VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum.

Lentiviral vectors transduce dividing and postmitotic cells and thus are being developed toward therapies for many diseases affecting diverse tissues. One essential requirement for efficacy will be that vector particles are resistant to inactivation by human serum complement. Most animal studies with lentiviral vectors have utilized VSV-G pseudotyped envelopes. Here we demonstrate that VSV-G pseudotyped HIV and FIV vectors produced in human cells are inactivated by human serum complement, suggesting that alternative envelopes may be required for therapeutic efficacy for many clinical applications of lentiviral vectors.

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.

The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent.

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430-7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1, 000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.

Evaluation of PCR and ELISA assays for screening clinical trial subjects for replication-competent retrovirus.

Gene delivery via murine-based recombinant retroviral vectors is currently widely used in gene therapy clinical trials. The vectors are engineered to be replication defective by replacing the structural and nonstructural genes of a cloned infectious retrovirus with a therapeutic gene of interest. The retroviral particles are currently generated in packaging cell lines, which supply all retroviral proteins in trans. Recombination between short homologous regions of the retroviral vector and packaging cell line elements can theoretically generate replication-competent retrovirus (RCR) and hence the Food and Drug Administration (FDA) requires the monitoring of clinical trial subjects for the presence of RCR. Sensitive polymerase chain reaction (PCR) assays have been used for the detection of murine leukemia virus (MLV) nucleotide sequences in peripheral blood mononuclear cells (PBMCs). A novel serological enzyme-linked immunosorbent assay (ELISA) for the detection of anti-MLV specific immunoglobulin (Ig) has been developed to be used as an alternative to the PCR assay. Both assays were used to monitor human immunodeficiency virus (HIV)-positive clinical trial subjects who had received multiple injections of HIV-IT (V), a retroviral vector encoding HIV-1 IIIBenv/rev. Western blot analysis and an in vitro vector neutralization assay were used to characterize further a subset of serum samples tested by ELISA. Results show no evidence of RCR infection in clinical trial subjects. PCR and ELISA assays are discussed in terms of their advantages and limitations as routine screening assays for RCR. The PCR assay is our current choice for monitoring clinical trial subjects receiving direct administration of vector, and the ELISA is our choice for those receiving ex vivo treatment regimens.

Aphidicolin-resistant polyomavirus and subgenomic cellular DNA synthesis occur early in the differentiation of cultured myoblasts to myotubes.

Small DNA viruses have been historically used as probes of cellular control mechanisms of DNA replication, gene expression, and differentiation. Polyomavirus (Py) DNA replication is known to be linked to differentiation of may cells, including myoblasts. In this report, we use this linkage in myoblasts to simultaneously examine (i) cellular differentiation control of Py DNA replication and (ii) an unusual type of cellular and Py DNA synthesis during differentiation. Early proposals that DNA synthesis was involved in the induced differentiation of myoblasts to myotubes were apparently disproved by reliance on inhibitors of DNA synthesis (cytosine arabinoside and aphidicolin), which indicated that mitosis and DNA replication are not necessary for differentiation. Theoretical problems with the accessibility of inactive chromatin to trans-acting factors led us to reexamine possible involvement of DNA replication in myoblast differentiation. We show here that Py undergoes novel aphidicolin-resistant net DNA synthesis under specific conditions early in induced differentiation of myoblasts (following delayed aphidicolin addition). Under similar conditions, we also examined uninfected myoblast DNA synthesis, and we show that soon after differentiation induction, a period of aphidicolin-resistant cellular DNA synthesis can also be observed. This drug-resistant DNA synthesis appears to be subgenomic, not contributing to mitosis, and more representative of polyadenylated than of nonpolyadenylated RNA. These results renew the possibility that DNA synthesis plays a role in myoblast differentiation and suggest that the linkage of Py DNA synthesis to differentiation may involve a qualitative cellular alteration in Py DNA replication.

E1A represses wild-type and F9-selected polyomavirus DNA replication by a mechanism not requiring depression of large tumor antigen transcription.

Polyomavirus (Py) DNA replication may be regulated to a low-level replication state in specific target cells in mice as well as in certain undifferentiated murine cell lines, such as embryocarcinoma (EC) cells. To investigate possible mechanisms by which such control may occur, we have examined the effects of E1A on Py DNA replication. Adenovirus E1A proteins repress transcriptional activation of various enhancers, including those of Py, and can stimulate DNA replication in quiescent cells, but E1A effects on Py DNA replication were unknown. We found that constitutive E1A expression in NIH 3T3 cells depressed Py DNA replication very strongly. Two F9 EC cell-selected Py enhancer variants, PyF441 and PyF101, were also examined because undifferentiated EC cells are hypothesized to have an E1A-like activity responsible for the Py restriction, and these variants activate Py DNA replication in cis in undifferentiated F9 cells. Both variants were repressed by E1A, indicating that E1A activity in 3T3 cells is not equivalent to undifferentiated F9 cell E1A-like activity. We also examined transient inducible E1A expression in cells supplying Py large tumor antigen (T-Ag). Py DNA replication was again repressed, and the inhibition increased with E1A induction. Analysis of T-Ag mRNA levels indicated that E1A repression of Py DNA replication was not an indirect result of depression of T-Ag transcription. This suggests that E1A may repress Py DNA replication by a more direct mechanism, possibly by blocking enhancer activation of DNA replication in a manner uncoupled with enhancer transcriptional control.

