Lipid Nanoparticle-Mediated Delivery of mRNA Into the Mouse and Human Retina and Other Ocular Tissues.

Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in LNPs with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA-carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and RPE. Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mouse eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge, we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time. Translational Relevance: Ocular gene-replacement therapies using non-viral vector methods are a promising alternative to adeno-associated virus (AAV) vectors. Our studies show that mRNA LNP delivery can be used to transfect retinal cells in both mouse and human tissues without inducing significant inflammation. This methodology could be used to transfect retinal cell lines, tissue explants, mice, or potentially as gene-replacement therapy in a clinical setting in the future.

The effect of exogenous ciliary neurotrophic factor on cell cycle and neural differentiation markers of in vitro model cells: New insights for future therapeutic approaches.

Retinoblastoma is known as childhood rare malignancy of the retina. Ciliary neurotrophic factor (CNTF) was previously found to reduce degeneration and promote retina survival. This work investigated the effects of CNTF supplementation on in-vitro model cells including retinoblastoma (Y79) and adipose-derived mesenchymal stem cells (AMSCs) viability, proliferation, gene expression and cell cycle. A drop of viability was detected in Y79 treated with CNTF in a dose-dependent manner (P < .05). However, the proliferation of AMSCs was increased at lower concentrations of CNTF (5 ng/mL), but declined in higher doses (50 and 100 ng/mL). The BrdU assay confirmed the MTT assay results. Cell cycle was arrested in both Y79 and AMSCs in the G0/G1 phase by CNTF treatment. A considerable down-regulation of Bcl2, CycD1 and N-Myc genes expression (P < .05) inversely, P15 and P21 genes up-regulation in treated Y79 cells was observed. Besides, stemness genes' transcription was reduced in AMSCs (P < .05), and levels of neuronal-specific markers such as neuron-specific enolase (NSE) and neuronal nuclei (NeuN) were increased (P < .05). The findings of this study suggest a promising potential of CNTF in terms of arresting Y79 retinoblastoma cells, and differentiation-inducing to AMSCs, which could be valuable for managing future innovative treatments targeting retinoblastoma. SIGNIFICANCE OF THE STUDY: We demonstrate that CNTF has the potential to reduce proliferation of Y79 cells and induce the cell cycle arrest of them. Also, down-regulation of oncogenes (such as N-Myc) while up-regulation of tumour suppressor genes (such as P21) was detected by exposure of Y79 cells to CNTF. Furthermore, we observed the cell cycle arrest, reduction of stemness gene and up-regulation of neural differentiation markers in AMSCs treated with CNTF. These results support the probable promising effects of CNTF for controlling retinoblastoma.

Localized co-delivery of CNTF and FK506 using a thermosensitive hydrogel for retina ganglion cells protection after traumatic optic nerve injury.

Following the traumatic axonal injury in the optic nerve, the failure of retrograde axonal transport to continuously supply neurotrophins from the brain to retina results in deprivation of neurotrophins in retinal ganglion cells (RGCs), which in turn can modulate the fate of RGCs toward apoptosis and thereby impede axon regeneration. In this study, a ciliary neurotrophic factor (CNTF) loaded thermo-sensitive hydrogel was designed and developed as a localized drug depot to restore neurotrophins supply following axon injury. Besides, following traumatic axon injury, overactive immune responses cause neurotoxicity and induce scar formation which together constitutes the major hindrances for axon regeneration. Thus, the FK506, a hydrophobic macrolide immunosuppressant, was co-loaded into the hydrogel after encapsulating it into a polymeric micelle. The materials can undergo sol-to-gel transition within minutes under a physiological pH of 37 °C. The release of drugs from the hydrogel exhibited a sustainable profile in vitro. The optic nerve was exposed by surgical procedure and the animal model was prepared by crushing the nerve with a reverse clamp. For the localized delivery to the optic nerve, a pre-hydrogel liquid containing chitosan, FK506 (in micelle), CNTF, and the gelling agent was directly smeared on the injured site, which gelled under physiological condition. This co-delivery system exhibited in vivo RGCs protective effect against the adverse effects caused by traumatic optic nerve injury, indicating the potential of this drug delivery system for effective optic nerve repair and this strategy may provide promising platforms for localized drug delivery in various other therapies.

Beyond Performance Metrics: Automatic Deep Learning Retinal OCT Analysis Reproduces Clinical Trial Outcome.

