Dendritic cells (DCs) are effective in stimulating and controlling the outcome of T cell responses. Human immunodeficiency virus type 1-based lentiviral vectors can achieve sustained transduction of genes/antigens in dividing and nondividing cells, thus representing a candidate vector for stable expression of antigens in DCs. We previously established conditions for transduction of purified cytokine mobilized rhesus CD34+ cells in vitro, and transplantation of the autologous transduced cells in a nonhuman primate model in vivo. In the present study, we transplanted DCs derived from EGFP-transduced CD34+ cells into nonmyeloablated rhesus macaques. Transplantation of DCs stably expressing EGFP into autologous animals induces persistent, long-lived (up to 100 weeks) EGFP-specific T cell responses. Of note, no humoral responses against EGFP are detected in the transplanted animals. These studies provide, to our knowledge, the first demonstration that lentiviral transduction of CD34+ progenitor cells subsequently differentiated to DCs is capable of priming a specific T cell response in a nonhuman primate in vivo. Taken together, our data provide formal in vivo evidence that lentivirus-transduced dendritic cells represent a potential approach in eliciting cellular immune responses in primates.

Specifically and effectively directing a therapeutic gene to its intended site of action is a critical issue for translation of basic genomics to clinical gene therapy. Delivering gene therapy vectors to specific cells or tissues through intravenous injection is the most desirable method for this purpose. In 2001, we reported successful targeted gene transduction in vitro utilizing both oncoretroviral and lentiviral vectors pseudotyped with a chimeric Sindbis virus envelope (ZZ SINDBIS). However, these pseudotypes mediated nonspecific gene transduction to liver and spleen in vivo. To address this problem we generated the modified ZZ SINDBIS ( termed m168) with significantly less nonspecific infectivity. To investigate the ability of m168 pseudotyped lentiviral vector to mediate targeted gene transduction in vivo, we utilized a metastatic tumor model by using mouse melanoma cells engineered to express human P-glycoprotein. We administered the m168 pseudotyped vector conjugated with anti-P-glycoprotein antibody into the mice intravenously to target metastatic melanoma. The m168 pseudotyped vector selectively infected metastatic melanoma cells demonstrating successful targeted gene transduction in vivo. Targeting technology based upon m168 can be further modified for application not only to cancer but also potentially to genetic, neurologic, infectious and immune diseases, thereby expanding the future application of gene therapy.

Live attenuated HIV vaccines offer a means to introduce exogenous sequences into the viral genome to target the virus elimination in vivo. Foreign genes inserted into the nef region of HIV-1 NL4-3 were found to be rapidly deleted following virus infection and/or replication, in a size dependent manner, in the human fetal Thymus/Liver implants of severe combined immunodeficient mouse (SCID-hu) model. When the murine heat stable antigen (HSA) of 283 bp was substituted into HIV-1 nef region, the viral loads in vivo were comparable to the negative control nef attenuated HIV-1, and the reporter HSA gene was not deleted upon infection. However, the murine Thy1.2 gene (505 bp) substituted into the nef attenuated HIV-1, upon infection and replication, deleted 441 bp in vitro and 437 bp in vivo, of the inserted Thy1.2 gene. When the enhanced green fluorescence protein (eGFP) gene (720 bp) was substituted for nef, virus replication was aborted in vivo in the Thy/Liv implants, as seen by the background levels of viral loads, comparable to mock infected implants, and the eGFP gene was deleted. When the herpes simplex virus thymidine kinase gene, HSV-TK (1.15 kbp), or HSA gene, was substituted into the viral vpr gene, TK but not HSA gene was deleted, upon infection in vitro. Moreover, NL-TKI reporter virus with both intact nef and vpr genes shows deletion of TK gene both in vitro and in vivo. Excision of foreign genes occurred within the exogenous segments but not in the viral own regions. These results suggest that larger "suicide" genes introduced via HIV-1 can be deleted upon infection. However, smaller size nucleotide sequences or genes (approximately 300 bp) inserted in place of viral nef or vpr gene may be used to target the virus or its components, for attack and elimination in vivo, and thus have implications for the development of live attenuated HIV vaccines.

