Use of adenoviral VAI small RNA as a carrier for cytoplasmic delivery of ribozymes.
The in vivo effectiveness of therapeutic RNAs, like antisense molecules and ribozymes, relies on several features: RNA molecules need to be expressed at high levels in the correct cellular compartment as stable and active molecules. The exploitation of "natural" small RNA coding genes as expressing cassettes gives high chances to fulfill these requirements. We have investigated the utilization of the adenoviral VAI RNA as a cytoplasmatic carrier for expressing ribozymes against HIV-1. The conserved 5' leader sequence of HIV was chosen as a target, because it is present in all the viral transcripts and is highly conserved. Hammerhead ribozymes were substituted to different portions of the VAI RNA and the resulting chimera were tested in the in vivo system of Xenopus laevis oocytes for their level of accumulation, cellular compartmentalization, and assembly in specific ribonucleoparticles containing the La antigen. Interesting differences in the activity of the different chimera were found in both in vitro cleavage assays and S100 extracts of injected oocytes where the catalytic activity of the ribozymes in the RNP context can be analyzed.
Prislei
S
S
Istituto Pasteur, Fondazione Cenci-Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Università La Sapienza, Roma, Italy.
Buonomo
S B
SB
Michienzi
A
A
Bozzoni
I
I
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D001324:Autoantigens / Q000378:metabolism; D002451:Cell Compartmentation; D003593:Cytoplasm / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D015497:HIV-1 / Q000187:drug effects; D008845:Microinjections; D009865:Oocytes; D016337:RNA, Catalytic / Q000235:genetics* / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics*; D012261:Ribonucleoproteins / Q000378:metabolism; D014158:Transcription, Genetic; D014982:Xenopus laevis; D000097585:SS-B Antigen
D019380:Anti-HIV Agents; D001324:Autoantigens; D016337:RNA, Catalytic; D012367:RNA, Viral; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; C052835:adenovirus associated RNA