Thymic regulatory T cells (tTregs) are potent inhibitors of autoreactive immune responses, and loss of tTreg function results in fatal autoimmune disease. Defects in tTreg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of Treg as cell therapies to establish immune tolerance. Because tTregs are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous Treg-like cells from bulk CD4+ T cells. We used homology-directed repair (HDR)–based gene editing to enforce expression of FOXP3, the master transcription factor for tTreg. Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edTregs, manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tTreg. Both human and murine edTregs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edTreg with robust in vitro and in vivo functional activity. Last, edTreg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edTreg production may permit broad future clinical application.

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bMultiresistant Acinetobacter baumannii, a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors aac(6=)-Ib. This gene specifies resistance to amikacin and other aminoglycosides, seriously limiting the effectiveness of these antibiotics. An antisense oligodeoxynucleotide (ODN4) that binds to a duplicated sequence on the aac(6=)-Ib mRNA, one of the copies overlapping the initiation codon, efficiently inhibited translation in vitro. An isosequential nuclease-resistant hybrid oligomer composed of 2=,4=-bridged nucleic acid-NC (BNA NC ) residues and deoxynucleotides (BNA NC -DNA) conjugated to the permeabilizing peptide (RXR) 4 XB ("X" and "B" stand for 6-aminohexanoic acid and ␤-alanine, respectively) (CPPBD4) inhibited translation in vitro at the same levels observed in testing ODN4. Furthermore, CPPBD4 in combination with amikacin inhibited growth of a clinical A. baumannii strain harboring aac(6=)-Ib in liquid cultures, and when both compounds were used as combination therapy to treat infected Galleria mellonella organisms, survival was comparable to that seen with uninfected controls.A cinetobacter baumannii is an opportunistic human pathogen, mainly nosocomial, that causes bacteremia, meningitis, urinary tract infections, pneumonia, and necrotizing fasciitis among other infections (1-4). Multidrug-resistant A. baumannii strains are increasingly found in hospitals, complicating treatment of the infections they cause (4). Antisense technologies could be a path for designing new therapeutic strategies to overcome this problem. Options include the silencing of one or more essential genes (5-12) or the silencing of one or more resistance genes to induce phenotypic conversion to susceptibility (13-16). In the latter case, the antisense compound would be administered in combination with the appropriate antibiotic. However, in spite of important advances, silencing of bacterial genes by antisense oligomers is far from reaching its full potential (10). The main antisense mechanisms of gene silencing include degradation of the target mRNA by double-stranded RNA (dsRNA)-specific RNase, RNase H, or RNase P and steric hindrance of translation (interference with assembly of the ribosome or translation arrest) (10, 17). Practical application of any of these strategies requires that the antisense compounds resist the action of the ubiquitous nucleases and reach the cytosol to exert their action.There are numerous nuclease-resistant nucleotide analogs available that are adequate for different antisense strategies (10,18,19). For example, hybrid molecules containing locked nucleic acid and deoxyribonucleotide residues (LNA-DNA) in different configurations have been successfully utilized in bacteria and eukaryotes (16,(20)(21)(22)(23). New analogs related to LNAs, the 2=,4=-bridged nucleic acid-NC (BNA NC ) analogs (Fig. 1), that exhibit advantages such as higher binding affinity to a cRNA and excellent single-mismatch discriminating ability, have been recently introduced (24). Furthermore,...

Previous studies have shown that keratin 6 (K6)-spermidine/spermine N1-acetyltransferase (SSAT) transgenic mice, which modestly over-express SSAT in the skin, are more sensitive to tumor induction by a two-stage tumorigenesis protocol using initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). To evaluate the role of altered levels of polyamines and oxidative stress in this increase, studies were carried out with pharmacologic and genetic manipulation of K6-SSAT mice subjected to DMBA/TPA carcinogenesis. The increased tumor incidence was partially prevented by treatment with 1,4-bis-[N-(buta-2,3-dienyl)amino]butane, an inhibitor of acetylpolyamine oxidase which prevented degradation of the acetylated polyamines. This result suggests that toxic products such as reactive oxygen species and aldehydes liberated by the action of polyamine oxidase on the acetylated polyamines formed by SSAT may enhance tumor development. Breeding of the K6-SSAT mice with K6-antizyme (AZ) mice [which express AZ, a negative regulator of ornithine decarboxylase (ODC)] blocked the development of tumors. In addition, treatment of tumor-bearing K6-SSAT mice with the ODC inhibitor, alpha-difluoromethylornithine, resulted in the complete regression of established tumors. In contrast, treatment with N1,N11-bis(ethyl)norspermidine which increased SSAT activity in the tumors did not enhance regression. These results indicate that the tumor progression in K6-SSAT mice is dependent on elevated ODC activity and increased putrescine levels and may be further enhanced by oxidative stress. They support the use of strategies to modulate polyamine levels through the inhibition of ODC activity or polyamine uptake, but not via increased SSAT expression, for cancer chemoprevention in individuals at high risk for skin tumor development.

