Hematopoietic stem cell gene therapy: selecting only the best

Abstract

Hematopoietic stem cell (HSC) gene therapy can potentially cure a variety of human hematopoietic diseases, such as sickle cell disease. Selection and expansion of gene-corrected HSCs has now been accomplished for the first time using HSC from large animals — dogs and humans — with a novel drug-resistance gene, MGMT, which is not expressed in normal HSCs (see the related articles beginning on pages 1561 and 1581). Highly efficient lentiviral transfer and expression of MGMT into relatively few HSCs led to repopulation of most of the hematopoietic compartment with gene-corrected cells following suitable drug treatment. This selection system may be useful in human clinical trials to permit gene therapy in autologous and allogeneic bone marrow transplantation settings.

Curing human diseases using gene transfer into human hematopoietic stem cells (HSCs) has long been a goal of scientists and physicians. HSCs remain among the most promising target cells for gene therapy because of their unique differentiation and expansion capabilities, which ensure that the transfer and expression of genes added to a small number of cells result in gene correction of much greater numbers of cells by self-renewal and differentiation. Targeting genes to cells more differentiated than HSCs does not result in long-term expression of the transferred genes, since these cells further differentiate and die rather than self-renew. In order to transduce HSCs it is necessary to use vectors that integrate into chromosomal DNA and are faithfully transferred to daughter cells. Currently, oncoretroviruses and lentiviruses are used as suitable vectors; adenoviruses and other transfer vectors are either carried in the cell as episomes or integrate poorly. HSCs are relatively rare populations and are often quiescent, which makes it difficult to transfer genes into these cells with high efficiency. Lentiviral vectors have an advantage over oncoretroviruses in that they target nondividing cells including quiescent HSC.

Lentiviral gene transfer

High-titer lentiviruses have been successful in curing mouse models of sickle cell disease and β-thalassemia (1, 2). Gene transfer into the HSCs of larger animals, including humans, has been much more problematic. Although human gene therapy trials have demonstrated successful transduction of human HSCs, the numbers of gene-corrected human HSCs observed following transplantation have been relatively low. To expand this population of gene-corrected cells, selectable marker genes have been used. The most successful clinical gene therapy trials to date used the human multidrug resistance 1 gene (MDR1), the P-glycoprotein product of which is required for the removal of certain drugs from cells (3, 4). P-glycoprotein expression permits the selection and expansion of the gene-corrected HSCs and their progeny by drug treatment. In these trials, selection with anthracyclines and taxanes, drugs toxic to untransduced cells without significant endogenous MDR1 activity, led to a modest selection and expansion of the MDR1-transduced human HSCs (3, 4).

A potent HSC selection system

More recently, a powerful selection system has been exploited to select for transduced HSCs using a mutant form of the enzyme O⁶-methylguanine-DNA methyltransferase (MGMT), encoded by the MGMT gene, the expression of which is markedly reduced in normal HSCs. MGMT confers resistance to the cytotoxic effects of the anticancer drugs the nitrosoureas (NUs). In murine studies, transduction of very few HSCs with the mutant MGMT vector results in profound selection and expansion of gene-corrected cells following exposure to NU and O⁶-benzylguanine (O⁶BG) (5–8). In this issue of the JCI, two independent studies demonstrate for the first time the power of lentiviral vectors expressing the mutant MGMT gene in HSC selection and expansion in dogs and in human HSC (9, 10). In the study by Neff et al. (9), donor dog HSCs transduced with mutant MGMT-expressing vectors were strongly selected in vivo after transfer into normal recipients in an allogeneic bone marrow setting after treatment with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and O⁶BG. In the second study, by Zielske et al. (10), human CD34⁺ cells (among which the majority of human HSCs are included) were transduced with the same mutant MGMT vector, and selection and expansion of gene-corrected human HSCs was demonstrated using NOD/SCID mice to measure engraftment and expansion of human HSCs and their progeny (Figure 1).

Figure 1

Gene therapy using HSCs. (a) Transduction of cells with lentiviral supernatants containing the transgene MGMT that integrates into chromosomal DNA. (b) Gene transfer into HSCs with subsequent injection of populations of hematopoietic cells containing gene-corrected HSCs into animals, either dogs or, for human HSCs, NOD/SCID mice. Cells given by vein home to the marrow. (c) In vivo selection. Before: Small numbers of MGMT-expressing gene-corrected cells. After: Almost complete killing of untransduced HSCs with BCNU and O⁶BG therapy. Self-renewal and expansion of MGMT gene-corrected HSCs repopulate all hematopoietic lineages.

