High-throughput integration site profiling has become a feasible tool to assess vector biosafety and to monitor the cell fate of the gene-corrected cell population in clinical gene therapy studies. Here we report a step-by-step protocol for universal genome-wide and comprehensive integrome analysis that can be performed on >10(2)-10(3) samples of interest in parallel. This assay is composed of fast and cost-efficient non-restrictive linear amplification-mediated PCR; optimized sample preparation for pyrosequencing; and automated bioinformatic data mining, including sequence trimming, alignment to the cellular genome and further annotation. Moreover, the workflow of this large-scale assay can be adapted to any PCR-based method aiming to characterize unknown flanking DNA adjacent to a known DNA region. Thus, in combination with next-generation sequencing technologies, large-scale integrome analysis of > 4 x 10(5)-1 x 10(6) integration site sequences can be accomplished within a single week.

Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the β-globin gene (β-LCR). We show that the β-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3-7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the β-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the β-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.