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In certain applications of gene therapy, regulated (rather than constitutive) gene expression will be crucial for proper function or optimal effect of the delivered gene. Therefore, an important goal of gene therapy is to be able to deliver genes so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Additionally, regulating transgene expression will be an important component of efforts for reducing risks of adverse advents associated with gene therapy with integrating vectors.Although tissue-specific promoters confer selectivity, in a vector- based system, their activity may be too weak to mediate sufficient levels of gene expression to achieve therapeutic efficacy in vivo. Additionally, amplification of a specific, but weak, signal would be beneficial to some applications, such as imaging. Iyer et al. (PNAS, 2001) described a two-step transcriptional activator (TSTA) system to amplify gene expression while preserving tissue specificity using adenoviral vectors. In this system, the tissue-specific promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA binding domain fused to the activation domain of the HSV-1 VP16 activator), which in turn activates a GAL4-responsive reporter.We have developed lentivirus-based vectors using this 2-tiered system with the constitutive but weak PGK housekeeping gene promoter and the human insulin promoter and have tested them in cell lines and primary cells. Compared to a vector carrying only the PGK promoter to drive eGFP expression, the PGK amplifiable- vector amplified eGFP expression from 2–11 fold, depending on the cell type. Vectors carrying the insulin promoter did not express in non-β cell lines (HT29, HEK293), but expressed in Min6 cells, a murine insulinoma cell line, indicating that the insulin promoter was capable of conferring cell specificity. Additionally, the insulin amplifiable-vector was able to amplify expression 4.6 times in these cells. In primary murine islets, no gene expression was detected from the vector carrying only the insulin promoter, but low eGFP expression was detected from the insulin-promoter amplifiable vector. Thus, we have demonstrated that it is possible to build lentivirus vectors carrying an amplifiable gene expression system that retains tissue specificity. These vectors will be useful in gene expression studies that require a detectable signal and tissue specificity.Molecular Therapy (2006) 13, S140–S140; doi: 10.1016/j.ymthe.2006.08.428 [ABSTRACT FROM AUTHOR]

An abstract of the article "In Vitro and In Vivo Gene Expression of Lentiviral Vectors in CD4+ T Cells," by Kit L. Shaw, Karen Pepper, Denise Petersen, Vesa Kaartinen and Donald B. Kohn is presented.

• Low-dose busulfan can open the bone marrow niche in the setting of gene therapy. • Body surface area- and weight-based dosing resulted in variable busulfan plasma exposure. • Therapeutic drug monitoring-based dosing resulted in consistent plasma exposure. • Busulfan clearance increased as subject age increased from birth to 18 months. The pharmacokinetics of low-dose busulfan (BU) were investigated as a nonmyeloablative conditioning regimen for autologous gene therapy (GT) in pediatric subjects with adenosine deaminase-deficient severe combined immunodeficiency disease (ADA SCID). In 3 successive clinical trials, which included either γ-retroviral (γ-RV) or lentiviral (LV) vectors, subjects were conditioned with BU using different dosing nomograms. The first cohort received BU doses based on body surface area (BSA), the second cohort received doses based on actual body weight (ABW), and in the third cohort, therapeutic drug monitoring (TDM) was used to target a specific area under the concentration-time curve (AUC). Neither BSA-based nor ABW-based dosing achieved a consistent cumulative BU AUC; in contrast, TDM-based dosing led to more consistent AUC. BU clearance increased as subject age increased from birth to 18 months. However, weight and age alone were insufficient to accurately predict the dose that would consistently achieve a target AUC. Furthermore, various clinical, laboratory, and genetic factors (eg, genotypes for glutathione-S-transferase isozymes known to participate in BU metabolism) were analyzed, but no single finding predicted subjects with rapid versus slow clearance. Analysis of BU AUC and the postengraftment vector copy number (VCN) in granulocytes, a surrogate marker of the level of engrafted gene-modified hematopoietic stem and progenitor cells (HSPCs), demonstrated gene marking at levels sufficient for therapeutic benefit in the subjects who had achieved the target BU AUC. Although many factors determine the ultimate engraftment following GT, this work demonstrates that the BU AUC correlated with the eventual level of engrafted gene-modified HSPCs within a vector group (γ-RV versus LV), with significantly higher levels of granulocyte VCN in the recipients of LV-modified grafts compared to recipients of γ-RV-transduced grafts. Taken together, these findings provide insight into low-dose BU pharmacokinetics in the unique setting of autologous GT for ADA SCID, and these dosing principles may be applied to future GT trials using low-dose BU to open the bone marrow niche. [ABSTRACT FROM AUTHOR]