High incidence of leukemia in large animals after stem cell gene therapy with a HOXB4-expressing retroviral vector
Xiao-Bing Zhang, Brian C. Beard, Grant D. Trobridge, Brent L. Wood, George E. Sale, Reeteka Sud, R. Keith Humphries, Hans-Peter Kiem
Xiao-Bing Zhang, Brian C. Beard, Grant D. Trobridge, Brent L. Wood, George E. Sale, Reeteka Sud, R. Keith Humphries, Hans-Peter Kiem
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Research Article

High incidence of leukemia in large animals after stem cell gene therapy with a HOXB4-expressing retroviral vector

Abstract

Retroviral vector–mediated HSC gene therapy has been used to treat individuals with a number of life-threatening diseases. However, some patients with SCID-X1 developed retroviral vector–mediated leukemia after treatment. The selective growth advantage of gene-modified cells in patients with SCID-X1 suggests that the transgene may have played a role in leukemogenesis. Here we report that 2 of 2 dogs and 1 of 2 macaques developed myeloid leukemia approximately 2 years after being transplanted with cells that overexpressed homeobox B4 (HOXB4) and cells transduced with a control gammaretroviral vector that did not express HOXB4. The leukemic cells had dysregulated expression of oncogenes, a block in myeloid differentiation, and overexpression of HOXB4. HOXB4 knockdown restored differentiation in leukemic cells, suggesting involvement of HOXB4. In contrast, leukemia did not arise from the cells carrying the control gammaretroviral vector. In addition, leukemia did not arise in 5 animals with high-level marking and polyclonal long-term repopulation following transplantation with cells transduced with an identical gammaretrovirus vector backbone expressing methylguanine methyltransferase. These findings, combined with the absence of leukemia in many other large animals transplanted with cells transduced with gammaretroviral vectors expressing genes other than HOXB4, show that HOXB4 overexpression poses a significant risk of leukemogenesis. Our data thus suggest the continued need for caution in genetic manipulation of repopulating cells, particularly when the transgene might impart an intrinsic growth advantage.

Authors

Xiao-Bing Zhang, Brian C. Beard, Grant D. Trobridge, Brent L. Wood, George E. Sale, Reeteka Sud, R. Keith Humphries, Hans-Peter Kiem

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Figure 2

Retroviral integration induced mutagenesis is associated with leukemogenesis.

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Retroviral integration induced mutagenesis is associated with leukemogen...
(A) Southern blot analyses of marrow samples from G374, G450, and K00339 demonstrate monoclonality. Marrow DNA was digested with BglII, which cuts the transgene once, releasing a unique band for each integrant. Digestion with SacI, which cuts the transgene twice, showed a 3.9-kb band for all the integrants. (B–D) Integration sites were determined by LAM-PCR, and the schematic representations of integration sites for G374 (B), G450 (C), and K00339 (D) are shown. Exons are represented by black boxes. MSCV indicates the integration site, and the arrow indicates the orientation. ATG denotes the translation start site. Note that exons and introns are not to scale. (E–G) Dysregulated expression of genes in close vicinity of the integration sites for G374 (E), G450 (F), and K00339 (G). SYBR Green real-time RT-PCR was performed to determine the expression levels of dog MYB (E), dog ZFP36L2 and thyroid adenoma associated (THADA) (F), and macaque PRDM16, SSBP2, and SOCS1 (G). Canine PRDM16 (E) and macaque MAGOH2 (G) expression was undetectable in samples from normal control animals. (G) Expression of NSMCE1 was undetectable in K00339 and control animals. Marrow mRNA samples from normal animals were used as controls. M, DNA ladder.