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Genetic Vaccines and Therapy

, 7:13

First Online: 30 December 2009Received: 08 April 2009Accepted: 30 December 2009


BackgroundMurine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase ADA expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus MuLV based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region LCR, ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.

MethodsA MuLV-based retroviral vector with a self-inactivating SIN backbone, the phosphoglycerate kinase promoter PGK and the enhanced green fluorescent protein eGFP, as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region LCR, introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting FACS. Non T-cell and T-cell lines were transduced at a multiplicity of infection MOI of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity MFI detection.

ResultsVectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.

ConclusionThese studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

AbbreviationsADAadenosine deaminase

MuLVMoloney murine leukemia virus

LCRlocus control region

PGKphosphoglycerate kinase promoter

eGFPenhanced green fluorescent protein


FACSfluorescence-activated cell sorting

MOImultiplicity of infection

MFImean fluorescent intensity

SCIDsevere combined immunodeficiency

WTwild type

Pphosphoglycerate kinase

Genhanced green fluorescent protein reporter gene


LADA locus control region

Ahuman ADA promoter

iLinverted ADA locus control region

Hhuman growth hormone polyadenylation signal


MCSmulticloning site.

Electronic supplementary materialThe online version of this article doi:10.1186-1479-0556-7-13 contains supplementary material, which is available to authorized users.

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Autor: Alice T Trinh - Bret G Ball - Erin Weber - Timothy K Gallaher - Zoya Gluzman-Poltorak - French Anderson - Lena A Basile


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