Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review.     

Genetic disruption of mammalian endoplasmic reticulum-associated protein degradation: Human phenotypes and animal and cellular disease models

Endoplasmic reticulum-associated protein degradation (ERAD) is a stringent quality control mechanism through which misfolded, unassembled and some native proteins are targeted for degradation to maintain appropriate cellular and organelle homeostasis. Several in vitro and in vivo ERAD-related studies have provided mechanistic insights into ERAD pathway activation and its consequent events; however, a majority of these have investigated the effect of ERAD substrates and their consequent diseases affecting the degradation process. In this review, we present all reported human single-gene disorders caused by genetic variation in genes that encode ERAD components rather than their substrates. Additionally, after extensive literature survey, we present various genetically manipulated higher cellular and mammalian animal models that lack specific components involved in various stages of the ERAD pathway.     

Monitoring growth and acetic acid secretion by a thermotolerantBacillus using conduction microcalorimetry

Summary A method is described to determine power of heat-time curves by conduction microcalorimetry in order to monitor the viability and ability of a thermotolerantBacillus strain to secrete acetic acid both during exponential growth and during stationary-phase. In this system secreted acetic acid is neutralized by an insoluble source of lime (dolime) which results in a poor correlation between optical density and culture dry weight. As an alternative, cells and residual dolime were rapidly resuspended in isothermal fresh medium with glucose in a conduction microcalorimeter. Heat evolution was rapid over a period of 200–800 s. Steady state heat evolution rate decreased as a function of culture time and did not correlate with: 1) specific growth rate: 2) viable cell number: 3) glucose consumption rate; or 4) acetic acid secretion rate. Glucose consumption and acetic acid secretion during the stationary growth phase were correlated with specific heat evolution rate. These initial results indicate that this technique may be useful for further development as an on-line flow or stopped-flow method to monitor the physiology of bacilli in response to nutrient depletion or growth inhibition.     

Cellular expansion of MSCs: Shifting the regenerative potential

Mesenchymal-derived stromal or progenitor cells, commonly called “MSCs,” have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.     

Ion-selective Microelectrodes: Their Use and Importance in Modern Plant Cell Biology

The exact ion gradients across cellular membranes and their changes due to metabolic or transport processes can be best studied with the use of ion-selective microelectrodes. The last decade of research using ion-selective microelectrodes in intact cells has proven this technique to be indispensable for the investigation of a variety of physiological questions of regulatory processes, membrane transport, cellular signalling, developmental biology and plant nutrition. Their application to selected problems has led to numerous exciting observations, many of which have changed our view concerning cellular responses to environmental stimuli and in many instances have led to a new understanding of plant cell physiology. Since, with these electrodes, intracellular as well as extracellular free ion concentrations can be simultaneously detected with electrical transport parameters such as membrane potential and membrane conductance, they can be powerful tools in the hands of many plant cell biologists.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

嵌合抗原受体T细胞治疗的现状与展望

肿瘤严重威胁着人类健康,当前肿瘤传统的治疗方法有手术治疗、化疗、放疗和靶向药物治疗等。近年来,肿瘤免疫治疗,尤其是嵌合抗原受体（chimeric antigen receptor,CAR） T细胞免疫疗法在基础研究与临床应用中蓬勃发展,并在治疗血液系统恶性肿瘤方面取得了巨大成功。然而,大量研究显示,细胞免疫治疗后可出现不同程度的毒副反应,且部分患者缓解后再次复发。因此,了解细胞治疗面临的挑战与局限性,寻找解决的办法,对继续发挥细胞免疫疗法的潜能具有重要意义。本文就免疫细胞的CAR结构、病毒载体的选择、细胞治疗面临的挑战及前景进行综述。     

DIEL CHANGES IN THE COMPOSITION OF PHOTOSYNTHETIC PIGMENTS AND CELLULAR CARBON AND NITROGEN IN CHATTONELLA ANTIQUA (RAPHIDOPHYCEAE)1

An axenic clonal culture of Chattonella antiqua (Hada) Ono was grown on a 12: 12 h LD cycle in a laboratory culture tank containing 1 m³ of f/2 medium. Diel changes in mean cell volume, cellular carbon (carbon content per cell), C/N ratio, cellular Chl a, Chl a/c ratio and carotenoid composition were observed. Mean cell volume and cellular C, N and pigments increased during the light period as a result of photosynthesis and decreased with increase of cell concentration by phased cell division during the dark period. These changes indicated that carbon assimilation and pigment synthesis occurred together during the light period. However, the patterns of increase were not the same since different diel patterns were also found in the ratios of C/N and chl a/c. Photosynthetic pigments were analyzed by reversed-phase high-performance liquid chromatography with ion-pairing solution. This analysis showed that the dominant carotenoids in C. antiqua were fucoxanthin, violaxanthin and β-carotene. Diel patterns of Chls a and c were similar to that of fucoxanthin but different from those of violaxanthin and β-carotene. The cellular contents of Chl a, fucoxanthin and carbon increased in a parallel manner during the light period. On the other hand, the increase of violaxanthin was restricted to only a few hours at the beginning of the light period during cell division cycles.