Barbara C. Engel

Expression from Second-Generation Feline Immunodeficiency Virus Vectors Is Impaired in Human Hematopoietic Cells

Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.

Gene Therapy for Pediatric AIDS

Abstract: Gene therapy is an experimental treatment modality under investigation for applications to HIV‐1 infection. We have developed retroviral vectors carrying anti‐HIV‐1 genes, demonstrated that these genes cause significant suppression of HIV‐1 replication in cultures of primary hematopoietic cells, and performed a clinical trial in pediatric AIDS patients. Four HIV‐1‐infected children and adolescents underwent bone marrow harvest from which CD34+ cells were isolated and transduced by a retroviral vector carrying an RRE decoy gene. The cells were reinfused into the subjects, without complications, showing that gene transfer in pediatric AIDS patients is safe and feasible. However, gene‐containing leukocytes in the peripheral blood were seen only at a low level and only in the first months following cell infusion. To attain some degree of efficacy, it will be necessary to achieve a higher level of gene transfer and to obtain sustained gene expression. We are currently developing new gene transfer methods and vectors designed to improve the results in future trials. If it becomes possible to reach the ideal goal of producing high percentages of T lymphocytes and monocytic cells that are resistant to HIV‐1 infection, gene therapy could serve as a complement to antiretroviral drug therapy and help to sustain immunologic function.

Clinical efficacy of gene-modified stem cells in adenosine deaminase–deficient immunodeficiency

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) &ggr;-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.

Gene Therapy for Inborn and Acquired Immune Deficiency Disorders

Gene therapy has been under development as a way to correct inborn errors for over 20 years. Immune deficiencies are favorable candidates for gene therapy because of the potential selective advantage of genetically corrected cells in these conditions. Gene therapy for immune deficiencies has been the only application to show incontrovertible benefit in clinical trials to date. Despite the success in treating the underlying disease, there have been two cases of insertional oncogenesis reported in one of these early phase trials. Gene therapy approaches and clinical trials for several inborn as well as acquired immune deficiencies will be reviewed.

We Have Met the Enemy and He Is Us

Numerous authors have demonstrated that when multiple institutional review boards (IRBs) review the same protocol, the results are quite variable (Dilts et al. 2009; Dziak et al. 2005; Green et al. 2006; Greene et al. 2006; Helfand et al. 2009; Kimberly et al. 2006; Mansbach et al. 2007; McWilliams et al. 2003; Silverman et al. 2001; Stair et al. 2001; Stark et al. 2010). However, the federal regulations governing the conduct of human subjects research are gray, by either design or accident, and it is therefore to be expected that reasonable people with differing interpretations will reach disparate conclusions about whether or not a given study meets the criteria for approval (Sayers 2007). Klitzman (2011) provides anecdotal evidence that lends credence to another aspect of IRB variability long known to clinical investigators, namely, that there are differences between the IRBs within institutions. The Klitzman paper is fundamentally based on anecdotes. There is no reason why clinical research into IRB decision making shouldn’t be held to the same standards as those for other types of clinical research. As Halpern (2005) advocated recently, there is a need for evidence-based bioethics to inform decisions. From an epidemiologist’s perspective, large randomized clinical trials rank highest in strength of evidence, followed by smaller clinical trials, cohort studies, case-control studies, and then anecdotal evidence at the bottom. Depending on one’s perspective, meta-analyses combining the results of several trials would be at or near the top of the hierarchy of evidence. Even when a study has a high degree of internal validity, the results from a study may not be generalizable to one’s own situation, patient population, and institution. Given the low position within the hierarchy of evidence occupied by this current report, the results and conclusions reported by Klitzman should be viewed with caution and skepticism. If cause and effect are beyond the realm of the anecdotal study design, then why bother with this type of research? Not all research needs to involve confirmatory designs with formal hypothesis testing. Other exploratory designs are valuable when aimed at generating hypotheses and directions for future research. The power of this paper, then, is not in the amalgamation of the results into a