We examined the potential of ex vivo gene therapy to enhance bone repair using lentiviral vectors encoding either enhanced green fluorescent protein (EGFP) as a reporter gene or bone morphogenetic protein-2 (BMP-2) downstream of either the cytomegalovirus immediate early (CMV) promoter or the murine leukemia virus long terminal repeat (RhMLV) promoter derived from a murine retrovirus adapted to replicate in a rhesus macaque. In vitro, rat bone marrow stromal cells (BMSCs) transduced with Lenti-CMV-EGFP or Lenti-RhMLV-EGFP demonstrated over 90% transduction efficiency at 1 week and continued to demonstrate stable expression for 8 weeks. ELISA results demonstrated that lentivirus-mediated gene transfer into BMSCs induced stable BMP-2 production in vitro for 8 weeks. Increased EGFP and BMP-2 production was noted with the RhMLV promoter. In addition, we implanted BMSCs transduced with Lenti-RhMLV-BMP-2 into a muscle pouch in the hind limbs of severe combined immune deficient mice. Robust bone formation was noted in animals that received Lenti-RhMLV-BMP-2 cells at 3 weeks. These results demonstrate that lentiviral vectors expressing BMP-2 can induce long-term gene expression in vitro and new bone formation in vivo under the control of the RhMLV promoter. Prolonged gene expression may be advantageous when developing tissue engineering strategies to repair large bone defects.

We read with great interest the article by Morris et al demonstrating the appearance of cytotoxic T lymphocytes specific to a transgene protein in baboons following myeloablative conditioning and transplantation of cytokine mobilized bone marrow (BM)–derived immunoselected CD34+ cells transduced

Oral mucosal cells can be infected by exogenous HIV during receptive oral sex or breast-feeding. The risk of oral mucosal infection depends on the infection efficiency of the HIV strains present in the oral cavity, the viral titers, and the defense mechanisms in the oral cavity environment. It is expected that alcohol can weaken the host defense mechanism against HIV infection in the oral cavity. We modified an HIV strain, NL4-3, by inserting the enhanced green fluorescent protein gene and used this virus to infect oral epithelial cells obtained from patients. Various concentrations of ethanol (0%-4%) were added to the infected cells. HIV-infected cells were detected by fluorescent microscopy or fluorescence-activated cell sorting. We found that ethanol significantly increases HIV infection of primary oral epithelial cells (POEs). POEs pretreated with 4% ethanol for less than 10 minutes demonstrated 3- to 6-fold higher susceptibility to infection by the CXCR-4 HIV strain NL4-3. Our studies also demonstrated that HIV infects POEs through a gp120-independent mechanism. We tested an HIV CCR5 strain, JRCSF, and also found its infection efficiency to be stimulated by alcohol. Our results indicate that in cell culture conditions, the ranges of concentrations of alcohol that are commercially available are able to stimulate the infection efficiency of HIV in POEs.

RNA interference is an evolutionarily conserved process of gene silencing that in plants serves as a natural defense mechanism against exogenous viral agents. RNA interference is becoming an important tool for the study of biological processes through reverse genetics and has potential for therapeutic applications in humans; however, effective delivery is still a major issue. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) have been introduced into cells by transfection of chemically synthesized and RNA expression via plasmid cassettes utilizing RNA polymerase III transcription. The employment of siRNA/shRNA for gene knockout requires an efficient stable transfection or transduction process. Here, we report the successful construction of lentiviral vectors to express shRNA stably in human cells. We demonstrate that lentiviral vectors expressing siRNA directed to the reporter gene luciferase, when stably transduced into human cells without drug selection, are capable of protecting the cells from infection by a lentiviral vector encoding humanized firefly luciferase as a reporter gene. We observed 16- to 43-fold reduction of gene expression in infected cells transduced with shRNA vectors relative to cells transduced with control vectors. This model system demonstrates the utility of lentiviral vectors to stably express shRNA as both a cellular gene knockout tool and as a means to inhibit exogenous infectious agents such as viruses in human cells.

