547. Antiviral RNA Interference Strategies That Resist HIV Escape
Authors: S. E. Braun, FE Wong, AL Chesebro, B. Nornen, D. J. Looney, F. Wong-Staal, R. P. Johnson
Journal: Molecular Therapy (2004)

The ribozyme 9456, a small RNA molecules having catalytic activity, has been designed to specifically cleave target sequences in the U3 region of SIV. Recent developments have indicated that lentiviral vectors may transduce quiescent cells and therefore provide more efficient gene transfer to important target populations for AIDS gene therapy. However, the optimal position within the lentiviral vector for expression of small RNA inhibitors, especially using internal polymerase III promoters, has not been determined. To compare expression of the tRNAval Pol III promoter/SIV-specific ribozyme 9456 inhibitor cassette, we generated a murine leukemia virus (MLV) vector with the inhibitor in the antisense orientation and a series of a self-inactivating lentiviral vectors (HRST) with the inhibitor in different positions in both the sense and antisense orientations (before RRE: P1/invP1, after CMV-GFP: P2/invP2, in the ΔU3 of the 3′ HIV LTR: P3/invP3). The CD4+ CEMx174 cell line was transduced with the MLV vector before selecting individual transduced clones or with the HRST vectors before sorting by green fluorescent protein (GFP) expression. Total RNA was isolated from transduced cells for analysis by quantitative, real-time RT-PCR for the expression of ribozyme 9456, MLV vector, HRST vector and β-actin sequences. To assess inhibition of viral replication, transduced cells were challenged with an MOI of 10−2 TCID50/cell of SIVmac239 and followed over fourteen days for viral replication. Expression of GFP from the internal CMV promoter with the HRST vectors was assessed by flow cytometry and transduced cells were >90% positive. Expression of the ribozyme 9456 inhibitor relative to expression of the β-actin gene in the MLV-transduced clones and the HRST-transduced populations ranged between 1.1 × 103 to 9.0 × 104 and 3.0x103 to 1.3 × 105 units, respectively. Expression of the ribozyme 9456 was highest in the sense orientation of P2 and P3 of the HRST vectors. The MLV vector backbone showed high levels of expression ranging from 2.2 × 106 to 2.9 × 107 units, while the HRST vector was lower but detectable ranging between 3.8 × 103 to 1.4 × 104 units, indicating that the ΔU3 promoter in the HIV LTR of the HRST vectors is not completely inactive. The MLV clones with the highest levels of 9456 expression also provided the strongest inhibition of viral replication. Many of the HRST vectors (P3, invP1, invP2, invP3) showed strong inhibition (>80%). Inhibition with P1 and P2 was modest even though P2 expressed the 9456 at the highest level. Inhibition with the HRST vectors was consistently stronger than in the MLV clones. These vectors demonstrate variable expression of the ribozyme inhibitor from a Pol III promoter in HRST and MLV vector backbones and may provide valuable insight into the expression of other small RNA molecules in transduced cells. Optimization of delivery of ribozymes by lentiviral vectors should facilitate analysis of the efficacy of ribozymes for stem cell gene therapy for AIDS in a nonhuman primate model.