Regulatory Aspects of Cell and Gene Therapy Products: The Japanese Perspective.

Regulations for regenerative medicine for human use, such as cell and gene therapy (CGT), have evolved in accordance with advancements in clinical experience, scientific knowledge, and social acceptance of these technologies. In November 2014, two acts, "The Act on the Safety of Regenerative Medicine" (ASRM) and the "Pharmaceuticals, Medical Devices, and Other Therapeutic Products Act" (PMD Act), came into effect in Japan. The responsibilities of medical institutions in ensuring the safety and transparency of such medical technologies are described under ASRM. The PMD Act provides the option of a new scheme for obtaining conditional and time-limited approval for CGT products. Overall, research and development on CGT products, especially gene therapy products, is progressing. New legislative frameworks have been designed to promote the timely development of new technologies and safe and effective CGT products for Japanese patients.

[Points to Consider for Continuous Manufacturing of Biotechnological Drug Products].

Continuous Manufacturing is a manufacturing method in which raw materials enter the manufacturing process continuously, and products are discharged continuously throughout the duration of the process including multiple unit operations. Continuous Manufacturing includes various options: Those with all stages of processing from charging raw materials to discharging final products that are continuous, and those with only certain parts of the manufacturing process that are continuous. It can be introduced in an appropriate range in consideration of the characteristics of products, manufacturing processes, robustness of processes, etc. Continuous Manufacturing is expected to have the following advantages: A wide range of manufacturing scales are available by adjusting the continuous operation time of the process; it is possible to use the same manufacturing equipment from the development stage to commercial production, eliminating the need to change to large scale manufacturing equipment; and it is possible to reduce the equipment installation area (footprint). While Continuous Manufacturing can be a major manufacturing technique of the pharmaceutical industry in the future, official documents such as guidelines for Continuous Manufacturing are limited. In particular, no official documents such as guidelines specialized in manufacturing of biotechnological drug products have been issued yet. In Japan Agency for Medical Research and Development (AMED) research project, we summarized the key points to consider for investigation of the control strategy for Continuous Manufacturing of biotechnological products as a document. In this paper, we introduce the document.

Identification of Potent In Vivo Autotaxin Inhibitors that Bind to Both Hydrophobic Pockets and Channels in the Catalytic Domain.

Autotaxin (ATX, also known as ENPP2) is a predominant lysophosphatidic acid (LPA)-producing enzyme in the body, and LPA regulates various physiological functions, such as angiogenesis and wound healing, as well as pathological functions, including proliferation, metastasis, and fibrosis, via specific LPA receptors. Therefore, the ATX-LPA axis is a promising therapeutic target for dozens of diseases, including cancers, pulmonary and liver fibroses, and neuropathic pain. Previous structural studies revealed that the catalytic domain of ATX has a hydrophobic pocket and a hydrophobic channel; these serve to recognize the substrate, lysophosphatidylcholine (LPC), and deliver generated LPA to LPA receptors on the plasma membrane. Most reported ATX inhibitors bind to either the hydrophobic pocket or the hydrophobic channel. Herein, we present a unique ATX inhibitor that binds mainly to the hydrophobic pocket and also partly to the hydrophobic channel, inhibiting ATX activity with high potency and selectivity in vitro and in vivo. Notably, our inhibitor can rescue the cardia bifida (two hearts) phenotype in ATX-overexpressing zebrafish embryos.

Structural basis for specific inhibition of Autotaxin by a DNA aptamer.

ATX is a plasma lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) and produces lysophosphatidic acid. To date, no ATX-inhibition-mediated treatment strategies for human diseases have been established. Here, we report anti-ATX DNA aptamers that inhibit ATX with high specificity and efficacy. We solved the crystal structure of ATX in complex with the anti-ATX aptamer RB011, at 2.0-Å resolution. RB011 binds in the vicinity of the active site through base-specific interactions, thus preventing the access of the choline moiety of LPC substrates. Using the structural information, we developed the modified anti-ATX DNA aptamer RB014, which exhibited in vivo efficacy in a bleomycin-induced pulmonary fibrosis mouse model. Our findings reveal the structural basis for the specific inhibition of ATX by the anti-ATX aptamer and highlight the therapeutic potential of anti-ATX aptamers for the treatment of human diseases, such as pulmonary fibrosis.

