Journal ArticleDOI
Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.
Manfred Schmidt,C von Kalle,Gesa Hoffmann,Manuela Wissler,N. Lemke,A. Müßig,H. Glimm,H. Glimm,D. A. Williams,D. A. Williams,Susanne Ragg,C. U. Hesemann +11 more
TLDR
EpTS/LM-PCR enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.Abstract:
By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.read more
Citations
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Journal ArticleDOI
Sustained Correction of X-Linked Severe Combined Immunodeficiency by ex Vivo Gene Therapy
Salima Hacein-Bey-Abina,Françoise Le Deist,Frédérique Carlier,Cécile Bouneaud,Christophe Hue,Jean-Pierre de Villartay,Adrian J. Thrasher,NM Wulffraat,Ricardo U. Sorensen,Sophie Dupuis-Girod,Alain Fischer,E. Graham Davies,Wietse Kuis,Lilly Leiva,Marina Cavazzana-Calvo +14 more
TL;DR: Ex vivo gene therapy with gamma(c) can safely correct the immune deficiency of patients with X-linked severe combined immunodeficiency and allow patients to have a normal life.
Journal ArticleDOI
HIV reproducibly establishes a latent infection after acute infection of T cells in vitro
TL;DR: Direct sequencing of integration sites demonstrated that latent clones frequently contain HIV integrated in or close to alphoid repeat elements in heterochromatin, demonstrating that HIV can reproducibly establish a latent infection as a consequence of integration in or near heterochROMatin.
Journal ArticleDOI
Murine leukemia induced by retroviral gene marking.
Zhixiong Li,Zhixiong Li,Jochen Düllmann,Bernd Schiedlmeier,Manfred Schmidt,Christof von Kalle,Johann Meyer,Martin Forster,Carol Stocking,Anke Wahlers,Oliver Frank,Wolfram Ostertag,Klaus Kühlcke,Hans Georg Eckert,Boris Fehse,Christopher Baum,Christopher Baum +16 more
TL;DR: Somatic gene transfer is a promising therapeutic strategy, but it may also evoke new types of side effects related to genetic damage or transgene activity.
Journal ArticleDOI
The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.
Eugenio Montini,Daniela Cesana,Manfred Schmidt,Francesca Sanvito,Cynthia C. Bartholomae,Marco Ranzani,Fabrizio Benedicenti,Lucia Sergi Sergi,Alessandro Ambrosi,Maurilio Ponzoni,Claudio Doglioni,Clelia Di Serio,Christof von Kalle,Luigi Naldini +13 more
TL;DR: It is determined that substantially greater LV integration loads are required to approach the same oncogenic risk as gammaRVs, which strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.
Journal ArticleDOI
Clonal Dominance of Hematopoietic Stem Cells Triggered by Retroviral Gene Marking
Olga S. Kustikova,Boris Fehse,Ute Modlich,Min Yang,Jochen Düllmann,Kenji Kamino,Nils von Neuhoff,Brigitte Schlegelberger,Zhixiong Li,Christopher Baum,Christopher Baum +10 more
TL;DR: It is demonstrated that retroviral integrations themselves may trigger nonmalignant clonal expansion in murine long-term hematopoiesis and have major implications for diagnostic gene marking and the discovery of genes regulating stem cell turnover.
References
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Journal ArticleDOI
A Third-Generation Lentivirus Vector with a Conditional Packaging System
Tom Dull,Romain Zufferey,Michael C. Kelly,Ronald J. Mandel,Matthew Nguyen,Didier Trono,Luigi Naldini +6 more
TL;DR: It is demonstrated that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript, and the improved design presented here should facilitate testing of lentivirus vectors.
Journal Article
Improved Retroviral Vectors for Gene Transfer and Expression
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TL;DR: A set of murine retrovirus-based vectors that include unique cloning sites for insertion of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter are described.
Journal ArticleDOI
Sustained Correction of X-Linked Severe Combined Immunodeficiency by ex Vivo Gene Therapy
Salima Hacein-Bey-Abina,Françoise Le Deist,Frédérique Carlier,Cécile Bouneaud,Christophe Hue,Jean-Pierre de Villartay,Adrian J. Thrasher,NM Wulffraat,Ricardo U. Sorensen,Sophie Dupuis-Girod,Alain Fischer,E. Graham Davies,Wietse Kuis,Lilly Leiva,Marina Cavazzana-Calvo +14 more
TL;DR: Ex vivo gene therapy with gamma(c) can safely correct the immune deficiency of patients with X-linked severe combined immunodeficiency and allow patients to have a normal life.
Journal ArticleDOI
In vivo footprinting of a muscle specific enhancer by ligation mediated PCR
Paul R. Mueller,Barbara J. Wold +1 more
TL;DR: In vivo protein-DNA interactions at the developmentally regulated enhancer of the mouse muscle creatine kinase gene were examined by a newly developed polymerase chain reaction (PCR) footprinting procedure and imply that additional regulatory mechanisms must restrict the interaction between this protein and its target site prior to differentiation.
Journal ArticleDOI
PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.
TL;DR: An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases.
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