Search

Non-premixed catalytic combustion in micro-combustors has not been reported so far. The present study demonstrates the feasibility of non-premixed CH4/(O2 + N2) catalytic combustion in a planar micro-combustor with a height less than the quenching distance. The impacts of inlet velocity, combustor height, nominal equivalence ratio and O2 concentration on the combustion characteristics were numerically investigated. First, it is found that with the increase of inlet velocity, the catalytic reaction zone moves downstream and the reaction intensity increases first and then decreases. Second, as the combustor height is increased, the catalytic reaction zone broadens and the largest heat release rate decreases, while the maximum temperature and combustion efficiency show non-monotonic variations. Thirdly, a slightly fuel-rich condition can increase Pt(s) coverage, enhance catalytic reaction on the oxidant side, and improve combustion efficiency. Finally, oxygen enrichment can significantly enhance both heterogeneous and homogeneous reactions, leading to a dramatic improvement in combustion efficiency.

Targeted gene therapy has been hampered by the inability to correct mutations in stem cells that can reconstitute the immune system after transplant into patients. De Ravin et al . now report that CRISPR, a DNA editing technology, corrected blood stem cells from patients with an immunodeficiency disorder (chronic granulomatous disease) caused by mutations in NOX2. CRISPR-repaired human stem cells engrafted in mice after transplant and differentiated into leukocytes with a functional NOX2 protein for up to 5 months. The authors did not detect off-target treatment effects, suggesting that this gene repair strategy may benefit patients with chronic granulomatous disease or other blood disorders.

For more than a decade, investigators have pursued methods to genetically engineer natural killer (NK) cells for use in clinical therapy against cancer. Despite considerable advances in viral transduction of hematopoietic stem cells and T cells, transduction efficiencies for NK cells have remained disappointingly low. Here, we show that NK cells can be genetically reprogramed efficiently using a cGMP-compliant mRNA electroporation method that induces rapid and reproducible transgene expression in nearly all transfected cells, without negatively influencing their viability, phenotype, and cytotoxic function. To study its potential therapeutic application, we used this approach to improve key aspects involved in efficient lymphoma targeting by adoptively infused ex vivo-expanded NK cells. Electroporation of NK cells with mRNA coding for the chemokine receptor CCR7 significantly promoted migration toward the lymph node-associated chemokine CCL19. Further, introduction of mRNA coding for the high-affinity antibody-binding receptor CD16 (CD16-158V) substantially augmented NK cell cytotoxicity against rituximab-coated lymphoma cells. Based on these data, we conclude that this approach can be utilized to genetically modify multiple modalities of NK cells in a highly efficient manner with the potential to improve multiple facets of their in vivo tumor targeting, thus, opening a new arena for the development of more efficacious adoptive NK cell-based cancer immunotherapies.

Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene, and can be cured by therapeutic β-globin gene addition into hematopoietic stem cells (HSCs) with lentiviral transduction. However, this method relies upon random integration, leaving the SCD mutation intact and potentially inducing insertional mutagenesis. Genome editing technologies have the potential to correct the SCD mutation without integration, producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. In this study, we investigated CRISPR/Cas9-based gene correction for SCD CD34+ cells. Plerixafor-mobilized SCD CD34+ cells were transfected by electroporation using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver SCD mutation-specific guide RNA at 200mg/ml, SpCas9 mRNA at 200mg/ml or protein at 120mg/ml, and single strand donor DNA with a normal β-globin sequence at 80, 120, or 200mg/ml. We chose Cas9 mRNA and single strand donor DNA due to the ease of clinical grade large-scale production and to avoid the need for viral vector manufacturing. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. Cell viability was reduced to 76-87% after electroporation, compared to 90% in the control. We observed high-efficiency genome editing (29-34% gene correction and 49-58% indels) with Cas9 mRNA, showing donor DNA concentration dependence, and editing levels were comparable to Cas9 protein (39% correction and 43% indels). 15-23% Biallelic and 17-26% monoallelic gene correction were detected at the clonal level by colony assay. After erythroid differentiation, up to 54% normal β-globin production was observed with Cas9 mRNA (Figure), comparable to Cas9 protein (67%), while βs-globin amounts were markedly reduced under both conditions (6-10%). Similar correction efficiencies were obtained from two additional SCD patients' CD34+ cells at DNA levels (28-35%) and protein levels (33-56%). These data demonstrate that Cas9 mRNA and single strand donor DNA allow for efficient gene correction in SCD CD34+ cells, exceeding the therapeutic threshold of 20% in SCD. We then evaluated off-target effects on the δ-globin gene, which was reported as a major off-target site in β-globin gene editing due to high homology; however, almost no off-target effects (0.6-1.3% indels) were detected. Interestingly, gene conversion in the 9T>C polymorphism (11bp upstream of SCD mutation) on the β-globin gene was observed, and this conversion always occurred with SCD gene correction (26-33% of SCD gene correction), suggesting that gene conversion is strongly affected by distance from the target site. In addition, we evaluated genome editing among subpopulations of CD34+ cells from 3 healthy donors under the same conditions (normal β-globin to SCD mutation). We observed similar editing efficiencies (conversion and indels) among more immature (CD34+CD133+CD90+) and relatively differentiated populations (CD34+CD133+CD90-, CD34+CD133-, and CD34-) as well as among cells at different phases of the cell cycle (G0/G1, S, and G2/M), suggesting that similar gene correction efficiencies are obtained in all CD34+ cell populations, including the HSC population. We have begun efforts to evaluate gene-corrected SCD CD34+ cell engraftment in the mouse xenograft model, as similarly corrected X-CGD CD34+ cells were engrafted in immunodeficient mice. To examine the effects of indels in the β-globin gene, we next evaluated Hb production from genome-edited SCD CD34+ cells (2 patients) without donor DNA. Editing without donor DNA resulted in 63-70% indels (compared to 26-29% correction and 46-53% indels with donor DNA) and increased non-adult Hb production (small amounts of fetal Hb and significant amounts of a Hb variant), which will require further investigation to characterize. In summary, we observed efficient gene correction in SCD CD34+ cells with a simple Cas9 mRNA, single strand donor DNA, and guide RNA method, resulting in ~30% gene correction and ~50% indels. After erythroid differentiation, the majority of Hb detected was adult Hb; we detected up to 54% normal β-globin production with a marked reduction of βs-globin to ~10%. Evaluation of engraftment potential is required for gene-corrected CD34+ cells, but these methods would be clinically applicable for gene correction in SCD. Figure. Figure. Disclosures Li: MaxCyte, Inc.: Employment. Allen:MaxCyte, Inc.: Employment. Peshwa:MaxCyte, Inc.: Employment.

Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N=5) or purified unstimulated NK cells from peripheral blood (N=4) resulted in good cell viability with eGFP expression in 85+/-6% and 86+/-4%, 24 h after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19(+) target cells resulting in > or =80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 h after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer.

Bulk materials with the general formula of Mn 3 (Cu 0.6 Nb x Ge 0.4 − x )N ( x = 0.05, 0.1, 0.15, 0.2, 0.25), Mn 3 (Cu 0.6 Ge 0.4 )N and Mn 3 (Cu 0.7 Ge 0.3 )N were fabricated by mechanical ball milling and solid state sintering. Their thermal expansion coefficients and electrical conductivities were investigated in the temperature range of 80–300 K. It is found that the temperature interval of negative temperature expansion behavior is about 95 K in the samples of Mn 3 (Cu 0.6 Nb 0.15 Ge 0.25 )N and Mn 3 (Cu 0.6 Nb 0.2 Ge 0.2 )N, which is twice as large as that of Mn 3 (Cu 0.7 Ge 0.3 )N. The negative thermal expansion of Mn 3 (Cu 0.6 Nb 0.15 Ge 0.25 )N can reach to − 19.5 × 10 −6 K − 1 in the temperature range of 165 to 210 K. The electrical conductivity of this series materials is in a level of about 2.5 × 10 6 (Ω m) − 1 .

High-voltage insulation and filling periodically with cryogen are key problems [Mitsuii H. Review of the research and development for insulation of superconducting fault-current limiters. Cryogenics 1998;38:1159–67] for the cryogenic system of the high Tc superconducting fault current limiters (HTC-SFCLs). The nitrogen thermo-siphon cooling system has been proposed, designed and tested for the 10.5 kV/1.5 kA HTC-SFCL cooled by liquid nitrogen. The problem of the high-voltage insulation gets to be solved by this cryogenic system, and also the period of the filling with liquid nitrogen can be obviously prolonged due to the closed self-circulation, even without further extra filling. In this paper, this unique cryogenic cooling system for the 10.5 kV/1.5 kA HTC-SFCLs has been described. The saturated nitrogen steam through the high-vacuum-jacked line from the HTC-SFCLs is cooled into the liquid nitrogen which returns again to the HTC-SFCLs. Two G–M refrigerators provide the cooling capacity for the condensation in the condenser. Test results have indicated that the 5–6 L/h of the saturated nitrogen vapor condensation can meet the cooling requirement of the 10.5 kV/1.5 kA HTC-SFCLs on each phase.

A 75 m, three-phase HTS power cable is successfully demonstrated in a distribution grid of Gansu Changtong Cable Science and Technology Joint Stock Co., Ltd. in northwest China. The HTS power cable operates at a local live distribution grid of 6.6 kV to supply electric power for Changtong’s factory in Baiyin, Gansu Province. The cable was rated for voltages up to 10.5 kV, and has conducted up to 1600 A at 400 V, which was limited only by the load available at Changtong’s factory. The critical current of cable was tested as high as 5300 A DC at 76 K. The cable was connected to a local distribution grid to supply the power for the workshops at Changtong’s factory. The cable operates stably and reliably over 5500 h since December 2004.

Superconducting fault current limiter (SFCL) is an attractive appliance for modern electrical power system. Institute of Electrical Engineering (IEE) at the Chinese Academy of Science (CAS) has successfully demonstrated a three-phase HTS bridge-type fault current limiter (FLC) at a substation in Hunan, China. The FCL suppressed the fault current below 635 A successfully in a 3-phase-to-ground short-circuit field test. Since August 2005, it has been going on a demonstrated long-term reliable operation for one year in the live 110 kV/10.5 kV substation.

This study was focused on the investigation of the transport properties of Bi 85 Sb 15 alloys prepared by three different methods. Bulk samples were prepared by a novel mechanical alloying and high pressing methods. By comparison, hot-pressing and melting samples were prepared, respectively. Techniques of SEM, TEM, were used to characterize the morphology, composition of alloys. Electrical conductivity, Seebeck coefficient and thermal conductivity were measured in the temperature range of 80–300 K. The influence of the different methods on thermoelectric properties was presented. The thermoelectric properties were calculated and compared with those of single crystals presented in previous studies.

The vibration problem of the large scale high speed motor itself has been widely researched in the motor product factory, however, the actual vibration is more larger after the installation in engineering spot. This paper is taking the practical problem of western oil power station project as the research background. It is aiming at solving the strong vibration problem of the system of the oil pipeline motors in the west of China. The researching methods of the field experiment and finite element simulation are both used to analysis the characteristics of mode for the fixing base bracket system. The natural frequencies of various structure bases including all-steel, fulfilling-grouting and semi-grouting are analyzed respectively, and the reason of the system over vibration problem has been found out, and it has been verified of the improved scheme of the base system.

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 4-11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.

Gene therapy using integrating viral vectors in hematopoietic stem cells (HSC) has shown clinical benefit in genetic diseases. The safety concerns associated with random integration and the lack of gene expression regulation remain key challenges with use of viral vectors. Efficient and target-site specific correction of mutation(s) in patient HSC genomic DNA using non-viral methods may improve safety and gene expression regulation. We and others have previously reported that loading of Nucleases in the form of messenger RNA (mRNA) results in low toxicity and efficient functional gene editing when using MaxCyte's rapid, automated, cGMP and regulatory compliant, and closed cell loading platform. This platform has been cleared for use in human clinical trials for multiple applications of targeted gene editing in stem cells and immune cells.We report herein that transfecting CRISPR (Cas9+gRNA) mRNA along with donor oligomer specific to a hotspot mutation in the gp91phox gene (CYBB) located on exon 7 associated with the X-linked form of Chronic Granulomatous Disease (X-CGD), using MaxCyte GT System, results in efficient repair of the 676T (stop codon) to 676C (Arginine) mutation, resulting in gp91 protein expression and associated oxidase activity.Plasmids encoding Cas9 and gRNA were purchased from the Genomic Engineering Center at Washington University (St. Louis, MO). The mRNA encoding Cas9 and gRNA were synthesized at MaxCyte using mMESSAGE mMACHINE® T7 Ultra kit, (Ambion, Austin, TX). Patient EBV-transformed B cell line (B-LCL) and patient HSC were obtained under NIAID IRB approved protocol after patient written informed consent. The cells were cultured in RPMI-1640+10% FBS+2mM L-glutamine and StemSpan medium (Stem Cell Technologies, BC, Canada) supplemented with 100ng/ml SCF, Flit3L and TPO, respectively. We first optimized transfection conditions with the X-CGD patient B-LCL. It was found that B-LCL can be efficiently transfected with CRISPR and donor oligomer. Transfected B-LCL exhibit 80±6% viability, and maintained cell proliferation rate identical to control cells. Cel-1 assay shows 30-40% (N=8) efficiency of specific-site gene editing. Using donor oligomer containing a HindIII restriction site, it was determined that the DNA integration efficiency in Exon 7 was 35±8% (N=3). Using donor oligomer with the removal of HindIII restriction site, the expression of functional gp91 protein as determine by flow cytometry was 9±3% (N=3). These developed protocols were used to transfect HSC obtained from the same CGD patient (N=1). Following in vitro myeloid differentiation of gene mutation-repaired CGD HSC, 6.3% of the differentiating cells containing neutrophils and monocytes were oxidase positive in the dihydrorhodamine (DHR) flow cytometry oxidase function assay. These observations demonstrated restoration of phagocyte oxidase activity from the gene repair. Work is in progress to optimize and achieve clinical scale-up of the process which may have broad applicability to the treatment of monogenic diseases.