The Role of Lysophosphatidic Acid on Airway Epithelial Cell Denudation in a Murine Heterotopic Tracheal Transplant Model.

BACKGROUND: Chronic rejection is the major leading cause of morbidity and mortality after lung transplantation. Obliterative bronchiolitis (OB), a fibroproliferative disorder of the small airways, is the main manifestation of chronic lung allograft rejection. However, there is currently no treatment for the disease. We hypothesized that lysophosphatidic acid (LPA) participates in the progression of OB. The aim of this study was to reveal the involvement of LPA on the lesion of OB. METHODS: Ki16198, an antagonist specifically for LPA1 and LPA3, was daily administered into the heterotopic tracheal transplant model mice at the day of transplantation. At days 10 and 28, the allografts were isolated and evaluated histologically. The messenger RNA levels of LPAR in microdissected mouse airway regions were assessed to reveal localization of lysophosphatidic acid receptors. The human airway epithelial cell was used to evaluate the mechanism of LPA-induced suppression of cell adhesion to the extracellular matrix (ECM). RESULTS: The administration of Ki16198 attenuated airway epithelial cell loss in the allograft at day 10. Messenger RNAs of LPA1 and LPA3 were detected in the airway epithelial cells of the mice. Lysophosphatidic acid inhibited the attachment of human airway epithelial cells to the ECM and induced cell detachment from the ECM, which was mediated by LPA1 and Rho-kinase pathway. However, Ki16198 did not prevent obliteration of allograft at day 28. CONCLUSIONS: The LPA signaling is involved in the status of epithelial cells by distinct contribution in 2 different phases of the OB lesion. This finding suggests a role of LPA in the pathogenesis of OB.

ENPP2 contributes to adipose tissue expansion and insulin resistance in diet-induced obesity.

Body weight is tightly regulated by food intake and energy dissipation, and obesity is related to decreased energy expenditure (EE). Herein, we show that nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, autotaxin) is an adipose-derived, secreted enzyme that controls adipose expansion, brown adipose tissue (BAT) function, and EE. In mice, Enpp2 was highly expressed in visceral white adipose tissue and BAT and is downregulated in hypertrophied adipocytes/adipose tissue. Enpp2(+/-) mice and adipocyte-specific Enpp2 knockout mice fed a high-fat diet showed smaller body weight gains and less insulin resistance than control mice fed the same diet. BAT was functionally more active and EE was increased in Enpp2-deficient mice. In humans, ENPP2 expression in subcutaneous fat and ENPP2 levels in serum were reduced in obese subjects. Taken together, our results establish ENPP2 as an adipose-derived, secreted enzyme that regulates adipose obesity and systemic metabolism. They also suggest ENPP2 could be a useful therapeutic target for the treatment of metabolic disease.

Potentials of the circulating pruritogenic mediator lysophosphatidic acid in development of allergic skin inflammation in mice: role of blood cell-associated lysophospholipase D activity of autotaxin.

Itching and infiltration of immune cells are important hallmarks of atopic dermatitis (AD). Although various studies have focused on peripheral mediator-mediated mechanisms, systemic mediator-mediated mechanisms are also important in the pathogenesis and development of AD. Herein, we found that intradermal injection of lysophosphatidic acid (LPA), a bioactive phospholipid, induces scratching responses by Institute of Cancer Research mice through LPA1 receptor- and opioid µ receptor-mediating mechanisms, indicating its potential as a pruritogen. The circulating level of LPA in Naruto Research Institute Otsuka Atrichia mice, a systemic AD model, with severe scratching was found to be higher than that of control BALB/c mice, probably because of the increased lysophospholipase D activity of autotaxin (ATX) in the blood (mainly membrane associated) rather than in plasma (soluble). Heparan sulfate proteoglycan was shown to be involved in the association of ATX with blood cells. The sequestration of ATX protein on the blood cells by heparan sulfate proteoglycan may accelerate the transport of LPA to the local apical surface of vascular endothelium with LPA receptors, promoting the hyperpermeability of venules and the pathological uptake of immune cells, aggravating lesion progression and itching in Naruto Research Institute Otsuka Atrichia mice.

Screening and X-ray crystal structure-based optimization of autotaxin (ENPP2) inhibitors, using a newly developed fluorescence probe.

Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), was originally identified as a tumor cell autocrine motility factor and was found to be identical to plasma lysophospholipase D, which is the predominant contributor to lysophosphatidic acid (LPA) production from lysophospholipids. ATX is therefore considered to regulate the physiological and pathological roles of LPA, including angiogenesis, lymphocyte trafficking, tissue fibrosis, and cancer cell invasion and metastasis. Thus, it is a potential therapeutic target. Here, we first developed a sensitive and specific ATX fluorescence probe, TG-mTMP, and used it to screen ATX inhibitors in a large chemical library. This probe, which is superior to previously available probes FS-3 and CPF4 in terms of sensitivity or specificity, enabled us to identify several novel ATX inhibitor scaffolds. We solved the crystal structures of ATX complexes with the hit compounds at high resolution (1.75-1.95 Å) and used this information to guide optimization of the structure of a selected inhibitor. The optimized compounds, 3BoA and its derivatives, exhibited potent ATX-inhibitory activity both in vitro and in vivo. These inhibitors are expected to be useful tools to understand the roles of ATX in vitro and in vivo and may also be candidate anti-ATX therapeutic agents.

Constitutive lymphocyte transmigration across the basal lamina of high endothelial venules is regulated by the autotaxin/lysophosphatidic acid axis.

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC's effect was abrogated by ATX inhibition, whereas LPA's effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte-HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.

Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling.

Enpp1 is a membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing extracellular nucleotide triphosphates to produce pyrophosphate. Enpp1 dysfunction causes human diseases characterized by ectopic calcification. Enpp1 also inhibits insulin signaling, and an Enpp1 polymorphism is associated with insulin resistance. However, the precise mechanism by which Enpp1 functions in these cellular processes remains elusive. Here, we report the crystal structures of the extracellular region of mouse Enpp1 in complex with four different nucleotide monophosphates, at resolutions of 2.7-3.2 Å. The nucleotides are accommodated in a pocket formed by an insertion loop in the catalytic domain, explaining the preference of Enpp1 for an ATP substrate. Structural mapping of disease-associated mutations indicated the functional importance of the interdomain interactions. A structural comparison of Enpp1 with Enpp2, a lysophospholipase D, revealed marked differences in the domain arrangements and active-site architectures. Notably, the Enpp1 mutant lacking the insertion loop lost the nucleotide-hydrolyzing activity but instead gained the lysophospholipid-hydrolyzing activity of Enpp2. Our findings provide structural insights into how the Enpp family proteins evolved to exert their diverse cellular functions.

Synthesis and biological evaluation of optically active Ki16425.

An enantionselective synthesis of both enantiomers of Ki16425, which possesses selective LPA antagonistic activity, was achieved. The isoxazole core was constructed by a 1,3-dipolar cycloaddition of nitrile oxide with alkyne and condensation with the optically active α-phenethyl alcohol segment, which was prepared by an enantioselective reduction of arylmethylketone. Biological evaluation of both enantiomers of Ki16425 revealed that the (R)-isomer showed much higher antagonistic activity for LPA(1) and LPA(3) receptors.

Identification and biochemical characterization of a novel autotaxin isoform, ATXδ, with a four-amino acid deletion.

Autotaxin (ATX) is lysophospholipase D, which converts lysophospholipids such as lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator with multiple biological roles. ATX is present in high concentrations in various biological fluids and is responsible for LPA production in these fluids. The plasma ATX level is altered in some patho-physiological conditions. Three splicing isoforms of ATX have been reported so far (ATXα, ß and γ). In this study, we identified and characterized ATXδ, a novel alternative splice variant of ATX, which has a four-amino acid deletion in the L2 linker region of ATXß. ATXδ was found to be the second major isoform following ATXß and fully active. ATXß and ATXδ showed similar divalent cation sensitivity and cell motility-stimulating activity. ATXß and ATXδ are present in wide range of organism from fish to mammals. Among them, only ATXδ was found in Gallus gallus and Xenopus laevis, suggesting the indispensable role of the isoform. ATXδ was expressed in various human tissues with different expression patterns from that of ATXß. These results show that ATXδ is a second major ATX isoform sharing similar biochemical characters with the major isoform, ATXß, and is a potential biomarker.

Autotaxin regulates vascular development via multiple lysophosphatidic acid (LPA) receptors in zebrafish.

