Chemical Inducer Of Dimerization Research Articles

A simeprevir-inducible molecular switch for the control of cell and gene therapies

Chemical inducer of dimerization (CID) modules can be used effectively as molecular switches to control biological processes, and thus there is significant interest within the synthetic biology community in identifying novel CID systems. To date, CID modules have been used primarily in engineering cells for in vitro applications. To broaden their utility to the clinical setting, including the potential to control cell and gene therapies, the identification of novel CID modules should consider factors such as the safety and pharmacokinetic profile of the small molecule inducer, and the orthogonality and immunogenicity of the protein components. Here we describe a CID module based on the orally available, approved, small molecule simeprevir and its target, the NS3/4A protease from hepatitis C virus. We demonstrate the utility of this CID module as a molecular switch to control biological processes such as gene expression and apoptosis in vitro, and show that the CID system can be used to rapidly induce apoptosis in tumor cells in a xenograft mouse model, leading to complete tumor regression.

Loss of optic nerve oligodendrocytes during maturation alters retinal organization

The myelin sheath facilitates signal conduction along axons in white matter tracts, and when disrupted, can result in significant functional deficits. Demyelination, observed in diseases like multiple sclerosis and optic neuritis, are associated with neural degeneration, however the extent of this damage on upstream circuitry is not well understood. Here we use the MBP-iCP9 mouse model to induce selective oligodendrocyte ablation in the optic nerve at P14 via a chemical inducer of dimerization (CID), resulting in partial demyelination of retinal ganglion cell (RGC) axons with minimal inflammation after two weeks. Oligodendrocyte loss reduced axon diameter and altered compound action potential waveforms, blocking conduction in the slowest-conducting axon populations. Demyelination resulted in disruptions to the normal composition of the retina, including reduced density of RBPMS+, Brn3a+, and OFF-transient RGCs, thinning of the IPL, and reduced density of displaced amacrine cells. The INL and ONL were unaffected by oligodendrocyte loss, suggesting that demyelination-induced deficits in this model are specific to the IPL and GCL. These results show that a partial demyelination of a subpopulation of RGC axons disrupts optic nerve function and affects the organization of the retinal network. This study highlights the significance of myelination in maintaining upstream neural connectivity and provides support for targeting neuronal degeneration in treatments of demyelinating diseases.

Inhibition of SHP2 and SHP1 Protein Tyrosine Phosphatase Activity by Chemically Induced Dimerization.

Protein tyrosine phosphatases (PTPs), the enzymes that catalyze the dephosphorylation of phosphotyrosine residues, are important regulators of mammalian cell signaling, whose activity is misregulated in numerous human diseases. PTPs are also notoriously difficult to selectively modulate with small molecules, and relatively few small-molecule tools for controlling their activities in the context of complex signaling pathways have been developed. Here, we show that a chemical inducer of dimerization (CID) can be used to selectively and potently inhibit constructs of Src-homology-2-containing PTP 2 (SHP2) that have been engineered to contain dimerization domains. Our strategy was inspired by the naturally occurring mechanism of SHP2 regulation, in which the PTP activity of SHP2’s catalytic domain is autoinhibited through an intramolecular interaction with the protein’s N-terminal SH2 (N-SH2) domain. We have re-engineered this inhibitory interaction to function intermolecularly by independently fusing the SHP2 catalytic and N-SH2 domains to protein domains that heterodimerize upon the introduction of the CID rapamycin. We show that rapamycin-induced protein dimerization leads to potent inhibition of SHP2’s catalytic activity, which is driven by increased proximity of the SHP2 catalytic and N-SH2 domains. We also demonstrate that CID-based inhibition of PTP activity can be applied to an oncogenic gain-of-function SHP2 mutant (E76K SHP2) and to the catalytic domain of the SHP2’s closest homologue, SHP1. In sum, CID-driven inhibition of PTP activity provides a broadly applicable tool for inhibiting dimerizable forms of the SHP PTPs and represents a novel paradigm for selective PTP inhibition through inducible protein–protein interactions.

