Efficient characterization of retro-, lenti-, and foamyvector-transduced cell populations by high-accuracy insertion site sequencing

The identification of unknown genomic flanking DNA sequences can be used for the molecular monitoring of retro-, lenti- and foamyviral integration, transgenes in early embryogenesis, insertional mutagenesis, cell fate, and stem cell plasticity. Most existing methods reflect shortcomings in sensitivity and or specificity, thus limiting genomic sequencing of unknown flanking DNA to clonal preparations. The application of linear amplification-mediated PCR (LAM-PCR), a recently developed direct sequencing technique for flanking DNA, should circumvent current limitations in different research fields. This technique combines preamplification of target DNA with a unique succession of enzymatic reactions on solid-phase. Using LAM-PCR, we show the previously unfeasible in vivo retro-, lenti- and foamyvirus integration site analysis in primate peripheral blood hematopoietic cells and human xenograft hematopoiesis. In light of two severe adverse events that occurred in a clinical SCID-X1 gene therapy trial, in vivo monitoring of the reinfused transduced cell pool by integration site analysis will be an important component of each gene transfer and therapy study aimed at clinical use.

Glimm, H (author)
Wissler, M (author)
Hoffmann, G (author)
Olsson, KarinLund University,Lunds universitet,Avdelningen för molekylärmedicin och genterapi,Institutionen för laboratoriemedicin,Medicinska fakulteten,Division of Molecular Medicine and Gene Therapy,Department of Laboratory Medicine,Faculty of Medicine(Swepub:lu)molm-kol (author)
Sellers, S (author)
Carbonaro, D (author)
Tisdale, JF (author)
Leurs, C (author)
Hanenberg, H (author)
Dunbar, CE (author)
Kiem, HP (author)
Karlsson, StefanLund University,Lunds universitet,Avdelningen för molekylärmedicin och genterapi,Institutionen för laboratoriemedicin,Medicinska fakulteten,Division of Molecular Medicine and Gene Therapy,Department of Laboratory Medicine,Faculty of Medicine(Swepub:lu)molm-ska (author)
Kohn, DB (author)
Williams, D (author)
von Kalle, C (author)
Avdelningen för molekylärmedicin och genterapiInstitutionen för laboratoriemedicin (creator_code:org_t)

In:Annals of the New York Academy of Sciences (HEMATOPOIETIC STEM CELLS 2002: GENETICS AND FUNCTION: Fourth International Symposium)996, s. 112-1210077-8923

By the author/editor: Schmidt, M; Glimm, H; Wissler, M; Hoffmann, G; Olsson, Karin; Sellers, S; show more...; Carbonaro, D; Tisdale, JF; Leurs, C; Hanenberg, H; Dunbar, CE; Kiem, HP; Karlsson, Stefan; Kohn, DB; Williams, D; von Kalle, C; show less...

About the subject

MEDICAL AND HEALTH SCIENCES: MEDICAL AND HEAL ...; and Clinical Medicin ...; and Hematology

Kungliga biblioteket hanterar dina personuppgifter i enlighet med EU:s dataskyddsförordning (2018), GDPR. Läs mer om hur det funkar här.
Så här hanterar KB dina uppgifter vid användning av denna tjänst.