Superconducting Fault Current Limiter (SFCL) is an attractive appliance for modern electrical power system. A 10.5kV/1.5 kA three-phase HTS fault current limiter was developed by IEE, CAS. This improved rectifier-type SFCL with HTS coil of 6.25 mH is going on a demonstrated long-term reliable operation in a real 10.5 kV substation located in Hunan, China. In a three-phase-to-ground short circuit test of grid, the prospective fault current of 3.5 kA was limited to 635 A at the pre-setup short-circuit point successfully

Using a nonviral, electroporation-based gene transfection approach, we demonstrate the efficient and consistent transfection of two poorly immunogenic tumor cell lines: B16F10 melanoma and renal carcinoma (RENCA). Three genes, IL-12, angiostatin (AS), and an endostatin:angiostatin fusion protein (ES:AS) were subcloned into a DNA plasmid containing EBNA1-OriP, which was then transfected into B16F10 and RENCA cells. Significant levels of protein were secreted into the culture supernatants of transfected cells in vitro. Transfected tumor cells were injected subcutaneously into mice. All the three transgenes were capable of significantly delaying and reducing the formation of primary B16F10 and RENCA tumors, as well as B16F10 lung metastases. By day 11 post-injection, all control mice that received either mock-transfected or empty vector DNA-transfected B16F10 tumor cells had developed large primary tumors. In contrast, mice that received IL-12-transfected B16F10 cells did not develop appreciable tumors until day 17, and these were significantly smaller than controls. Similar results were observed for the RENCA model, in which only one of the IL-12 mice had developed tumors out to day 31. Expression of AS or ES:AS also significantly delayed and reduced primary tumors. Overall, ES:AS was more effective than AS alone. Furthermore, 25% of the AS mice and 33% of the ES:AS mice remained tumor-free at day 17, by which point all control mice had significant tumors. Mouse survival rates also correlated with the extent of tumor burden. Importantly, no lung metastases were detected in the lungs of mice that had received either AS or ES:AS-transfected B16F10 tumor cells and significantly fewer metastases were found in the IL-12 group. The consistency of our transfection results highlight the feasibility of directly electroporating tumor cells as a means to screen, identify, and validate in vivo potentially novel antiangiogenic and/or antineoplastic genes.

Electroporation is widely used to transfect and load cells with various molecules. Traditional electroporation using a static mode is typically restricted to volumes less than 1 mL, which limits its use in clinical and industrial bioprocessing applications. Here we report efficient, large volume transfection results by using a scalable-volume electroporation system. Suspended (Jurkat) and adherent cells (10T1/2 and Huh-7) were tested. A large macromolecule, FITC-conjugated dextran (MW=500 kD) was used to measure cell uptake, while a plasmid carrying the gene coding for enhanced green fluorescence protein (eGFP) was used to quantitate the flow electrotransfection efficiency as determined by flow cytometry. The flow electroloading efficiency of FITC-dextran was >90%, while the cell viability was highly maintained (>90%). High flow electrotransfection efficiency (up to 75%) and cell viability (up to 90%) were obtained with processing volumes ranging from 1.5 to 50 mL. No significant difference of electrotransfection efficiency was observed between flow and static electrotransfection. When 50 mL of cell volume was processed and samples collected at different time points during electroporation, the transgene expression and cell viability results were identical. We also demonstrated that DNA plasmid containing EBNA1-OriP elements from Epstein-Barr virus were more efficient in transgene expression than standard plasmid without the elements (at least 500 too 1000-fold increase in expression level). Finally, to examine the feasibility of utilizing flow electrotransfected cells as a gene delivery vehicle, 10T1/2 cells were transfected with a DNA plasmid containing the gene coding for mIL12. mIL12 transfected cells were injected subcutaneously into mice, and produced functional mIL12, as demonstrated by anti-angiogenic activity. This is the first demonstration of efficient, large volume, flow electroporation and the in vivo efficacy of flow electrotransfected cells. This technology may be useful for clinical gene therapy and large-scale bioprocesses.

CD19-targeted chimeric antigen receptor (CAR)-engineered T/NK-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been less successful in solid cancers. This is partly due to specificity for shared tumor antigens also present on normal host tissues that leads to ‘on-target/off-tumor’ toxicity. We therefore developed a non-viral approach using repeated infusions of mesothelin-specific messenger RNA (mRNA) CAR-transfected T cells to permit prospective control of ‘on-target/off-tumor’ toxicity. Early trials provided preliminary evidence of the safety and anti-tumor activity of this strategy, but the ex vivo selection, activation and expansion of lymphocytes is laborious and expensive. We therefore explored the feasibility of using a rapid, automated, closed system for cGMP-compliant mRNA CAR transfection into freshly isolated peripheral blood mononuclear cells for clinical scale manufacture (CARMA). The resulting cryopreserved cellular product expressed CAR in >95% of cells, and recognized and lysed tumor cells in an antigen-specific manner. Expression of CAR was detectable for 5-7 days in vitro, with a progressive decline of CAR expression related to in vitro cell expansion. In a murine ovarian cancer model, a single intra-peritoneal (IP) injection of CARMA resulted in the dose-dependent inhibition of tumor growth and prolonged the overall survival (OS) of mice. Multiple weekly IP injections of the optimal CARMA dose enhanced disease control and further prolonged OS, both of which improved with an increasing number of injections. No significant off-tumor toxicities were observed. These data support further investigation of serial IP CARMA administration as a potential treatment for ovarian cancer and other mesothelin-expressing tumors involving the peritoneum, and provide preclinical proof of principle of CARMA for solid tumors. Citation Format: Chien-Fu Hung, Xuequn Xu, Linhong Li, Ying Ma, Qiu Jin, Angelia Viley, Cornell Allen, Pachai Natarajan, Rama Shivakumar, Madhusudan V. Peshwa, Leisha A. Emens. Development of anti-human mesothelin chimeric antigen receptor (CAR) messenger RNA (mRNA) transfected peripheral blood mononuclear cells (CARMA) for the treatment of mesothelin-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3748. doi:10.1158/1538-7445.AM2017-3748

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patient's own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.

