HUMAN IMMUNODEFICIENCY VIRUS TYPE I AS A TARGET FOR GENE THERAPY

Magnús Gottfredsson and Paul R. Bohjanen

Division of Infectious Diseases and International Health, Department of Medicine, Duke University Medical Center, Durham, NC 27710

Received 11/17/97 Accepted 11/24/97

5. COMBINATIONS OF ANTIVIRAL STRATEGIES

Although most studies have focused only on single antiviral genes, combinations of antiviral elements in a single vector often confers added benefit as discussed below. Given the success of antiviral drugs, future clinical trials gene therapeutic strategies are likely to be used in combination with already established antiviral drug regimens. Relatively few reports, however have focused on the efficacy of such combinations. The addition of pharmacologic inhibitors of NF-kB has been recently studied in cells stably expressing an anti-Tat sFv antibody (179). Theoretically this combination should result in cooperative suppression in LTR-driven gene expression and replication. On challenge with HIV-1 it was shown in both T cell lines and primary T cells that the combined treatment resulted in more profound and durable antiviral action than was seen with either treatment alone (179).

5.1. Ribozymes and RNA decoys

A combination of ribozymes and RNA decoys has recently been described. A ribozyme with an RRE or TAR sequence coupled to the stem loop of each ribozyme was shown to exhibit dual function in vitro (180). A combination of TAR decoys and gag-specific ribozymes has been shown to suppress HIV-1 and simian immunodeficiency virus in T cell lines (181). Another chimeric molecule consisting of an RRE RNA decoy fused with a ribozyme directed at the HIV-1 U5 sequence has been constructed. Subsequent challenge of T cells transduced with this construct revealed much greater protection in cells expressing the chimeric construct as compared to the ribozyme molecule alone (182). A triple-copy vector encoding the U5 ribozyme, a ribozyme against the env/rev region, and a RRE decoy was recently shown to confer an even higher level of resistance to T cells against diverse clades of HIV-1 (183).

5.2. Trans-dominant mutant combinations

Increased suppression of HIV-1 replication may be achieved by combinations of two trans-dominant mutant proteins. A novel fusion protein, Trev, which joins Tat and Rev trans-dominant mutant sequences has been constructed and shown to inhibit both Tat and Rev activities in T-cell lines and primary T-cells challenged with HIV-1 (184, 185). When expressed simultaneously, Tat and Rev trans-dominant mutants resulted in additive or even synergistic inhibition of HIV-1 when compared to the Rev mutant alone (186). This paradigm also seems to apply to other combinations; for example Gag dominant mutants and gag antisense sequences are more effective with combined expression of TAR-decoys (107).

5.3. Multitarget ribozymes

A novel approach to HIV-1 inhibition has been taken by Chen and colleagues who constructed several multitarget ribozymes with variable number of hammerhead motifs (mono-, di-, tetra-, penta-, and nonaribozymes), each of which was targeted to cleave HIV-1 env RNA at up to nine conserved sites (162). In cotransfection experiments in cell lines, activity of the ribozyme construct roughly paralleled the number of catalytic motifs on the molecules. More recent work in stably transduced T-cell lines has confirmed that the multimeric ribozymes confer an added resistance to HIV replication (187). Another modification of ribozymes using so-called "shotgun" ribozymes against HIV has been described (188). These are multiple ribozymes, flanked by cis-acting ribozymes at both their 3' and 5' ends. Upon transcription multiple ribozyme molecules are trimmed at their 3' and 5' ends, resulting in release of multiple independent ribozymes, each of which can be targeted differently. RNA decoys (RRE and TAR) can be added to the cis-acting ribozymes, further enhancing the antiviral activity of this technique by sequestering Tat and Rev in vitro (180). Despite promising results in vitro, however, these ribozymes have still not been studied in vivo (188).

5.4. Other combinations

Dual inhibition by antisense oligonucleotides to both the TAR and the polyadenylation signal has been shown to reduce the production of infectious HIV-1 by more than 99% in cell lines (99). Similarly, expression of multitargeting tat/rev antisense sequences has been shown to result in stable resistance to HIV in monocytic cells (189).

Recent data from primary T-cells and alveolar macrophages shows that combination of anti-Rev single chain immunoglobulin (SFv) and RRE RNA decoys delivered by AAV vectors results in synergistic inhibition of both laboratory and clinical isolates of HIV-1 (138). Although proviral sequences could be detected following challenge with HIV-1, viral production (p24) was 1000-fold lower in the cells harboring the combination.

The combination of an anti-Rev SFv which targets the Rev activation domain and a ribozyme which specifically targets RRE has recently been developed (190). A modest additive effect of the combination was demonstrated in T-cell lines.