Inducible caspase-9 suicide gene under control of endogenous oct4 to safeguard mouse and human pluripotent stem cell therapy

Pluripotent stem cells (PSCs) are promising in regenerative medicine. A major challenge of PSC therapy is the risk of teratoma formation because of the contamination of undifferentiated stem cells. Constitutive promoters or endogenous SOX2 promoters have been used to drive inducible caspase-9 (iCasp9) gene expression but cannot specifically eradicate undifferentiated PSCs. Here, we inserted iCasp9 gene into the endogenous OCT4 locus of human and mouse PSCs without affecting their pluripotency. A chemical inducer of dimerization (CID), AP1903, induced iCasp9 activation, which led to the apoptosis of specific undifferentiated PSCs in vitro and in vivo. Differentiated cell lineages survived because of the silence of the endogenous OCT4 gene. Human and mouse PSCs were controllable when CID was administrated within 2 weeks after PSC injection in immunodeficient mice. However, an interval longer than 2 weeks caused teratoma formation and mouse death because a mass of somatic cells already differentiated from the PSCs. In conclusion, we have developed a specific and efficient PSC suicide system that will be of value in the clinical applications of PSC-based therapy.

Engineered osteoclasts resorb necrotic alveolar bone in anti-RANKL antibody-treated mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of antiresorptive medications such as denosumab (humanized anti-RANKL antibody), yet its pathophysiology remains elusive. It has been posited that inhibition of osteoclastic bone resorption leads to the pathological sequelae of dead bone accumulation, impaired new bone formation, and poor wound healing in MRONJ, but this hypothesis has not been definitively tested. We previously engineered myeloid precursors with a conditional receptor activator of nuclear factor kappa-Β intracellular domain (iRANK cells), which differentiate into osteoclasts in response to a chemical inducer of dimerization (CID) independently of RANKL. In this study, we showed that CID-treated iRANK cells differentiated into osteoclasts and robustly resorbed mineralized surfaces even in the presence of anti-RANKL antibody in vitro. We then developed a tooth extraction-triggered MRONJ model in nude mice using anti-RANKL antibody to deplete osteoclasts. This model was used to determine whether reconstitution of engineered osteoclasts within sockets could prevent specific pathological features of MRONJ. Locally delivered iRANK cells successfully differentiated into multinucleated osteoclasts in response to CID treatment in vivo as measured by green fluorescent protein (GFP), tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, matrix metallopeptidase 9 (MMP-9), and cathepsin K staining. Sockets treated with iRANK cells + CID had significantly more osteoclasts and less necrotic bone than those receiving iRANK cells alone. These data support the hypothesis that osteoclast deficiency leads to accumulation of necrotic bone in MRONJ.

Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Inducible MyD88 Signaling Enhances the Anti-AML Activity of BiTE-Secreting ENG T-Cells

