Nanocarriers for Delivery of Oligonucleotides to the CNS

Nanoparticles with oligonucleotides bound to the outside or incorporated into the matrix can be used for gene editing or to modulate gene expression in the CNS. These nanocarriers are usually optimised for transfection of neurons or glia. They can also facilitate transcytosis across the brain endothelium to circumvent the blood-brain barrier. This review examines the different formulations of nanocarriers and their oligonucleotide cargoes, in relation to their ability to enter the brain and modulate gene expression or disease. The size of the nanocarrier is critical in determining the rate of clearance from the plasma as well as the intracellular routes of endothelial transcytosis. The surface charge is important in determining how it interacts with the endothelium and the target cell. The structure of the oligonucleotide affects its stability and rate of degradation, while the chemical formulation of the nanocarrier primarily controls the location and rate of cargo release. Due to the major anatomical differences between humans and animal models of disease, successful gene therapy with oligonucleotides in humans has required intrathecal injection. In animal models, some progress has been made with intraventricular or intravenous injection of oligonucleotides on nanocarriers. However, getting significant amounts of nanocarriers across the blood-brain barrier in humans will likely require targeting endothelial solute carriers or vesicular transport systems.

Impediment of Cerebrospinal Fluid Drainage Through Glymphatic System in Glioma

BackgroundCerebrospinal fluid (CSF) plays an important role in maintaining tissue homeostasis in the central nervous system. In 2012, the new CSF outflow pathway, “the glymphatic system,” was discovered. The glymphatic system mediates CSF and interstitial fluid exchange through the perivascular pathway, which eliminates harmful solutes in the brain parenchyma. In recent studies, the importance of the glymphatic system has been demonstrated in healthy and neurodegenerative disease brains. However, there is limited research on the function of the CSF in brain tumors. Intracranial hypertension caused by glioma can affect CSF drainage, which impacts the delivery of chemotherapy drugs via intrathecal injection. This study focused on changes in the glymphatic system and the role of aquaporin 4 (AQP4) in glymphatic transport in glioma.MethodsIn glioma-bearing rats, the effect of tracer infusion on the intracranial pressure (ICP) was evaluated using an ICP microsensor. In vivo magnetic resonance imaging and ex vivo bright field were used to monitor CSF tracer distribution after cisterna magna injection. AQP4 expression was quantitatively detected, and AQP4 in the astrocytes around the vessels was observed using immunofluorescence.ResultsThe ICP of the tumor group was higher than that of the control group and the infusion rate of 2 µl/min did not affect ICP. In vivo and ex vivo imaging showed that the circulation of CSF tracers was significantly impaired in the tumor. High-power confocal microscopy revealed that, in the tumor, the surrounding of AQP4 by Evans Blue was decreased. In both tumor and contralateral areas, data indicated that the number of cluster designation 34 (CD34+) alpha-smooth muscle actin (α-SMA−) veins were more than that of CD34+α-SMA+ arteries. Moreover, in the tumor area, AQP4 in the astrocytes around the vessels was decreased.ConclusionsThese findings indicate that the para-arterial influx of subarachnoid CSF is limited in glioma, especially in those with reduced levels of the fundamental protein AQP4. Our results provide evidence toward a potential new treatment method for glioma in the future.

Constitutively active SARM1 variants that induce neuropathy are enriched in ALS patients

Background In response to injury, neurons activate a program of organized axon self-destruction initiated by the NAD+ hydrolase, SARM1. In healthy neurons SARM1 is autoinhibited, but single amino acid changes can abolish autoinhibition leading to constitutively active SARM1 enzymes that promote degeneration when expressed in cultured neurons. Methods To investigate whether naturally occurring human variants might disrupt SARM1 autoinhibition and potentially contribute to risk for neurodegenerative disease, we assayed the enzymatic activity of all 42 rare SARM1 alleles identified among 8507 amyotrophic lateral sclerosis (ALS) patients and 9671 controls. We then intrathecally injected mice with virus expressing SARM1 constructs to test the capacity of an ALS-associated constitutively active SARM1 variant to promote neurodegeneration in vivo. Results Twelve out of 42 SARM1 missense variants or small in-frame deletions assayed exhibit constitutive NADase activity, including more than half of those that are unique to the ALS patients or that occur in multiple patients. There is a > 5-fold enrichment of constitutively active variants among patients compared to controls. Expression of constitutively active ALS-associated SARM1 alleles in cultured dorsal root ganglion (DRG) neurons is pro-degenerative and cytotoxic. Intrathecal injection of an AAV expressing the common SARM1 reference allele is innocuous to mice, but a construct harboring SARM1V184G, the constitutively active variant found most frequently among the ALS patients, causes axon loss, motor dysfunction, and sustained neuroinflammation. Conclusions These results implicate rare hypermorphic SARM1 alleles as candidate genetic risk factors for ALS and other neurodegenerative conditions.

