Gene targeting as a therapeutic avenue in diseases mediated by the complement alternative pathway.

The complement alternative pathway (AP) is implicated in numerous diseases affecting many organs, ranging from the rare hematological disease paroxysmal nocturnal hemoglobinuria (PNH), to the common blinding disease age-related macular degeneration (AMD). Critically, the AP amplifies any activating trigger driving a downstream inflammatory response; thus, components of the pathway have become targets for drugs of varying modality. Recent validation from clinical trials using drug modalities such as inhibitory antibodies has paved the path for gene targeting of the AP or downstream effectors. Gene targeting in the complement field currently focuses on supplementation or suppression of complement regulators in AMD and PNH, largely because the eye and liver are highly amenable to drug delivery through local (eye) or systemic (liver) routes. Targeting the liver could facilitate treatment of numerous diseases as this organ generates most of the systemic complement pool. This review explains key concepts of RNA and DNA targeting and discusses assets in clinical development for the treatment of diseases driven by the alternative pathway, including the RNA-targeting therapeutics ALN-CC5, ARO-C3, and IONIS-FB-LRX, and the gene therapies GT005 and HMR59. These therapies are but the spearhead of potential drug candidates that might revolutionize the field in coming years.

Novel Selection Approaches to Identify Antibodies Targeting Neoepitopes on the C5b6 Intermediate Complex to Inhibit Membrane Attack Complex Formation.

The terminal pathway of complement is implicated in the pathology of multiple diseases and its inhibition is, therefore, an attractive therapeutic proposition. The practicalities of inhibiting this pathway, however, are challenging, as highlighted by the very few molecules in the clinic. The proteins are highly abundant, and assembly is mediated by high-affinity protein-protein interactions. One strategy is to target neoepitopes that are present transiently and only exist on active or intermediate complexes but not on the abundant native proteins. Here, we describe an antibody discovery campaign that generated neoepitope-specific mAbs against the C5b6 complex, a stable intermediate complex in terminal complement complex assembly. We used a highly diverse yeast-based antibody library of fully human IgGs to screen against soluble C5b6 antigen and successfully identified C5b6 neoepitope-specific antibodies. These antibodies were diverse, showed good binding to C5b6, and inhibited membrane attack complex (MAC) formation in a solution-based assay. However, when tested in a more physiologically relevant membrane-based assay these antibodies failed to inhibit MAC formation. Our data highlight the feasibility of identifying neoepitope binding mAbs, but also the technical challenges associated with the identification of functionally relevant, neoepitope-specific inhibitors of the terminal pathway.

Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.

Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).

Understanding chronic inflammatory and neuropathic pain.

This meeting report highlights the main topics presented at the conference "Chronic Inflammatory and Neuropathic Pain," convened jointly by the New York Academy of Sciences, MedImmune, and Grünenthal GmbH, on June 2-3, 2011, with the goal of providing a conducive environment for lively, informed, and synergistic conversation among participants from academia, industry, clinical practice, and government to explore new frontiers in our understanding and treatment of chronic and neuropathic pain. The program included leading and emerging investigators studying the pathophysiological mechanisms underlying neuropathic and chronic pain, and experts in the clinical development of pain therapies. Discussion included novel issues, current challenges, and future directions of basic research in pain and preclinical and clinical development of new therapies for chronic pain.

Biologics: the next-generation therapeutics for analgesia?

There is a huge unmet need for novel medicines for the treatment of chronic pain. In this article, we briefly examine some of the issues associated with traditional approaches to the treatment of these conditions. We go on to present some of the current evidence for three of the most advanced approaches to the treatment of pain using a biological approach and briefly review some of the future trends that may lead to better treatments for this debilitating condition.

IL-17 induces hyperalgesia via TNF-dependent neutrophil infiltration.

