Advances in HIV Gene Therapy Rose Kitawi, Scott Ledger, Anthony D. Kelleher, and Chantelle L. Ahlenstiel summarize recent developments in HIV gene therapy. Early gene therapy studies showed promise for curing heritable diseases but faced setbacks due to genotoxic events. Recent advances in genetic engineering have renewed interest, leading to the approval of the first gene therapy product targeting genetic mutations in 2017. Gene therapy (GT) can be delivered in vivo or ex vivo. Ex vivo approaches allow for cell characterization and selection before administration, reducing immune rejection risks. This review highlights ex vivo gene therapy stages, current research, and HIV gene therapy studies, which predominantly use ex vivo methods. HIV cure challenges include the latent viral reservoir, which is resistant to current therapy. Antiretroviral therapy (ART) may not effectively reach tissues harboring latent virus, leading to viral persistence. HIV also impairs immune function, reducing CD4+ T cell reconstitution. Effective cure strategies aim to inactivate or remove latent virus and restore immune function. Gene-modified cells should resist infection and avoid severe adverse effects. Strategies include stem cell transplantation, the 'Shock and Kill' approach, gene editing with nuclease-based tools, and the 'Block and Lock' approach. Ex vivo gene therapy involves modifying cells, typically hematopoietic stem cells (HSCs), which can be sourced from bone marrow, peripheral blood, umbilical cord, or placenta. HSCs are characterized by markers like CD34 and are crucial for blood cell production. Various methods are used to isolate and purify HSCs, including magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Culturing HSCs for more than 48 hours reduces their engraftment capacity, so cultures are typically limited to 36 hours. Gene delivery vectors include lentiviral vectors, which can transduce both dividing and non-dividing cells, and adeno-associated virus (AAV) vectors, which are used for transient expression. Lentiviral vectors face challenges like transgene silencing and insertional mutagenesis, which are addressed through modifications like self-inactivating (SIN) vectors and altering integrase function. AAVs are limited by pre-existing immunity to their capsid proteins. Gene-modified HSCs are expanded and selected for engraftment, often using cell surface markers or antibiotic resistance genes. Expansion is achieved with serum-free media and growth factors to maintain stemness. Conditioning regimens, including myeloablative (MAC), non-myeloablative (NMA), and reduced intensity conditioning (RIC), are used to clear the hematopoietic niche and support engraftment. MAC regimens are highly toxic, while NMA and RIC are less toxic but less effective for gene therapy. In vivo selection techniques use drug-resistance genesAdvances in HIV Gene Therapy Rose Kitawi, Scott Ledger, Anthony D. Kelleher, and Chantelle L. Ahlenstiel summarize recent developments in HIV gene therapy. Early gene therapy studies showed promise for curing heritable diseases but faced setbacks due to genotoxic events. Recent advances in genetic engineering have renewed interest, leading to the approval of the first gene therapy product targeting genetic mutations in 2017. Gene therapy (GT) can be delivered in vivo or ex vivo. Ex vivo approaches allow for cell characterization and selection before administration, reducing immune rejection risks. This review highlights ex vivo gene therapy stages, current research, and HIV gene therapy studies, which predominantly use ex vivo methods. HIV cure challenges include the latent viral reservoir, which is resistant to current therapy. Antiretroviral therapy (ART) may not effectively reach tissues harboring latent virus, leading to viral persistence. HIV also impairs immune function, reducing CD4+ T cell reconstitution. Effective cure strategies aim to inactivate or remove latent virus and restore immune function. Gene-modified cells should resist infection and avoid severe adverse effects. Strategies include stem cell transplantation, the 'Shock and Kill' approach, gene editing with nuclease-based tools, and the 'Block and Lock' approach. Ex vivo gene therapy involves modifying cells, typically hematopoietic stem cells (HSCs), which can be sourced from bone marrow, peripheral blood, umbilical cord, or placenta. HSCs are characterized by markers like CD34 and are crucial for blood cell production. Various methods are used to isolate and purify HSCs, including magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Culturing HSCs for more than 48 hours reduces their engraftment capacity, so cultures are typically limited to 36 hours. Gene delivery vectors include lentiviral vectors, which can transduce both dividing and non-dividing cells, and adeno-associated virus (AAV) vectors, which are used for transient expression. Lentiviral vectors face challenges like transgene silencing and insertional mutagenesis, which are addressed through modifications like self-inactivating (SIN) vectors and altering integrase function. AAVs are limited by pre-existing immunity to their capsid proteins. Gene-modified HSCs are expanded and selected for engraftment, often using cell surface markers or antibiotic resistance genes. Expansion is achieved with serum-free media and growth factors to maintain stemness. Conditioning regimens, including myeloablative (MAC), non-myeloablative (NMA), and reduced intensity conditioning (RIC), are used to clear the hematopoietic niche and support engraftment. MAC regimens are highly toxic, while NMA and RIC are less toxic but less effective for gene therapy. In vivo selection techniques use drug-resistance genes