Jayan GC, Bouhamdan M, Hyman P, Lamothe M, Johnson RP, Lisziewicz J, Strayer DS; Conference on Retroviruses and Opportunistic Infections.
7th Conf Retrovir Oppor Infect Jan 30 Feb 2 2000 Conf Retrovir Oppor Infect 7th 2000 San Franc Calif. 2000 Jan 30-Feb 2; 7: 194 (abstract no. 636).
Jefferson Med. Coll., Philadephia, PA.
Tat protein encoded by HIV-1 is a trans-activator of transcription of the viral genome, and is essential for virus replication. Therefore, inhibition of Tat function is an ideal strategy for gene therapy of HIV-1 infection. We constructed a bifunctional antitat gene expressing a polymeric TAR (Tat Activation Response element) sequence and an antisense-tat sequence, driven by the HIV-1 LTR promoter. This construct would both sequester Tat protein and block Tat translation from mRNA. The antitat gene was delivered into target cells using a simian-virus 40 (SV40) vector. Prior work indicated that SV40 is an effective gene delivery vector for human lymphocytes, and that it provided a high enough level of transduction that selection of transduced cells was unnecessary. The HIV-1 LTR promoter followed by 25 tandem repeats of TAR cDNA, and a cDNA sequence antisense to the tat gene was cloned into the SV40 genome in place of large T antigen (Tag). To make the vector (SV[PolyTAR]), the viral genome was excised from carrier plasmid, gel-purified, recircularized, and then transfected into COS-7 cells. COS-7 cells are the packaging cell line for recombinant SV40 vectors. They supply the Tag necessary to produce the viral vector in trans. Crude virus stocks were prepared as cell lysates, then purified by sucrose-equilibrium density gradient centrifugation. Infectivity of replication-defective SV40 stocks, assayed by in situ PCR, was 1011 infectious units/ml. For delivery of the polyTAR + antitat gene into lymphocytes (SupT1 cells), the cells were treated with SV[PolyTAR] for 24 h at an MOI of 10. This step was repeated two more times on subsequent days, at an MOI of 3. No selection was used. Expression of antitat in transduced cells was detected by RT-PCR, using primers specific to the antitat sequence. Transduced cells were challenged with cell-free HIV (strain pNL-4-3) at two different doses (0.1 pg/ml and 0.2 pg/ml). After challenge, cells were maintained at a density of approximately 10(6)/ml, and culture supernatants were collected for HIV-1 p24 antigen analyses, which were determined by ELISA. Cells were also examined for the presence of syncytia. We found that HIV-1 replication as measured by p24 content and HIV1-induced syncytia formation was significantly inhibited in cells expressing the polyTAR decoy + antitat gene delivered by the SV40 vector. Therefore, antitat gene delivery mediated by the SV40 vector is a promising strategy for inhibiting HIV-1 replication and for gene therapy of HIV-1.
Publication Types:
Keywords:
- Acquired Immunodeficiency Syndrome
- Cell Line
- Delivery, Obstetric
- Female
- Gene Products, tat
- Gene Therapy
- Genes, tat
- Genetic Vectors
- HIV Infections
- HIV Long Terminal Repeat
- HIV-1
- Humans
- Simian virus 40
- Virus Replication
- genetics
- surgery
- virology
Other ID:
UI: 102243510
From Meeting Abstracts