Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression of viral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (L1, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 10(6) transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the E1 region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helper-dependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.