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Exploiting the phenomenon of RNA interference (RNAi), recent studies established the utility of monospecific small interfering RNAs (siRNAs) in suppressing HIV-1 infection. However, because of the high mutation rate of the HIV genome, there are considerable challenges in the design of fully efficacious gene therapeutic constructs. Therefore, approaches that simultaneously target different stages of the viral life cycle are desirable. In our current studies, we designed bispecific siRNA constructs against HIV-1 cell surface receptors to inhibit viral entry. Dual specific short hairpin siRNA constructs, containing an 8-nucleotide intervening spacer, targeted against either CXCR4 and CD4 or CCR5 and CXCR4 were synthesized by in vitro transcription. Cleavage of the bispecific constructs yielding monospecific siRNAs was shown to occur in cell extracts. Magi-CXCR4 and CCR5 cells transfected with bispecific siRNAs showed significant downregulation of their respective coreceptors, as determined by FACS analysis. This suggested that combinatorial constructs comprising multiple effector motifs were processed in transfected cells into their respective functional siRNAs. Transfected cells were challenged with either X4 (NL4-3) or R5-tropic (BaL-1) strains of HIV-1. Downregulation of the cell surface receptors coincided with resistance to in vitro viral challenge in both Magi cell lines and peripheral blood mononuclear cells (PBMCs). These results demonstrated the practical utility of short hairpin siRNA bispecific constructs synthesized as a single transcript. Because the short hairpin design will permit tandem assembly of multiple effector motifs, it is now possible to introduce promising multivalent siRNA constructs into retroviral and lentiviral vectors for in vivo gene therapeutic applications.

The phenomenon of RNA interference (RNAi) sparked a new surge in the area of posttranscriptional gene silencing methodologies and their potential application for HIV-1 gene therapy. A potentially promising strategy is to exploit siRNAs to prevent viral entry at the cell surface by down-regulating essential cell surface HIV-1 coreceptors. In the present studies we targeted the CXCR4 coreceptor for disruption with siRNA to inhibit HIV-1 entry as a first step toward the ultimate goal of translating this to gene therapy for AIDS. A stem-loop hairpin structured anti-CXCR4 siRNA was designed and synthesized in vitro by transcription with T7 polymerase. Down-regulation of the coreceptor was assayed in U373-Magi-CXCR4 cells. FACS analysis showed marked down-regulation of CXCR4 on the cell surface and Western blot analysis confirmed the reduced levels of intracellular synthesis. When challenged with X4-tropic HIV-1 NL4-3, the siRNA-transfected cells exhibited marked viral resistance. Consistent with these results, siRNA-transfected primary lymphocytes also exhibited significant resistance to HIV-1 entry. These proof-of-concept studies demonstrated the efficacy of an siRNA targeted to an essential cellular coreceptor CXCR4 in protecting from HIV-1 infection. Delivery of this siRNA into hematopoietic stem cells via lentiviral vectors may have potential gene therapeutic applications.

Lentiviral vectors are attractive candidates for gene therapy because of their ability to integrate into nondividing cells. To date, conventional HIV-1-based vectors can be produced at higher titers, but concerns regarding their safety for human use exist because of the possibility of recombination leading to production of infectious virions with pathogenic potential. Development of lentivirus vectors based on nonhuman lentiviruses constitutes an active area of research. We described a novel HIV-SIV hybrid vector system in which an HIV-1-derived transfer vector is encapsidated by SIVmac1A11 core particles and pseudotyped with VSV glycoprotein G. In an effort to further develop this vector system, we modified the packaging plasmid by deletion of the SIV accessory genes. Specifically, versions of the packaging plasmid (SIVpack) lacking vif, vpr, vpx, and/or nef were constructed. Our results indicate that, as with HIV-1-based packaging plasmids, deletion of accessory genes has no significant effect on transduction in either dividing or nondividing cells. The SIV packaging plasmid was also modified with regard to the requirement for RRE and rev. Deletion of the RRE and rev from SIVpack led to dramatic loss of transduction ability. Introduction of the 5′ LTR from the spleen necrosis virus to packaging plasmids lacking RRE/Rev was then sufficient to fully restore vector titer. A minimal SIV transfer vector was also developed, which does not require RRE/Rev and exhibits no reduction in transduction efficiency in two packaging systems. The SIV-based vector system described here recapitulates the biological properties of minimal HIV-1-derived systems and is expected to provide an added level of safety for human gene transfer. We suggest that the SIV-derived vector system will also be useful to deliver anti-HIV-1 gene therapy reagents that would inhibit an HIV-1-derived vector.

