Development of Improved Adenosine Deaminase Retroviral Vectors

A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a “simplified” vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA⁻) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA⁻ severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.Retroviral vectors have been the most common gene transfer vehicles in clinical gene therapy trials (15). These vectors can integrate into the host genome to provide permanent transgene expression in the targeted cells (20). The first generation of retroviral vectors have been useful in demonstrating the feasibility of gene therapy approaches, but vectors capable of higher levels of gene transfer and transgene expression would be beneficial. For example, gene transfer levels achieved by first-generation retroviral vectors in large mammals (28) and in human gene therapy trials (7, 13) have been disappointing. There are at least two avenues for improving retroviral vectors. First, molecular changes can be made in the retroviral vector sequence. Second, different packaging cell lines could be tested to modify the host range, increase transduction in a given cell type, and/or render the virions resistant to inactivation by human complement.A clinically useful model for improving retroviral vector design is the vector LASN packaged in the amphotropic line PA317. PA317/LASN was the first therapeutic vector used in a gene therapy clinical trial (1). This vector has yielded gene transfer levels of generally less than 10% in peripheral blood T cells of adenosine deaminase-deficient (ADA⁻) severe combined immunodeficiency (SCID) patients. Two possibilities to improve this vector include eliminating the dominant selectable marker gene and changing the long terminal repeat (LTR) promoter to optimize expression. LASN, like many of the retroviral vectors used in clinical trials to date, contains two genes: the therapeutic gene (the ADA gene) and a dominant selectable marker gene (the bacterial neomycin phosphotransferase II gene; neo). Dominant selectable marker genes have historically been included to facilitate the generation, isolation, and titration of retroviral producer cell clones and to permit the evaluation and selection of successfully targeted cells. neo is the most commonly used selectable marker gene, although other genes have been used, including a mutant dihydrofolate reductase gene (dhfr) (19), the multidrug resistance gene (mdr) (10), and genes for cell surface markers such as cd24 (24) and the human nerve growth factor receptor (2). Vectors carrying dominant selectable marker genes, particularly those of nonhuman origin, have two theoretical disadvantages. First, careful analysis of some patients has revealed an immune response directed against the dominant selectable marker protein expressed from the retroviral integrant (20a, 25). Second, the more complex retroviral genomes required to express two separate genes may result in lower titers or suboptimal expression of the therapeutic gene product due to promoter interference (8, 29). On the other hand, cloning and determining the titers of useful retroviral vectors without selectable markers have been laborious. Using a recently developed rapid-screening procedure, we have been able to identify a number of “simple” ADA retroviral vectors which lack dominant selectable markers (23).Different packaging cell lines may also improve gene transfer of retroviral vectors into specific target cells. Retroviral vectors are limited by the host range specified by the envelope protein on the surface of the retrovirus. Most gene therapy trials have used retroviruses with a murine amphotropic (4070A) host range. However, packaging cell lines with the gibbon ape leukemia virus (GALV) envelope (PG13 cells) (18) and the cat endogenous virus RD114 envelope (FLYRD18 cells) (5) have become available; these may improve transduction frequencies into various target cell populations. For example, there is evidence that GALV-pseudotyped retroviral vectors may facilitate gene transfer into human peripheral blood T cells with greater efficiency than vectors with an amphotropic envelope (3). Packaging cell lines derived from murine cells have the additional disadvantage that they produce retroviruses which are inactivated by complement in human sera. Packaging cell lines of human origin (FLYA13 and FLYRD18) (5) produce vectors which are complement resistant. Testing both new simple retroviral vector designs and new packaging cells may therefore improve retrovirus-mediated gene transfer.We report the construction and characterization of three simplified ADA vectors by using either the Moloney murine leukemia virus (MLV) LTR, the myeloproliferative sarcoma virus (MPSV) LTR, or the SL3-3 LTR. We tested these vectors to determine which LTR provided the highest level of ADA expression in our target cells of interest: human ADA⁻ lymphohematopoietic cells. The ADA retroviral vector with the highest level of transduction/expression was then evaluated in different packaging cell lines including PG13, FLYA13, and FLYRD18.