Mesenchymal stem cells as vehicles for gene delivery.

Mesenchymal stem cells contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and marrow stroma. Transduction of mesenchymal stem cells from species other than humans is required for the development of disease models in which mesenchymal stem cells-based gene delivery is evaluated. Attempts to transduce mesenchymal stem cells from some species with amphotropic retroviral vectors were unsuccessful, leading to comparative mesenchymal stem cells transductions with xenotropic and gibbon-ape leukemia virus envelope-pseudotyped retroviral vectors. Human, baboon, canine, and rat mesenchymal stem cells were transduced optimally with amphotropic vector supernatants. In contrast, sheep, goat, and pig mesenchymal stem cells showed highest transduction levels with xenotropic retroviral vector supernatant, and rabbit mesenchymal stem cells were transduced optimally with gibbon-ape-enveloped vectors. Using a myeloablative canine transplantation model and gene-marked canine mesenchymal stem cells, the biodistribution of infused and ex vivo expanded mesenchymal stem cells were examined. The majority of transduced canine mesenchymal stem cells were found in the bone marrow samples. The current study shows the use of mesenchymal stem cells as a delivery vehicle for gene transfer studies, and validates the feasibility of delivering mesenchymal stem cells to the marrow compartment for stromal regeneration after cancer-associated cytotoxic therapies.

Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer.

We report increased transduction of human hematopoietic progenitor cells through a combination of novel retroviral vector packaging cell lines, and improved vector supernatant production. The new ProPak packaging cell lines produce either murine leukemia virus (MLV) xenotropic (ProPak-X cells) or amphotropic particles (ProPak-A cells), and ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing. Vector supernatants from ProPak or existing packaging cell lines producing different pseudotyped particles (amphotropic MLV, xenotropic MLV or gibbon ape leukemia virus) were compared for the ability to transduce clinically relevant human hematopoietic cells. All vector types transduced primary human CD34-positive or CD4-positive cells, regardless of tropism. However, consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector. The highest transduction of human hematopoietic progenitor cells was achieved with vector supernatant generated from a coculture of the ProPak-X and ProPak-A cell lines. This ping-pong amplification yielded supernatant containing vector targeted to two distinct receptors present on human cells, and did not result in detectable RCR formation. In addition, we describe conditions for improved vector supernatant production in a packed-bed bioreactor.

Improved detection of replication-competent retrovirus.

Recombinant retroviral vectors are the predominant delivery system in human gene therapy protocols. Since contaminating replication-competent retrovirus (RCR) can arise during the production of retroviral vector supernatants, sensitive assays for the screening of supernatants are necessary. In this study, we present a marker rescue assay based upon a Mus dunni cell line stably transduced with a lacZ gene. We show that detection of RCR in vector supernatants by the M. dunni lacZ marker rescue assay or PG-4 S+ L- focus-forming assay is equally sensitive. By inoculating test supernatants under centrifugation (which we term spinoculation), we increased the sensitivity of detection of RCR 10 to 100-fold with the PG-4 S+ L- and lacZ marker rescue assays. While the spinoculation protocol had no adverse effects on cells, spinoculation of high titer vector supernatants onto PG-4 cells resulted in some cytotoxicity, making identification of RCR positive cultures difficult. However, spinoculation of vector supernatants onto M. dunni lacZ cells resulted in no cytotoxicity, and also partially overcame inhibition of detection of low levels of RCR due to the presence of high titer replication-incompetent vector.

A novel human amphotropic packaging cell line: high titer, complement resistance, and improved safety.

Successful retroviral-mediated gene therapy will depend on safe, efficient packaging cell lines for vector particle production. Existing packaging lines for murine leukemia virus (MLV)-based vectors are predominantly derived from NIH/3T3 cells which carry endogenous MLV sequences that could participate in recombination to form replication-competent retrovirus (RCR). To identify cells devoid of such sequences, we screened genomic DNA from eight cell lines. DNA from the human 293 cell line did not cross-hybridize with MLV sequences, and these cells were able to secrete Gag particles after transfection. We derived a stable amphotropic packaging cell line (called ProPak-A) in 293 cells in which the Gag-Pol and Env (packaging) functions are expressed separately from a heterologous (non-MLV) promoter, to maximally reduce homology between packaging and vector sequences. ProPak-A-based producer cells are efficient, yielding higher stable titers than PA317-based producers. In addition, a vector that consistently gave rise to RCR in PA317 cells never resulted in detectable RCR in ProPak-A-based producer cultures. We have also shown that ProPak-A-packaged particles are not inactivated by human serum. Thus, the packaging cells we describe are as efficient and safer than the amphotropic packaging cells most commonly used in clinical gene therapy work and are also more appropriate for in vivo gene delivery.

Retroviral end-point titer is not predictive of gene transfer efficiency: implications for vector production.

