Edward Otto

Breeding of retroviruses by DNA shuffling for improved stability and processing yields.

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 × 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.

Molecular Evaluation of Biopsy and Autopsy Specimens from Patients Receiving in Vivo Retroviral Gene Therapy

We used the polymerase chain reaction (PCR) to assay for the presence of retroviral vector and replication-competent retrovirus (RCR) in autopsy and biopsy specimens from patients who received inoculations of retroviral vector producer cells (VPCs) into brain tumors or apparently normal tissues surrounding resected tumors. The PCR assays were capable of detecting 1 or more proviral copies of vector or RCR in 500,000 cells. Of 113 patients treated in clinical trials between 1994 and 1997, autopsy specimens were available from 32 patients. Brain tumor biopsies were also available from 24 patients. A total of 346 specimens was analyzed. Vector DNA was detected in 55% of tumor samples and 22% of brain samples obtained from resection margins. In contrast, most of the nonbrain tissues were negative for vector DNA; only low levels (<0.03%) of vector sequence were detected in 6 of 240 (2.5%) nonbrain tissues. Vector DNA was not detected in gonadal tissues from 12 men and 10 women. More importantly, RCR was not detected in any of the 134 biopsy and autopsy tissues tested, including all brain tumor, brain, and gonadal specimens. These results comprise the largest data set on molecular analysis of autopsy specimens from patients receiving retroviral gene therapy and indicate that distribution of retroviral vectors following injection of high doses of VPCs is limited to the site of inoculation.

In vitro analysis of transformation potential associated with retroviral vector insertions

While replication-defective retroviral vectors provide excellent vehicles for the long-term expression of therapeutic genes, they also harbor the potential to induce undesired genetic changes by random insertions into the host genome. The rate of insertional mutagenesis for retroviral vectors has been determined in several different assay systems; however, the rate at which such events induce cellular transformation has not been directly determined. Such measurements are critical to determining the actual risk of carcinogenesis resulting from retroviral gene therapy. In this study, the ability of a replication-defective retroviral vector, GlnBgSvNa, to induce cellular transformation in the BALB/c-3T3 in vitro transformation assay was assessed. The transformation frequency observed in vector-transduced BALB/c-3T3 cells, which contained one to six copies of integrated provirus, was not significantly different from that of untreated control cells. The finding that GlnBgSvNa was nontransforming in this assay indicates that the rate of transformation induced by retroviral insertions is less than the spontaneous rate of cellular transformation by BALB/c-3T3 cells, or less than 1.1 x 10(-5). These results are the first to define an upper limit for the rate of transformation induced by retroviral vectors.

Quantitative detection of cell culture Mycoplasmas by a one step polymerase chain reaction method

A rapid, sensitive assay was developed that can detect the six species of Mycoplasmas that account for the vast majority of cell culture infections. This assay, a modification of the method published by Wong-Lee & Lovett [1], allows direct evaluation of culture medium by a single-step PCR method that utilizes primers complementary to conserved 16S rRNA sequences. Extensive testing of medium from uninfected cultures spiked with purified Mycoplasma DNAs showed that the method described in this report can detect the equivalent of one Mycoplasma in 15 μl culture medium; thus, evaluation of a single culture sample allows detection of Mycoplasmas in cultures infected with the equivalent of 10 or more Mycoplasmas per 15 μl (or ≥6.7×102 Mycoplasma equivalents/ml) with greater than 99.99% confidence. Comparison of results obtained with this PCR-based assay and a standard biological colony-forming assay revealed that the PCR assay is capable of detecting 0.0015-0.015 colony forming units, suggesting that the PCR assay may also be detecting nonviable Mycoplasmas. The high level of amplification achieved with this method allows direct detection of amplification products by ethidium bromide staining of agarose gels, and thus allows rapid screening of cell cultures.