Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008:434:183-203.
doi: 10.1007/978-1-60327-248-3_12.

Reducing the genotoxic potential of retroviral vectors

Ali Ramezani  1 , Teresa S Hawley, Robert G Hawley
Affiliations

Reducing the genotoxic potential of retroviral vectors

Ali Ramezani et al. Methods Mol Biol. 2008.

Abstract

The recent development of leukemia in gene therapy patients with X-linked severe combined immunodeficiency disease because of retroviral vector insertional mutagenesis has prompted reassessment of the genotoxic potential of integrating vector systems. In this chapter, various strategies are described to reduce the associated risks of retroviral genomic integration. These include deletion of strong transcriptional enhancer-promoter elements in the retroviral long terminal repeats, flanking the retroviral transcriptional unit with enhancer blocking sequences and designing vectors with improved RNA 3' end processing. Protocols are provided to evaluate the relative biosafety of the modified vectors based on their ability to immortalize hematopoietic progenitor cells and propensity to trigger clonal hematopoiesis or leukemogenesis following hematopoietic stem cell transplantation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Schematic representation of integrated forms of prototypical LTR and SIN retroviral vectors. For simplicity, gammaretroviral vectors are illustrated. Comparable lentiviral vectors contain additional components such as a central polypurine tract and the Rev response element involved in import of the preintegration complex into the nucleus and export of vector RNA into the cytoplasm, respectively (72,73). (Top) LTR vector The flanking intact LTRs comprise: U3, sequence unique to the 3' vector RNA and repeated in the integrated vector DNA; R, short sequence repeated at both termini of the vector RNA; and U5, sequence unique to the 5' vector RNA and repeated in the integrated vector DNA. Vector RNA transcripts initiate at the 5' boundary of the R region in the 5' LTR (arrow) and are polyadenylated ((A)n) at the 3' boundary of the R region in the 3' LTR. The packaging signal (ψ+) allows the full-length vector RNA to be efficiently encapsidated into budding vector particles. The protein coding region (cDNA) of the gene of interest is expressed as a spliced transcript (SD, splice donor; SA, splice acceptor). (Bottom) SIN vector The enhancer-promoter elements are deleted from the U3 regions of the LTRs (ΔU3) and an internal enhancer (E)-promoter (P) is used to drive transgene expression. The transgene pre-mRNA has been optimized for improved splicing and 3' end formation by inclusion of an intron and a posttranscriptional regulatory element (PRE). Enhancer-mediated interactions with cellular promoters are modulated by inclusion of enhancer blocking sequences formula image in the deleted U3 regions of the LTRs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Stewart AK, Dubé ID, Hawley RG. Gene marking and the biology of hematopoietic cell transfer in human clinical trials. In: Fairbairn LJ, Testa N, editors. Blood Cell Biochemistry8, Hematopoiesis and Gene Therapy. Kluwer Academic/Plenum Publishers; NY: 1999. pp. 243–268.
    1. Kohn DB, Sadelain M, Dunbar C, Bodine D, Kiem HP, Candotti F, et al. American Society of Gene Therapy (ASGT) ad hoc subcommittee on retroviral-mediated gene transfer to hematopoietic stem cells. Mol. Ther. 2003;8:180–187. - PubMed
    1. Kiem HP, Sellers S, Thomasson B, Morris JC, Tisdale JF, Horn PA, et al. Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: no progression to clonal hematopoiesis or leukemia. Mol. Ther. 2004;9:389–395. - PubMed
    1. Hacein-Bey-Abina S, von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415–419. - PubMed
    1. von Kalle C, Fehse B, Layh-Schmitt G, Schmidt M, Kelly P, Baum C. Stem cell clonality and genotoxicity in hematopoietic cells: gene activation side effects should be avoidable. Semin. Hematol. 2004;41:303–318. - PubMed

Publication types

Substances