Autotaxin (ATX) is a multifunctional ecto-type phosphodiesterase that converts lysophospholipids, such as lysophosphatidylcholine, to lysophosphatidic acid (LPA) by its lysophospholipase D activity. LPA is a lipid mediator with diverse biological functions, most of which are mediated by G protein-coupled receptors specific to LPA (LPA1-6). Recent studies on ATX knock-out mice revealed that ATX has an essential role in embryonic blood vessel formation. However, the underlying molecular mechanisms remain to be solved. A data base search revealed that ATX and LPA receptors are conserved in wide range of vertebrates from fishes to mammals. Here we analyzed zebrafish ATX (zATX) and LPA receptors both biochemically and functionally. zATX, like mammalian ATX, showed lysophospholipase D activity to produce LPA. In addition, all zebrafish LPA receptors except for LPA5a and LPA5b were found to respond to LPA. Knockdown of zATX in zebrafish embryos by injecting morpholino antisense oligonucleotides (MOs) specific to zATX caused abnormal blood vessel formation, which has not been observed in other morphant embryos or mutants with vascular defects reported previously. In ATX morphant embryos, the segmental arteries sprouted normally from the dorsal aorta but stalled in midcourse, resulting in aberrant vascular connection around the horizontal myoseptum. Similar vascular defects were not observed in embryos in which each single LPA receptor was attenuated by using MOs. Interestingly, similar vascular defects were observed when both LPA1 and LPA4 functions were attenuated by using MOs and/or a selective LPA receptor antagonist, Ki16425. These results demonstrate that the ATX-LPA-LPAR axis is a critical regulator of embryonic vascular development that is conserved in vertebrates.

Serum autotaxin measurements in pregnant women: application for the differentiation of normal pregnancy and pregnancy-induced hypertension.

BACKGROUND: The bioactive lipid lysophosphatidic acid (LPA) exerts multiple effects in the female reproductive system. Serum/plasma LPA is mainly produced by the lysophospholipase D activity of autotaxin (ATX). Previous studies have suggested that ATX has critical roles in cancer, reproduction, and vascular development. In the present study, we evaluated the usefulness of serum ATX measurements in pregnant women. METHODS: We measured the serum ATX antigen levels in 32 normal pregnant women, 15 patients with pregnancy-induced hypertension (PIH), and 7 patients with preterm delivery using a recently developed automated enzyme immunoassay. RESULTS: The serum ATX antigen levels in normal pregnant women were significantly higher than those in non-pregnant women (P<0.001). The serum ATX antigen levels in normal pregnant women were significantly and positively correlated with the gestational week (r=0.809, P<0.001). During the third trimester, the serum ATX antigen levels of the patients with PIH (3.299 ± 1.720 mg/l) were significantly lower than those of the normal pregnant women (4.915 ± 2.323 mg/l) (P=0.04). CONCLUSIONS: The serum ATX antigen level increases with the progression of pregnancy. The serum ATX level may be a serological marker for the prediction of PIH.

Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor.

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80,000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC(50) value of 0.21 µM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.

Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.

Autotaxin (ATX, also known as Enpp2) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine to generate lysophosphatidic acid (LPA), a lipid mediator that activates G protein-coupled receptors to evoke various cellular responses. Here, we report the crystal structures of mouse ATX alone and in complex with LPAs with different acyl-chain lengths and saturations. These structures reveal that the multidomain architecture helps to maintain the structural rigidity of the lipid-binding pocket, which accommodates the respective LPA molecules in distinct conformations. They indicate that a loop region in the catalytic domain is a major determinant for the substrate specificity of the Enpp family enzymes. Furthermore, along with biochemical and biological data, these structures suggest that the produced LPAs are delivered from the active site to cognate G protein-coupled receptors through a hydrophobic channel.

Glucagon regulates intracellular distribution of adipose differentiation-related protein during triacylglycerol accumulation in the liver.

