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The advent of combinatorial antiretroviral therapy proved to be highly effective in controlling HIV-1 disease progression, transforming AIDS from a deadly to a chronic condition. However, major current issues are systemic drug toxicity, generation of drug resistant viral mutants and persistence of a latent viral reservoir. In addition, drug-based regimens require daily intake and many patients cannot maintain the high level of adherence necessary for viral control. Given the limitations of the current therapeutic approaches and the absence of any effective vaccination strategy against HIV-1 infection, there is a pressing need to develop a curative treatment. The first reported cure of HIV-1 infection was provided by the apparent eradication of the virus in a patient transplanted with hematopoietic stem cells (HSCs) from a donor lacking the functional CCR5 co-receptor, which is used by most HIV-1 strains to enter target cells. However, while an important proof-of-principle, few individuals could benefit from such procedure due to toxicities of allogeneic rejection and limitations of finding compatible CCR5-negative donors. Therefore, gene therapy approaches aimed to modify autologous HSCs in order to render them resistant to HIV-1 infection have emerged as a promising direction. If successfully engrafted, these cells would offer continuous, long-term production of virus-resistant immune cells. To this end, several anti-HIV-1 genes have been developed and tested both in preclinical and clinical settings and, among these, RNA interference (RNAi)-based approaches represent one of the most powerful tools. Starting from these considerations, this study was aimed to develop lentiviral vectors expressing anti-HIV-1 RNAi triggers and to test their efficacy in relevant human primary cells for HIV-1 infection, including CD4+ T lymphocytes and macrophages. The final goal of the research project is to use the most effective and safe vector(s) to genetically modify HSCs harvested from AIDS-related lymphoma (ARL) patients, that offer a unique opportunity to evaluate anti-HIV-1 gene therapy strategies in an ethically acceptable clinical setting, as they often undergo HSC transplantation. To account for HIV-1 variability, we selected multiple RNAi triggers, including a short hairpin RNA (shRNA), generating one single small interfering RNA (siRNA) against either the CCR5 cellular gene (shCCR5) or the vif viral gene (shvif), and a long hairpin RNA, giving rise to two different siRNAs against the viral tat and rev overlapping first exons (lhtat/rev). Overall these molecules inhibit different steps of the HIV-1 life cycle, including entry into target cells (shCCR5), gene expression (lhtat/rev) and infectivity of the newly produced particles (shvif). In the first part of the work, we constructed vectors expressing the shCCR5, the shvif or the lhtat/rev as a single transcriptional unit under the control of different human polymerase III promoters (i.e. U6, 7SK or H1). These vectors allowed us to investigate promoter influence on the siRNA silencing activity, finding out the best combination of promoter-RNAi trigger for the development of a combinatorial antiviral approach. As a next step, we obtained combinatorial vectors simultaneously expressing the above described siRNAs, as independent transcriptional units within the same vector backbone. To optimize vector design, a number of different vectors were developed by using either the same or distinct promoters driving the expression of each RNAi trigger. Moreover, the transcriptional units were cloned in different position with respect to each other within the vector framework. Considering that the use of multiple highly active RNA polymerase III promoters can potentially saturate the endogenous microRNA biogenesis pathway, we also explored an alternative combinatorial strategy, based on the use of an extended shRNA (e-shRNA). This molecule simultaneously expresses the three siRNAs targeting the CCR5, the vif and the tat/rev transcripts, under the control of the U6, the 7SK or the H1 promoter. When comparing the antiviral activity of all the different combinatorial platforms, we could identify two vectors (i.e. U6shCCR5-7SKshvif-H1lhtat/rev and H1e-shRNA) conferring highly potent protection against HIV-1 infection in both cell lines and human primary cells, in the absence of cytotoxicity. Overall, our findings highlighted some important strengths and pitfalls of different approaches used for multiple siRNAs delivery, providing valuable insights for the design and application of reliable combinatorial RNAi to counteract HIV-1 replication. In addition, this study contributed to the identification of new anti-HIV-1 combinatorial platforms that, once shown to be effective and safe in vivo, may be next in line for clinical testing.