Sean P. Forestell

Improved detection of replication-competent retrovirus

Recombinant retroviral vectors are the predominant delivery system in human gene therapy protocols. Since contaminating replication-competent retrovirus (RCR) can arise during the production of retroviral vector supernatants, sensitive assays for the screening of supernatants are necessary. In this study, we present a marker rescue assay based upon a Mus dunni cell line stably transduced with a lacZ gene. We show that detection of RCR in vector supernatants by the M. dunni lacZ marker rescue assay or PG-4 S+ L- focus-forming assay is equally sensitive. By inoculating test supernatants under centrifugation (which we term spinoculation), we increased the sensitivity of detection of RCR 10 to 100-fold with the PG-4 S+ L- and lacZ marker rescue assays. While the spinoculation protocol had no adverse effects on cells, spinoculation of high titer vector supernatants onto PG-4 cells resulted in some cytotoxicity, making identification of RCR positive cultures difficult. However, spinoculation of vector supernatants onto M. dunni lacZ cells resulted in no cytotoxicity, and also partially overcame inhibition of detection of low levels of RCR due to the presence of high titer replication-incompetent vector.

Addition of the Human Interferon β Scaffold Attachment Region to Retroviral Vector Backbones Increases the Level of in Vivo Transgene Expression among Progeny of Engrafted Human Hematopoietic Stem Cells

Absence of durable high-level expression of transgenes from Moloney murine leukemia (Mo-MuLV) retroviral vectors has been a hurdle in bringing effective gene therapy to the clinic. In this study we have analyzed transgene expression among the progeny of mobilized hematopoietic stem cells (HSCs), comparing Mo-MuLV and mouse stem cell virus (MSCV) vectors, with or without addition of a scaffold attachment region (SAR) from the human interferon beta gene. Retroviral (RV) vector supernatant quality was assessed by comparing NGFR transgene expression by HEL cells, and transgene delivery and expression by CD34(+) cells 72 hr after transduction, using real-time PCR and FACS analysis. This is the first description of the effect of SAR within both Mo-MuLV and MSCV vector backbones on long-term RV transgene expression among in vivo HSC progeny in HSC repopulation assays (SCID-hu bone and NOD/SCID). After transduction of mobilized CD34(+) cells with MSCV-SAR vector, transgene expression was observed among a mean of 10% of donor HSC progeny in the SCID-hu bone (range, 0.6-43%). The predominant effect of SAR was to increase the mean fluorescence intensity (MFI) of transgene expression among HSC progeny in both in vivo bone repopulation models (three- to fourfold), and after long-term stromal cultures (twofold).