The ecdysone-inducible gene switch is a useful tool for modulating gene expression in mammalian cells and transgenic animals. We have identified inducers derived from plants as well as certain classes of insecticides that increase the versatility of this gene regulation system. Phytoecdysteroids share the favorable kinetics of steroids, but are inert in mammals. The gene regulation properties of one of these ecdysteroids have been examined in cell culture and in newly developed strains of ecdysone-system transgenic mice. Ponasterone A is a potent regulator of gene expression in cells and transgenic animals, enabling reporter genes to be turned on and off rapidly. A number of nonsteroidal insecticides have been identified that also activate the ecdysone system. Because the gene-controlling properties of the ecdysone switch are based on a heterodimer composed of a modified ecdysone receptor (VgEcR) and the retinoid X receptor (RXR), we have tested the effect of RXR ligands on the VgEcR͞RXR complex. Used alone, RXR ligands display no activity on the ecdysone switch. However, when used in combination with a VgEcR ligand, RXR ligands dramatically enhance the absolute levels of induction. This property of the heterodimer has allowed the development of superinducer combinations that increase the dynamic range of the system.

Lentiviral vectors have undergone several generations of design improvement to enhance their biosafety and expression characteristics, and have been approved for use in human clinical studies. Most preclinical studies with these vectors have employed easily assayed marker genes for the purpose of determining vector titers and transduction efficiencies. Naturally, the adaptation of these vector systems to clinical use will increasingly involve the transfer of genes whose products may not be easily measured, meaning that the determination of vector titer will be more complicated. One method for determining vector titer that can be universally employed on all human immunodeficiency virus type 1-based lentiviral vector supernatants involves the measurement of Gag (p24) protein concentration in vector supernatants by immunoassay. We have studied the effects that manipulation of several variables involved in vector design and production by transient transfection have on vector titer and infectivity. We have determined that manipulation of the amount of transfer vector, packaging, and envelope plasmids used to transfect the packaging cells does not alter vector infectivity, but does influence vector titer. We also found that modifications to the transfer vector construct, such as replacing the internal promoter or transgene, do not generally alter vector infectivity, whereas inclusion of the central polypurine tract in the transfer vector increases vector infectivity on HEK293 cells and human umbilical cord blood CD34 ؉ hematopoietic progenitor cells (HPCs). The infectivities of vector supernatants can also be increased by harvesting at early time points after the initiation of vector production, collection in serum-free medium, and concentration by ultracentrifugation. For the transduction of CD34 ؉ HPCs, we found that the simplest method of increasing vector infectivity is to pseudotype vector particles with the RD114 envelope instead of vesicular stomatitis virus G glycoprotein (VSV-G). OVERVIEW SUMMARYWe have analyzed several variables involved in the design and production of lentiviral vectors for the effect they have on vector titer and infectivity. Wide variability in the amounts of input transfer vector, packaging, and envelope plasmids, as well as changes to the transfer vector construct, are tolerated with no significant change in vector infectivity. We found that inclusion of the central polypurine tract, concentration by ultracentrifugation, and collection in serum-free medium increase the infectivity of vectors on HEK293 cells and human umbilical cord blood CD34 ؉ HPCs. In addition, use of the RD114 envelope instead of VSV-G dramatically increases the infectivity of lentiviral vectors when they are used to transduce CD34 ؉ HPCs, but the titers and infectivities of RD114-pseudotyped vectors as determined on HEK293 cells is misleading, because of poor performance of the RD114 envelope on these cells.

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34 ؉ CD38 ؊ hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.Retroviral vectors based upon the Moloney murine leukemia virus (MLV) have been used widely due to their high efficiency of stable gene transfer. Transcription from the MLV long terminal repeat (LTR) is severely repressed in mouse embryonal carcinoma (EC) cells (7,23,42,45) and mouse embryonal stem (ES) cells (13,21). Characteristics of MLV that have been shown to mediate poor expression in undifferentiated ES and EC cell lines include the inadequate function of the enhancer in the 5ЈLTR due to the lack of transcriptional activator binding sites (9,20,21,27,41,47), the negative control region (NCR) located in the U3 region of the LTR (1, 5, 10, 12, 47), and the repressor binding site (RBS) (2, 9).The RBS is an 18-bp DNA element located downstream of the 5ЈLTR which directly overlaps the MLV primer binding site (PBS) by 17 of its 18 bp (25). The MLV PBS functions by binding a proline tRNA molecule that primes reverse transcription. The existence of the overlapping RBS was first determined due to a spontaneously occurring single-base G-to-A mutation that occurred within the PBS sequence that allowed expression by MLV in F9 EC cells (2). This single base mutation was called the B2 mutation (2). Like the MLV PBS, the B2 PBS binds to a proline tRNA molecule, but with a single base-pair mismatch (4). Several other mutations that have been made within the MLV PBS have also been shown to relieve the r...