Fracture experiments were done on 3 mol% Y-TZP ceramics from room temperature to 4.2 K. X-ray diffraction analysis was used to study the phase contents of the ceramics. The monoclinic phase content and fracture toughness, K Ic , all increase with decreasing temperature, which is mainly due to the stress-induced transformation. On the other hand, the spontaneous transformation also takes a part in toughening.

Genetic modification for enhancing cellular function has been continuously pursued for fighting diseases. Messenger RNA (mRNA) transfection is found to be a promising solution in modifying hematopoietic and immune cells for therapeutic purpose. We have developed a flow electroporation-based system for large volume electroporation of cells with various molecules, including mRNA. This allows robust and scalable mRNA transfection of primary cells of different origin. Here we describe transfection of chimeric antigen receptor (CAR) mRNA into NK cells to modulate the ability of NK cells to target tumor cells. High levels of CAR expression in NK cells can be maintained for 3-7 days post transfection. CD19-specific CAR mRNA transfected NK cells demonstrate targeted lysis of CD19-expressing tumor cells OP-1, primary B-CLL tumor cells, and autologous CD19+ B cells in in vitro assays with enhanced potency:80% lysis at effector-target ratio of 1:1. This allows current good manufacturing practices (cGMP) and regulatory compliant manufacture of CAR mRNA transfected NK cells for clinical delivery.

The microstructural changes associated with the tetragonal to monoclinic martensitic transformation at cryogenic temperatures in sintered CeO2-ZrO2 ceramics containing 15.5-16.5 mol% CeO2 have been studied by means of TEM observations. X-ray diffraction analysis indicates that the stress-induced martensitic phase increases with decreases in both temperature and CeO2 content. The effects of martensitic morphologies, anti-phase boundaries (APBs) and various dislocation features on mechanical properties are also discussed in the paper.

A pile group consisted of in-plane hinge-jointed, rigid, sloping piles embedded in a homogeneous, isotropic elastic half space under in-plane loading is analysed by Line-Loaded Integral Equation Method (LLIEM). Based on the analysis of a single pile, where the problem is reduced to a set of Fredholm integral equations of the first kind and solved numerically by LLIEM, the analysis of a pile group consisted of two hinge-jointed, rigid, sloping piles under in-plane loading at a joint is reduced to a set of 10 simultaneous nonlinear equations with 10 unknowns and solved by group iteration method. A program is designed for the solution of this problem. The accuracy of the numerical solution can be checked by the reciprocal theorem of work.

Gene therapy using integrating viral vectors in hematopoietic stem cells (HSC) has shown clinical benefit in genetic diseases. However, there remain safety concerns associated with random integration and the lack of regulation of gene expression. Efficient and site-specific correction of mutation(s) in HSC using non-viral methods may improve safety and regulation of gene expression. Chronic granulomatous disease (CGD) due to defective phagocyte NADPH oxidase complex and lack of bactericidal superoxide and other reactive oxidative species (ROS) is characterized by severe infections and hyperinflammation. Although the X-linked form of CGD with gp91phox deficiency results from mutations that span the CYBB gene, we identified a ‘hotspot’ mutation at Exon 7 c. 676C>T, causing a premature stop codon in 17 out of 285 patients with X-linked CGD at the NIH. Here we report the result of the efficient correction of the hotspot CYBB mutation using highly efficient CRISPR (Cas9 and sgRNA) system with an oligomer as donor repair template, using MaxCyte's commercially/clinically validated cGMP/regulatory compliant and closed platform technology. Plasmids encoding Cas9 and gRNA were purchased from the Genomic Engineering Center at Washington University (St. Louis, MO). The mRNA encoding Cas9 and gRNA were in vitro transcribed at MaxCyte using mMESSAGE mMACHINE® T7 Ultra kit, (Ambion, Austin, TX). We screened and selected best gRNA from four gRNA candidates for correction and then optimized transfection conditions with EBV-transformed B cell line (B-LCL) derived from an adult patient (P1) with the hotspot CYBB mutation. Transfected B-LCL exhibit 80±6% viability, minimal detectable toxicity as determined by cell proliferation rate referenced to control cells, and efficient site-specific gene correction with 20-50% WT gp91 expression. These developed protocols were used to treat G-CSF and pleraxifor mobilized CD34+ HSC from P1. Following optimization, in vitro treated HSC from P1 achieved 20-30% WT gp91 expression, with >50% viability, and minimal loss of cell proliferation capacity compared with control cells. CD34+ HSCs undergo myeloid differentiation in DMEM supplemented with G-CSF prior to functional evaluation using flow cytometric dihydrorhodamine (DHR) assay, demonstrating ~20% ROS+ cells in treated samples compared to ~80% in normal controls. P1's HSC treated the same way were transplanted into immunodeficient mice, and analyzed 8 weeks later. Bone marrow from mice transplanted with P1 treated cells showed CD45+ human cell engraftment rates at 50-80%, and of the forward/side scatter-gated granulocytes, 11-26% express gp91phox relative to 68% in normal control. Peripheral blood from mice demonstrated 11-23% human CD45+ cells, of which 9-21% expressed gp91phox, compared to 79% in normal controls. Deep sequencing of human CD45+ cells sorted from mouse bone marrow confirmed high rates (up to 21%) of genetic correction from the ‘T’ mutation to the wildtype ‘C’. Since female carriers of X-CGD with ~10-15% normal functioning neutrophils appear to have normal resistance to infections, this level of correction at 10-20% in human CD45+ cells from transplanted mice suggest CRISPR/oligo approach a feasible therapeutic option for treatment of CGD patients with the Ex7, c. 676C>T mutation.

