Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.

Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 10(7) transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

Interactions within the ubiquitin pathway of Caenorhabditis elegans.

The ubiquitin system is a well-conserved and pervasive process for post-synthetic modification of proteins. Three key components of the pathway are required for ubiquitination to occur: the E1 ubiquitin activating enzyme, the E2 ubiquitin conjugating enzyme, and the E3 ubiquitin ligase. There are several different E2 ubiquitin conjugating enzymes and an even greater number of E3 ubiquitin ligases. Interactions between these two groups are critical for substrate ubiquitination. This study reports a two-hybrid analysis of interactions within the ubiquitin system of Caenorhabditis elegans. Forty-three RING finger proteins (presumed E3 ubiquitin ligases) and 14 predicted E2 ubiquitin conjugating enzymes were included in the screen. A total of 31 E2-E3 interactions were uncovered. In addition, the UBC-13 conjugating enzyme was observed to interact with two different E2s, UEV-1 and UBC-1. The interaction of UBC-1 and UBC-13 was confirmed with in vitro ubiquitination reactions. Using NHL-1 as the E3 in the assays, ubiquitination was observed when both UBC-1 and UBC-13 were present but not with either alone. These data imply that some E2s require dimerization in order to function.