Abstract 3770

Gene therapy allows correction of a number of monogenic diseases affecting the hematopoietic system. This approach can be conducted by two different strategies which are either semi-random integration of the therapeutical gene using self-inactivating (SIN) retroviral vectors or homologous recombination (HR). The feasibility and efficacy of the first approach has been demonstrated in different forms of severe combined immunodeficiency (SCID) (SCID-X1 and Adenosine Deaminase) but the occurrence of a number of severe adverse events leads to further consider the HR approach.

Homologous recombination (HR) is a highly specific DNA-repair mechanism, allowing to selectively replace a DNA sequence by another of interest. DNA double strand breaks (DSB) can enhance the frequency of HR. Specific DSB can be induced by meganucleases. These meganucleases are endonucleases with long DNA-recognition sites (12-30bp). They thus principally allow to target gene specific positions in the genome. In view of that, engineering gene specific meganucleases represents a tempting novel approach for gene therapy and targeting.

To further develop this approach, we chose the Artemis deficiency model. This variant of SCID combines the phenotype of radio-sensitivity and lymphocyte developmental arrest at the double negative CD44-CD25+ T-progenitor and the B220+CD43+ B-progenitor stage. In this model, successful gene targeting can thus be assessed by reversion of the pathological phenotype in a number of assays used to study radio-sensitivity, B- or T-cell development in vitro.

The Artemis deficient mouse model was created by substitution of exon 12 of the murine Artemis gene by a restriction site for the I-Sce 1. The I-Sce 1 specific meganuclease would thus allow site specific induction of DSB at the Artemis gene. To deliver the I-Sce 1 meganuclease and to provide the Artemis DNA matrix we designed two SIN-integrase-defective lentiviral vectors (SIN-IDLV): IDLV-CMVI-Sce 1 and IDLV-Art. SIN-IDLV were used to avoid non-specific integration and transgene over-expression.

Transduction of bone marrow derived Sca1+ hematopoietic stem cells (HSC) from Art-/- mice with both vectors, IDLV-CMVI-Sce 1 and IDLV-Art, resulted in a transduction efficiency of about 30%. Reversion of the Artemis deficient phenotype indicating successful gene targeting of the Artemis exon 12 was observed in 2 out 10 experiments by means of appearance of CD4/CD8 double positive T cells after co-culture on the OP9-DL1 system. Site specific insertion of Artemis exon 12 by HR was confirmed by nested-PCR and genomic DNA sequence analysis.

These preliminary results demonstrate that meganucleases and repair matrixes can induce site specific DNA-repair by HR in HSC. Even though the efficiency of genomic reparation needs to be further improved and its potential genotoxicity remains to be evaluated, this new approach of gene therapies is very promising for the future.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.