As regulatory T cell (Treg) adoptive therapy continues to develop clinically, there is a need to determine which immunomodulatory agents pair most compatibly with Tregs to enable persistence and stabilize suppressor function. Prior work has shown that mechanistic target of rapamycin inhibition can increase the stability of thymic Tregs. In this study, we investigated the transcriptomic signatures of ex vivo–expanded Tregs after adoptive transfer in the setting of clinically relevant immunosuppression using a nonhuman primate (NHP) model as a prelude to future transplant studies. Here, we found that adding interleukin-2 (IL-2) to rapamycin in vivo supported a logarithmic increase in the half-life of adoptively transferred carboxyfluorescein diacetate succinimidyl ester–labeled, autologous NHP Tregs, effectively doubling the number of cells in the peripheral blood Treg compartment compared with Treg infusion when rapamycin was given alone. Using single-cell transcriptomics, we found that transferred ex vivo–expanded Tregs initially exhibit a gene expression signature consistent with an activated state. Moreover, those cells with the highest levels of activation also expressed genes associated with p53-mediated apoptosis. In contrast, transferred Tregs interrogated at day +20 posttransfer demonstrated a gene signature more similar to published profiles of resting Tregs. Together, these preclinical data further support combining IL-2 and rapamycin in vivo as adjunctive therapy for ex vivo–expanded adoptively transferred Tregs and suggest that the activation status of ex vivo–expanded Tregs is critical to their persistence.

There are hundreds of essential genes in multidrug-resistant bacterial genomes, but only a few of their products are exploited as antibacterial targets. An example is the electron transfer flavoprotein (ETF), which is required for growth and viability in Burkholderia cenocepacia. Here, we evaluated ETF as an antibiotic target for Burkholderia cepacia complex (Bcc). Depletion of the bacterial ETF during infection of Caenorhabditis elegans significantly extended survival of the nematodes, proving that ETF is essential for survival of B. cenocepacia in this host model. In spite of the arrest in respiration in ETF mutants, the inhibition of etf expression did not increase the formation of persister cells, when treated with high doses of ciprofloxacin or meropenem. To test if etf translation could be inhibited by RNA interference, antisense oligonucleotides that target the etfBA operon were synthesized. One antisense oligonucleotide was effective in inhibiting etfB translation in vitro but not in vivo, highlighting the challenge of reduced membrane permeability for the design of drugs against B. cenocepacia. This work contributes to the validation of ETF of B. cenocepacia as a target for antibacterial therapy and demonstrates the utility of a C. elegans liquid killing assay to validate gene essentiality in an in vivo infection model.

As regulatory T cell (Treg) adoptive therapy continues to develop clinically, there is a need to determine which immunomodulatory agents pair most compatibly with Tregs to enable persistence and stabilize suppressor function. Prior work has shown that mechanistic target of rapamycin (mTOR) inhibition can increase stability of thymic Tregs. In this study we investigated the transcriptomic signatures of ex-vivo expanded Tregs after adoptive transfer in the setting of clinically relevant immunosuppression using a non-human primate (NHP) model as a prelude to future transplant studies.Here, we found that adding interleukin-2 (IL2) to rapamycin in vivo supported a logarithmic increase in the half-life of adoptively transferred CFSE-labeled, autologous NHP Tregs, effectively doubling the number of cells in the peripheral blood Treg compartment compared to Treg infusion with rapamycin alone. Using single cell transcriptomics, we found that transferred ex-vivo expanded Tregs initially exhibit a gene expression signature consistent with an activated state. Moreover, those cells with the highest levels of activation also expressed genes associated with p53-mediated apoptosis. In contrast, transferred Tregs interrogated at Day +20 post-transfer demonstrated a gene signature more similar to published profiles of resting Tregs.Together, these preclinical data further support combining IL2 and rapamycin in vivo as adjunctive therapy for ex-vivo expanded adoptively transferred Tregs and suggest that the activation status of ex-vivo expanded Tregs is critical to their persistence.

Engineered long lived plasma cells have the potential to be a new area of cell therapy. A key step in developing this cell therapy is testing in a model with an intact immune system similar to humans. To that end, we have developed methods to purify, expand, and differentiate non-human primate (NHP; rhesus macaque) B cells ex vivo. By comparing several media types and conditions, we consistently achieved 10-fold expansion of NHP B cells using a readily available commercial supplement. After only seven days in culture, large percentages of cells in NHP B cell cultures were differentiated. These cells expressed surface markers found in human antibody secreting cells (CD38 and CD138) and secreted immunoglobulin G. We also identified the serotypes (2.5 and D-J) and conditions necessary for efficient transduction of NHP B cells with AAV vectors for the purposes of producing a secreted protein (BAFF). We hope that this work will accelerate proof-of-concept in vivo studies using engineered protein-secreting B cells in an NHP model.