Interleukin-17 (IL-17) and tumour necrosis factor-α (TNF) are critical in the pathogenesis of arthritis but their relationship during inflammatory pain has received limited attention. We aimed to establish whether IL-17 can induce hyperalgesia in acute conditions, and investigated the role of TNF in mediating the pain response. Hyperalgesia was elicited in C57BL/6 mice by injection of recombinant IL-17, TNF or vehicle into the plantar tissue. Elevated pain was measured by the Hargreaves test for thermal hyperalgesia and Linton incapacitance tester for weight-bearing change. Cellular infiltration during hyperalgesia was determined by histological analysis and myeloperoxidase assay. IL-17 was found to induce hyperalgesia, but this was dependent on neutrophil migration and TNF binding to TNF receptor 1 (TNFR1). Because TNF-induced hyperalgesia was also dependent on neutrophil migration, the relationship between the resident fibroblasts, the cytokines and the migrating neutrophils was further investigated. By means of an air pouch model of cell migration, it was established that IL-17-induced neutrophil infiltration was dependent of TNF/TNFR1 as this interaction was required for the induction of the chemokine keratinocyte chemoattractant. These findings suggest that IL-17 causes acute hyperalgesia indirectly by inducing TNF from resident cells. The subsequent production of keratinocyte chemoattractant then triggers neutrophil chemotaxis to the plantar tissue, releasing algesic mediators locally to sensitise the nerve.

Apoptosis-associated tyrosine kinase and neuronal cell death.

Apoptosis-associated tyrosine kinase (AATYK) is up-regulated by phosphorylation in cultured cerebellar granule neurons (CGN) undergoing apoptosis upon switch to low KCl-containing medium. However, the underlying signaling pathways remain to be fully characterized. When CGN at culture day 7 were switched from 25 mM KCl (K25) to 5 mM (K5) medium, AATYK band migration on SDS-PAGE shifted to a more slowly migrating position expected for the hyperphosphorylated protein. The apoptosis-inducing agent C(2)-ceramide also caused a mobility shift of the AATYK protein. Exposing CGN (K25) to L-type voltage-dependent Ca(2+) channel antagonists shifted the AATYK band to the K5-induced position, while the Ca(2+) channel activator FPL-64176 had the contrary effect. FK-506, a calcineurin inhibitor caused AATYK hyperphosphorylation under high KCl conditions. CGN death in K5 medium is linked to inhibition of the PI 3-kinase/Akt survival pathway and concomitant activation of the pro-apoptotic downstream target glycogen synthase kinase-3 (GSK-3). GSK-3 inhibitors blocked the K5-induced mobility shift of AATYK. Moreover, CGN cultured from AATYK-deficient mice remained sensitive to death in K5 medium. Thus, AATYK activation may not be a physiologically relevant principal regulatory target of the GSK-3 death pathway in KCl-deprived CGN.

Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain.

ATP is a known mediator of inflammatory and neuropathic pain. However, the mechanisms by which specific purinergic receptors contribute to chronic pain states are still poorly characterized. Here, we demonstrate that in response to peripheral nerve injury, P2X(4) receptors (P2X(4)R) are expressed de novo by activated microglia in the spinal cord. Using in vitro and in vivo models, we provide direct evidence that P2X(4)R stimulation leads to the release of BDNF from activated microglia and, most likely phosphorylation of the NR1 subunit of NMDA receptors in dorsal horn neurons of the spinal cord. Consistent with these findings, P2X4-deficient mice lack mechanical hyperalgesia induced by peripheral nerve injury and display impaired BDNF signaling in the spinal cord. Furthermore, ATP stimulation is unable to stimulate BDNF release from P2X(4)-deficient mice microglia in primary cultures. These results indicate that P2X(4)R contribute to chronic pain through a central inflammatory pathway. P2X(4)R might thus represent a potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain injury and neurodegenerative disorders.

The putative cannabinoid receptor GPR55 plays a role in mechanical hyperalgesia associated with inflammatory and neuropathic pain.

