Transgene optimization significantly improves SIN vector titers, gp91phox expression and reconstitution of superoxide production in X-CGD cells

Moreno-Carranza, Bibiana; Gentsch, Marcus; Stein, Stefan; Schambach, Axel; Santilli, Giorgia; Rudolf, E; Ryser, Martin; Haria, S; Thrasher, Adrian J.; Baum, Christopher; Brenner, Sebastian; Grez, Manuel

doi:10.1038/gt.2008.143

Cited by 52 publications

(36 citation statements)

References 45 publications

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“…For both BTK and RAG1 gene (for which we develop gene therapy in another project), 10-100 fold increased gene expression was obtained after lentiviral transduction with codon-optimized human sequences. Very recently, a similar observation was made by Moreno-Carranza et al 50 in the gp91 phox gene, which is involved in chronic granulomatous disease. In fact, in our initial efforts using BTK cDNA without codon optimization, no restoration of B-cell development was found 16 weeks after transplantation; neither could we observe the correction of B1 cells in the peritoneal cavity.…”

Section: Discussionsupporting

confidence: 48%

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK

Baert

Pike-Overzet

et al. 2010

Leukemia

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X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. BtkÀ/À mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK 4100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary BtkÀ/À recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in BtkÀ/À mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.

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Section: Discussionsupporting

confidence: 48%

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK

Baert

Pike-Overzet

et al. 2010

Leukemia

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“…As we did not observe malignant events in our experimental animals, it is possible that the use of such SIN vectors in our experimental setting provides safer alternatives for future gene therapy trials. Of note, SIN gammaretroviral vectors are currently being developed for CGD therapy (Moreno-Carranza et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Stable Marking and Transgene Expression Without Progression to Monoclonality in Canine Long-Term Hematopoietic Repopulating Cells Transduced with Lentiviral Vectors

et al. 2010

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Lentiviral gene transfer vectors have a number of potential advantages over gammaretroviral vectors including more efficient transduction of nondividing cells, a more favorable integration site profile, and the ability to accommodate large transgenes. Here, we present long-term follow-up data of animals that received lentivirustransduced CD34-enriched cells. Six long-term surviving dogs were available for analysis. Transgene expression was analyzed from at least 12 months to more than 5 years after transplantation in peripheral blood cells and multiple cell lineages. All animals demonstrated long-term stable transgene expression in peripheral blood myeloid, lymphoid, and red blood cells as well as in platelets. Vector integration sites were analyzed by linear amplification-mediated polymerase chain reaction and showed a polyclonal repopulation pattern in all animals. There was no evidence of any development of monoclonality or leukemia in the animals. The stable long-term multilineage transgene expression, together with detection of the same integration site in myeloid and lymphoid cells, strongly suggests the transduction of long-term repopulating stem cells. Our data demonstrate safe and efficient transduction of multilineage long-term repopulating cells with lentiviral vectors and support the use of such vectors for gene therapy studies in patients.

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“…For this purpose, two therapeutic vectors aimed at the correction of CGD (see MorenoCarranza et al 31 and M Grez, unpublished data) and Xlinked severe combined immunodeficiency (SCID-X1 32 ) were inserted into pEMTAR-1 and targeted into PG368 packaging cells. The internal P1 promoter in the SIN11-SF vector was replaced by either the EFS promoter or the c-fes promoter (M Grez, unpublished data), and eGFP was replaced by either a codon-optimized gp91phox cDNA (for CGD) 31 or the wt IL2Rgc-chain cDNA (for SCID-X1) 32 ( Figure 5a). The titer of the best PG368 producer clones for SIN11(fes-gp91) and SIN11(EFS-gc) was similar and ranged between 1.0 Â 10 5 and 4.1 Â 10 5 IP ml…”

Section: Generation Of Stable Pg368 Packaging Cells For Clinically Rementioning

confidence: 99%

“…The SIN g-retroviral vectors, SIN11(fes-gp91) and SIN11(EFS-gp91), were derived from SERS11.SF.gp91.W 31 by replacing the SFFV LTR by a 500-bp DNA fragment obtained from the human c-fes promoter and the intronless version of the elongation factor 1a promoter (REF), respectively. The SERS11(EFS-gc) viral vector has been described previously, 32 and was used for construction of the SIN11(EFS-gc) retroviral vector transferred by pEM-TAR-1 (introduction of vectors into pEMTAR or pEMTAR-1 was done using standard molecular biology techniques).…”

Section: Plasmid Constructsmentioning

confidence: 99%

A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating γ-retroviral vectors using targeted integration

et al. 2009

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The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN g-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinasemediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN g-retroviral vectors for clinical trials.

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Transgene optimization significantly improves SIN vector titers, gp91phox expression and reconstitution of superoxide production in X-CGD cells

Cited by 52 publications

References 45 publications

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK

Stable Marking and Transgene Expression Without Progression to Monoclonality in Canine Long-Term Hematopoietic Repopulating Cells Transduced with Lentiviral Vectors

A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating γ-retroviral vectors using targeted integration

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