ALBERT

Description: Background: Driving fatigue affects the reaction ability of a driver. The aim of this research is to analyze the relationship between driving fatigue, physiological signals and driver’s reaction time. Methods: Twenty subjects were tested during driving. Data pertaining to reaction time and physiological signals including electroencephalograph (EEG) were collected from twenty simulation experiments. Grey correlation analysis was used to select the input variable of the classification model. A support vector machine was used to divide the mental state into three levels. The penalty factor for the model was optimized using a genetic algorithm. Results: The results show that α/β has the greatest correlation to reaction time. The classification results show an accuracy of 86%, a sensitivity of 87.5% and a specificity of 85.53%. The average increase of reaction time is 16.72% from alert state to fatigued state. Females have a faster decrease in reaction ability than males as driving fatigue accumulates. Elderly drivers have longer reaction times than the young. Conclusions: A grey correlation analysis can be used to improve the classification accuracy of the support vector machine (SVM) model. This paper provides basic research that online detection of fatigue can be performed using only a simple device, which is more comfortable for users.

Description: 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.

Description: 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.

Description: 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

Description: 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.