7th Conference - Abstract 549

A Novel Retrovirus-Based Transducing Vector That Transduces CD34+ Enriched Leukocytes

D. LIU¹, J. NG¹, J. HO¹, Q. WU¹, G. NUOVO², E. WREN³, M. COWAN³, M. CONANT³, B. THALENFELD¹, and D. ENGELHARDT*¹. ¹Enzo Therapeutics, Inc., Farmingdale, NY ²Ohio State Univ., Columbus; and ³Univ. of California at San Francisco

A retrovirus-based transducing vector and producing cell line have been developed. This vector, HGTV43 is made up of modified Moloney Murine Leukemia Virus LTR's and three U1 genes in their entirety each containing a genetic antisense sequence nested within the RNA coding portion of the gene. The three genetic antisense sequences are complementary to three separate sequences of the HIV-1 genome including one against the TAR region and two against two separate sites of the TAT region. The producing cell line contains a sequence coding for the envelope protein of HTLV II and a separate sequence coding for the MMLV gag-pol region. When transfected with the clone of the pro-HGTV43 vector DNA, a stable producer cell line is made. Transducing vector is produced from this cell line with consistent titers of greater than 10^7 per ml.
HGTV43 was grown in a large lot and tested on CD34+ enriched cell populations isolated from subjects, both infected and uninfected with HIV-1 virus. The CD34+ enriched cell populations were isolated after promotion using GCSF. The cells were removed after leukophersis and enriched on an Isolex 300 column. The purity was at or above 85% and the viability at the beginning of the transduction was >90%. These cells were transduced both in the presence and absence of various cytokines and in the presence and absence of a feeder layer. Most noteworthy the cells were transduced in medium without feeder layers and in the absence of added cytokines for less than 1 day. In all instances the cells were successfully transduced as measured by in situ RT/PCR and the presence of total RNA in a population of cells. The amount of antisense RNA (measured by RT-PCR) per cell of the population of transduced cells was greater than 1000 copies. In addition 30 to 80% of the colonies of these transduced cells were positive for antisense RNA after 3 weeks of growth in culture.
We conclude we have developed a transducing vector that works to deliver the antisense genes efficiently and quickly to cells in vitro.

Key Words: Blood Stem Cell, Gene therapy, Genetic antisense