Efforts to improve gene transfer (transduction) efficiency achieved with retroviral vectors often focus on increasing the end-point titer. In this study, we assayed more than 70 retroviral vector supernatants for end-point titer and for the ability to transfer reporter genes into cell populations (referred to as transduction efficiency). We found no correlation between end-point titer and transduction efficiency. We also show that increasing end-point titer by ultrafiltration does not necessarily increase transduction efficiency. Evidence presented shows that nontransducing retroviral particles interfere with transducing virions and reduce transduction efficiency without reducing end-point titer. We have investigated production parameters and stability of retroviral vector particles using transduction efficiency as a measure of supernatant potency. Analysis of the production kinetics showed that the rate of virus production was marginally higher at 37 degrees C than at 32 degrees C. However, recombinant amphotropic retrovirus particles are significantly more stable at 32 degrees C than at 37 degrees C. In addition, we show that short incubation periods are sufficient to yield supernatants with high transduction efficiencies. We have implemented improved culture conditions, including short incubation periods, by continually perfusing medium over producer cells in a packed-bed bioreactor incubated at 32 degrees C. By operating the packed-bed bioreactor in perfusion mode, retroviral vector supernatants with a high transduction efficiency can be routinely produced in large quantities.

Detection of intracellular HIV-1 Rev protein by flow cytometry.

The Rev trans-activator protein plays a pivotal role in human immunodeficiency virus type 1 (HIV-1) replication by allowing expression of the viral structural proteins. We have developed a protocol to quantitatively assay intracellular steady state levels of Rev Ag (Rev wild type and RevM10 proteins) by flow cytometry. Three fixation and permeabilization techniques were compared. These protocols varied in the magnitude of the signal which could be detected, and in the ability to distinguish between Rev Ag positive and negative populations. This technology is applicable to a variety transduced or transfected cell types (species, lineage), and for cell lines and primary cells acutely infected with HIV-1. The assay is therefore a valuable tool both to analyze Rev protein expression levels in HIV-infected cells and to optimize delivery of the dominant-negative RevM10 gene for clinical gene therapy applications. In addition, a second, independent intracellular protein (HIV-Tat) has been detected using the same approach.

Duck hepatitis B virus infection of hepatocytes is not dependent on low pH.

The pH dependency for initiation of infection by the hepadnavirus duck hepatitis B virus (DHBV) was investigated in primary duck hepatocytes. First, an infection assay was developed using a radioimmunoblot to measure DHBV e antigen secreted into tissue culture fluid from infected hepatocytes. The quantity of this viral marker was proportional to the duration of inoculation and the amount of DHBV used as inoculum. The role of pH in initiation of DHBV infection was investigated by using this assay, but no dependence on low pH was found. DHBV was able to infect hepatocytes in the presence of NH4Cl and monensin, agents that raise the pH in intracellular vesicles and prevent penetration of viruses dependent on low pH in endosomes. In control experiments, infection by Semliki Forest virus, which is low pH dependent, was inhibited, whereas herpes simplex virus type 1 infection, which is pH independent, occurred. Attempts to trigger DHBV-cell fusion by exposure of DHBV prebound to hepatocytes to mildly acidic pH were unsuccessful. In these experiments, it was also observed that internalization of DHBV occurred only between pH 6.8 and 8.0. Additionally, in the absence of cells, infectivity of DHBV was stable at pH 4.6 to 4.8, which is lower than the pH encountered in endosomes (pH 5 to 6.6). Thus, no evidence for a role for mildly acidic pH in the initiation of DHBV infection was found. Therefore, we propose that the infection route followed by DHBV resembles that of the group of enveloped viruses, including herpesviruses, that fuse with their host cells at neutral pH.

IgG-neutralized influenza virus undergoes primary, but not secondary uncoating in vivo.

Even when neutralized by saturating amounts of monoclonal IgG directed against the haemagglutinin, influenza virus attaches to cells with kinetics similar to those of infectious virus. It then enters those cells and is uncoated; its RNA becomes localized within the nucleus and its lipid envelope and associated proteins remain in the cytoplasm. In this report we show that despite the apparent normality of these early stages of virus-cell interaction, neutralized virus underwent no detectable primary transcription. In contrast, there was only a slight inhibition of transcription by neutralized virus in vitro which was insufficient to account for the loss in infectivity, despite using mRNA to measure the production of capped oligonucleotides or to prime the elongation step. To test whether the absence of primary transcription in vivo resulted from non-accessibility of the genome rather than an effect on the transcriptase complex itself, we examined the susceptibility to RNase of virion RNA after inoculation of cells with neutralized virus. Data clearly show that, unlike RNA of infectious virus, RNA of neutralized virus did not become sensitive to RNase and we conclude that neutralization of influenza virus by IgG results in failure of virus to undergo a secondary uncoating process which is necessary for the activity of the virion transcriptase complex. Finally we show that by treatment of virions in vitro with detergent it is possible to produce a core structure which is stable and has some of the properties expected of a structure resulting from primary uncoating.