Background: Pediatric and adult acute myeloid Leukemia (AML) is a disease with poor prognosis due to its high relapse rate and treatment related mortality. Adoptive immunotherapy has the potential to improve outcomes and CD123 present a promising immunotherapy target for AML. CD123-specific engager (ENG) T-cells are cells genetically modified to secrete bispecific T-cell engagers (BiTEs) that recognize CD3+ on T-cells and CD123+ on AML. We had previously shown that inclusion of an inducible MyD88/CD40 costimulatory molecule, which is activated by a chemical inducer of dimerization (CID), improved anti-tumor effect against CD123+ targets in vitro. We now wanted to determine, which component of the costimulatory molecule is critical for the improved effector function of CD123-ENG T cells and perform in vivo studies. Methods: We generated a panel of retroviral vectors encoding CD20 as a safety switch, CD123-ENG, and an inducible costimulatory molecule consisting of a myristoylation sequence, FKBP dimerizer domains and one of three signaling costimulatory domains (MyD88 (iM) ,CD40(iC) or MyD88/CD40(iMC)). A vector specific for CD19 (CD20.CD19-ENG.iMC) and non-transduced (NT) T-cells served as controls. We genetically modified T-cells using a retroviral transduction protocol and evaluated their effector function +/- CID. We assessed the effector function of transduced T-cells using flow cytometry, ELISA and luciferase based cytotoxicity assays. We evaluated antitumor activity and persistence in vivo in a xenograft AML model. Results: We successfully generated CD20.CD123-ENG.iM, CD20.CD123-ENG.iC and CD20.CD123-ENG.iMC T-cells. Transduction efficiency ranged from 45 to 70% (±5). The immunophenotype of CD123-ENG T-cells was predominandly CD8+ effector memory. CD20.CD123-ENG T-cells expressing inducible costimulatory molecules secreted higher levels of CD123-specific BiTEs, IL2 and IFNγ in comparison to CD20.CD123-ENG T-cells when cocultured with of MOLM-13 (CD123+) and CID (p<0.005, N=3). In the presence of CID, CD20.CD123-ENG.iM or CD20.CD123-ENG.iMC T-cells were able to sequentially kill AML cells for up to 5 stimulations, as opposed to CD20.CD123 ENG, CD20.CD123 ENG.iC and NT T-cells (n=3, E:T=1:1). We evaluated the in vivo anti-AML activity of ENG T cells using an AML cell line expressing firefly luciferase (MOLM-13.ffluc) to allow for serial bioluminescence imaging. MOLM-13.ffluc bearing mice were treated with one i.v. dose of effector cells (CD20.CD123-ENG, CD20.CD123-ENG iM, CD20.CD123-ENG.iC, CD20.CD123-ENG.iMC or NT T-cells ) and 2 doses/week of CID for 2 weeks (100mg/mice, i.p.). CD20.CD123-ENG.iMC and iM T-cells had potent anti-leukemic activity, resulting in a significant survival advantage in comparison to CD20.CD123-ENG T-cells group (P<0.005, n=10 animals per group). Conclusion: We demonstrate here that inducible MyD88/CD40 costimulation significantly enhances the anti-AML activity of CD123-ENG T cells in vitro and in xenograft models. Mechanistic studies revealed that MyD88 costimulation is critical for the observed benefit. Thus, genetically modifying T-cells to express BiTEs and an inducible MyD88 costimulatory molecule may present a promising cell product for the immunotherapy of CD123+ hematological malignancies. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:TESSA Therapeutics: Other: Research Collaboration; Tidal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; EMD Serono: Honoraria; ViraCyte: Consultancy; NHLBI: Research Funding; ASSISI fundation of Memphis: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; America Lebanese Syrian Associated Charities: Research Funding. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties: Patent Applications in the Fields of Cell and Gene Therapy .

Combination Suicide Gene Delivery with an Adeno-Associated Virus Vector Encoding Inducible Caspase-9 and a Chemical Inducer of Dimerization Is Effective in a Xenotransplantation Model of Hepatocellular Carcinoma.

Current treatment approaches for hepatocellular carcinoma (HCC) have a narrow therapeutic index and alternate modes of treatment are thus required. We have utilized a gene delivery vector containing inducible caspase 9 (iCasp9) gene, which is a synthetic analogue based on the mammalian caspase 9 and fused to a human FK506 binding protein that allows its conditional dimerization to a synthetic, small molecule [chemical inducer of dimerization, AP20187] and results in target cell apoptosis. In our studies, we have tested these synthetic vectors based on an adeno-associated virus platform for their potential anti-tumorigenic effect in human HCC cells in vitro and in a HCC tumor model developed in nude mice. Our data demonstrates that the iCasp9-AP20187 bioconjugate is able to trigger terminal effectors of cellular apoptosis and presents a viable approach for the potential treatment of HCC.

Regulated ex vivo regional gene therapy for bone repair using an inducible caspase-9 suicide gene system.

In order to adapt ex vivo regional gene therapy for clinical applications in orthopaedic surgery, safety issues must be considered. In this study we developed a suicide approach using a dual gene expression two step transcriptional amplification lentiviral vector (LV-TSTA) encoding BMP-2 and an inducible caspase 9 (iC9) system that selectively induces apoptosis upon activation with a chemical inducer of dimerization (CID). Transduction of rat bone marrow stromal cells (RBMSCs) with LV-TSTA-iC9/BMP-2 led to abundant BMP-2 production (90.3 ± 7.9 ng/24 h/106 cells) in vitro and stimulated bone formation in a mouse muscle pouch in the absence of CID. Moreover it was shown that CID could be used to selectively induce apoptosis in iC9-transduced cells both in vitro and in vivo. Double exposure to serial dilutions of CID decreased in vitro production of BMP-2 by 85-87% and Luc activity by 97-99% in iC9/BMP-2 or iC9/Luc-transduced cells respectively. Early administration of CID (Days 0-1 post-op) in mice implanted with iC9/BMP-2-transduced RBMSCs was effective in blocking bone formation, indicating that CID was toxic to the transduced cells. In iC9/Luc-implanted mice, late administration of two doses of CID (Days 27-28 post-op) significantly reduced the luciferase signal. The current study provides proof of concept for the potential clinical application of regulated gene therapy to promote bone repair.

