A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense, decoy, and sense RNAs, and trans-dominant negative mutant Gag and Env proteins.
Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Psi(e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Psi(e) signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.
Ding
Shi-Fa
SF
Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
Lombardi
Rocco
R
Nazari
Reza
R
Joshi
Sadhna
S
D002460:Cell Line; D015686:Gene Products, env / Q000235:genetics* / Q000378:metabolism; D015683:Gene Products, gag / Q000235:genetics* / Q000378:metabolism; D015694:Gene Products, rev / Q000494:pharmacology; D015696:Gene Products, tat / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D015658:HIV Infections / Q000628:therapy; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008979:Moloney murine leukemia virus / Q000235:genetics; D009154:Mutation; D016366:Open Reading Frames; D016372:RNA, Antisense / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis; D013601:T-Lymphocytes / Q000821:virology; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014779:Virus Replication; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015686:Gene Products, env; D015683:Gene Products, gag; D015694:Gene Products, rev; D015696:Gene Products, tat; D016372:RNA, Antisense; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus