes; D038301:Virus Inactivation
chemical_list: D008562:Membrane Glycoproteins; D013481:Superoxides; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases