Haematologica
HOME HELP FEEDBACK TABLE OF CONTENTS ARCHIVE SUBSCRIPTIONS
QUICK SEARCH:   [advanced]


     

Haematologica, Vol 92, Issue 1, 129-130 doi:10.3324/haematol.10560
Copyright © 2007 by Ferrata Storti Foundation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Colosimo, A.
Right arrow Articles by Dallapiccola, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Colosimo, A.
Right arrow Articles by Dallapiccola, B.

Red Cell Disorders

Sequence-specific modification of a ß-thalassemia locus by small DNA fragments in human erythroid progenitor cells

Alessia Colosimo*,°,+,, Valentina Guida*,#,+, Ivana Antonucci@, Tiziana Bonfini^, Liborio Stuppia@, Bruno Dallapiccola*,#

* IRCCS-CSS San Giovanni Rotondo, Italy and CSS-Mendel Institute, Rome, Italy;
° Department of Comparative Biomedical Sciences, University of Teramo, Italy;
# Department of Experimental Medicine and Pathology, University "La Sapienza", Rome, Italy;
@ Department of Biomedical Sciences, "G. D’Annunzio" University Foundation, Chieti-Pescara, Italy;
^ Department of Transfusional Medicine, A.S.L.-Pescara, Italy

Correspondence: Alessia Colosimo, Department of Comparative Biomedical Sciences, University of Teramo, piazza Aldo Moro, 64100 Teramo, Italy. Phone: international +39.0861.266862. E-mail: acolosimo{at}unite.it


   ABSTRACT
TOP
 ABSTRACT
Appendix
 References
 
Gene therapy has been proposed as a definitive cure for ß-thalassemia. We applied a gene targeting approach, based on the introduction of small DNA fragments (SDF) into erythroid progenitor cells, to specifically modify the ß-globin gene sequence at codon 39. The strategy was first tested in normal individuals by delivering mutant SDF that were able to produce the ß39 (C->T) mutation. Secondly, wild-type SDF were electroporated into target cells of ß39/ß39. ß-thalassemic patients to correct the endogenous mutation. In both cases, gene modification was assayed by allele-specific polymerase chain reaction of DNA and mRNA, by restriction fragment length polymorphism analysis and by direct sequencing.

Key words: gene targeting, gene therapy, ß-thalassemia, HBB gene.

ß-thalassemia is an autosomal recessive form of chronic anemia and can be caused by over 200 mutations in the ß-globin gene (HBB). Since blood transfusions and bone marrow transplantation are limited by shortage of compatible donors and by significant costs and risks, efforts have been focused on the development of alternative clinical treatment, including gene therapy. So far, conventional gene therapy approaches, based on virus-mediated delivery of functional copies of the gene, have been relatively unsuccessful.1 In contrast, gene targeting methodologies that repair the mutant gene in situ seem particularly attractive since theoretically they allow appropriate expression of the target gene, which remains in its native chromosomal environment.2 In this study, we used a gene targeting approach that employed small DNA fragments (SDF) to directly modify several disease-causing loci.3–7 We evaluated this approach in terms of its effectiveness in introducing or eliminating the most common Italian HBB mutation (ß39 C->T) in primary erythroid progenitor cells of normal or ß-thalassemic individuals, respectively. Differentiation into erythroid progenitor cells was allowed to light-density mononuclear cells (MNC) and enriched hematopoietic CD34+ cells, separated from umbilical cord blood or peripheral blood of normal donors, and from peripheral blood or bone-marrow of ß-thalassemic patients, as described elsewhere.8 Two different SDF of 517-bp and 190-bp were generated by polymerase chain reaction (PCR) using intronic primers and genomic DNA from normal subjects (wild type fragment) or from homozygous ß39/ß39 patients (mutant fragment).