HIV-1 infection of susceptible cells is mediated by the specific interaction of viral envelope glycoproteins with the cell surface CD4 receptor and a chemokine coreceptor, CCR5 or CXCR4. Individuals with a CCR5 genetic defect show resistance to HIV-1 infection, indicating that downregulation of CCR5 expression on target cells can prevent viral infection. In previous studies we demonstrated the utility of an anti-CCR5 ribozyme targeted to a single cleavage site in downregulating CCR5 expression and consequently providing resistance to viral infection. To improve on the level of downregulation we designed a construct containing an anti-CCR5 ribozyme heterotrimer (R5RbzTM) targeted to three different cleavage sites in CCR5 mRNA. In vitro tests showed that the anti-CCR5 ribozyme heterotrimer could effectively cleave the CCR5 RNA substrates to yield products of the expected sizes. This construct was introduced into various retroviral vectors for stable gene transduction. HOS.CD4/R5 cells stably transduced with this anti-CCR5 heterotrimer showed a marked reduction in the surface expression of CCR5 and a concomitant 70% reduction in macrophage-tropic viral infection. In addition, a retroviral vector containing the anti-CCR5 ribozyme heterotrimer and an anti-HIV-1 tat-rev ribozyme heterodimer was constructed. This construct also showed a similar inhibition of CCR5 surface expression and reduced infectability by the macrophage-tropic HIV-1 vector in HOS.CD4/R5 cells. The trimeric and multimeric ribozyme constructs were transduced into CD34+ hematopoietic progenitor cells to determine their effects on lineage-specific differentiation. We show that multivalent ribozyme gene-transduced hematopoietic progenitors differentiated normally into mature macrophages that bear CD14 and CD4 surface markers. Macrophages containing the transgenes expressed ribozymes, and showed resistance to M-tropic HIV-1 infection. These results provide strong support for the use of the trimeric anti-CCR5 ribozyme approach in a gene therapy setting for the treatment of HIV infection.

The cellular entry of HIV is mediated by the specific interaction of viral envelope glycoproteins with the cell-surface marker CD4 and a chemokine receptor (CCR5 or CXCR4). Individuals with a 32-base-pair (bp) deletion in the CCR5 coding region, which results in a truncated peptide, show resistance to HIV-1 infection. This suggests that the downregulation of CCR5 expression on target cells may prevent HIV infection. Therefore, ribozymes that inhibit the CCR5 expression offer a novel approach for anti-HIV gene therapy. To assess the effect of an anti-CCR5 ribozyme (R5Rbz) on macrophage differentiation, CD34+hematopoietic progenitor cells were transduced with a retroviral vector carrying R5Rbz and allowed to differentiate in the presence of appropriate cytokines. R5Rbz-transduced CD34+cells differentiated normally into mature macrophages that carried CD14 and CD4 surface markers, expressed the anti-CCR5 ribozyme, and showed significant resistance to viral infection upon challenge with the HIV-1 BaL strain. Using an in vivo thymopoiesis model, the effect of R5Rbz on stem cell differentiation into thymocytes was evaluated by reconstituting SCID-hu mice thymic grafts with ribozyme-transduced CD34+cells. FACS analysis of cell biopsies at 4 and 6 weeks postengraftment for HLA, CD4, and CD8 markers showed comparable levels of reconstitution and similar percentages of subpopulations of thymocytes between grafts receiving R5Rbz-transduced and control CD34+cells. RT-PCR assays demonstrated the expression of the anti-CCR5 ribozyme in CD4+, CD8+, and CD4+/CD8+thymocyte subsets derived from R5Rbz-transduced CD34+cells. These results indicate that anti-CCR5 ribozyme can be introduced into hematopoietic stem cells without adverse effects on their subsequent lineage-specific differentiation and maturation. The expression of anti-CCR5 ribozymes in HIV-1 target cells offers a novel gene therapy strategy to control HIV infection.Molecular Therapy (2000) 1, 244–254; doi: 10.1006/mthe.2000.0038

