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 x 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.

Distribution of retroviral vectors and vector producer cells using two routes of administration in rats.

The clinical use of retroviral vector producer cells (VPCs) to deliver retroviral vectors efficiently to target cells has been investigated as a method to increase efficiency of gene delivery, presumably as a result of continued vector production in vivo. Studies were conducted in rats to evaluate the distribution of vector to distal organs and tissues as measured by transduction. Rats were treated with two doses of VPCs using two routes of administration: (1) subcutaneous injection, chosen to maximize both the dose and exposure of animals, thereby enabling identification of potential target organs under worst-case conditions; and (2) direct injection into brain parenchyma, chosen to mimic the intended clinical route of administration and provide an estimate of risk to patients receiving this therapy. Twelve organs or tissues were collected 7 days after administration of VPCs and analyzed by PCR for the presence of vector and vector producer cell sequences. Vector was detected most frequently at the site of injection by either route of administration. Less frequently, vector was detected in draining lymph nodes at the higher dose only using either route of injection. Single specimens of lung and contralateral skin were positive for vector following subcutaneous administration only. Vector was detected in gonadal tissue from a single low-dose male following subcutaneous administration, but this finding was not reproduced in any high-dose male or any males injected intracerebrally. In contrast, VPCs were detected only at the site of administration. The frequency of detection of VPCs 7 days after administration was higher when rats were injected by the intracerebral route. Based on these studies, gene transfer to distal organs or gonadal tissue following intracerebral administration of VPCs is not considered to be a risk to patients undergoing retroviral vector gene therapy for the treatment of brain cancer (glioblastoma multiforme; GBM).

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.

Efficacy of antiretroviral agents against murine replication-competent retrovirus infection in human cells.

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.

In vivo evaluation of an adenoviral vector encoding canine factor VIII: high-level, sustained expression in hemophiliac mice.

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

An improved retroviral vector encoding the herpes simplex virus thymidine kinase gene increases antitumor efficacy in vivo.

Brain tumors have been treated clinically by intratumoral injection of cells that produce retroviral vectors encoding the herpes simplex virus thymidine kinase (HSV-TK) gene followed by systemic administration of the antiviral drug ganciclovir. In vitro and in vivo comparisons of two different HSV-TK vector producer clones, which were made using standard transfection and transinfection techniques, were conducted. The two clones, PA317/G1TkSvNa.53 (TK.53) and PA317/G1Tk1SvNa.7 (TK1.7), both used in clinical trials, differ with respect to sequences 3' to the HSV-TK stop codon. The retroviral construct used to generate the TK.53 vector producer cell clone contains an open reading frame encoding a portion of the herpes simplex virus glycoprotein H (gH), a potential polyadenylation site and a putative splice site in this region. These sequences were removed from the retroviral construct used to create the TK1.7 vector producer cell clone. Supernatants obtained from TK1.7 vector producer cells had 100- to 1000-fold higher titers (G418 or HAT) than did corresponding supernatants from TK.53 vector producer cells. A murine subcutaneous tumor model was used to assess transduction efficiency and antitumor activity of each vector producer cell clone. In vivo tumor cell transduction was 13- to 18-fold more efficient with TK1.7 cells as compared with TK.53 cells at equivalent doses. Complete tumor ablation was achieved using a 10-fold lower dose of TK1.7 cells as compared with TK.53 cells. These results suggest that TK1.7 cells combined with ganciclovir may provide a more potent antitumor response in humans.

Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis.

Rapidly progressive T-cell lymphomas were observed in 3 of 10 rhesus monkeys several months after autologous transplantation of enriched bone marrow stem cells that had been transduced with a retroviral vector preparation containing replication-competent virus (R. E. Donahue, S. W. Kessler, D. Bodice, K. McDonagh, C. Dunbar, S. Goodman, B. Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis, J. Exp. Med. 176:1124-1135, 1992). The animals with lymphoma appeared to be tolerant to retroviral antigens in that their sera lacked antibodies reactive with viral proteins and contained 10(4) to 10(5) infectious virus particles per ml. By molecular cloning and DNA sequencing, we have now demonstrated that the serum from one of the monkeys contained a replication-competent retrovirus that arose by recombination between vector and packaging encoding sequences (vector/helper [V/H] recombinant) in the producer clone used for transduction of bone marrow stem cells. Southern blot analysis demonstrated 14 or 25 copies of this genome per cell where present in two animals. The genome of a second replication-competent virus was also recovered by molecular cloning; it arose by recombination involving the genome of the V/H recombinant and endogenous murine retroviral genomes in the producer clone. Twelve copies of this amphotropic virus/mink cell focus-forming virus genome were present in tumor DNA of one animal, but it was not found in tumor DNA of the other two animals with lymphoma. Southern blot analysis of DNA from various tissues demonstrated common insertion site bands in several samples of tumor DNA from one animal, suggesting clonal origin of the lymphoma. Our data are most consistent with a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis of critical growth control genes, leading to cell transformation and clonal tumor evolution.