To evaluate the optimal method of intracellular delivery of exogenous DNA into the erythroid progenitor cells, the pEGFP reporter plasmid (Clontech) was used to compare chemical (Lipofectamine, SuperFect) and physical (electroporation) systems assessed by flow cytometric analysis. While both Lipofectamine and Superfect were relatively ineffective at delivering the reporter plasmid to this subset of hematopoietic cells, electroporation (using BTX ECM 830 apparatus) was more efficient with a transfection efficiency of about 10–12%. However, the transfection efficiency decreased to levels below 0.6% when fluoroscein isothiocyanate (FITC)-labeled SDF were electroporated into the target cells. Initially, semi-nested PCR were carried out on gel-purified genomic DNA and cDNA isolated from erythroid progenitor cells from normal donors, using primers external to the fragments (HB3/HB4 for DNA and HB5/HB6 for cDNA), and then allele-specific primers (B39N or B39M). Mutant SDF were able to introduce the ß39 mutation both at DNA and mRNA levels. Direct sequence analysis of PCR products also showed a heterozygous C to T transition in all samples (data not shown). Because the mutation creates a novel BfaI restriction site, site-specific modification could also be assayed by restriction fragment length polymorphism (RFLP) analysis of DNA, showing the appearance of the heterozygous additional bands (275- and 150-bp) after digestion of 527-bp PCR products (Figure 1). To obtain an approximate quantification of the frequency of targeted cells in this model system, we cloned semi-nested PCR products into pGEM vector (Promega) and then screened 123 individual bacterial colonies by allele-specific PCR. The detection of three mutant colonies carrying the ß39 sequence suggested that the efficiency of short-fragment homologous replacement (SFHR)-mediated modification was approximately 2.4%. Secondly, erythroid precursor cells, expanded from peripheral blood and bone marrow of homozygous patients, were electroporated with wild-type fragments. Targeted correction was detected in eight of ten different experiments, as determined by allele-specific amplification performed on both genomic DNA and mRNA (Figure 2). Direct sequence analysis carried out on positive PCR samples showed the presence of both wild-type and ß39 sequences.


Figure 10920129
View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Figure 1. BfaI digestion analysis of PCR products of genomic DNA from normal individuals following the SFHR strategy. BfaI digestion analysis of PCR products of genomic DNA of normal (+/+) cells following SFHR strategy. M={Phi}X/HaeIII, molecular marker. 1: undigested PCR product of normal (+/+) cells electroporated with SDF; 2: BfaI-digested PCR product of normal (+/+) cells electroporated with SDF; 3: BfaI-digested PCR product of a heterozygous subject (ß39/+); 4: BfaI-digested PCR product of normal (+/+) cells electroporated without SDF; 5: undigested PCR product of normal (+/+) cells electroporated without SDF. 527 bp: undigested product using R37 and R47 primers; 425 bp and 102 bp: BfaI digested products of normal cells; 425 bp, 275 bp, 150 bp and 102 bp: BfaI digested products of heterozygous cells.

 

Figure 20920129
View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Allele-specific PCR on cDNA from ß-thalassemic (ß39/ß39) patients following the SFHR strategy. The primers used in 1n, 2n, and 3n (HB5 +ß39N) are specific for the normal sequence. The primers used in 1m ß2m, and 3m (HB5+ß39M) are specific for the ß39 sequence. 1n-1m: ß-thalassemic (ß39/ß39) cells electroporated without SDF; 2n-2m: ß-thalassemic (ß39/ß39) cells electroporated with SDF; 3n-3m: total RNA of ß-thalassemic (ß39/ß39) cells electroporated with SDF, without reverse transcriptase. M: 100 bp, molecular marker.

 
Different gene targeting strategies have already shown that site-specific correction of a single point mutation in the HBB gene can be achieved, but with variable efficiencies. 7,9 In this study, successful correction was routinely observed when 106 cells were electroporated with 1 or 2 µg of 517bp SDF. When the number of cells or the amount of fragment was reduced, no sequence-specific modification was detected, indicating that the ratio of SDF/cellular target is a fundamental factor for successful gene repair.7 Molecular analysis showed that both the 517- and 190-bp long SDF were able to mediate sequence-specific nucleotide exchange of DNA and mRNA in primary erythroid progenitor cells. The origin of the target cells did not appear to be relevant to the success of the experiments, although samples obtained from bone marrow generally showed better results. The possibility of PCR artifacts, due to residual contaminating SDF, was ruled out using gel-purified genomic DNA, DNase-digested RNA and external primers for PCR analysis, as recently suggested.7,10 Unfortunately, the number of corrected cells remaining from each experiment was insufficient to carry out protein studies by MALDI-TOF analysis. More importantly, improvements in delivery approaches and/or design of SDF are clearly required to yield sufficient quantities of corrected cells for a significant therapeutic benefit.