Gene therapy of human T-lymphocyte disorders, including acquired immunodeficiency syndrome (AIDS), would be greatly facilitated by the development of an in vivo system in which transduced human hematopoietic stem cells can be used to reconstitute the T-lymphoid compartment. Here we use the SCID-hu mouse asa recipient for human CD34+ hematopoietic progenitor cells transduced in vitro with a retroviral vector carrying the neomycin resistance gene (neoR).The transduced cells engraft and reconstitute the lymphoid compartments of the human thymus implant with as few as 5 × 104CD34+cells. The neoRgene was expressed at low levels in human thymocytes and there was noapparent effect on thymocyte differentiation as a result of vector transduction. Thus, this SCID-hu mouse system is the first in vivo model showing human thymopoiesis after transduction of exogenous vectors, and should allow preclinical testing of gene therapeutic reagents designed to function in human cells of the T-lymphoid lineage. Because human immunodeficiency virus type 1 infection induces depletion of human thymocytes in SCID-hu mice, this system may be particularly valuable in evaluating efficacy of gene therapies to combat AIDS.

Adequate antiretroviral (ARV) concentrations in lymphoid tissues are critical for optimal antiretroviral therapy (ART). While the spleen contains 25% of the body’s lymphocytes, there are minimal data on ARV penetration in this organ. This study quantified total and protein-unbound splenic ARV concentrations and determined whether drug transporters, sex, or infection status were modifiers of these concentrations in animal models and humans. Two humanized mice models (hu-HSC-Rag [n= 36; 18 HIV-positive (HIV+) and 18 HIV-negative (HIV−)] and bone marrow-liver-thymus [n= 13; 7 HIV+and 6 HIV−]) and one nonhuman primate (NHP) model (rhesus macaque [n= 18; 10 SHIV+and 8 SHIV−]) were dosed to steady state with ARV combinations.

For HIV cure strategies like “kick and kill” to succeed, antiretroviral (ARV) drugs must reach effective concentrations in putative viral reservoirs. We characterize penetration of six ARVs in three preclinical animal models and humans. We found that standard dosing strategies in preclinical species closely mimicked tissue concentrations in humans for some, but not all, ARVs.

Imaging reveals that antiretroviral drugs are not distributed evenly in the mammalian gut, with potential implications for HIV replication.

ABSTRACTHuman noroviruses (HuNoVs) cause significant morbidity and mortality worldwide. However, despite substantial efforts, a small-animal model for HuNoV has not been described to date. Since “humanized” mice have been successfully used to study human-tropic pathogens in the past, we challenged BALB/c mice deficient in recombination activation gene (Rag) 1 or 2 and common gamma chain (γc) (Rag-γc) engrafted with human CD34+hematopoietic stem cells, nonengrafted siblings, and immunocompetent wild-type controls with pooled stool isolates from patients positive for HuNoV. Surprisingly, both humanized and nonhumanized BALB/c Rag-γc-deficient mice supported replication of a GII.4 strain of HuNoV, as indicated by increased viral loads over input. In contrast, immunocompetent wild-type BALB/c mice were not infected. An intraperitoneal route of infection and the BALB/c genetic background were important for facilitating a subclinical HuNoV infection of Rag-γc-deficient mice. Expression of structural and nonstructural proteins was detected in cells with macrophage-like morphology in the spleens and livers of BALB/c Rag-γc-deficient mice, confirming the ability of HuNoV to replicate in a mouse model. In summary, HuNoV replication in BALB/c Rag-γc-deficient mice is dependent on the immune-deficient status of the host but not on the presence of human immune cells and provides the first genetically manipulable small-animal model for studying HuNoV infection.IMPORTANCEHuman noroviruses are a significant cause of viral gastroenteritis worldwide, resulting in significant morbidity and mortality. Antivirals and vaccines are currently not available, in part due to the inability to study these viruses in a genetically manipulable, small-animal model. Herein, we report the first mouse model for human noroviruses. This model will accelerate our understanding of human norovirus biology and provide a useful resource for evaluating antiviral therapies.

