Denise Petersen

Lentivirus Vectors Incorporating the Immunoglobulin Heavy Chain Enhancer and Matrix Attachment Regions Provide Position-Independent Expression in B Lymphocytes

ABSTRACT In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (Eμ) with and without associated matrix attachment regions (EμMAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34+ progenitors in vitro transduced with Eμ- and EμMAR-containing lentivectors. Lastly, we evaluated the expression from the EμMAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the Eμ and EμMAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the EμMAR-containing vector and not other cells types or vectors. Proviral genomes with the EμMAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the EμMAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies.

Expression from Second-Generation Feline Immunodeficiency Virus Vectors Is Impaired in Human Hematopoietic Cells

Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.

IL-3 Increases Production of B Lymphoid Progenitors from Human CD34+CD38− Cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38− cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38− cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34−CD19+ B cells. B cell production was increased whether CD34+CD38− cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Rα expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Rα protein expression was largely restricted to myeloid progenitors. CD34+CD38− cells had low to undetectable levels of IL-3Rα by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Rα protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38− cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.

37. A Novel Form of Enzyme Replacement Therapy for ADA-Deficiency: In Vivo Transduction by Neonatal Injection of Lentivirus Expressing ADA

Genetic deficiency of adenosine deaminase (ADA) is responsible for approximately 20% of human Severe Combined Immune Deficiency (SCID). A murine model of ADA-deficient SCID was produced by Blackburn and Kellems (U. of Texas) by ADA gene knock-out. These mice die from non-infectious pulmonary insufficiency between days 19|[ndash]|20 post-natal, unless maintained on enzyme replacement therapy (ERT) with PEG-ADA. We previously reported that neonatal mice injected with a lentiviral vector expressing hu ADA have increased survival (ASGT 7th annual meeting, June 2004). We have continued our efforts to characterize the effects of systemic ADA enzyme expression in this model of ADA-deficient SCID, using a SIN HIV-1-based lentivirus, SMPU-R-MND-ADA, pseudotyped with VSV-G and given via the temporal vein on day 2 of life. Viral supernatant was produced at high titer (1|[times]| 10^10 TU/ml). When mice received a dosage of 1.0 |[times]| 10^7 TU/kg surivial was not prolonged, but when mice received a dosage of 1.6 |[times]| 10^8 TU/kg, survival was significantly prolonged. An initial cohort of mice received PEG-ADA ERT until day 45 after neonatal injection of vector (n=4). A second cohort of mice was injected with the lentiviral vector as neonates and never given ERT (n=5). Both cohorts had survival greater than 180 days without further treatment. Mice were either sacrificed at 2 or 6 months of age. Immunophenotype analysis revealed the absence of a SCID phenotype with normal numbers of T and B lymphocytes and adequate lymphocyte proliferation in response to stimulation with ConA. Vector proviral copy number was determined by Q-PCR in peripheral blood, lung, liver, thymus, spleen and bone marrow. Proviral marking was highest in the liver (1.2+/|[minus]|0.18 copies/cell) and lung (0.12+/|[minus]|0.013 copies/cell), as has been reported for multiple bio-distribution studies of lentivirus (and AAV) given by tail vein to mice. Proviral marking in the thymus (0.0024+/|[minus]|0.0001 copies per cell), spleen (0.0037+/|[minus]|0.0001 copies/cell) and bone marrow (0.0023+/|[minus]|0.0001 copies/cell) were at least 10|[ndash]|100 fold lower than the level of marking observed in the liver and lung tissues analyzed. T-cells (CD4/8) sorted from thymus or B-cells (CD19) sorted from spleen had proviral marking similar to the marking observed in the organs from which the cells were isolated. Peripheral blood marking was very low at 2 mo (0.00007+/|[minus]|.0006 copies/cell) and undetectable by 6 mo. To determine if there was transduction of hematopoietic stem cells in surviving mice, bone marrow was transplanted into lethally irradiated, secondary recipients (n=6). There was no marking in the peripheral blood, bone marrow or colony forming units (CFUs) from bone marrow. Thus, the mice had restoration of immune function, despite very low levels of gene correction of T lymphocytes and hematopoietic cells. The findings suggest that ADA enzyme made in highly transduced organs (e.g. liver, lung) may be rescuing ADA-deficient T lymphocytes in trans; this approach of intravenous lentiviral vector delivery of the ADA gene may provide a novel form of in vivo enzyme replacement therapy.

408. Developing Lentiviral Vectors for Specific Gene Expression in CD4+ T Cells

In certain applications of gene therapy, regulated (rather than constitutive) gene expression will be crucial for proper function or optimal effect of the delivered gene. Therefore, an important goal of gene therapy is to be able to deliver genes so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Additionally, regulating transgene expression will be an important component of efforts for reducing risks of adverse advents associated with gene therapy with integrating vectors.