Retroviral vectors. From laboratory tools to molecular medicine.

The majority of clinical trials for gene therapy currently employ retroviral-mediated gene delivery. This is because the life cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently and safely packaged. Here, we review the molecular technology behind the generation of recombinant retroviral vectors. We also highlight the problems associated with the use of these viruses as gene therapy vehicles and discuss future developments that will be necessary to maintain retroviral vectors at the forefront of gene transfer technology.

Detection and significance of bcr-abl mRNA transcripts and fusion proteins in Philadelphia-positive adult acute lymphoblastic leukemia.

Blast cells from an unselected consecutive series of 84 adults presenting with acute lymphoblastic leukemia (ALL) to St Bartholomew's Hospital over a seven year period were tested prospectively by cytogenetic and retrospectively by RT-PCR analysis for the presence of the Ph translocation and bcr-abl mRNA. This combination gave an overall figure of 20.3% for bcr-abl-positive and/or Ph-positive ALL. The incidence of bcr-abl-positive/Ph-positive ALL was most common between the ages of 31 and 50 years, becoming less common after the age of 50. Eight out of ten bcr-abl-positive patients expressed the e1a2 mRNA transcript, the other two expressed the b3a2 and b2a3 transcripts respectively. Cells from all patients with bcr-abl mRNA transcripts expressed the appropriate p190 or p210 bcr-abl protein and all were Ph-positive.

Expression of the ABL-BCR fusion gene in Philadelphia-positive acute lymphoblastic leukemia.

We have previously shown that the chimeric gene ABL-BCR, formed on the derivative chromosome 9q+ as a result of the t(9;22) translocation, is transcriptionally active in 65% of chronic myeloid leukemia patients. We have now used the same technique, reverse transcription/polymerase chain reaction amplification of ABL-BCR transcripts, to study nine patients with Philadelphia (Ph) chromosome-positive acute lymphoblastic leukemia (ALL); seven expressed the P190 and two the P210 type of BCR-ABL fusion protein. All seven patients with P190 had ABL-BCR transcripts containing a junction between ABL exon Ib and BCR exon 2 (Ib-e2); in two cases, ABL-BCR transcripts with the Ia-e2 junction type were also present. Of the two P210 ALL patients, one had a Ib-b4 ABL-BCR transcript and the other showed no detectable ABL-BCR expression. Although the BCR-ABL gene is probably fundamental in the pathogenesis of the Ph+ leukemias, differential expression of the ABL-BCR gene could contribute to the biologic heterogeneity of the disease.

Detection of additional JH/BCL2 translocations in follicular lymphoma.

In vitro enzymatic amplification and direct sequencing were used to detect and characterize t(14;18) recombination junctions in peripheral blood and bone marrow mononuclear cell preparations from patients with follicular lymphoma in remission. Samples from 24/44 patients were found to be positive for translocations involving the major breakpoint region of the BCL2 gene. In samples from seven patients two distinct t(14;18) translocations were shown to be present simultaneously; in one case the second translocation involved the minor cluster region of the BCL2 gene. Biopsy tissue obtained earlier in the course of the disease was available from five of these patients and was shown to contain one of the translocations in each case, but both translocations in only one. Further remission blood and bone marrow samples from the group were also examined. This led to the detection of both translocations in separate samples obtained at different times in a total of four out of the seven cases. In two of the remaining three patients the second translocation could not be amplified from further samples, but in both cases the search led to the identification of a third translocation, again only detectable in a single sample. These findings demonstrate that JH/BCL2 translocations can occur more than once during the course of follicular lymphoma. They suggest that biclonal follicular lymphoma may be more common than has previously been recognized but also raise the possibility that the translocation arises sporadically in the normal lymphoid cells of this group of patients.

Two radioassays for serum vitamin B12 and folate determination compared in a reference interval study.

For 154 subjects, we verified that vitamin B12 and folate status was normal, using as criteria the average polymorphonuclear lobe count, mean corpuscular volume, and hemoglobin concentration. We then used blood from these subjects to compare values obtained with two radioassay kits, each designed for simultaneous vitamin B12 and folate determination. Although regression analysis showed reasonable correlation between the folate (r = 0.87) and vitamin B12 (r = 0.94) kits, we observed significant differences in the overall mean values for vitamin B12 (p less than 0.01) and folate (p less than 0.001) as measured with the kits in this population. Radioassay standard-curve data for the folate assays were similar, but these data indicated greater sensitivity at low concentrations for one vitamin B12 kit than the other. Using reference intervals recommended in the kit inserts, we found that the vitamin B12 status for 9% of these subjects would have been misclassified by one kit, 2% by the other.