Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Transplantation of hematopoietic stem/progenitor cells (HSPC) modified with a lentiviral vector bearing a potent nontoxic short hairpin RNA (sh1005) directed to the HIV coreceptor CCR5 is capable of continuously producing CCR5 downregulated CD4+ T lymphocytes. Here, we characterized HIV-1 resistance of the sh1005-modified CD4+ T lymphocytes in vivo in humanized bone marrow/liver/thymus (hu BLT) mice. The sh1005-modified CD4+ T lymphocytes were positively selected in CCR5-tropic HIV-1-challenged mice. The sh1005-modified memory CD4+ T lymphocytes (the primary target of CCR5-tropic HIV-1) expressing sh1005 were maintained in lymphoid tissues in CCR5-tropic HIV-1-challenged mice. Frequencies of HIV-1 p24 expressing cells were significantly reduced in the sh1005-modified splenocytes by ex vivo cell stimulation confirming that CCR5 downregulated sh1005 modified cells are protected from viral infection. These results demonstrate that stable CCR5 downregulation through genetic modification of human HSPC by lentivirally delivered sh1005 is highly effective in providing HIV-1 resistance. Our results provide in vivo evidence in a relevant small animal model that sh1005 is a potent early-step anti-HIV reagent that has potential as a novel anti-HIV-1 HSPC gene therapeutic reagent for human applications.

Background

The use of shRNAs to downregulate the expression of specific genes is now relatively routine in experimentation but still hypothetical for clinical application. A potential therapeutic approach for HIV-1 disease is shRNA mediated downregulation of the HIV-1 co-receptor, CCR5. It is increasingly recognized that siRNAs and shRNAs can have unintended consequences such as cytotoxicities in cells, particularly when used for long term therapeutic purposes. For the clinical use of shRNAs, it is crucial to identify a shRNA that can potently inhibit CCR5 expression without inducing unintended cytotoxicities.

Results

Previous shRNAs to CCR5 identified using conventional commercial algorithms showed cytotoxicity when expressed using the highly active U6 pol III promoter in primary human peripheral blood derived mononuclear cells. Expression using the lower activity H1 promoter significantly reduced toxicity, but all shRNAs also reduced RNAi activity. In an effort to identify shRNAs that were both potent and non-cytotoxic, we created a shRNA library representing all potential CCR5 20 to 22-nucleotide shRNA sequences expressed using an H1 promoter and screened this library for downregulation of CCR5. We identified one potent CCR5 shRNA that was also non-cytotoxic when expressed at a low level with the H1 promoter. We characterized this shRNA in regards to its function and structure. This shRNA was unique that the use of commercial and published algorithms to predict effective siRNA sequences did not result in identification of the same shRNA. We found that this shRNA could induce sequence specific reduction of CCR5 at post transcriptional level, consistent with the RNA interference mechanism. Importantly, this shRNA showed no obvious cytotoxicity and was effective at downregulating CCR5 in primary human peripheral blood derived mononuclear cells.

Conclusion

We report on the characterization of a rare shRNA with atypical structural features having potent RNAi activity specific to CCR5. These results have implications for the application of RNAi technology for therapeutic purposes.

Background

Current understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment.

Methods

Humanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias.

Results

A total of 897 HSPC clones were longitudinally tracked in hu-BLT mice-providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions.

Conclusions

Human repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.

Investigations of anti-HIV-1 human hematopoietic stem/progenitor cell (HSPC)-based gene therapy have been performed by HIV-1 challenge after the engraftment of gene-modified HSPCs in humanized mouse models. However, the clinical application of gene therapy is to treat HIV-1-infected patients. Here, we developed a new method to investigate an anti-HIV-1 HSPC-based gene therapy in humanized mice previously infected with HIV-1. First, humanized mice were infected with HIV-1. When plasma viremia reached >10⁷ copies/mL 3 weeks after HIV-1 infection, the mice were myeloablated with busulfan and transplanted with anti-HIV-1 gene-modified CD34⁺ HSPCs transduced with a lentiviral vector expressing two short hairpin RNAs (shRNAs) against CCR5 and HIV-1 long terminal repeat (LTR), along with human thymus tissue under the kidney capsule. Anti-HIV-1 vector-modified human CD34⁺ HSPCs successfully repopulated peripheral blood and lymphoid tissues in HIV-1 previously infected humanized mice. Anti-HIV-1 shRNA vector-modified CD4⁺ T lymphocytes showed selective advantage in HIV-1 previously infected humanized mice. This new method will be useful for investigations of anti-HIV-1 gene therapy when testing in a more clinically relevant experimental setting.

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4(+) T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+) T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+) T-cells ex vivo. Furthermore, levels of gene-marked CD4(+) T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease.

The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34⁺ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ^-/-) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.

We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.