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Unique risk factors for insertional mutagenesis in a mouse model of XSCID gene therapy.

ABSTRACT: Although gene therapy can cure patients with severe combined immunodeficiency (SCID) syndromes, the clinical occurrence of T cell malignancies due to insertional mutagenesis has raised concerns about the safety of gene therapy. Several key questions have remained unanswered: (i) are there unique risk factors for X-linked SCID (XSCID) gene therapy that increase the risk of insertional mutagenesis; (ii) what other genetic lesions may contribute to transformation; and (iii) what systems can be used to test different vectors for their relative safety? To address these questions, we have developed an XSCID mouse model in which both the Arf tumor-suppressor gene and the gammac gene were ablated. Gene therapy in this animal model recapitulates the high incidence of integration-dependent, T cell tumors that was seen in the clinical trial. Ligation-mediated PCR analysis showed integration sites near or within established protooncogenes (Chd9, Slamf6, Tde1, Camk2b, and Ly6e), demonstrating that T cell transformation was associated with targeting of oncogene loci; however, no integrations within the Lmo2 locus were identified. The X-SCID background in transplanted cells was required for high rate transformation and was associated with expansion of primitive hematopoietic cells that may serve tumor precursors. This model should be useful for testing safety-modified vectors and for further exploring the risk factors leading to insertional mutagenesis in gene therapy trials.

SUBMITTER: Shou Y

PROVIDER: S-EPMC1518804 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

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Unique risk factors for insertional mutagenesis in a mouse model of XSCID gene therapy.

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