PURPOSE: To validate the efficacy of a fully automatic, deep learning-based segmentation algorithm beyond conventional performance metrics by measuring the primary outcome of a clinical trial for macular telangiectasia type 2 (MacTel2). DESIGN: Evaluation of diagnostic test or technology. PARTICIPANTS: A total of 92 eyes from 62 participants with MacTel2 from a phase 2 clinical trial (NCT01949324) randomized to 1 of 2 treatment groups METHODS: The ellipsoid zone (EZ) defect areas were measured on spectral domain OCT images of each eye at 2 time points (baseline and month 24) by a fully automatic, deep learning-based segmentation algorithm. The change in EZ defect area from baseline to month 24 was calculated and analyzed according to the clinical trial protocol. MAIN OUTCOME MEASURE: Difference in the change in EZ defect area from baseline to month 24 between the 2 treatment groups. RESULTS: The difference in the change in EZ defect area from baseline to month 24 between the 2 treatment groups measured by the fully automatic segmentation algorithm was 0.072±0.035 mm2 (P = 0.021). This was comparable to the outcome of the clinical trial using semiautomatic measurements by expert readers, 0.065±0.033 mm2 (P = 0.025). CONCLUSIONS: The fully automatic segmentation algorithm was as accurate as semiautomatic expert segmentation to assess EZ defect areas and was able to reliably reproduce the statistically significant primary outcome measure of the clinical trial. This approach, to validate the performance of an automatic segmentation algorithm on the primary clinical trial end point, provides a robust gauge of its clinical applicability.

Ciliary neurotrophic factor signaling in the rat orbitofrontal cortex ameliorates stress-induced deficits in reversal learning.

Deficits in cognitive flexibility, i.e. the ability to modify behavior in response to changes in the environment, are present in several psychiatric disorders and are often refractory to treatment. However, improving treatment response has been hindered by a lack of understanding of the neurobiology of cognitive flexibility. Using a rat model of chronic stress (chronic intermittent cold stress, CIC) that produces selective deficits in reversal learning, a form of cognitive flexibility dependent on orbitofrontal cortex (OFC) function, we have previously shown that JAK2 signaling is required for optimal reversal learning. In this study we explore the molecular basis of those effects. We show that, within the OFC, CIC stress reduces the levels of phosphorylated JAK2 and of ciliary neurotrophic factor (CNTF), a promoter of neuronal survival and an activator of JAK2 signaling, and that neutralizing endogenous CNTF with an intra-OFC microinjection of a specific antibody is sufficient to produce reversal-learning deficits similar to stress. Intra-OFC delivery of recombinant CNTF to CIC-stressed rats, at a dose that induces JAK2 and Akt but not STAT3 or ERK, ameliorates reversal-learning deficits, and Akt blockade prevents the positive effects of CNTF. Further analysis revealed that CNTF may exert its beneficial effects by inhibiting GSK3ß, a substrate of Akt and a regulator of protein degradation. We also revealed a novel mechanism of CNTF action through modulation of p38/Mnk1/eIF4E signaling. This cascade controls translation of select mRNAs, including those encoding several plasticity-related proteins. Thus, we suggest that CNTF-driven JAK2 signaling corrects stress-induced reversal learning deficits by modulating the steady-state levels of plasticity-related proteins in the OFC.

Effect of Ciliary Neurotrophic Factor on Retinal Neurodegeneration in Patients with Macular Telangiectasia Type 2: A Randomized Clinical Trial.

PURPOSE: To test the effects of an encapsulated cell-based delivery of a neuroprotective agent, ciliary neurotrophic factor (CNTF), on progression of macular telangiectasia type 2, a neurodegenerative disease with no proven effective therapy. DESIGN: Randomized sham-controlled clinical trial. PARTICIPANTS: Ninety-nine study eyes of 67 eligible participants were enrolled. METHODS: Single-masked randomized clinical trial of 24 months' duration conducted from May 2014 through April 2017 in 11 clinical centers of retinal specialists in the United States and Australia. Participants were randomized 1:1 to surgical implantation of intravitreal sustained delivery of human CNTF versus a sham procedure. MAIN OUTCOME MEASURES: The primary outcome was the difference in the area of neurodegeneration as measured in the area of the ellipsoid zone disruption (or photoreceptor loss) measured on spectral-domain (SD) OCT images at 24 months from baseline between the treated and untreated groups. Secondary outcomes included comparison of visual function changes between treatment groups. RESULTS: Among the 67 participants who were randomized (mean age, 62±8.9 years; 41 women [61%]; 58 white persons [86%]), 65 (97%) completed the study. Two participants (3 study eyes) died and 3 participants (4 eyes) were found ineligible. The eyes receiving sham treatment had 31% greater progression of neurodegeneration than the CNTF-treated eyes. The difference in mean area of photoreceptor loss was 0.05±0.03 mm2 (P = 0.04) at 24 months. Retinal sensitivity changes, measured using microperimetry, were correlated highly with the changes in the area of photoreceptor loss (r = 0.86; P < 0.0001). The mean retinal sensitivity loss of the sham group was 45% greater than that of the treated group (decrease, 15.81±8.93 dB; P = 0.07). Reading speed deteriorated in the sham group (-13.9 words per minute) with no loss in the treated group (P = 0.02). Serious adverse ocular effects were found in 2 of 51 persons (4%) in the sham group and 2 of 48 persons (4%) in the treated group. CONCLUSIONS: In participants with macular telangiectasia type 2, a surgical implant that released CNTF into the vitreous cavity, compared with a sham procedure, slowed the progression of retinal degeneration. Further research is needed to assess longer-term clinical outcomes and safety.