It has been postulated that the G protein-coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB(1) and CB(2) receptors are effective analgesic and anti-inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well-validated GPR55 tool compounds, a GPR55 knockout (GPR55(-/-)) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55(-/-) mice revealed no obvious primary differences, compared with wild-type (GPR55(+/+)) littermates. GPR55(-/-) mice were then tested in the models of adjuvant-induced inflammation and partial nerve ligation. Following intraplantar administration of Freund's complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55(-/-) mice up to 14 days post-injection. Cytokine profiling experiments showed that at 14 days post-FCA injection there were increased levels of IL-4, IL-10, IFN gamma and GM-CSF in paws from the FCA-injected GPR55(-/-) mice when compared with the FCA-injected GPR55(+/+) mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55(-/-) mice. In the model of neuropathic hypersensitivity, GPR55(-/-) mice also failed to develop mechanical hyperalgesia up to 28 days post-ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.

Demonstration of a novel technique to quantitatively assess inflammatory mediators and cells in rat knee joints.

BACKGROUND: The inflammation that accompanies the pain and swelling associated with osteo- and rheumatoid arthritis is mediated by complex interactions of inflammatory mediators. Cytokines play a pivotal role in orchestrating many of these processes, including inflammatory cell recruitment, adhesion and activation. In addition, prostaglandins are secreted into the synovial cavity and are involved in perpetuation of local inflammation, vasodilatation and vasoconstriction, and also with bone resorption. Pre-clinical models have been developed in order to correlate to the human disease and principle among these is the adjuvant-induced arthritis model in the rat. METHODS: We have developed a technique to quantitatively assess the contents of synovial fluid samples from rat joints. Two needles joined together are inserted into the knee joint of anaesthetised rats and connected to a Watson-Marlow perfusion pump. Sterile saline is infused and withdrawn at 100 microl min-1 until a 250 microl sample is collected. RESULTS: Our results demonstrate up to 125 fold increases in synovial IL1alpha and IL1beta concentrations, approximately 30 fold increases in levels of IL6 and IL10 and a 200-300 fold elevation in synovial concentrations of TNFalpha during FCA-induced experimental arthritis. Finally, this novel technique has demonstrated a dose-response relationship between FCA and the total cell counts of synovial perfusates. CONCLUSION: In summary, this new technique provides a robust method for quantifying inflammatory mediators and cells from the synovial cavity itself, thereby detailing the inflammatory processes from within the capsule and excluding those processes occurring in other tissues surrounding the entire articulation.

The role of P2X7 in pain and inflammation.

The P2X 7purinoceptor is unique amongst the P2X receptor family in that its activation is able to stimulate the release of mature, biologically active interleukin-1beta (IL-1ß), as well as a variety of other proinflammatory cytokines. Coupled with the predominate localisation of this receptor to immunocytes of haemopoetic origin, this receptor is an obvious candidate to play a major and pivotal role in processes of pain and inflammation. Using genetically modified animals that lack the P2X7 receptor, several investigators have shown that these mice do indeed demonstrate a blunted inflammatory response, and fail to develop pain following both inflammatory and neuropathic insult. These animals also show altered cytokine production in response to inflammatory stimulus, which is far broader than merely modulation of IL-1ß release. In this short article, we review the role of the P2X7 receptor in modulating the release of cytokines and other mediators, and discuss the findings made from P2X7 receptor-deficient animals. As well as highlighting outstanding questions regarding this intriguing receptor, we also speculate as to the potential therapeutic benefit of P2X7 receptor modulation.

Isolation of genes controlling apoptosis through their effects on cell survival.

The identification of the most suitable molecular targets for gene and drug therapy is the crucial first step in the development of new disease treatments. The rational identification of such targets depends on a detailed understanding of the pathological changes occuring at the molecular level. We have applied forward genetics approaches to the identification of the critical genes involved in the control of apoptosis in mammalian cells, since defective control of apoptosis underlies many diseases, including cancer and neurodegenerative diseases. We have identified two groups of genes by their effects on cell survival using retroviral cDNA functional expression cloning and retroviral insertional mutagenesis. The identification of these novel genes opens up new areas for apoptosis research and subsequently for the development of new gene and drug therapies.

Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain.

The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients. We hypothesise that the P2X(7) receptor, via regulation of mature IL-1beta production, plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential to deliver broad-spectrum analgesia.

TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase.

1. TRPM2 (melastatin-like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and cell death. We investigated the role of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on hydrogen peroxide (H(2)O(2))-mediated TRPM2 activation using a tetracycline-inducible TRPM2-expressing cell line. 2. In whole-cell patch-clamp recordings, intracellular adenine 5'-diphosphoribose (ADP-ribose) triggered an inward current in tetracycline-induced TRPM2-human embryonic kidney (HEK293) cells, but not in uninduced cells. Similarly, H(2)O(2) stimulated an increase in [Ca(2+)](i) (pEC(50) 4.54+/-0.02) in Fluo-4-loaded TRPM2-expressing HEK293 cells, but not in uninduced cells. Induction of TRPM2 expression caused an increase in susceptibility to plasma membrane damage and mitochondrial dysfunction in response to H(2)O(2). These data demonstrate functional expression of TRPM2 following tetracycline induction in TRPM2-HEK293 cells. 3. PARP inhibitors SB750139-B (patent number DE10039610-A1 (Lubisch et al., 2001)), PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide) and DPQ (3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone) inhibited H(2)O(2)-mediated increases in [Ca(2+)](i) (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively), increases in mitochondrial dysfunction (pIC(50) vs 300 microm H(2)O(2): 7.32+/-0.23; 6.69+/-0.22; 5.44+/-0.09, respectively) and decreases in plasma membrane integrity (pIC(50) vs 300 microm H(2)O(2): 7.45+/-0.27; 6.35+/-0.18; 5.29+/-0.12, respectively). The order of potency of the PARP inhibitors in these assays (SB750139>PJ34>DPQ) was the same as for inhibition of isolated PARP enzyme. 4. SB750139-B, PJ34 and DPQ had no effect on inward currents elicited by intracellular ADP-ribose in tetracycline-induced TRPM2-HEK293 cells, suggesting that PARP inhibitors are not interacting directly with the channel. 5. SB750139-B, PJ34 and DPQ inhibited increases in [Ca(2+)](i) in a rat insulinoma cell line (CRI-G1 cells) endogenously expressing TRPM2 (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively). 6. These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.

Mitogen and stress response kinase-1 (MSK1) mediates excitotoxic induced death of hippocampal neurones.

Activation of the mitogen-activated protein kinase (MAPK/ERK) signal transduction pathway may mediate excitotoxic neuronal cell death in vitro and during ischemic brain injury in vivo. However, little is known, of the upstream regulation or downstream consequences of ERK activation under these conditions. Magnesium removal has been described to induce hyperexcitability and degeneration in cultured hippocampal neurones. Here, we show that neurotoxicity evoked by Mg2+ removal in primary hippocampal neurones stimulates ERK, but not p38, phosphorylation. Removal of Mg2+ also resulted in induction of the MAPK/ERK substrate mitogen- and stress-response kinase 1 (MSK1) and induced phosphorylation of the MSK1 substrate, the transcription factor cAMP response element binding protein (CREB). Neuronal death and phosphorylation of components in this cascade were inhibited by the Raf inhibitor SB-386023, by the MEK inhibitor U0126, or by the MSK1 inhibitors H89 and Ro318220. Importantly, this form of cell death was inhibited in hippocampal neurones cultured from MSK1-/- mice and inhibitors of Raf or MEK had no additive neuroprotective effect. Together, these data indicate that MSK1 is a physiological kinase for CREB and that this activity is an essential component of activity-dependent neuronal cell death.