Myd88/CD40 Costimulation to Enhance the Antitumor Activity of CD123-ENG T-Cells for the Immunotherapy of AML

Background: Acute myeloid leukemia (AML) represents a treatment challenge due to its poor prognosis and high associated morbidity and mortality rates. Thus, new therapies are needed. CD123-ENG T-cells are cells genetically modified to secrete bispecific antibodies that recognize CD3 (T-cells) and CD123 (AML). Preclinical studies show that these cells recruit bystander T-cells to kill CD123+ blasts in vitro and in vivo. However, CD123-ENGs are unable to maintain sequential killing capability of CD123+ targets. To overcome this limitation, we have devised an approach that provides inducible Myd88/CD40 costimulation activated by a chemical inducer of dimerization (CID). This strategy should increase the persistence, expansion and anti-AML activity of CD123-ENG T-cells.Methods: We generated a retroviral vector encoding a CD20 safety switch, CD123-ENG, and the inducible costimulatory molecule MyD88.CD40 (iMC) linked by 2A sequences (CD20.2A.CD123-ENG.2A.iMC). We used a vector specific for CD19 (CD20.2A.CD19-ENG.2A.iMC) and non-transduced (NT) cells as controls for non-specific effects of the iMC construct. We genetically modified T-cells using a retroviral transduction protocol and evaluated their effector function +/- CID. We assessed the effector function of transduced T-cells using standard immunological assays and a flow cytometry based sequential killing assay.Results: We successfully generated CD20.CD123-ENG.iMC T-cells, which maintained a transduction efficiency above 50% throughout our study period. We performed coculture and cytotoxicity assays using NT, CD20.CD123-ENG, CD20.CD19-ENG.iMC and CD20.CD123-ENG.iMC T-cells +/-CID as effectors and MOLM13 (CD123+), Kg1a (CD123+) and K562 (CD123-) as targets. Cocultures were performed +/- CID. CD20.CD123-ENG.iMC T-cells maintained CD123 antigen specificity, as evidenced by cytotoxicity and cytokine assays. CD20.CD123-ENG.iMC T-cells + CID secreted increased IL-2 and IFN-γ in the presence of CD123+ targets (KG1a and MOLM13) when compared to baseline and to CD20.CD123-ENG T-cells. In addition, CD20.CD123-ENG.iMC T-cells + CID displayed enhanced sequential killing capabilities at a 1:1 ratio, compared to CD20.CD123-ENG T-cells.Conclusion: CD20.CD123-ENG.iMC T-cells recognize and kill CD123+ AML blasts in an antigen dependent manner. In addition, control ENG.iMC T-cells have no antitumor activity, indicating that activation of Myd88/CD40 does not induce nonspecific AML blast killing. CD20.CD123-ENG.iMC T-cells have improved effector function in the presence of CID as judged by increased cytokine production and their ability to sequentially kill CD123+ target cells in vitro. Based on our promising in vitro experiments, we have initiated in vivo studies in AML xenograft models, which are currently in progress. DisclosuresLeen:Marker: Equity Ownership.