Genetic modification for enhancing cellular function has been continuously pursued for fighting diseases. Messenger RNA (mRNA) transfection is found to be a promising solution in modifying hematopoietic and immune cells for therapeutic purpose. We have developed a flow electroporation-based system for large volume electroporation of cells with various molecules, including mRNA. This allows robust and scalable mRNA transfection of primary cells of different origin. Here we describe transfection of chimeric antigen receptor (CAR) mRNA into NK cells to modulate the ability of NK cells to target tumor cells. High levels of CAR expression in NK cells can be maintained for 3-7 days post transfection. CD19-specific CAR mRNA transfected NK cells demonstrate targeted lysis of CD19-expressing tumor cells OP-1, primary B-CLL tumor cells, and autologous CD19+ B cells in in vitro assays with enhanced potency: >80% lysis at effector-target ratio of 1:1. This allows current good manufacturing practices (cGMP) and regulatory compliant manufacture of CAR mRNA transfected NK cells for clinical delivery.

Resettlement is one of the most important major issues of the hydropower development. Based on GPS multi-antenna, wireless bridge, 3rd Generation telecommunication technologies and single epoch solution algorithm, a near real-time landslide monitoring system of Pubugou hydropower engineering resettlement zone, new Hanyuan County, is under construction. GPS has also been proven an effective mean to detect atmospheric water vapor and the precipitable water (PW) over the new town was obtained. Results show the GPS system provides deformation with 3–4 mm accuracy which can meet the requirements of landslide monitoring and the atmospheric water vapor changes is useful and meaningful to precipitation forecast as well as the landslide early warning system.

Background aims. Several studies have demonstrated that the immunogenicity of chronic lymphocytic leukemia (CLL) cells can be increased by manipulation of the CD40/CD40-ligand (CD40L) pathway. Although immunologic, and perhaps clinical, benefits have been obtained with an autologous CLL tumor vaccine obtained by transgenic expression of CD40L and interleukin (IL)-2, there is little information about the optimal gene transfer strategies. Methods. We compared two different CLL vaccines prepared by adenoviral gene transfer and plasmid electroporation, analyzing their phenotype and immunostimulatory activity. Results. We found that higher expression of transgenic CD40L was mediated by adenoviral gene transfer than by plasmid transduction, and that adenoviral transfer of CD40L was associated with up-regulation of the co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54. In contrast, transgenic IL-2 secretion was greater following plasmid transduction. These phenotypic differences in the vaccines were associated with different functionality, both ex vivo and following administration to patients. Thus adenoviral vaccines induced greater activation of leukemia-reactive T cells ex vivo than plasmid vaccines. In treated patients, specific T-cell (T helper 1 (Th1) and T helper 2 (Th2)) and humoral anti-leukemia responses were detected following administration of the adenoviral vaccine (n = 15), while recipients of the plasmid vaccine (n = 9) manifested only a low-level Th2 response. Progression-free survival at 2 years was 46.7% in the adenoviral vaccine recipients, versus 11.1 % in those receiving plasmid vaccine. Conclusions. CLL vaccines expressing the same transgenes but produced by distinct methods of gene transfer may differ in the polarity of the immune response they induce in patients

Many monogenic recessive diseases of blood can, in principle, be cured by transfer of functional therapeutic transgene to the genome of the hematopoietic stem cell (HSC) – a strategy proven successful for multiple rare diseases using current integrating vector gene therapy. With a focus on X-linked chronic granulomatous disease (X-CGD), we report a directed approach orthogonal to randomly integrating retrovector gene therapy: the highly specific targeted placement of the curative transgene into a validated safe harbor locus in human HSCs via human genome editing with zinc finger nucleases (ZFNs) and donor insert delivery using an AAV6 vector.We describe here an integrated targeted delivery platform customized for targeted addition to human HSCs using a cGMP-compliant electroporation system compatible with clinical scale production. Using next-generation, highly optimized ZFNs against the AAVS1/PPP1R12C gene locus, we optimized conditions for addition of the fluorescent Venus cDNA into human peripheral blood G-CSF mobilized CD34+ HSCs. Venus expression in manipulated human HSCs in vitro reached >50% efficiency, while earlier experiments demonstrated persistence of gene-modified cells in NSG mice with 12-15% Venus+ human CD45+ cells retrieved from transplanted mouse bone marrow and overall human HSC engraftment levels of >15%. Targeted integration (TI) rates achieved in human CD45+ cells from mouse bone marrow were 28-57%. Similar levels of Venus+ (~10%) are observed in spleen and peripheral blood CD45+ cells, indicating differentiation of gene-modified CD34 HSCs into circulating blood cells.X-CGD patients suffer from severe bacterial and fungal infections with excessive inflammation due to a defect in the gp91phox subunit of phagocyte oxidase. To extend the results above to CGD, we therefore used the same approach to target addition of the relevant curative transgene, gp91phox, into the AAVS1 safe harbor locus of HSCs from patients with X-linked CGD. In vitro levels of gp91phox expression in gene-modified patient CD34+ HSCs population achieve 12-16% gp91phox expression by flow cytometric analysis, with an NSG xenograft study demonstrating 3-5% of the engrafted human CD45+ cells expressing gp91phox. Of note, the MND-driven gp91phox expression from the safe harbor locus in human neutrophils differentiating from CD34+ cells transplanted into the NSG mouse model parallels wildtype gp91phox levels produced at the native locus.Our studies demonstrate the feasibility of targeted addition of different genes at the AAVS1 safe harbor site of the genome in human HSCs at an unprecedented efficiency and specificity; we demonstrate the efficient correction of the enzymatic defect in neutrophils arising from patient-derived HSCs in vivo. In sum with the advances in GMP-scale cell processing for genome editing, and the charted regulatory path for ZFNs to the clinic provided by ongoing trials in HIV, our studies represent the foundation for a rapid translation of ZFN-driven targeted addition as a clinical modality for X-linked CGD.

