Optimized human CYP4B1 in combination with the alkylator prodrug 4-ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies.

Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4-ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4-ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene.

The role of intron sequences in high level expression from CD45 cDNA constructs.

Consistent expression from CD45 cDNA constructs has proven difficult to achieve. Through the use of new CD45 cDNA constructs and reporter genes, the role 5', 3', and intron sequences play in CD45 expression was determined. The CD45 polyadenylation signal sequence was fully functional in a beta-galactosidase reporter construct. Furthermore, the CD45 3'-untranslated region and downstream sequences were shown to contain no negative regulatory elements. Several new CD45 cDNA constructs were designed that contain either the cytomegalovirus promoter, the leukocyte function-associated antigen (LFA-1; CD11a) promoter, or various CD45 5' regions. Neither the cytomegalovirus nor the LFA-1 promoter was capable of generating detectable levels of expression in constructs with CD45 cDNA. However, when CD45 intron sequences between exons 3 and 9 were inserted in the cDNA construct to generate a CD45 minigene, the LFA-1 promoter was able to drive reproducible, significant expression of CD45. CD45 minigenes using the CD45 5' sequences up to 19 kilobases upstream of the transcriptional start produced very little protein. The LFA-1 CD45 minigene construct produced correct cell type-specific isoforms when expressed in T and B lymphocyte lines. Therefore, we conclude that the regulation of CD45 expression and cell type-specific splicing requires elements within the intron sequences.

Expression of CD45 isoforms lacking exons 7, 8 and 10.

The CD45 exon usage pattern of various CD8+ and CD4+ T cell lines was studied. By using the reverse transcription-polymerase chain reaction (RT PCR) and Southern analysis with exon specific or exon junction probes, we showed that all of the cytotoxic T cell lines and the majority of the helper T cells expressed the 789 isoform as a major splice variant. Expression of the splice product lacking exons 4-7 (isoform 89) was not as ubiquitous. All Th lines produced mRNA encoding this isoform, but in only three of the Tc lines was the 89 isoform detectable by RT/PCR. RNase protection assays with RNA isolated from normal CD8+ splenic cells demonstrated the 89 splice product was present in low abundance. The relative abundance of the different isoforms in the thymic lymphoma, BW5147, was determined through RNase protection analysis. The 789 isoform predominates, representing approximately 75% of the CD45 mRNA whereas the 89 form constitutes about 24%. In addition, an isoform lacking exons 4-8 (isoform 9) also was detected and comprises approximately 1% of the total CD45 mRNA in this cell line. Finally, these studies demonstrate that exon 10 is also used as an alternatively spliced exon.

Murine mast cells and monocytes express distinctive sets of CD45 isoforms.

CD45 isoform expression patterns were examined in various mast and monocyte cell populations. The reverse transcription-polymerase chain reaction (RT/PCR) and Southern analysis showed these myeloid cells express characteristic sets of CD45 isoforms. Mast cells produce mRNA for two splice variants, one containing exons 5, 7 and 8 of the alternatively expressed exons (therefore lacking exons 4 and 6) and another containing variable exons 7 and 8. Monocytes express three prominent CD45 mRNA species, one which includes exons 5, 7 and 8, another with exons 7 and 8 and the third containing only exon 8 of the variable exons. These results show that there are clear differences within the myeloid lineage sub-populations with respect to CD45 exon usage which appear to delineate mast cell and monocyte specific patterns.

Common regulatory elements control symbiotic and microaerobic induction of nifA in Rhizobium meliloti.

We have previously demonstrated that the nifA promoter (nifAp) of Rhizobium meliloti is inducible under microaerobic conditions in the absence of alfalfa. Here we show that microaerobic activation of nifAp involves both cis- and trans-acting regulatory controls identical to those used symbiotically. The start site for nifA mRNA synthesis was found to be the same during symbiosis and microaerobiosis, and a deletion analysis of nifAp demonstrated that DNA between positions -62 and -45 is essential for induction. Mutants isolated as being unable to induce nifA microaerobically also were found to be defective in symbiotic nitrogen fixation with alfalfa. Such mutants form nodules that are equivalent cytologically to those induced by nifA::Tn5 mutants. Genetic and structural studies have localized the mutations to a cluster of fix genes 200 kilobases distant from the nod-nif region on the pSym megaplasmid [Renalier, M.-H., Batut, J., Ghai, J., Terzaghi, B., Gherardi, M., David, M., Garnerone, A.-M., Vasse, J., Truchet, G., Huguet, T. & Boistard, P. (1987) J. Bacteriol. 169, 2231-2238].

The nifA gene of Rhizobium meliloti is oxygen regulated.

Experiments using plasmid-borne gene fusions and direct RNA measurements have revealed that expression from the nifA gene is induced in Rhizobium meliloti when the external oxygen concentration is reduced to microaerobic levels. Induction occurs in the absence of alfalfa and in the presence of fixed nitrogen and does not require ntrC. The production of functional nifA gene product (NifA) can be demonstrated by its ability to activate the nitrogenase promoter P1. Aerobic induction of nifA can also occur during nitrogen starvation at low pH, but in this case induction is dependent on ntrC and does not lead to P1 activation. The data indicate that reduced oxygen tension is potentially a major trigger for symbiotic activation of nitrogen fixation in Rhizobium species.

Analysis of transfer of tumor-inducing plasmids from Agrobacterium tumefaciens to Petunia protoplasts.

Petunia protoplasts were infected with the virulent Agrobacterium tumefaciens strain A348 or the avirulent strain A136 (lacking a Ti plasmid). The infection process was stopped at various time intervals up to 24 h after inoculation, and the DNA from the plant cells was isolated. Southern blot analysis indicated that the DNA isolated from infected Petunia cells was not detectably contaminated by bacterial DNA from lysed Agrobacterium cells. Analysis of the DNA from the virulent infections suggested that the transferred DNA (T-DNA) may be transferred to the plant cell rapidly (within 2 to 6 h) after the bacteria bind to the cell wall and that the T-DNA may exist in a rearranged state which is stable over the time period investigated. Dot blot analysis indicated that regions far outside the T-DNA may be transferred to the plant cell. Most of the DNA transferred to the plant cell during the initial hours of infection is rapidly degraded.