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author:("hislop, HE")
1.  The immunogenicity of virus-derived 2A sequences in immunocompetent individuals 
Gene therapy  2013;20(9):958-962.
Genetic engineering of T cells for adoptive immunotherapy in cancer patients has shown significant promise. To ensure optimal antitumor activity and safety, the simultaneous expression of multiple genes is frequently required, and short viral-derived 2A sequences are increasingly preferred for this purpose. Concerns exist, however, that these virus-derived sequences may induce unwanted immune responses, and thus diminish persistence of the gene-modified cells after adoptive transfer. Whereas such responses were absent in immunocompromised recipients, potential immunogenicity in immunocompetent individuals remains a concern. We now address whether ex vivo T cell responses can be elicited against the most widely used 2A sequences (2A-Thosea asigna virus (TAV) or 2A-equine rhinitis virus (ERAV), specifically) in immunocompetent individuals. We used a potent ex vivo culture system previously validated to induce T cell responses even against weakly immunogenic antigens. Of the sixteen donors tested, only five released very low levels of interferon-γ in response to 2A-TAV peptide mixtures (single peptide specificity in three donors, adjacent self-antigen peptide specificity in one donor and nonspecific reactivity in one donor). None of them produced cytotoxic activity or responded to 2A-ERAV. These results suggest that exposure to viral-derived 2A sequences is unlikely to produce unwanted T cell responses in immunocompetent individuals and further supports their continued use for studies of human gene therapy.
doi:10.1038/gt.2013.25
PMCID: PMC3766470  PMID: 23698740
2A sequences; polycistronic vectors; T cell gene transfer; immunogenicity
2.  OUTCOME OF HEMATOPOIETIC STEM CELL TRANSPLANTATION AS SALVAGE THERAPY FOR HODGKIN’S LYMPHOMA IN ADOLESENCE AND YOUNG ADULTS AT A SINGLE INSTITUTION 
Leukemia & lymphoma  2010;51(4):664-670.
For patients with relapsed Hodgkin Lymphoma, high dose chemotherapy with stem cell rescue may improve survival over chemotherapy alone. We assessed outcomes of HDCT-SCT in 37 consecutive adolescent and young adult patients with relapsed HL whose malignancy was categorized based on sensitivity to chemotherapy. We determined whether current outcomes supported the use of HDCT-SCT in all of our patients or just those patients with lower risk characteristics such as chemosensitivity. With a median follow up of 6.5 years, the 2 year overall survival was 89% (95% CI: 62%–97%) for the chemo-sensitive patients (n=21). Whereas for patients with resistant disease (n=16) OS was 53% (95%CI: 25%–74%). Both autologous and allogeneic transplants were well tolerated, with 100 day treatment related mortality under 10%. Our data show encouraging outcomes for patients with chemosensitive relapsed HL who receives HSCT and supports the value of the procedure even when the disease is chemoresistant.
doi:10.3109/10428190903580410
PMCID: PMC2932472  PMID: 20367182
Hodgkin’s Lymphoma; Stem Cell Transplant; Salvage Therapy
3.  Flanking-sequence exponential anchored–polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant–host–junction sequences 
Cytotherapy  2008;10(5):526-539.
Background
Retroviral vectors are regularly used to transduce stem cells and their derivatives for experimental and therapeutic purposes. Because these vectors integrate semi-randomly into the cellular genome, analysis of integranated retroviral DNA/host cell DNA junctions (IHJ) facilitates clonality studies of engrafted cells, allowing their differentiation, survival and fate to be tracked. In the case of any adverse events, IHJ analysis can allow the identification of potentially oncogenic integration sites. At present, most measures to assess IHJ are complex, insensitive and may be subject to IHJ selection bias inherent to the technology used.
Methods
We have developed and validated a simple but effective technique for generating libraries of IHJ, which we term flanking-sequence exponential anchored–polymerase chain reaction (FLEA-PCR). Flanking-sequence random anchoring is used as an alternative to restriction enzyme digestion and cassette ligation to allow consistent detection of IHJ and decrease bias.
Results
Individual clones from plasmid libraries can be sequenced and assembled using custom-written software, and FLEA-PCR smears can be analyzed by capillary electrophoresis after digestion with restriction enzymes.
Discussion
This approach can readily analyze complex mixtures of IHJ, allowing localization of these sequences to their genomic sites. This approach should simplify analysis of retroviral integration.
doi:10.1080/14653240802192636
PMCID: PMC2749733  PMID: 18821360
gene therapy; integration-site analysis; LAM-PCR; polymerase chain reaction; retrovirus

Results 1-3 (3)