Viral vectors show promise as gene therapy treatments for a number of diseases. However, it has been difficult to develop a robust system for generating large volumes of high-titer viral vectors based on lentivirus, alphavirus and adeno-associated virus (AAV) that are needed for clinical applications. Problems stem from the lack of stable producer cell lines and inherent inefficiencies associated with introducing multiple plasmids into adherent monolayer cells by conventional transient transfection techniques (i.e., calcium phosphate precipitation or cationic lipids). Other researchers have demonstrated that suspension cells of lymphoid origin, which can be grown at much higher density than adherent cells, can be converted into efficient retroviral vector producer cell lines (Chan et al., 2001. Gene Ther. 8: 697). However, commercially available transient transfection systems are not optimal for large volume co-transfection of suspension cells, especially hematopoietic cells. MaxCyte's proprietary high-throughput electroporation technology provides a closed, robust, scalable system for high efficiency transfection of both adherent and suspension cells. The MaxCyte system previously was used to produce high titer alphaviral vectors from >10 x109 Vero cells and lentiviral vectors from >1 x109 adherent HEK 293T cells. To improve the efficiency of lentiviral vector production at large scale and to demonstrate the feasibility of lentivector production in suspension cells, we used the MaxCyte system to transfect large volumes (38 x109 cells in 380 mL) of K562 human hematopoietic cells rapidly (within 20 minutes) with a plasmid encoding the green fluorescent protein (GFP) marker gene. The transfected cells were collected in fractions of 35-40 mL. Cells were cultured from each fraction or pooled samples to monitor consistency of the high- throughput electroporation. Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 48 hrs post-electroporation for all fractionated and pooled cell samples. Propidium iodide (PI) exclusion revealed the cell viability of all the fractionated and pooled cell samples to be greater than 90%. In addition, we were able to produce lentivirus in K562 cells by small- scale electroporation of 10 x106 cells with four plasmids encoding components of a lentiviral vector derived from HIV carrying the eGFP marker gene. In conclusion, these studies will demonstrate that use of suspension cells processed by MaxCyte's high- throughput electroporation-based transfection system is a feasible approach for generating large volumes of high-titer viral vectors suitable for clinical / commercial applications in human gene therapy.

Electroporation of dendritic cells (DCs) with tumor lysate elicited greater antitumor responses in vitro and in vivo, using less lysate than standard coincubation. Electroloaded DCs had normal surface marker expression and matured into competent antigen-presenting cells. In a renal carcinoma (RENCA) model, mice were pretreated with lysate-loaded DCs before tumor challenge. Mice that received DCs electroloaded with RENCA lysate had significantly smaller tumors (9 ± 6 mm 2 ) than mice given DCs coincubated with the same lysate (23 ± 5 mm 2 ). To evaluate a metastatic therapeutic tumor model, mice were first injected with Lewis lung carcinoma (LLC) and then given 2 doses of cryopreserved LLC lysate-loaded DCs. Mice treated with electroloaded DCs had a 50% reduction in lung metastases compared with control mice that received no DCs or DCs loaded with liver lysate. In contrast, DCs coincubated with LLC lysate were indistinguishable from controls. Tumor lysate-electroloaded but not-coincubated DCs also primed syngeneic mouse splenocytes in vitro to produce interferon-γ and, specifically, lyse tumor cells. The electroloaded DCs elicited specific T-cell responses with less lysate than the amount reported in standard coincubation procedures. This approach may be particularly useful when small amounts of tumor material are available.

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

Natural killer (NK) cells are immune cells involved in the defense against cancer. They have also been shown to induce strong anti-tumor responses in the setting of hematopoietic stem cell transplantation and in early clinical trials on adoptive NK cell transfer. Several methods to expand large numbers of clinical-grade NK cells have been developed for trials exploring adoptive NK cell immunotherapy for cancer. However, long-term culturing of NK cells often lead to undesirable phenotypic changes that may compromise their homing capacity and cytotoxic function, and can also lead to senescence compromising in vivo longevity. Introducing genes into NK cells that improve their in vivo viability, cytotoxicity, and ability to home to disease sites could improve the efficacy of NK cell-based immunotherapy. Previously, we and others have shown that genetic manipulation of NK cells through viral transduction is challenging, typically resulting in substantial reduction in NK cell viability and low transduction efficiency. mRNA transfection is an alternative strategy to genetically modify ex vivo expanded NK cells that may overcome limitations of viral transduction. Here we present data characterizing the transgene expression, viability, proliferative capacity, phenotype and cytotoxic function of clinical-grade ex vivo expanded human NK cells following mRNA electroporation using the GMP compliant MaxCyte system. Using unmodified mRNA coding for GFP and the cell surface marker CD34, we established that this technology resulted in rapid and highly efficient protein expression in NK cells without compromising their viability and cytotoxic function (Figure). NK cells electroporated with GFP mRNA rapidly became GFP positive and remained fluorescent for more than two weeks. Following transfection of CD34 mRNA, nearly 100% of NK cells expressed CD34 that remained detectable on the cell surface for up to five days, without affecting viability amongst transfected cells. With the exception of a slight reduction in proliferative capacity compared to controls, no negative impacts of mRNA electroporation using the MaxCyte platform were observed. Transfection of expanded NK cells did not alter expression of twenty cellular markers as assessed by flow cytometry, including activating and inhibitory NK cell receptors and death receptor ligands such as TRAIL. Further, electroporated NK cells maintained high cytotoxic function against K562 cells and multiple myeloma cells (Figure). In conclusion, mRNA electroporation of ex vivo expanded NK cells using the clinical-grade MaxCyte transfection system is highly efficient and opens numerous new possibilities to advancethe field of NK cell-based cancer immunotherapy. Figure 1 Figure 1. Disclosures Li: MaxCyte Inc.: Employment, Patents & Royalties. Peshwa:MaxCyte Inc.: Employment, Patents & Royalties; Indian Biomedical Association: Membership on an entity's Board of Directors or advisory committees; Epidarex Capital: Membership on an entity's Board of Directors or advisory committees; BioMetrx LLC: Membership on an entity's Board of Directors or advisory committees.

Primary pulmonary hypertension (PPH) is a progressive disease of unknown pathogenesis with a bleak prognosis for affected patients. The available treatment options utilize vasorelaxants such as continuously infused prostaglandin analogs or inhaled nitric oxide (NO). We are developing a cell-based gene therapy approach that administers NO in situ and utilizes a non-viral gene delivery system. We present here preclinical in vivo data on a model system that utilizes PH induced by a chemical agent, monocrotaline (MCT), in the rat and intravenous (IV) administration of fibroblasts transfected with endothelial nitric oxide synthase (eNOS).