The MGMT cell selection system is already being tested for protection of marrow from the toxic effects of NU treatment in a human phase 1 clinical trial in patients being treated with NUs (S. Gerson, unpublished observations). One future possibility is the use of this system to permit successful human allogeneic transplantation by MGMT gene transfer into umbilical cord blood that may have insufficient HSCs to fully engraft adults. Subsequent in vivo selection and expansion of the transduced donor HSCs would be achieved using NU and O⁶BG administration, analogous to the experiment performed in dogs and described in this issue by Neff et al. (9). Another potential application in humans would be the addition of the MGMT gene to retroviral or lentiviral vectors containing a nonselectable gene such as the human β-globin gene in an autotransplantation setting, followed by selection for the gene-corrected cells after transplantation.

Gene therapy today

Despite the recent development of leukemia in two of the nine subjects enrolled in a human clinical trial of retroviral gene therapy for X-linked SCID (11), the benefits afforded to almost all patients in this trial, in addition to the lack of significant toxicity reported in other similar trials, lend weight to the general opinion that human retroviral gene therapy trials using HSCs should continue in the hope of increasing therapeutic efficacy and safety (12, 13). Both reports in this issue of the JCI also show that relatively low numbers of integrations of the MGMT gene result in sufficient expression of the enzyme to successfully select and expand the gene-corrected HSCs (9, 10). This is important because of the presumed potential for increased insertional mutagenesis with increased numbers of integrations. In the X-linked SCID gene therapy trial, both patients who developed leukemia demonstrated insertion of an unregulated γC receptor gene into the locus of a known proliferative gene, LMO2. In both cases, there was a 2- to 3-year interval between the gene-correction event and the development of leukemia. These circumstances suggest that the leukemic clones were produced by the rare event of gene insertion at the LMO2 locus and were selected for proliferation over time. Methods that may be employed in future studies to reduce the type of insertional mutagenesis associated with these two individuals include the use of (a) insulators, which would prevent the upstream or downstream activation of the inserted gene(s); and (b) gene- and tissue-specific promoters, such as the human β-globin gene promoter in human β-globin gene therapy trials, as alternatives to more generally expressed promoters such as the retroviral long-terminal repeats used in the X-linked SCID trial. These modifications and the use of HSC selection as described in the two papers in this issue of the JCI (9, 10) may permit safe and effective HSC gene therapy of hematological disorders in the near future.

Footnotes

See the related article beginning on page 1561.

Conflict of interest: The author has an equity interest in Genetix Pharmaceuticals Inc., which is active in the development of effective gene therapy technologies.

Nonstandard abbreviations used: hematopoietic stem cell (HSC); multidrug resistance 1 (MDR1); O⁶-methylguanine-DNA methyltransferase (MGMT); nitrosourea (NU); O⁶-benzylguanine (O⁶BG); 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU).

References

1. May, C, et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature. 2000. 406:82-86.
   View this article via: PubMed CrossRef Google Scholar
2. Pawliuk, R, et al. Correction of sickle cell disease in transgenic mouse models by hematopoietic gene therapy. Science. 2001. 294:2368-2371.
   View this article via: PubMed CrossRef Google Scholar
3. Moscow, JA, et al. Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy. Blood. 1999. 94:52-61.
   View this article via: PubMed Google Scholar
4. Abonour, R, et al. Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells. Nat. Med. 2000. 6:652-658.
   View this article via: PubMed CrossRef Google Scholar
5. Allay, JA, Dumenco, LL, Koc, ON, Liu, L, Gerson, SL. Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells. Blood. 1995. 85:3342-3351.
   View this article via: PubMed Google Scholar
6. Allay, JA, Davis, BM, Gerson, SL. Human alkyltransferase-transduced murine myeloid progenitors are enriched in vivo by BCNU treatment of transplanted mice. Exp. Hematology. 1997. 25:1069-1076.
7. Gerson, SL. Clinical relevance of MGMT in the treatment of cancer. J. Clin. Oncol. 2002. 20:2388-2399.
   View this article via: PubMed CrossRef Google Scholar
8. Persons, DA, et al. Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells. Blood. 2003. 102:506-513.
   View this article via: PubMed CrossRef Google Scholar
9. Neff, T, et al. Methylguanine methyltransferase–mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model. J. Clin. Invest. 2003. 112:1581-1588. doi:10.1172/JCI200318782.
   View this article via: JCI PubMed Google Scholar
10. Zielske, SP, et al. In vivo selection of MGMT(P140K) lentivirus–transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning. J. Clin. Invest. 2003. 112:1561-1570. doi:10.1172/JCI200317922.
    View this article via: JCI PubMed Google Scholar
11. Kaiser, J. Gene therapy: seeking the cause of induced leukemias in X-SCID trial. Science. 2003. 299:495.
    View this article via: PubMed CrossRef Google Scholar
12. Friedmann, T. Gene therapy’s new era: a balance of unequivocal benefit and unequivocal harm. Mol. Ther. 2003. 8:5-7.
    View this article via: PubMed CrossRef Google Scholar
13. Kohn, DB, 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.
    View this article via: PubMed CrossRef Google Scholar