Human cytochrome P450 2B6 ( CYP2B6 ) metabolizes the prodrug cyclophosphamide ( CPA ) to produce phosphoramide mustard that cross -links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 ( designated``MetXia -P450'' ) and used it to transduce the human tumor cell lines HT29 and T47D. MetXia -P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide -treated cells transduced with MetXia -P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3 -D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed -enzyme prodrug therapy ( GDEPT ). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450 -based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. Cancer Gene Therapy ( 2001 ) 8, 473 ± 482
Two siblings (case 1 and case 2) with homozygous C1q deficiency are described. Both presented with a photosensitive rash, and during follow-up case one developed SLE with nephrotic range proteinuria. Case 2 had microscopic hematuria with a past history of macroscopic hematuria. Renal biopsies revealed mesangioproliferative glomerulonephritis in case 1 and IgA nephropathy in case 2, a new finding in association with C1q deficiency. Since the classical pathway of complement plays a role in the development of antibody responses, the family was also evaluated for the immune response to hepatitis B vaccine. Antibody response to hepatitis B vaccine was normal in both affected members and the rest of the family. The A-, B- and C- chain genes of C1q were amplified by PCR and directly sequenced. A homozygous C to T point mutation was identified in genomic DNA isolated from the patients at codon 186 in the A chain that resulted in a premature stop codon. This mutation was present in both parents and both unaffected sibs in the heterozygous state. This mutation was identical to that previously described in a Slovakian family with C1q deficiency. Because of this finding, a series of 92 genomic DNA samples was screened from ethnically distinct patient groups with SLE to test the hypothesis that this mutation of C1q may be a widespread disease susceptibility gene. No further examples of this mutation were found.
A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells.
Objective. To describe a new kindred with Clq deficiency and to identify the molecular lesions responsible for complete functional Clq deficiency in this and 2 other previously described kindreds.Methods. The A-, B-, and C-chain genes of Clq were amplified by polymerase chain reaction, cloned, and sequenced. The DNA sequence was checked for mutations.
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