A variety of gene therapeutic approaches have begun to be developed to combat human immunodeficiency virus type I (HIV-1) infections. One classical conception of intracellular immunization is to express in the host a mutant form of a viral protein which can interfere with the replication of the virus. We now propose novel methodologies in the development of new intracellular immunization techniques against HIV-1 replication. The isolated immunoglobulin variable regions (light and heavy chains) of monoclonal antibodies directed against select HIV-1 proteins will be directly cloned, utilizing reverse transcriptase-initiated polymerase chain reactions (RT-PCR), from hybridoma cell-lines. Single chain variable regions (SFv) (or single chain antibodies) will be constructed, using designed linker peptides. SFv moieties incorporate the complete antigen- binding domain (Fv) into a single polypeptide. Preliminary data are presented in which an anti-Rev SFv molecule is cloned and expressed in bacteria, as well as in mammalian cells. This anti-Rev SFv, when intracellularly expressed, is demonstrated to have potent anti-HIV-1 activity. Anti-HIV-1 SFv molecules will be constructed against select HIV-1-encoded proteins (e.g., Tat, Rev, Integrase, Gag - p24 and - p17), binding affinities will be evaluated in vitro and the constructs will be intracellularly expressed, with and without nuclear localization signals, in various target cell-types. The SFvs' anti-HIV-1-activities will be evaluated. In addition, retroviral shuttle vectors and adeno-associated virus vectors will be developed to efficiently transduce these SFv moieties into specific cells. Importantly, in collaborative efforts, studies using various anti-HIV-1 SFv moieties in primary CD4-positive PBMC, bone marrow stem cells and thymocytes, ex vivo, will be highlighted. These efforts will focus on inhibition of low passage primary HIV-1 isolates, as model systems for genetic therapy of HIV-1 infection in humans, utilizing targeted SFv constructs. Systems for constitutive and inducible expression of intracellular SFv molecules will be explored. Finally, the severe combined immunodeficiency mouse reconstituted with human peripheral blood lymphocytes (Human - PBL-SCID mouse) will be used as an in vivo model to assess the efficacy of anti-HIV-1 SFv molecules, expressed intracellularly, as a therapeutic modality against HIV-1 infection. Thus, the exquisite binding specificity and nearly limitless diversity of monoclonal antibodies will be brought to bear in gene therapy against HIV-1.
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