W. Jay Ramsey

T Lymphocyte-Directed Gene Therapy for ADA− SCID: Initial Trial Results After 4 Years

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA− SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

The Release of Inflammatory Cytokines from Human Peripheral Blood Mononuclear Cells In Vitro Following Exposure to Adenovirus Variants and Capsid

Preclinical and clinical studies with adenoviral vectors have clearly illustrated the potential advantages of this gene transfer system. However, many studies have also demonstrated potent immune responses directed at both vector and transduced cells. We examined in vitro responses of human peripheral blood mononuclear cells (PBMC) to virus exposure as a model for this host response. PBMC were isolated from normal donors and incubated with wild-type adenovirus (Ad5), Ad5 variants deleted for segments of E1 and/or E3, and empty viral capsids. Proinflammatory cytokine release was monitored for 96 hr. Induction of TNF-alpha by intact virions was low although stimulation by empty capsid gave a significant and sustained response. Induction of IL-6, GM-CSF, and a panel alpha- and beta-chemokines by intact virions was prominent, often approaching results obtained with 2.5 microg/ml of lipopolysaccharide (LPS). Responses were generally independent of virion genetic composition and were only partially blunted when UV-inactivated virus was used. Dose-response data showed 100-fold increases in virion concentration produced a maximum 3-fold increase in cytokine release, suggesting saturation. Surprisingly, prominent stimulation occurred after addition of empty capsid, which typically provoked responses equivalent to those seen with LPS stimulation. We present arguments that cellular signal transduction mechanisms activated by binding of virions/capsids stimulate transcription of proinflammatory cytokine genes.

Modified Human Immunodeficiency Virus-Based Lentiviral Vectors Display Decreased Sensitivity to Trans-Dominant Rev

As a first step toward the development of HIV-based conditionally replicating defective interfering particles expressing trans-dominant Rev (TdRev), we studied whether mutation of the splicing signals and replacement of the RRE by the SRV-1 CTE would render these vectors less sensitive to TdRev. Vectors with mutations in the splicing signals (SD-/RRE+) yielded high titers (5 X 10(6) CFU/ml) and showed higher levels of cytoplasmic unspliced mRNA than the corresponding SD+/RRE+ vectors either in the absence of Rev, in the presence of TdRev, or in the presence of both TdRev and Rev. Proviral copies of SD-/RRE+ vectors were rescued more efficiently than SD+/RRE+ vectors when TdRev was expressed. Vectors with the SRV-1 CTE (SD+/CTE+ and SD-/CTE+) expressed high levels of cytoplasmic unspliced mRNA in the absence of Rev expression. Titers obtained with the SD-/CTE+ vectors (10(6) CFU/ml) were higher than the titers obtained with SD+/CTE+ vectors. We also tested the effect of other structural modifications such as the orientation of the expression cassette and the presence of the central polypurine tract (cPPT/CTS). We show that an expression cassette cloned in the reverse orientation with respect to the LTRs or elimination of the cPPT/CTS element severely affected vector titers. We also demonstrated that these vectors can be efficiently mobilized from their proviral state by HIV trans-complementing functions, and transduced into secondary target cells without suffering any genomic rearrangement.

In situ generation of pseudotyped retroviral progeny by adenovirus‐mediated transduction of tumor cells enhances the killing effect of HSV‐tk suicide gene therapy in vitro and in vivo

Hybrid adeno‐retroviral vector systems utilize the high efficiency of adenovirus transduction to direct the in situ production of retroviral progeny. In this study, we show that a single‐step transduction of glioma cells with trans‐complementing hybrid adeno‐retroviral vectors effectively turns these cells into retrovirus vector‐producing cells, which in turn facilitates the transduction of adjacent cells.

Chapter 29 – HyperAcute Vaccines: A Novel Cancer Immunotherapy

The hyperacute rejection of a xenotransplant is characterized by a complement-antibody mediated immune response dependent on αGal epitopes. Animal studies confirm that αGal epitopes expressed on allogeneic tumor vaccines elicit a potent T-cell-dependent antitumor immunity. Based on these immunologic reactions, we hypothesized that the hyperacute rejection mechanism could be exploited to alter antigen processing resulting in a novel therapeutic approach to treat human malignancies. Clinical trials data confirm that an immediate hypersensitivity response directed toward a vaccine composed of genetically modified allogeneic tumor cells expressing the xenoantigen αGal (HyperAcute vaccines) constitutes a polyvalent tumor cell vaccine with signs of clinical efficacy, concomitant to eliciting both a humoral IgG response as well as T-cell-mediated antitumor immunity. This conceptually innovative immunotherapy degrades tumoral immune escape and portends a promising genetic engineering tactic for the cost-effective development of a generally applicable human cancer vaccine principle with minimal toxicity. Encouraging results support additional clinical immunotherapy studies using HyperAcute vaccines.