   Appendix
TOP
 ABSTRACT
 Appendix
References
 
Sequences of the primers used for the analyses:

HB1L (5'-CACTAGCAACCTCAAACAGAC-3')

HB2L (5'-CCATAGAAAAGAAGGGGAAAG-3') used to generate the 517-bp fragment.

HB1S (5'-TCTGATAGGCACTGACTCTCT-3')

HB2S (5'-AGCCAGGCCATCACTAAAGG-3') used to generate the 190-bp fragment.

HB3 (5'-ATAAAAGTCAGGGCAGAGCCA-3')

HB4 (5'-CCACACTGATGCAATCATTCG-3') used for the first PCR on genomic DNA.

HB 5 (5'-CATTTGCTTCTGACACAACTG-3')

HB6 (5'-AAAGTGATGGGCCAGCACAC-3') used for the first PCR on cDNA

B39N (5'-CAGATCCCCAAAGGACTCAAAGAACCTGTG-3', normal allele)

B39M (5'-CAGATCCCCAAAGGACTCAAAGAACCTGTA-3', ß-thal allele)

R37 (5'-CCAATCTACTCCCAGGAGCA-3')

R47 (5'-CACTCAGTGTGGCAAAGGTG-3') used for the BfaI digestion.


   Acknowledgments
 
we would also like to thank Ida Villanova for her outstanding technical assistance, Drs. Antonio Iacone and Paolo Cianciulli for providing the thalassemic samples and Prof. Dieter C. Gruenert for very useful comments


   Footnotes
 
+ AC and VG contributed equally to this work and should be considered co-first authors. Back

Funding: this work was supported by grants from the Italian Ministry of Health (Ricerca Corrente 2005).


   References
TOP
 ABSTRACT
 Appendix
 References
 

  1. Persons DA, Nienhuis AW. Gene therapy for the hemoglobin disorders. Curr Hematol Rep 2003;2:348-55.[Medline]
  2. Sullenger BA. Targeted genetic repair: an emerging approach to genetic therapy. J Clin Invest 2003;112:310-11.[CrossRef][ISI][Medline]
  3. Goncz KK, Colosimo A, Dallapiccola B, Gagne L, Hong K, Novelli G, et al. Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR. Gene Ther 2001;8:961-5.[CrossRef][ISI][Medline]
  4. Kapsa R, Quigley A, Lynch GS, Steeper K, Kornberg AJ, Gregorevic P, et al. In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement. Hum Gene Ther 2001;12:629-42.[CrossRef][ISI][Medline]
  5. Gruenert DC, Bruscia E, Novelli G, Colosimo A, Dalla - piccola B, Sangiuolo F, et al. Sequence-specific modification of genomic DNA by small DNA fragments. J Clin Invest 2003;112:637-41.[CrossRef][ISI][Medline]
  6. Sangiuolo F, Filareto A, Spitalieri P, Scaldaferri ML, Mango R, Bruscia E, et al. In vitro restoration of functional SMN protein in human trophoblast cells affected by spinal muscular atrophy by small fragment homologous replacement. Hum Gene Ther Jul 2005;16:869-80.[CrossRef]
  7. Goncz KK, Prokopishyn NL, Abdolmohammadi A, Bedayat B, Maurisse R, Davis BR, et al. Small fragment homologous replacement-mediated modification of genomic beta-globin sequences in human hematopoietic stem/progenitor cells. Oligonucleotides 2006;16:213-24.[CrossRef][ISI][Medline]
  8. Migliaccio G, Di Pietro R, Di Giacomo V, Di Baldassarre A, Migliaccio AR, Maccioni L, et al. In vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients. Blood Cells Mol Dis 2002;28:169-180.[CrossRef][ISI][Medline]
  9. Liu H, Agarwal S, Kmiec E, Davis BR. Targeted beta-globin gene conversion in human hematopoietic CD34(+) and Lin(–) CD38(–)cells. Gene Ther 2002;9:118-26.[CrossRef][ISI][Medline]
  10. Maurisse R, Fichou Y, De Semir D, Cheung J, Ferec C, Gruenert DC. Gel purification of genomic DNA removes contaminating small DNA fragments interfering with PCR analysis of SFHR. Oligonucleotides 2006;16:375-86.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Colosimo, A.
Right arrow Articles by Dallapiccola, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Colosimo, A.
Right arrow Articles by Dallapiccola, B.

HOME HELP FEEDBACK TABLE OF CONTENTS ARCHIVE SUBSCRIPTIONS