Ligand gated split Lysine Acetyl Transferases(KATs) and Kinases

Post translational modifications such as phosphorylation, methylation, acetylation, ubiquitination control activity of many proteins. Knowledge of one third of cellular proteins are covalently modified via phosphorylation, marks it as an important post translational modification. The >500 protein kinases in humans catalyze the phosphorylation of Tyr, Ser and Thr residues on proteins and thereby control their function. The structural similarity of kinases makes it challenging to selectively turn‐on or turn‐off desired kinases using small molecule inhibitors or activators. Genetic approaches are powerful, but cells and organisms respond and adapt to long term changes in gene expression levels, thus making it challenging to understand the true role of any enzyme in time and space. Towards a potential solution to this problem, we have developed a method to control the activity of individual enzymes utilizing small molecules. This approach was successfully applied to protein kinases and phosphatases, leading to the control of their activity in vitro and in cellulo. The strategy entails sequence alignment and the identification of regions that harbor significant dissimilarities. The sites are subsequently targeted for 25 amino acid loop insertion to investigate whether a specific protein can tolerate the new loop without greatly compromising its activity. The loop insertion mutants that retain the activity are used as fragmentation sites to generate ligand‐gated split proteins. The well‐studied chemical inducer of dimerization (CID), rapamycin dependent heterodimerization of FKBP/FRB was used as a first test of a successful ligand gated split‐enzyme. We have successfully demonstrated the feasibility of this approach with members of the tyrosine kinase group. Moreover, orthogonal CIDs rapamycin and abscisic acid were successfully used for regulating the activity of Kinases. Then the activity of these two‐different split‐enzyme systems were tested in HEK293T cells. split‐protein counterparts expressing stable cell lines are used to reproduce orthogonal kinase activation accurately. Furthermore, quantitative mass‐spectrometry based phosphoproteomics experiments will be used for validating the system and will be useful for demonstrating the potential of the split‐Kinases for answering relevant biological question or creating a novel signaling system. Extending the trend, we have used this approach for Lysine Acetyl Transferases(KATs). (KATs) were formerly known as Histone Acetyl Transferases (HATs) as they acetylated histones and thereby control transcription. However, recent studies strongly suggest that the acetylation mark is far more common protein modification with possibly over 7,000 acetylated proteins in the human proteome. There are numerous KATs and currently available small molecule based inhibition methods are not uniquely specific while RNAi based gene knock down studies can fail to provide details related to the true role of any enzyme as compensatory acetylation may occur. To address this problem, we have successfully created the first generation of ligand inducible split‐KATs, GCN5 and PCAF. We are currently using this approach for designing several new KATs, Myst2 and HAT1, which belong to different families, with the intention of exploring the possibility to establish temporal and spatial control over multiple KATs simultaneously in relevant cell lines.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optogenetic Manipulation of Mouse Oocytes.

Like many biological processes, oocyte development depends on careful orchestration of protein localization. Optogenetic approaches have the potential to manipulate this dynamic system with spatial and temporal precision and molecular specificity. This chapter describes the use of a photocaged chemical inducer of dimerization to control localization of genetically tagged proteins with light. As an example, we recruit a fluorescently tagged protein to one spindle pole in metaphase.

Inducible Activation of MyD88 and CD40 in CAR T Cells Results in Controllable and Potent Antitumor Activity in Preclinical Solid Tumor Models.

Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CAR) has had limited success for solid tumors in early-phase clinical studies. We reasoned that introducing into CAR T cells an inducible costimulatory (iCO) molecule consisting of a chemical inducer of dimerization (CID)-binding domain and the MyD88 and CD40 signaling domains would improve and control CAR T-cell activation. In the presence of CID, T cells expressing HER2-CARζ and a MyD88/CD40-based iCO molecule (HER2ζ.iCO T cells) had superior T-cell proliferation, cytokine production, and ability to sequentially kill targets in vitro relative to HER2ζ.iCO T cells without CID and T cells expressing HER2-CAR.CD28ζ. HER2ζ.iCO T cells with CID also significantly improved survival in vivo in two xenograft models. Repeat injections of CID were able to further increase the antitumor activity of HER2ζ.iCO T cells in vivo Thus, expressing MyD88/CD40-based iCO molecules in CAR T cells has the potential to improve the efficacy of CAR T-cell therapy approaches for solid tumors.Significance: Inducible activation of MyD88 and CD40 in CAR T cells with a small-molecule drug not only enhances their effector function, resulting in potent antitumor activity in preclinical solid tumors, but also enables their remote control post infusion. Cancer Discov; 7(11); 1306-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

Development of a Cell-Based Gene Therapy Approach to Selectively Turn Off Bone Formation.

