Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer.

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

A chimeric Ty3/Moloney murine leukemia virus integrase protein is active in vivo.

This report describes the results of experiments to determine whether chimeras between a retrovirus and portions of Ty3 are active in vivo. A chimera between Ty3 and a Neo(r)-marked Moloney murine leukemia virus (M-MuLV) was constructed. The C-terminal domain of M-MuLV integrase (IN) was replaced with the C-terminal domain of Ty3 IN. The chimeric retroviruses were expressed from an amphotrophic envelope packaging cell line. The virus generated was used to infect the human fibrosarcoma cell line HT1080, and cells in which integration had occurred were selected by G418 resistance. Three independently integrated viruses were rescued. In each case, the C-terminal Ty3 IN sequences were maintained and short direct repeats of the genomic DNA flanked the integration site. Sequence analysis of the genomic DNA flanking the insertion did not identify a tRNA gene; therefore, these integration events did not have Ty3 position specificity. This study showed that IN sequences from the yeast retrovirus-like element Ty3 can substitute for M-MuLV IN sequences in the C-terminal domain and contribute to IN function in vivo. It is also one of the first in vivo demonstrations of activity of a retrovirus encoding an integrase chimera. Studies of chimeras between IN species with distinctive integration patterns should complement previous work by expanding our understanding of the roles of nonconserved domains.

Gene expression from transcriptionally disabled retroviral vectors.

Retroviral vectors are used for the efficient transfer of foreign genes into mammalian cells. We report here the construction of murine retrovirus-based vectors carrying the full-length cDNA for human hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and from which the enhancer sequences, the "CAAT box," and the "TATA box" in the long terminal repeats (LTRs) have been deleted. After infection of HPRT-deficient rat cells by the vectors, transcriptional activity from the 5' LTR was undetectable and expression of the HPRT cDNA was dependent on an internal promoter. Removal of the LTR regulatory elements increased HPRT gene expression from an internal promoter, indicating interference between the two sets of transcriptional signals. Such disabled vectors may reduce the likelihood of undesirable genetic changes through insertional mutagenesis in cells infected with retroviral vectors.

Expression of retrovirally transduced genes in primary cultures of adult rat hepatocytes.

Differentiated primary rat hepatocyte cultures have been infected with retroviral vectors expressing human hypoxanthine/guanine phosphoribosyltransferase or the transposon Tn5 neomycin-resistance gene. Expression of the markers was detected only after infection of the cells during a short period of cell replication and transient dedifferentiation from days 1 to 5 of culture. Provirus integrated during that period remains fully expressed during the entire subsequent stationary period of culture up to at least 25 days. Selection with the neomycin analogue G418 of cells infected with the neomycin vector led to the appearance of cells with hepatocyte morphology in which newly synthesized albumin was detectable by immunoprecipitation, indicating successful infection of hepatocytes.

The human ribosomal RNA genes: structure and organization of the complete repeating unit.

The complete repeating unit of the human ribosomal RNA gene has been reconstructed by the cloning of approximately 27 kilobases (kb) of non-transcribed spacer. The structure of this tandemly repeated gene can now be studied in its entirety. We report the analysis of spacer DNA by molecular cloning and its organization in the genome by Southern transfer analysis. These studies reveal both length and sequence variation of the spacer. Sequence variations are distributed throughout the spacer while the length variations exist near the 5' end of the transcript and just beyond the 3' end. The human spacer shares extensive homology with primates but little with other mammals. Within the primates the degree of homology reflects the rapid evolutionary changes characteristic of the primate group.

The organization of the human HPRT gene.

The organization of the X-linked gene for human hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8.) has been determined by a combination of restriction endonuclease mapping, heteroduplex analysis and DNA sequence analysis of overlapping genomic clones. The entire gene is 42 kilobases in length and split into 9 exons. The sizes of the 7 internal exons and the exon-intron boundaries are identical to those of mouse HPRT gene. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements but contains extremely GC-rich sequences and five GC hexanucleotide motifs (5'-GGCGGG-3'). These structural features are very similar to those found in the mouse HPRT gene and to some of the regulatory signals common to a class of constitutively expressed "housekeeping" genes. Several transcriptional start sites have been identified by nuclease protection studies. Extensive sequence homology between the mouse and human genes is found in the 3' non-coding portion of the gene.

Molecular analysis of rearrangements in human ribosomal RNA gene clones.

Human placental DNA, enriched for ribosomal sequences, was cloned in the phage vector lambda Charon 16A. Recombinants containing 28S rDNA sequences were isolated, and all were found to have deletions in the insert and/or vector DNA. Electron microscopic analysis was used to map the deletions and provide evidence that unstable forms of the recombinants can revert to the original vector or undergo further rearrangements. Specific deletions are manifested as previously unreported plaque phenotypes.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate on concanavalin A-stimulated sterol and fatty acid synthesis in mouse spleen lymphocytes.

Substantial increases in both 3beta-OH sterol and fatty acid synthesis were observed after concanavalin A addition to mouse spleen lymphocytes cultured in serum-free media. The rate of sterol synthesis increased linearly up to 60 h. The rate of fatty acid synthesis increased up to 20 h, reaching a plateau in synthetic activity which was the maintained. CO2 production from acetate was slightly stimulated by concanavalin A. In contrast to sterol and fatty acid synthesis, the rate of CO2 production in both mitogen-stimulated and resting cultures declined with time. Dibutyryl cyclic AMP had a strong inhibitory effect on concanavalin A-stimulated sterol and fatty acid synthesis from acetate, but only a slight effect on CO2 production. Delayed addition of dibutyryl cyclic AMP resulted in reduced inhibition. The data suggest a sequence of initiation for fatty acid and sterol synthesis prior to DNA synthesis and a possible regulatory role of cyclic AMP in this initiation. The results support the hypothesis that lymphocyte activation is sequential within the spleen cell population and is accompanied by fatty acid and sterol synthesis.