Neurotensin Attenuates Nociception by Facilitating Inhibitory Synaptic Transmission in the Mouse Spinal Cord

Neurotensin (NT) is an endogenous tridecapeptide in the central nervous system. NT-containing neurons and NT receptors are widely distributed in the spinal dorsal horn (SDH), indicating their possible modulatory roles in nociception processing. However, the exact distribution and function of NT, as well as NT receptors (NTRs) expression in the SDH, have not been well documented. Among the four NTR subtypes, NTR2 is predominantly involved in central analgesia according to previous reports. However, the expression and function of NTR2 in the SDH has not yet been directly elucidated. Specifically, it remains unclear how NT-NTR2 interactions contribute to NT-mediated analgesia. In the present study, by using immunofluorescent histochemical staining and immunohistochemical staining with in situ hybridization histochemical staining, we found that dense NT- immunoreactivity (NT-ir) and moderate NTR2-ir neuronal cell bodies and fibers were localized throughout the superficial laminae (laminae I-II) of the SDH at the light microscopic level. In addition, γ-aminobutyric acid (GABA) and NTR2 mRNA were colocalized in some neuronal cell bodies, predominantly in lamina II. Using confocal and electron microscopy, we also observed that NT-ir terminals made both close contacts and asymmetrical synapses with the local GABA-ir neurons. Second, electrophysiological recordings showed that NT facilitated inhibitory synaptic transmission but not glutamatergic excitatory synaptic transmission. Inactivation of NTR2 abolished the NT actions on both GABAergic and glycinergic synaptic release. Moreover, a behavioral study revealed that intrathecal injection of NT attenuated thermal pain, mechanical pain, and formalin induced acute inflammatory pain primarily by activating NTR2. Taken together, the present results provide direct evidence that NT-containing terminals and fibers, as well as NTR2-expressing neurons are widely distributed in the spinal dorsal horn, GABA-containing neurons express NTR2 mainly in lamina II, GABA coexists with NTR2 mainly in lamina II, and NT may directly increase the activity of local inhibitory neurons through NTR2 and induce analgesic effects.

IL-23 Enhances C-Fiber-Mediated and Blue Light-Induced Spontaneous Pain in Female Mice

The incidence of chronic pain is especially high in women, but the underlying mechanisms remain poorly understood. Interleukin-23 (IL-23) is a pro-inflammatory cytokine and contributes to inflammatory diseases (e.g., arthritis and psoriasis) through dendritic/T cell signaling. Here we examined the IL-23 involvement in sexual dimorphism of pain, using an optogenetic approach in transgenic mice expressing channelrhodopsin-2 (ChR2) in TRPV1-positive nociceptive neurons. In situ hybridization revealed that compared to males, females had a significantly larger portion of small-sized (100-200 μm2) Trpv1+ neurons in dorsal root ganglion (DRG). Blue light stimulation of a hindpaw of transgenic mice induced intensity-dependent spontaneous pain. At the highest intensity, females showed more intense spontaneous pain than males. Intraplantar injection of IL-23 (100 ng) induced mechanical allodynia in females only but had no effects on paw edema. Furthermore, intraplantar IL-23 only potentiated blue light-induced pain in females, and intrathecal injection of IL-23 also potentiated low-dose capsaicin (500 ng) induced spontaneous pain in females but not males. IL-23 expresses in DRG macrophages of both sexes. Intrathecal injection of IL-23 induced significantly greater p38 phosphorylation (p-p38), a marker of nociceptor activation, in DRGs of female mice than male mice. In THP-1 human macrophages estrogen and chemotherapy co-application increased IL-23 secretion, and furthermore, estrogen and IL-23 co-application, but not estrogen and IL-23 alone, significantly increased IL-17A release. These findings suggest a novel role of IL-23 in macrophage signaling and female-dominant pain, including C-fiber-mediated spontaneous pain. Our study has also provided new insight into cytokine-mediated macrophage-nociceptor interactions, in a sex-dependent manner.

Heparin-Binding Growth-Associated Molecule (Pleiotrophin) Affects Sensory Signaling and Selected Motor Functions in Mouse Model of Anatomically Incomplete Cervical Spinal Cord Injury

Heparin-binding growth-associated molecule (pleiotrophin) is a neurite outgrowth-promoting secretory protein that lines developing fiber tracts in juvenile CNS (central nervous system). Previously, we have shown that heparin-binding growth-associated molecule (HB-GAM) reverses the CSPG (chondroitin sulfate proteoglycan) inhibition on neurite outgrowth in the culture medium of primary CNS neurons and enhances axon growth through the injured spinal cord in mice demonstrated by two-photon imaging. In this study, we have started studies on the possible role of HB-GAM in enhancing functional recovery after incomplete spinal cord injury (SCI) using cervical lateral hemisection and hemicontusion mouse models. In vivo imaging of blood-oxygen-level-dependent (BOLD) signals associated with functional activity in the somatosensory cortex was used to assess the sensory functions during vibrotactile hind paw stimulation. The signal displays an exaggerated response in animals with lateral hemisection that recovers to the level seen in the sham-operated mice by injection of HB-GAM to the trauma site. The effect of HB-GAM treatment on sensory-motor functions was assessed by performance in demanding behavioral tests requiring integration of afferent and efferent signaling with central coordination. Administration of HB-GAM either by direct injection into the trauma site or by intrathecal injection improves the climbing abilities in animals with cervical hemisection and in addition enhances the grip strength in animals with lateral hemicontusion without affecting the spontaneous locomotor activity. Recovery of sensory signaling in the sensorimotor cortex by HB-GAM to the level of sham-operated mice may contribute to the improvement of skilled locomotion requiring integration of spatiotemporal signals in the somatosensory cortex.