Cell and gene therapy approaches are safer when they possess a system that enables the therapy to be rapidly halted. Human mesenchymal stem cells were transduced with an adenoviral vector containing the cDNA for bone morphogenetic protein 2 (AdBMP2) to induce bone formation. To make this method safer, a system to quickly kill these virally transduced cells was designed and evaluated. Cells were encapsulated inside poly(ethylene glycol) diacrylate (PEG-Da) hydrogels that are able to shield the cells from immunological destruction. The system involves an inducible caspase-9 (iCasp9) activated using a specific chemical inducer of dimerization (CID). Delivering AdBMP2-transduced human mesenchymal stem cells encapsulated in PEG-Da hydrogel promoted ectopic ossification in vivo, and the iCasp9 system allowed direct control of the timing of apoptosis of the injected cells. The iCasp9-CID system enhances the safety of delivering AdBMP2-transduced cells, making it a more compelling therapeutic for bone repair and spine fusion. J. Cell. Biochem. 118: 3627-3634, 2017. © 2017 Wiley Periodicals, Inc.

Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent Kd of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates.

Clonal Dynamics In Vivo of Virus Integration Sites of T Cells Expressing a Safety Switch

Safety switches are becoming relevant for the clinical translation of T-cell-based immunotherapies. In patients receiving an allogeneic hematopoietic stem cell transplant, the inducible caspase-9 gene (iC9) safety switch expressed by donor-derived T lymphocytes efficiently controls acute graft versus host disease (GvHD). However, in vivo elimination of iC9-T cells by the chemical inducer of dimerization (CID) that activates the iC9 protein is incomplete. To study this effect, we characterized the clonal diversity and dynamics of vector insertion sites (VIS) in iC9-T cells pre- and post-CID administration in four patients who developed GvHD. We identified 3,203 VIS among four patients and followed their in vivo clonal dynamics up to 161 days post-CID. VIS were categorized by their proximity to host genome elements, gene associations, and cis-modulatory relationship to mapped promoters. We found that VIS are preferentially located near open chromatin and promoter regions; furthermore, there was no evidence for selection bias among VIS surviving the CID treatment. The majority of iC9-T cells with high normalized VIS copy number at the time of GvHD onset were eliminated by CID, while iC9-T cells detectable post-CID generally have low normalized VIS copy number. We propose that suboptimal iC9 transgene expression is responsible for the incomplete elimination of iC9-T cells and illustrate here by simple model how cis-modulatory influences of local genome context and T-cell receptor activation status at time of CID treatment contribute to stochastic sparing of iC9-T cells.

Serial Activation of the Inducible Caspase 9 Safety Switch After Human Stem Cell Transplantation

Activation of the inducible caspase 9 (iC9) safety gene by a dimerizing drug (chemical inducer of dimerization (CID) AP1903) effectively resolves the symptoms and signs of graft-versus-host disease (GvHD) in haploidentical stem cell transplant (HSCT) recipients. However, after CID treatment, 1% of iC9-T cells remain and can regrow over time; although these resurgent T cells do not cause recurrent GvHD, it remains unclear whether repeat CID treatments are a safe and feasible way to further deplete residual gene-modified T cells should any other adverse effects associated with them occur. Here, we report a patient who received an infusion of haploidentical iC9-T cells after HSCT and subsequently received three treatments with AP1903. There was a mild (grade 2) and transient pancytopenia following each AP1903 administration but no non-hematological toxicity. Ninety five percent of circulating iC9-T cells (CD3(+)CD19(+)) were eliminated after the first AP1903 treatment. Three months later, the residual cells had expanded more than eightfold and had a lower level of iC9 expression. Each repeated AP1903 administration eliminated a diminishing percentage of the residual repopulating cells, but elimination could be enhanced by T-cell activation. These data support the safety and efficiency of repeated CID treatments for persistent or recurring toxicity from T-cell therapies.