Effective immunotherapies rely on triggering specific cytotoxic responses against target cells. Chimeric Antigen Receptors (CAR) composed of antigen recognition domains in the form of scFv portions of monoclonal antibodies and immune cell activation domains provide non-MHC restricted, KIR-independent, immune cellmediated cytotoxicity directed toward target cells expressing specific antigens. It is well demonstrated that CAR transfection into immune cells using viral vectors redirects their specificity and enhances their cytotoxicity. Translation of these findings into clinically feasible treatment strategies is challenged by the labor/costs associated with extended cell processing, and by safety concerns associated with viral vectors. We describe here the use of non-viral methods to deliver a nucleotide sequence encoding CAR into freshly isolated immune cells obtained from peripheral blood leukapheresis collection. Protocols for loading mRNA encoding eGFP (irrelevant control) and a previously described anti-CD19 CAR (Imai et al. Blood 2005) into peripheral blood mononuclear cells (PBMC) and cell subsets (T cells, NK cells, and monocyte-depleted PBMC) were developed and optimized using a clinically validated, automated, closed-system, flow electroporation technology. PBMC were obtained from 5 normal healthy donors and 1 patient with B-cell chronic lymphocytic leukemia (B-CLL). Expression levels of eGFP or anti-CD19-BB-z were analyzed by flow cytometry. Specific cytotoxicity of target cells after 4 hours was measured by flow cytometry using acetoxymethyl-calcein labeled CD19+ target cells which included acute lymphoblastic leukemia (ALL) cell lines, primary B-CLL cells and autologous B cells. Following loading of mRNA encoding eGFP, cell viability was consistently ≥80% for all cell populations. Most cells expressed eGFP: 97±1% (day 1) and 83±2% (day 7). Following loading of mRNA encoding anti-CD19-BB-z, cell viability was 96%±1% for resting T cells (n=2), 93%±10% for PBL (n=2), 94%±8% for PBMC (n=3), 80%±15% for NK cells (n=4). Expression of anti-CD19-BB-z protein receptor was 54%±11% (day 1) in PBMC (n=6). CAR expression decreased over time and was essentially similar to baseline on day 7 post-loading. Cells expressing anti-CD19-BB-z protein (but not those expressing eGFP only), lysed 60%–80% ALL or B-CLL target cells at an effector-to-target ratio (E:T) of 8:1. A shigher E:T ratio of 20:1 was required for 60%–80% lysis of autologous B-cell targets. There was no change in cytolytic activity of CAR mRNA loaded-PBMC during the first 3 days post-loading. Subsequently, cytolytic activity dropped and on day 7 (post-loading) was approximately half of the activity measured on day 3 (post-loading). In vivo testing was performed in a sub-cutaneous tumor grafting model in Beige SCID mice. The mice (N = 5 per group) were transplanted subcutaneously with CD19+ tumor (HS Sultan) cells (1 × 106) co-mixed with PBMC loaded with anti-CD19-BB-z mRNA (doses = 0.74, 2.22, 6.67 and 20 ×106). Controls were tumor cells only or tumor cells co-mixed with unmodified PBMC. Tumors developed and grew in 3/5 animals in each control group and in 2/5 in lowest anti-CD19-BB-z mRNA-loaded dose. No tumor formation was observed in any animal in each of the 3 higher cell doses. More comprehensive animal studies and investigation of engineering additional constructs for chimeric signal receptor with tumor affinities other than anti-CD19 are in progress. These results indicate that CAR can be effectively transfected into immune cells with the methodology that we developed. Because of its simplicity and effectiveness, this methodology is well suited for clinical application and holds promise for improving the specificity and widening the feasibility of cell therapy.

B-chronic lymphocytic leukemia (B-CLL) cells express tumor associated antigens that may generate a T cell mediated immune response, but present these antigens poorly. Moreover, patients with B-CLL often have poor immune function due to the disease or its treatment. We have shown that expression of transgenic CD40L increases the immunogenecity of human B-CLL cells ex vivo and in vivo, and that this effect can be potentiated by co-expression of transgenic IL2. Previous studies described outcomes when adenoviral vectors were used to obtain gene transfer, but because of the complexities and expense of manufacture of viral vectors, and their lingering safety concerns, we determined whether it was possible to use electroporation (with the MaxCyte device) as a physical means of transferring CD40L and IL2 plasmids to produce vaccines with similar biological properties in vitro and in vivo. Table 1 compares the phenotype of the vaccines using each vector. Table 1. Comparision of immunogenic characteristics and viability of the adenoviral and plasmid vaccines Type of Vaccine CD40L (%) CD80 (%) CD86 (%) IL-2 (pg/ml/10e6 cells) Viability (%) IL2 CD40L All the values are given as mean ± SE. * P< 0.01, Paired Student’s t test. Adenoviral Pre 0.2 ± 0.01 2.6 ± 2.4 7.5 ± 3.9 Post 66.1 ± 5.5* 50.2 ± 7.8* 69.5 ±11* 253.5 ± 82.6 93.6 94.2 Plasmid Pre 1.3 ± 0.85 11.5 ± 6.2 19.7 ± 6.8 Post 55.5 ± 5.1* 19.2 ± 9.3 26.4 ± 9.7 4806.6 ±1398.9 84.4 88.4 Vaccines made by both approaches met the release criteria for CD40L and IL2 expression (CD40L ≥20% and IL-2 ≥ 150 pg/ml/1x10e6 cells ), but expression of IL2 was higher in the plasmid vaccines, expression of CD40L was equivalent in each and expression of the additional co-stimulatory molecules CD80 and CD86 (induced after CD40 activation by transgenic CD40L) was higher in the adenoviral vaccines. Fourteen patients were given adenoviral-vaccines and nine the plasmid transduced cells. Each of these patients received up to 18 s.c. injections of IL-2 secreting and CD40L expressing tumor cells. Both types of vaccine were well tolerated. Table 2 shows the results of culturing patient T cells with autologous B-CLL tumor cells. Table 2. Comparision of anti-B-CLL T cell responses induced by adenoviral and plasmid vaccines Type of Vaccine Pre-vaccine After 3rd vaccine After 6th vaccine All the values were are given as mean ± SE. *P After 3 and 6 injections, both the adenoviral and plasmid vaccines had induced a rise in spot forming cells (SFC) for IL5, a cytokine associated with Th2 cells, but the rise was greatest in the recipients of the electroporated plasmid vaccine. By contrast, only the adenoviral vaccine induced a rise in SFC that produced IFN-γ, a cytokine associated with Th1 cells. Studies using MHC class I and II blocking antibodies showed that the IL5 and IFN-γ responses to both types of vaccine were mediated by HLA restricted T lymphocytes. The 1-year progression-free survival rates (PFS) for adenoviral vaccine group and plasmid vector group were 43% and 22% respectively. Figure 1 shows 1-year PFS rates for each group. Hence electroporation provides a more rapid and simpler means of preparing IL2/CD40L expressing B-CLL vaccines, but the cells express higher levels of IL2 and lower levels of “secondary” co-stimulator molecules than adenoviral vaccines, and produce an anti-tumor immune response of different polarity. Currently, we are evaluating electroporation of mRNA encoded CD40L which appears to augment upregulation of additional costimulatory molecules. Figure Figure

