370. CRM1 Enhances the Lentiviral Vector Production
Authors: Hsin-Lung Lo, Tammy Chang, Paola M. Marcovecchio, Priscilla Yam, John A. Zaia, Jiing-Kuan Yee
Journal: Molecular Therapy (2006)

Lentiviral vectors derived from human immunodeficiency virus 1 (HIV-1) are increasingly used in the delivery of transgenes into mammalian cells for gene function studies and for potential treatment of human diseases. One of the major barriers of applying HIV vector in disease treatment is to provide sufficient amounts of infectious vectors at high concentration. HIV vectors were generated by transient transfection of human 293T cells with the packaging plasmids and the vector DNA. The exceptionally high transfection efficiency of this cell line leads to production of vector with titers ranging between 105 and 107 infectious particles/ml. We have shown that delivery of small hairpin RNA (shRNA) genes into human hematopoietic cells with HIV vectors led to efficient inhibition of HIV-1 replication. However, the presence of the anti-HIV shRNA gene resulted in at least a 10–100 fold reduction in vector titer. For efficient delivery of shRNA genes into human hematopoietic cells, methods to enhance vector production from 293T cells therefore need to be developed.Since transient transfection likely results in the production of large quantities of viral transcripts in the nucleus of the transfected cells, we hypothesized that the process of viral transcript export into cytoplasm might become a rate-limiting step for efficient vector production. HIV replication requires the expression of un-spliced, partially spliced and fully spliced viral RNA transcripts. Several studies have demonstrated that cellular factors CRM1, DDX3, SAM68 and hRIP participate in the transport of partial-spliced or un-spliced viral RNA. We therefore tested the ability of these cellular factors to enhance the HIV vector production.Our studies showed that Rev-dependent expression of the firefly luciferase gene was increased 2–3 fold by all four factors with CRM1 demonstrated the highest activity. However, only CRM1 showed consistent ability to enhance the vector titer during vector production from 293T cells. Over-expression of CRM1 boosted the production of a vector containing no shRNA gene by an average of 6 fold. CRM1 was able to enhance the titer of a vector containing the anti- HIV shRNA gene up to 19 fold. Our results demonstrate viral RNA export from nucleus into cytoplasm can be a rate-limiting step for efficient production of HIV vectors from transiently transfected 293T cells, and CRM1 expression can partially compensate the poor titer of HIV vectors containing an anti-HIV shRNA gene. Lentiviral vectors derived from human immunodeficiency virus 1 (HIV-1) are increasingly used in the delivery of transgenes into mammalian cells for gene function studies and for potential treatment of human diseases. One of the major barriers of applying HIV vector in disease treatment is to provide sufficient amounts of infectious vectors at high concentration. HIV vectors were generated by transient transfection of human 293T cells with the packaging plasmids and the vector DNA. The exceptionally high transfection efficiency of this cell line leads to production of vector with titers ranging between 105 and 107 infectious particles/ml. We have shown that delivery of small hairpin RNA (shRNA) genes into human hematopoietic cells with HIV vectors led to efficient inhibition of HIV-1 replication. However, the presence of the anti-HIV shRNA gene resulted in at least a 10–100 fold reduction in vector titer. For efficient delivery of shRNA genes into human hematopoietic cells, methods to enhance vector production from 293T cells therefore need to be developed. Since transient transfection likely results in the production of large quantities of viral transcripts in the nucleus of the transfected cells, we hypothesized that the process of viral transcript export into cytoplasm might become a rate-limiting step for efficient vector production. HIV replication requires the expression of un-spliced, partially spliced and fully spliced viral RNA transcripts. Several studies have demonstrated that cellular factors CRM1, DDX3, SAM68 and hRIP participate in the transport of partial-spliced or un-spliced viral RNA. We therefore tested the ability of these cellular factors to enhance the HIV vector production. Our studies showed that Rev-dependent expression of the firefly luciferase gene was increased 2–3 fold by all four factors with CRM1 demonstrated the highest activity. However, only CRM1 showed consistent ability to enhance the vector titer during vector production from 293T cells. Over-expression of CRM1 boosted the production of a vector containing no shRNA gene by an average of 6 fold. CRM1 was able to enhance the titer of a vector containing the anti- HIV shRNA gene up to 19 fold. Our results demonstrate viral RNA export from nucleus into cytoplasm can be a rate-limiting step for efficient production of HIV vectors from transiently transfected 293T cells, and CRM1 expression can partially compensate the poor titer of HIV vectors containing an anti-HIV shRNA gene.