Acquired immune deficiency syndrome (AIDS) caused by human immunodeficiency virus type 1 (HIV-1) infection remains one of the most important global public health threats 
. Advances in anti-HIV-1 therapy such as highly active antiretroviral therapy (HAART) have markedly improved patient survival. However, most of these treatments are likely never to be curative and are limited by toxicity, cost, and, especially, the emergence of resistant viral strains 
. Thus, alternative methods that decrease or eliminate the need for such lifelong continuous treatments and combat HIV-1 resistance to current therapies are greatly desired. Gene therapy which could provide lifelong therapeutic interventions with one or few administrations is a potentially promising approach 
HIV-1 utilizes the CD4 receptor and, primarily, chemokine receptors CXCR4 and CCR5, as co-receptors that are crucial for viral entry into host cells 
. As such, inhibition of the co-receptor-virion interaction has elicited much therapeutic interest 
. Landmark population genetic and molecular studies demonstrated that individuals homozygous for a defective CCR5 gene, CCR5Δ32, are protected from HIV-1 infection 
. Recently, an HIV-1 positive individual with concurrent acute myeloid leukemia (AML) was treated by transplant of allogeneic hematopoietic stem progenitor cells (HSPCs) isolated from a CCR5Δ32 homozygous donor. Remarkably, the CCR5Δ32 donor cells nearly completely replaced the recipient's cells within a rapid 61 days and HIV-1 virus has remained undetectable in the patient despite discontinuation of HAART for more than 4 years 
. This striking evidence supports the idea that stable down-regulation of CCR5 expression could result in reduced viral load and prevent the progression to AIDS in HIV-1 infected patients. However, the difficulty of finding a human leukocyte antigen matched CCR5Δ32 homozygous donor considerably limits widespread use of this strategy.
RNA interference (RNAi) is a highly evolutionarily conserved mechanism of post-transcriptional gene silencing that can be triggered by small interfering RNAs (siRNAs) 
. In addition to its experimental utility in loss-of-function studies, RNAi emerges as a potentially powerful therapeutic approach towards human diseases. We and others have developed lentiviral vectors encoding short hairpin RNA (shRNA), commonly driven by RNA polymerase III promoters H1 or U6, which are processed by cellular machinery into siRNA for stable inhibition of HIV-1 co-receptors and HIV-1 gene expression 
. We previously demonstrated efficient knock-down of CCR5 expression via H1 promoter-driven expression of a highly potent CCR5-specific shRNA (sh1005) in human primary T-cells 
and macrophages 
, resulting in strong inhibition of HIV-1 infection in vitro
. Importantly, we found it necessary to screen extensively for shRNAs that maintain potency at low expression levels to avoid cytotoxic effects associated with shRNA overexpression 
Use of various humanized small animal models, where immunocompromised mice are reconstituted with either HSPCs or differentiated T-cells to confer susceptibility to HIV-1 infection, have facilitated preclinical assessments of in vivo
efficacy of various CCR5-targeted HIV-1 therapies. While several humanized mouse model studies have focused on systemically delivered methods such as CCR5-specific RNAi inducers, coupled with aptamers 
, nanoparticles 
, or peptides 
, as well as small molecule CCR5 antagonists 
which require repeated doses, potentially longer-lasting strategies using genetically modified HSPCs have also been explored in vivo
. While others have employed zinc-finger nuclease-mediated genome editing 
, we have demonstrated continuous RNAi-mediated down-regulation of CCR5 expression via vector-transduced HSPC transplantation 
. To our knowledge, we reported the first application of the humanized bone marrow/liver/thymus (hu-BLT) mouse model 
as an in vivo
AIDS gene therapy model.
The hu-BLT mouse model provides robust peripheral reconstitution of human T-cells, B-cells, and macrophages and importantly, unlike other models, efficient repopulation of many lymphoid tissue compartments including highly CCR5-expressing bone marrow and gut-associated lymphoid tissue (GALT), a primary target site of CCR5-tropic HIV-1 infection 
. Hence, the hu-BLT mouse has become a model of choice to investigate HIV-1 infection and pathogenesis. Previously, we showed in hu-BLT mice successful engraftment of transplanted fetal-liver-derived CD34+
) cells transduced with the sh1005-encoding vector and differentiation into CCR5-down-regulated T-cells and monocytes/macrophages in peripheral blood and systemic lymphoid organs 
. Similar observations were seen in our nonhuman primate rhesus macaque studies 
. Cells transduced with this vector showed excellent protection against CCR5 (R5)-tropic 
, but not CXCR4 (X4)-tropic 
, viral strains. Therefore, CCR5 down-regulation, although promising against infection by R5-tropic viral strains, would be ineffective against pre-existing X4-tropic and dual tropic strains or the emergence of viral escape mutant strains, necessitating the incorporation of additional therapeutic reagents.
To confer protection against HIV-1 strains unimpeded by sh1005-mediated CCR5 down-regulation, we evaluated the anti-HIV-1 effects of selected previously published shRNAs targeting conserved regions of the HIV-1 genomic sequence. After screening for candidates with high anti-viral effects at low shRNA expression levels, we selected sh516, which targets the long terminal repeat (LTR) R region of HIV-1. Following extensive vector characterization in vitro, we evaluated the in vivo reconstitution and stability of HSPCs engineered with our novel sh1005/sh516 combination vector and assessed conferred anti-viral potency of transplanted HSPC-derived T lymphocytes. Here, we report that transplantation of sh1005/sh516-transduced HSPCs resulted in efficient engraftment, stable marking in resultant hematopoietic lineages and potent inhibition of HIV-1-mediated depletion of modified CD4+ T-cells in vivo. This work describes a novel and safe combination vector which may provide both potent anti-viral inhibition and high reconstitution efficiencies for effective control of HIV infection.