Multiple mechanisms determine the sensitivity of human-induced pluripotent stem cells to the inducible caspase-9 safety switch

Expression of the inducible caspase-9 (iC9) suicide gene is one of the most appealing safety strategies for cell therapy and has been applied for human-induced pluripotent stem cells (hiPSC) to control the cell fate of hiPSC. iC9 can induce cell death of over 99% of iC9-transduced hiPSC (iC9-hiPSC) in less than 24 hours after exposure to chemical inducer of dimerization (CID). There is, however, a small number of resistant cells that subsequently outgrows. To ensure greater uniformity of the hiPSC response to iC9 activation, we purified a resistant population by culturing iC9-hiPSC with CID and analyzing the mechanisms by which the cells evade killing. We found that iC9-resistant hiPSC have significant heterogeneity in terms of their escape mechanisms from caspase-dependent apoptosis including reduced expression of iC9 by promoter silencing and overexpression of BCL2. As a consequence, modifying a single element alone will be insufficient to ensure sustained susceptibility of iC9 in all cells and prevent the eventual outgrowth of a resistant population. To solve this issue, we propose to isolate an iC9-sensitive population and show that this hiPSC line has sustained a uniform responsiveness to iC9-mediated growth control.

Induction of Fetal Hemoglobin and Reduction of Disease Pathology in Sickle Cell Mice By a Synthetic Zinc Finger Gamma-Globin Activator

A plethora of research has established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the only FDA-approved treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Thus, research has focused on identifying agents that specifically reactivate γ-globin gene expression during adult definitive erythropoiesis, with minimal off-target effects. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in K562 cells, murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene, in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients, and in vivo, in gg1-VP64 β-YAC double transgenic (bigenic) mice. Transgenic mice with enforced expression of the gg1-VP64 fusion protein only in the erythroid-megakaryocytic compartment were crossed into the Townes sickle cell knock-in mouse (Jackson Laboratory) background. Compared with control sickle cell (HbSS) mice, gg1-VP64 ATF sickle cell (gg1-HbSS) mice had hematological values at levels found in wild-type homozygous or heterozygous adult hemoglobin (HbAA or HbAS, respectively) mice. For example, average RBC (106/mm3) was 11.7 for wild-type mice and 12.9 for gg1 HbSS, compared to 8.2 for HbSS mice. Average HGB (g/dl) was 15.1 for wild-type mice and gg1 HbSS mice, versus 10.0 for HbSS mice. Average HCT was 52.5% for wild-type mice, 53.7% for gg1 HbSS mice, but only 41.5% for HbSS mice. Finally, average WBC (103/mm3) was 9.4 for wild-type mice, 9.0 for gg1 HbSS mice and 91.0 for HbSS mice. HPLC and Western blot analysis to determine the effect of gg1-VP64 on HbF synthesis are underway. In addition, we are examining mice for numbers of HbF-positive cells, mature cells, and reticulocytes, as well as looking at organ damage. Our results demonstrate that the ATF class of reagent may be an effective gene therapy for treatment of SCD. DisclosuresMakala:Georgia Regents University: Employment.

Engineering macrophages to control the inflammatory response and angiogenesis

Macrophage (MΦ) dysregulation is increasingly becoming recognized as a risk factor for a number of inflammatory complications including atherosclerosis, cancer, and the host response elicited by biomedical devices. It is still unclear what roles the pro-inflammatory (M1) MΦ and pro-healing (M2) MΦ phenotypes play during the healing process. However, it has been shown that a local overabundance of M1 MΦs can potentially lead to a chronically inflamed state of the tissue; while a local over-exuberant M2 MΦ response can lead to tissue fibrosis and even promote tumorigenesis. These notions strengthen the argument that the tight temporal regulation of this phenotype balance is necessary to promote inflammatory resolution that leads to tissue homeostasis. In this study, we have engineered pro-inflammatory MΦs, MΦ-cTLR4 cells, which can be activated to a M1-like MΦ phenotype with a small molecule, the chemical inducer of dimerization (CID) drug. The MΦ-cTLR4 cells when activated with the CID drug, express increased levels of TNFα, IL-6, and iNOS. Activated MΦ-cTLR4 cells stay stimulated for at least 48h; once the CID drug is withdrawn, the MΦ-cTLR4 cells return to baseline state within 18h. Further, in vitro CID-activated MΦ-cTLR4 cells induce upregulation of VCAM-1 and ICAM-1 on endothelial cells (EC) in a TNFα-dependent manner. With the ability to specifically modulate the MФ-cTLR4 cells with the presence or absence of a small molecule, we now have the tool necessary to observe a primarily M1 MФ response during inflammation. By isolating this phase of the wound healing response, it may be possible to determine conditions for ideal healing.