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma with the worst long-term prognosis of any NHL subtype. Current therapeutic options are unsatisfactory. MCL patients’ malignant B cells are ineffective antigen-presenting cells (APCs), perhaps resulting from low level expression of the immune co-stimulatory molecules that are essential to activate T cells upon interaction with the T-cell receptor. The MCL cells can be engineered to be effective APCs and thereby function as a therapeutic cellular vaccine in combination with chemotherapy and/or stem cell transplantation to eradicate residual disease. However, primary MCL cells are difficult targets for gene transfer by both viral and non-viral methodologies. Ligation of CD40 resulting from co-culturing with hCD40L expressing murine fibroblasts was shown to be superior to a panel of other immune stimulants and cytokines in upregulating co-stimulatory markers and inducing anti-tumor T cell responses (Hoogendoorn et al. 2005). We now report on a technology platform, based on electroporation of mRNA for CD40L, for the introduction of CD40L protein expression and subsequent induction of immune co-stimulatory molecules by MCL tumor cells. Primary MCL malignant B cells were obtained from patients’ lymph node biopsies by mechanical dissociation, placed in single cell suspension and cryopreserved prior to modification. Full-length 5′-end capped hCD40L mRNA transcript was generated by in vitro transcription with a commercially available T7 polymerase kit. The transfected MCL cells were immunostained with fluorophore-conjugated monoclonal antibodies against hCD40L, hCD80 and 86 then analyzed by FACS. Data showed hCD40L could be detected in ≥ 80% of the transfected MCL cells as early as 2 hrs post transfection. At 3 days post manipulation, hDC40L expression could be detected on approximately 30% of the transfected MCL cells. Cell viability remained at approximately 80% during the 3 day in vitro culturing. FACS analysis of the immune co-stimulatory molecules revealed that forced expression of hCD40L caused an up-regulation of CD80/86, which was increased approximately 10 fold compared to the expression levels in naïve, non modified cells. The increased expression level of CD80/86 was maintained for 3 days. Furthermore, when the hCD40L modified MCL cells were mixed with allogeneic PBMC, they stimulated IFN-γ production at a level 4 fold higher than was observed with naïve, non modified MCL cells mixed with allogeneic PBMC. This provides proof-of-concept that MCL cells modified by mRNA-hCD40L transfection have the potential to be used as a cellular vaccine. Such transduced cells function to protect animals from tumor challenge. The process can be scaled up to produce >2×1010 modified tumor cells. This simple, non-viral cell manipulation system is practical and will be a useful tool for immunotherapy of human hematopoietic malignancies such as MCL.

Previously we reported (Li LH et al, 2006, Cancer Gene Therapy13:215–224) rapid and efficient production of human CD40L+ (hCD40L) B-Chronic lymphocytic leukemia (B-CLL) tumor vaccine by electrotransfecting the cells with a DNA plasmid encoding hCD40L. The hCD40L-transfected B-CLL cells cryopreserved at 3 hrs post transfection showed cell viability ≥50%, and CD40L expression level ≥50% (N=10). Although costimulatory molecule upregulation was not detected at 3 hrs, we hypothesized the vaccine would upregulate costimulatory molecules in vivo, emulating levels seen in vitro among viable cells after 12–24h culture. The suboptimal immunologic and clinical results of our previous vaccine preparation reported last year (Fratantoni JC et al, 2005, Blood106:136a) suggested that costimulatory molecule upregulation in vivo was insufficient. Simply increasing culture time of hCD40L-transfected B-CLL cells is limited by the resulting low cell viability caused by DNA uptake-mediated toxicity. In this study, we report a simple modified vaccine manufacturing method that yields a vaccine with good cell viability and expression of co-stimulatory molecules prior to injection. During vaccine production, a portion of the B-CLL cells were first transfected with pCMV-hCD40L via electroporation (provider cells) and then mixed with non-transfected autologous B-CLL cells (naïve recipients) followed by coculture for 12 to 24hrs. Our data show that hCD40L expression levels on the transfected provider cells and the ratio of provider cells to naïve recipient cells directly correlate with hCD40L molecule expression on the naïve recipient cells. The naïve recipient cells in the mixture maintained high cell viability, 80%–90%, when normalized by the input naïve cell number, while cell viability of the provider cells declined to 19 ± 9% at 1d and further down to 2 ± 1% at 7ds post transfection (n=4). The percentage of cells expressing hCD40L depended on the mixing ratio. When a 10:1 ratio (provider: naïve) was used, the hCD40L expression level in naïve cells was up to 80%. In order to make an hCD40L+ B-CLL vaccine with high cell viability, a 1:1 ratio was applied. The viability of the final tumor vaccine product including both provider and recipient cells was 56 ± 6%, while hCD40L was detected among 34%±12% of the cells at 12–24h post mixing (n=10). Expression of CD80, CD86 and CD54 in the mixed cells were increased by 16 ± 8, 10 ± 5 and 24 ± 17 folds respectively, when compared to those of the naïve B-CLL cells (3 patients). Furthermore, we examined the capacity of the vaccine product to present antigen using an allo MLR, and monitored IFN-g secretion and proliferation of CFSE-labeled allo PBL. Data from 3 CLL patients’ samples showed that vaccine prepared by the mixing process could induce 6.8 ± 0.01, 2.1 ± 0.35 and 2.5 ± 1 fold more allo PBL proliferation and ≥25 folds higher IFN-g production than the control B-CLL cells (p2x1010 modified cancer cells. This simple, non-viral vaccine manufacturing process is practical and currently under evaluation in Phase I/II clinical study.

The identification of the chemokine receptor CCR5 as the major co-receptor required for HIV entry into CD4 T-cells has made the targeted disruption of CCR5 an attractive potential therapeutic approach for treating HIV-infected patients. Yet, no current method permits the efficient therapeutic disruption of a chosen gene in the human genome. We have developed designed zinc-finger protein nucleases (ZFNs) that specifically target the coding sequence of the CCR5 gene. Using standard delivery methods, we have shown these ZFNs can efficiently stimulate the formation of a double strand break (DSB) in the CCR5 locus to allow the natural DNA repair pathways, including Non-Homologous End Joining (NHEJ), to subsequently repair the DSB. Importantly, however, NHEJ is error prone and thus can result in frameshift mutations, leading to permanent disruption in >10% of K562 cells.