Cellular lipid droplets (LD) are organelles involved in cellular lipid metabolism. When liver cellular components were fractionated using sucrose density gradient centrifugation, adipose differentiation-related protein (ADRP) was distributed in both the top and bottom fractions, which correspond to the LD and membranous fractions, respectively, in the mouse liver under normal feeding conditions. After overnight fasting, triacylglycerol and ADRP increased nearly 2.5-fold in the mouse liver, and a portion appeared in the intermediate-density LD (iLD) fractions. ADRP in the iLD fractions was also increased in a mouse nonalcoholic steatohepatitis model induced by methione/choline-deficient diet. When HuH-7 human hepatoma cells were incubated with oleic acid for 24 h, the amount of ADRP increased, and it was distributed in both the LD and membrane fractions. However, ADRP appeared in the iLD fractions upon treatment of HuH-7 cells with glucagon. This behavior of ADRP was cAMP-dependent, as the ADRP-positive iLD fractions were induced by dibutylyl cAMP and were blocked by protein kinase A inhibitors. A portion of ADRP colocalized microscopically with calnexin, which is present in the iLD fractions, by treatment of HuH-7 cells or human primary hepatocytes with oleic acid and glucagon, but not by treatment with oleic acid alone. Glucagon has a role in the reorganization of endoplasmic reticulum membranes to generate ADRP-associated lipid-poor particles in hepatic cells, which is related to LD formation during lipid storage.

Biological roles of lysophosphatidic acid signaling through its production by autotaxin.

Lysophosphatidic acid (LPA) exhibits a wide variety of biological functions as a bio-active lysophospholipid through G-protein-coupled receptors specific to LPA. Currently at least six LPA receptors are identified, named LPA(1) to LPA(6), while the existence of other LPA receptors has been suggested. From studies on knockout mice and hereditary diseases of these LPA receptors, it is now clear that LPA is involved in various biological processes including brain development and embryo implantation, as well as patho-physiological conditions including neuropathic pain and pulmonary and renal fibrosis. Unlike sphingosine 1-phosphate, a structurally similar bio-active lysophospholipid to LPA and produced intracellularly, LPA is produced by multiple extracellular degradative routes. A plasma enzyme called autotaxin (ATX) is responsible for the most of LPA production in our bodies. ATX converts lysophospholipids such as lysophosphatidylcholine to LPA by its lysophospholipase D activity. Recent studies on ATX have revealed new aspects of LPA. In this review, we highlight recent advances in our understanding of LPA functions and several aspects of ATX, including its activity, expression, structure, biochemical properties, the mechanism by which it stimulates cell motility and its pahto-physiological function through LPA production.

Adiponectin suppresses tumorigenesis in Apc(Min)(/+) mice.

Recent reports have shown that adiponectin has a suppressive effect on various types of malignancy. In order to clarify the role of adiponectin in colorectal carcinogenesis, we examined the effect of exogenous administration of adiponectin on intestinal polyp formation in C57BL/6J-Apc(Min)(/+) mice, which possess a point mutation in the Apc gene. And we found that adiponectin treatment significantly decreased the number of adenomatous polyps, especially polyps larger than 2mm in diameter, in the small intestine. Two major receptors for adiponectin, AdipoR1 and AdipoR2, were expressed in adenomatous polyps, and their expression levels were not altered by adiponectin injection. In conclusion, adiponectin suppresses the growth of intestinal adenomas in the Apc(Min)(/+) mice. Increasing the adiponectin level may be a new strategy for the prevention of colorectal cancer at an early step of carcinogenesis.

Expression of autotaxin, an ectoenzyme that produces lysophosphatidic acid, in human placenta.

PROBLEM: Lysophosphatidic acid (LPA) is a bioactive lipid mediator and thought to play an important role in pregnancy. Plasma LPA is produced by autotaxin (ATX), and ATX activity in plasma increases during pregnancy paralleled with gestational weeks and decreases to near the non-pregnant level soon after delivery. However, the source of increased ATX during pregnancy is still uncertain. We hypothesized that the source of increased ATX might be placenta. METHOD OF STUDY: We investigated the protein and mRNA expression of ATX in human placenta using immunohistochemistry and RT-PCR, respectively. RESULTS: At all 3 gestational trimesters, immunohistochemical staining for placenta tissues revealed the most marked positive staining of ATX protein in trophoblasts. Real-time PCR revealed that mRNA amounts of ATX in placenta tissues paralleled with gestational weeks, i.e. ATX level in plasma. CONCLUSION: These findings suggest that trophoblasts might produce ATX and its bioactive resultant substance, LPA, paralleled with gestational weeks.