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

Modeling human hematopoietic progenitor cell gene therapy in nonhuman primates allows long-term evaluation of safety, maintenance of gene expression, and potential immune response against transgene products. We transplanted autologous G-CSF/SCF-mobilized CD34+ cells transduced with lentiviral vectors expressing EGFP into myeloablated rhesus macaques. To date, more than 4 years posttransplantation, 0.5–8% EGFP expression is maintained in multiple cell lineages. The animals remain healthy with no evidence of hematopoietic abnormalities or malignancies. To assess immune functions, we actively immunized two of our transplanted animals with purified rEGFP proteins and CpG adjuvant and demonstrated stable levels of EGFP+ cell populations maintained for over 29 months despite four active immunizations. We did not detect a persistent anti-EGFP antibody response or anti-EGFP T cell response in these immunized animals. Immune response to an irrelevant antigen was normal. Taken together, our data provide formal support that transplantation of lentivirally transduced CD34+ progenitor cells in myeloablated rhesus macaques induces specific immunological tolerance toward a foreign transgene.

Infection of CD4- cells by HIV-1 is well documented, but the mechanism responsible remains a matter of discussion. Previously we modified an HIV-1 virus strain, NL4-3, by deleting the Env proteins (gp41 and gp120) and inserting the enhanced green fluorescent protein (EGFP), and found that the Env(-) virus infects several types of CD4- cells. Here, we have prepared Env(-) virus from both the CD4- cell line, 293T, and the CD4+ cell lines, CEM and SUPT1, and found that HIV-1 Env(-) virus from either cell type is infectious for both CD4+ and several CD4- cell lines. Replication of HIV-1 Env(-) virus-infected cells was demonstrated by p24 gag protein assays and real-time reverse transcriptase polymerase chain reaction (RT-PCR) of the culture medium from infected cells. Virus collected from the HIV-1-Env(-) infected cultures proved infectious to several CD4- cell lines. Our results suggest that HIV-1 infects both CD4- and CD4+ cells using a gp120-independent mechanism. This infection mechanism may provide new explanations for HIV-1 latency and persistent infection in patients.

Although Nef has been proposed to effect the escape of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTL) through downmodulation of major histocompatibility complex class I molecules, little direct data have been presented previously to support this hypothesis. By comparing nef-competent and nef-deleted HIV-1 strains in an in vitro coculture system, we demonstrate that the presence of this viral accessory gene leads to impairment of the ability of HIV-1-specific CTL clones to suppress viral replication. Furthermore, inhibition by genetically modified CTL that do not require major histocompatibility complex class I-presented antigen (expressing the CD4 T-cell receptor [TCR] zeta-chain hybrid receptor) is similar for both nef-competent and -deleted strains, indicating that Nef does not impair the effector functions of CTL but acts at the level of TCR triggering. In contrast, we note that another accessory gene, vpr, does not induce resistance of HIV-1 to suppression by CTL clones. We conclude that Nef (and not Vpr) contributes to functional HIV-1 immune evasion and that this effect is mediated by diminished antigen presentation to CTL.

Mucosal tissue is the main portal of entry for HIV-1 infection and, in macaques, has been demonstrated to be a significant compartment for viral replication and CD4+ T lymphocyte depletion. Quantitating tissue viral burden in addition to plasma viral load provides insights into HIV-1 pathogenesis and an additional means to gauge antiretroviral response. The aim of this study was to develop reliable, reproducible, and sensitive assays to quantitate tissue viral burden of HIV-1 RNA and DNA using 1–3 endoscopically acquired, rectosigmoid biopsies. Total DNA and RNA were simultaneously extracted following homogenization from the same tissue samples. Quantitative polymerase chain reaction (PCR) assay in the HIV-1 LTR region was used to detect viral DNA and RT-PCR for viral RNA. It was determined that HIV-1 RNA and DNA can be reproducibly quantified from a single rectosigmoid biopsy with minimal intra-assay or intra-patient variability. These results reflect high recovery of extracted nucleic acids with calculated results accurately reflecting in vivo levels. The techniques outlined differ from currently available approaches by incorporating control standards to identify loss or degradation of RNA and DNA from acquisition through the in vitro assay and permit extraction with high yields of RNA and DNA from the same tissue sample.