A dual-function aptamer that targets both a HIV-1 surface protein and a critical messenger RNA can inhibit HIV infection in humanized mice.

Background: Optimal therapy of ARL relies on both effective anti-tumor chemotherapy and successful control of the underlying HIV infection. Management of HIV using HAART has been hampered by patient non-compliance with complex regimens, drug resistance and ongoing low level viral replication. Multiplexed RNA based anti-HIV gene transfer strategies to confer intrinsic cellular resistance may help circumvent these problems. Autologous stem cell transplantation for ARL is an ideal clinical platform for delivery of SiRNA transduced stem cells as combining gene transfer strategy with high dose antilymphoma therapy could provide control of both the HIV infection and the ARL. Methods: Gene transfer - anti-HIV RNA elements, including short hairpin RNA (shRNA) targeted to HIV tat/rev, a TAR-specific decoy sequence, and a ribozyme targeted to CCR5 were combined into a lentivirus vector (LV, rHIV7-shI-TAR-CCR5RZ). Using LV transduction methods, these anti-HIV RNAs were delivered into CD34+ hematopoietic progenitor cells (HPC). Results: Preclinical vector development - LV transduction allowed differentiation in liquid culture and in a SCID-hu model which produced macrophage and T cell progeny that were resistant to the HIV virus. Although HIV resistance can be induced in vitro with single anti-HIV shRNAs, no resistance was found in multiply passaged HIV in rHIV7-shI-TAR-CCR5RZ-transduced cells. In addition, cells were analyzed by microarray for mRNA and miRNA alterations and no significant changes were noted between CD34 transduced and untransduced cells. Copy number in transduced cells was 1–2/cell, and integration site analysis localized to transcriptionally active sites, usually away from terminal portions of gene sequences. This vector is proposed for use in ASCT for ARL. Update of ASCT in ARL: Between 1998 and 2006, 28 patients with high-risk ARL underwent ASCT at the City of Hope. Conditioning regimens included CBV (cyclophosphamide (Cy)100mg/kg, Carmustine150 mg/m2, VP16 60mg/kg) in 24 and FTBI/Cy/VP16 in 4. All patients engrafted, median time to ANC>500 was 11 days (range, 8–19). Regimen related toxicities included grade 3–4 hepatic toxicity n=3 and interstitial pneumonitis n=2. OI’s included PCP pneumonia n=2, CMV infection (2 viremias, 1 retinitis) and 2 cases of VZV. Therapy-related MDS was seen in one patient who ultimately died of MDS while in remission from his ARL. Median HIV viral load (VL) at ASCT was 6113 gc/ml with 22 having an undetectable VL. At two years only 9 pts had an undetectable VL. Median CD4 count at ASCT was 164 (range 25–1064), this rose to 263 (95–1164) at two years post ASCT. With a median f/u of 41 months, 2yr OS is 78% (95% CI 63–87) and PFS 78% (95% CI 63–87). Conclusions: ASCT can lead to durable remission for ARL. The fluctuation in VL seen post ASCT reflects the natural history of HIV infection and limitations of current antiviral therapy. Ultimately the system of gene transfer outlined above combined with ASCT as part of our next generation clinical trial could provide the optimal chance of cure for pts with high-risk ARL by offering both effective antilymphoma and anti-HIV therapy.

Background: Optimal therapy of ARL relies on both effective anti-tumor chemotherapy and successful control of the underlying HIV infection. Management of HIV using HAART has been hampered by patient non-compliance with complex regimens, drug resistance and ongoing low level viral replication. Multiplexed RNA based anti-HIV gene transfer strategies to confer intrinsic cellular resistance may help circumvent these problems. Autologous stem cell transplantation for ARL is an ideal clinical platform for delivery of SiRNA transduced stem cells as combining gene transfer strategy with high dose antilymphoma therapy could provide control of both the HIV infection and the ARL.