Abstract
Chimeric oligonucleotides have been used successfully to correct point and frameshift mutations in several cell types, as well as in animal and plant models. However, their application to primitive human blood cells has been limited. In this investigation, chimeric oligonucleotides designed to direct a site-specific nucleotide exchange in the human β-globin gene (an A to T substitution within codon 6) were introduced into normal human CD34+ and Lin−CD38- cells via microinjection. This A to T nucleotide exchange introduces the single site mutation responsible for sickle cell anemia. In 23% of experimental samples, gene conversion was detected in the progeny of microinjected CD34+ and Lin−CD38− cells that were cultured for at least 4 weeks. In addition, gene conversion was detected in the erythroid progeny of Lin−CD38− cells at the mRNA level. Conversion rates as high as 10–15% in 11% (five of 44) of experimental samples were confirmed by allele-specific PCR and sequence analysis of genomic DNA from the progeny of microinjected Lin−CD38− cells. Given that as few as 10% normal hematopoietic cells are sufficient to keep patients free of sickle cell disease, the level of conversion we have achieved in some samples may well be of therapeutic benefit in patients with sickle cell disease.
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References
Kmiec E.B. . Targeted gene repair (editorial) Gene Therapy 1999 6: 1 1
Cole-Strauss A. et al. Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide Science 1996 273: 1386 1386
Kren B.T., Cole-Strauss A., Kmiec E.B., Steer C.J. . Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide Hepatology 1997 25: 1462 1462
Kren B.T., Bandyopadhyay P., Steer C.J. . In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides Nat Med 1998 4: 285 285
Alexeev V., Yoon K. . Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA-DNA oligonucleotide Nat Biotechnol 1998 16: 1343 1343
Lai L.W., Lien Y.H. . Homologous recombination based gene therapy Exp Nephrol 1999 7: 11 11
Beetham P.R. et al. A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations Proc Natl Acad Sci USA 1999 96: 8774 8774
Zhu T. et al. Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides Proc Natl Acad Sci USA 1999 96: 8768 8768
Kren B.T. et al. Correction of the UDP-glucuronosyltransferase gene defect in the gunn rat model of Crigler–Najjar syndrome type I with a chimeric oligonucleotide Proc Natl Acad Sci USA 1999 96: 10349 10349
Rando T.A., Disatnik M.H., Zhou L.Z. . Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides Proc Natl Acad Sci USA 2000 97: 5363 5363
Alexeev V. et al. Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide Nat Biotechnol 2000 18: 43 43
Bartlett R.J. et al. In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide Nat Biotechnol 2000 18: 615 615
Rice M.C., Bruner M., Czymmek K., Kmiec E.B. . In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae Mol Microbiol 2001 40: 857 857
Davis B.R. et al. Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells Blood 2000 95: 437 437
Xu R., Reems J.A. . Umbilical cord blood progeny cells that retain a CD34+ phenotype after ex vivo expansion have less engraftment potential than unexpanded CD34+ cells Transfusion 2001 41: 213 213
Bhatia M. et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice Proc Natl Acad Sci USA 1997 94: 5320 5320
Bandyopadhyay P. et al. Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor J Biol Chem 1999 274: 10163 10163
Cole-Strauss A. et al. Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract Nucleic Acids Res. 1999 27: 1323 1323
Harrison G.S. et al. Optimization of gene transfer using cationic lipids in cell lines and primary human CD4+ and CD34+ hematopoietic cells Biotechniques 1995 19: 816 816
Van Tendeloo V.F. et al. High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery Gene Therapy 2000 7: 1431 1431
Xiang Y. et al. Targeted gene conversion in a mammalian CD34+-enriched cell population using a chimeric RNA/DNA oligonucleotide J Mol Med 1997 75: 829 829
Walters M.C. et al. Bone marrow transplantation for sickle cell disease N Engl J Med 1996 335: 369 369
Gamper H.B. et al. A plausible mechanism for gene correction by chimeric oligonucleotides Biochemistry 2000 39: 5808 5808
Saiki R.K. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 1985 230: 1350 1350
Malik P. et al. An in vitro model of human red blood cell production from hematopoietic progenitor cells Blood 1998 91: 2664 2664
Acknowledgements
The work was funded in part by NIH grant R01HL58583 (to EBK). We thank David B Brown, ND Victor Carsrud, Judith Yannariello-Brown, Aqing Yao and Tamara V Tsulaia for their excellent scientific and technical guidance, Dr Nicole L Prokopishyn and Barbara L Chow for their critical review of this manuscript. We also thank Hetal Parekh-Olmedo and Alyson Cole-Strauss for their technical assistance during the early stages of the work.
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Liu, H., Agarwal, S., Kmiec, E. et al. Targeted beta-globin gene conversion in human hematopoietic CD34+ and Lin−CD38− cells. Gene Ther 9, 118–126 (2002). https://doi.org/10.1038/sj.gt.3301610
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DOI: https://doi.org/10.1038/sj.gt.3301610
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