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Background: Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study.Methods: Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution.Results: With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant.Conclusion: These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile.Trial Registration: ClinicalTrials.gov NCT00794508.Funding: Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124. [ABSTRACT FROM AUTHOR]

Foamy viruses (FVs) are classified in the family Retroviridae, but recent data have shown that they are not conventional retroviruses. Notably, several characteristics of their particle replication strategies are more similar to those of hepatitis B virus (HBV) than those of typical retroviruses. Compared to conventional retroviruses, which require only Gag proteins for budding and release of virus-like particles (VLPs), both FV and HBV require Env proteins. In the case of HBV, Env (S protein) alone is sufficient to form subviral particles (SVPs). Because FVs also depend on Env for budding, we tested whether FV Env alone could produce SVPs. The Env proteins of FV and murine leukemia virus (MuLV) were both released into cell culture supernatants and migrated into isopycnic gradients; however, unlike MuLV Env, FV Env displayed characteristics of SVPs. FV Env particles were of greater density than those of MuLV (1.11 versus 1.07 g/ml, respectively), which strongly suggested that the released proteins of FV Env were particulate. When we examined FV SVPs by immunoelectron microscopy, we found particles that were consistent in morphology, size, and staining with gold beads, similar to FV VLPs and unlike the particle-like structures of MuLV Env, which were more consistent with vesicles produced from nonspecific membrane "blebbing." Taken together, our results demonstrated that FV Env alone is sufficient for particle budding. This finding is unique among retroviruses and further demonstrated the similarities between FV and HBV. [ABSTRACT FROM AUTHOR]

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]