Continuing coevolution of virus and defective interfering particles and of viral genome sequences during undiluted passages: virus mutants exhibiting nearly complete resistance to formerly dominant defective interfering particles.

We quantitatively analyzed the interference interactions between defective interfering (DI) particles and mutants of cloned vesicular stomatitis virus passaged undiluted hundreds of times in BHK-21 cells. DI particles which predominated at different times in these serial passages always interfered most strongly (and very efficiently) with virus isolated a number of passages before the isolation of the DI particles. Virus isolated at the same passage level as the predominant DI particles usually exhibited severalfold resistance to these DI particles. Virus mutants (Sdi- mutants) isolated during subsequent passages always showed increasing resistance to these DI particles, followed by decreasing resistance as new DI particles arose to predominate and exert their own selective pressures on the virus mutant population. It appears that such coevolution of virus and DI particle populations proceeds indefinitely through multiple cycles of selection of virus mutants resistant to a certain DI particle (or DI particle class), followed by mutants resistant to a newly predominant DI particle, etc. At the peak of resistance, virus mutants were isolated which were essentially completely resistant to a particular DI particle; i.e., they were several hundred thousand-fold resistant, and they formed plaques of normal size and numbers in the presence of extremely high multiplicities of the DI particle. However, they were sensitive to interference by other DI particles. Recurring population interactions of this kind can promote rapid virus evolution. Complete sequencing of the N (nucleocapsid) and NS (polymerase associated) genes of numerous Sdi- mutants collected at passage intervals showed very few changes in the NS protein, but the N gene gradually accumulated a series of stable nucleotide and amino acid substitutions, some of which correlated with extensive changes in the Sdi- phenotype. Likewise, the 5' termini (and their complementary plus-strand 3' termini) continued to accumulate extensive base substitutions which were strikingly confined to the first 47 nucleotides. We also observed addition and deletion mutations in noncoding regions of the viral genome at a level suggesting that they probably occur at a high frequency throughout the genome, but usually with lethal or debilitating consequences when they occur in coding regions.

The intracellular half-lives of nonreplicating nucleocapsids of DI particles of wild type and mutant strains of vesicular stomatitis virus.

We have used defective interfering (DI) particles purified free of all infectious virus to determine the intracellular biological stability of nonreplicating nucleocapsids of vesicular stomatitis virus (VSV). Following infection of BHK-21 cells or tc-7 cells with a low multiplicity of pure DI particles, we superinfected them at varying times afterward with a high multiplicity of infectious helper virus to allow replication of those DI particle nucleocapsids retaining biological activity. Careful quantitation of DI particles in the yields from each time point showed that the biological half-life of intracellular VSV Indiana wild type DI particle nucleocapsids was 6 hr in BHK-21 cells and 5.3 hr in tc-7 cells (not significantly different). However, a DI particle from a temperature-sensitive mutant (tsG31) of VSV exhibited a biological nucleocapsid half-life of 12.5 hr and a DI particle isolated following 5 years of persistent infection had a half-life of 18 hr. These findings have significance for the stability of DI particle activity in vivo during acute infections where virus and DI particles are not always present together in the same cells due to cycling interactions. The increased half-life of DI nucleocapsids after years of persistent infection contrasts with the decreased stability and debilitation generally observed in infectious virus from persistent infection. Finally, transcapsidation studies showed that the intracellular half-life differences between DI particles are due mainly to their RNA genomes rather than to the N protein which encapsidates them.

Very rapid generation/amplification of defective interfering particles by vesicular stomatitis virus variants isolated from persistent infection.

Multiply cloned variants of vesicular stomatitis virus (VSV) were found to generate/amplify defective interfering (DI) particles at a rate greatly exceeding the rates normally observed for wild-type VSV (or for other mutants of VSV). A single undiluted passage of the first clonal pool of this variant virus produced concentrated visible bands of DI particles on sucrose gradients whereas wild-type and other mutant strains of VSV required from three to six or more serial undiluted passages. Since DI particle amplification by wild-type VSV at each undiluted passage can exceed 10,000-fold enrichment, these variant virus clones were generating/amplifying DI particles many millions of times more rapidly than were wild-type and other mutant strains of VSV. This rate of generation/amplification is so high that it was not feasible to obtain accurate estimates of the rates of generation (or amplification) of these DI particles.