Efficacy of AAV serotypes to target Schwann cells after intrathecal and intravenous delivery

To optimize gene delivery to myelinating Schwann cells we compared clinically relevant AAV serotypes and injection routes. AAV9 and AAVrh10 vectors expressing either EGFP or the neuropathy-associated gene GJB1/Connexin32 (Cx32) under a myelin specific promoter were injected intrathecally or intravenously in wild type and Gjb1-null mice, respectively. Vector biodistribution in lumbar roots and sciatic nerves was higher in AAVrh10 injected mice while EGFP and Cx32 expression rates and levels were similar between the two serotypes. A gradient of biodistribution away from the injection site was seen with both intrathecal and intravenous delivery, while similar expression rates were achieved despite higher vector amounts injected intravenously. Quantified immune cells in relevant tissues were similar to non-injected littermates. Overall, AAV9 and AAVrh10 efficiently transduce Schwann cells throughout the peripheral nervous system with both clinically relevant routes of administration, although AAV9 and intrathecal injection may offer a more efficient approach for treating demyelinating neuropathies.

Effects of Upregulation of TNFAIP3 on Diabetic Neuropathic Pain in Mice

Globally, diabetes has assumed epidemic proportions with the neuropathic complications attributed to the malady emerging as a substantial burden on patients and society. DNP has greatly affected the daily life of patients, the effect of traditional treatment methods is not ideal, and it is easy to produce drug resistance. This work is aimed at scrutinizing the effect of upregulating the expression of TNFAIP3 on diabetic neuralgia in mice. This work entailed ascertaining the effects of TNFAIP3 on a murine DNP system. This inspired us to observe the analgesic effect via high expression of lentivirus-mediated TNFAIP3 by intrathecal injection in the animal model to explore its regulatory impacts, symptom relief, and mechanistic role in pain. The results displayed an attenuation of hind paw pain hypersensitivity by LV-TNFAIP3 in the animals. The spinal cord and dorsal root ganglion of mice with neuropathic pain displayed an evident dip in TNFAIP3. Inhibition of the ERK/NF-κB signaling pathway employing LV-TNFAIP3 conspicuously suppressed this pathway while the diabetic pain hypersensitivity was quelled. This effect was also seen with insulin treatment evidently. In conclusion, according to the above analyses, the interaction between DNP and extracellular signal-regulated kinase signal transduction pathway is one of the key factors of pathogenesis.

DDRE-02. PATIENTS WITH LEPTOMENINGEAL METASTASES FROM NSCLC BENEFITED FROM INTRATHECAL PEMETREXED AFTER FAILURE TO TYROSINE KINASE INHIBITORS: TWO CASE REPORTS

BACKGROUND The incidence of leptomeningeal metastasis (LM) is 3%-5% in NSCLC patients; the incidence is even higher in patients with EGFR mutations or ALK gene rearrangement. The prognosis of NSCLC patients with LM is poor with an overall survival (OS) of 3 months with contemporary treatment. METHODS Here we report two cases of patients with leptomeningeal metastases from NSCLC who benefited from intrathecal pemetrexed after failure to tyrosine kinase inhibitors(TKIs). RESULTS A 53-year-old woman, diagnosed with adenocarcinoma of the lung with ALK rearrangement, suffered form right limb weakness, dysphagia after multi-line targeted therapy and radiotherapy for brain metastases. Evaluation revealed a rapidly progressing right brainstem mass and diffuse leptomeningeal enhanced. And cytological examination of CSF showed neoplastic cells, which definitely diagnosed as LM. After first intrathecal injection of 30mg pemetrexed every three weeks, the patient's symptoms improved. There were no significant treatment side effects and the quality of life was not affected during the five subsequent treatments. Another patient was a 32-year-old man, diagnosed with adenocarcinoma of the lung with EGFR E24-RAD51E4 fusion mutation with KPS score 30. LM was confirmed after multiline therapy. He suffered form blurred vision and drooping eyelid. Similarly, after first intrathecal injection of 20mg pemetrexed every week, the patient's symptoms improved and KPS score increased gradually. And level II leukopenia was detected during subsequent treatments. Combined with intrathecal pemetrexed progression-free survival (PFS) were 3 months in two NSCLC patients with LM harboring ALK/EGFR mutation . And the quality of life of patients were effectively increased. CONCLUSION Combination therapy third generation EGFR/ALK agents with intrathecal chemotherapy might be benefited in overall survival.