Controlled release strategy designed for intravitreal protein delivery to the retina.

Therapeutic protein delivery directly to the eye is a promising strategy to treat retinal degeneration; yet, the high risks of local drug overdose and cataracts associated with bolus injection have limited progress, requiring the development of sustained protein delivery strategies. Since the vitreous humor itself is a gel, hydrogel-based release systems are a sensible solution for sustained intravitreal protein delivery. Using ciliary neurotrophic factor (CNTF) as a model protein for ocular treatment, we investigated the use of an intravitreal, affinity-based release system for protein delivery. To sustain CNTF release, we took advantage of the affinity between Src homology 3 (SH3) and its peptide binding partners: CNTF was expressed as a fusion protein with SH3, and a thermogel of hyaluronan and methylcellulose (HAMC) was modified with SH3 binding peptides. Using a mathematical model, the hydrogel composition was successfully designed to release CNTF-SH3 over 7 days. The stability and bioactivity of the released protein were similar to those of commercial CNTF. Intravitreal injections of the bioengineered thermogel showed successful delivery of CNTF-SH3 to the mouse retina, with expected transient downregulation of phototransduction genes (e.g., rhodopsin, S-opsin, M-opsin, Gnat 1 and 2), upregulation of STAT1 and STAT3 expression, and upregulation of STAT3 phosphorylation. This constitutes the first demonstration of intravitreal protein release from a hydrogel. Immunohistochemical analysis of the retinal tissues of injected eyes confirmed the biocompatibility of the delivery vehicle, paving the way towards new intravitreal protein delivery strategies.

Pronounced synergistic neuroprotective effect of GDNF and CNTF on axotomized retinal ganglion cells in the adult mouse.

Neuroprotection is among the potential treatment options for glaucoma and other retinal pathologies characterized by the loss of retinal ganglion cells (RGCs). Here, we examined the impact of a neural stem (NS) cell-based intravitreal co-administration of two neuroprotective factors on the survival of axotomized RGCs. To this aim we used lentiviral vectors to establish clonal NS cell lines ectopically expressing either glial cell line-derived neurotrophic factor (GDNF) or ciliary neurotrophic factor (CNTF). The modified NS cell lines were intravitreally injected either separately or as a 1:1 mixture into adult mice one day after an optic nerve lesion, and the number of surviving RGCs was determined in retinal flat-mounts two, four and eight weeks after the lesion. For the transplantation experiments, we selected a GDNF- and a CNTF-expressing NS cell line that promoted the survival of axotomized RGCs with a similar efficacy. Eight weeks after the lesion, GDNF-treated retinas contained 3.8- and CNTF-treated retinas 3.7-fold more RGCs than control retinas. Of note, the number of surviving RGCs was markedly increased when both factors were administered simultaneously, with 14.3-fold more RGCs than in control retinas eight weeks after the lesion. GDNF and CNTF thus potently and synergistically rescued RGCs from axotomy-induced cell death, indicating that combinatorial neuroprotective approaches represent a promising strategy to effectively promote the survival of RGCs under pathological conditions.

3D Visualization of Individual Regenerating Retinal Ganglion Cell Axons Reveals Surprisingly Complex Growth Paths.

Retinal ganglion cells (RGCs), the sole output cells of the retina, are a heterogeneous population of neurons that project axons to visual targets in the brain. Like most CNS neurons, RGCs are considered incapable of mounting long distance axon regeneration. Using immunolabeling-enabled 3D imaging of solvent-cleared organs (iDISCO) in transgenic mice, we tracked the entire paths of individual RGC axons and show that adult RGCs are highly capable of spontaneous long-distance regeneration, even without any treatment. Our results show that the Thy1-H-YFP mouse sparsely labels RGCs, consisting predominantly of regeneration-competent α-type RGCs (αRGCs). Following optic nerve crush, many of the YFP-labeled RGC axons extend considerable distances proximal to the injury site with only a few penetrating through the lesion. This tortuous axon growth proximal to the lesion site is even more striking with intravitreal ciliary neurotrophic factor (CNTF) treatment. We further demonstrate that despite traveling more than 5 mm (i.e., a distance equal to the length of mouse optic nerve), many of these circuitous axons are confined to the injury area and fail to reach the brain. Our results re-evaluate the view that RGCs are naturally incapable of re-extending long axons, and shift the focus from promoting axon elongation, to understanding factors that prevent direct growth of axons through the lesion and the injured nerve.

Prevention of dendritic and synaptic deficits and cognitive impairment with a neurotrophic compound.

BACKGROUND: The use of neurotrophic factors to treat Alzheimer's disease (AD) is hindered by their blood-brain barrier impermeability, short half-life, and severe side effects. Peptide 021 (P021) is a neurotrophic/neurogenic tetra-peptide that was derived from the most active region of the ciliary neurotrophic factor (CNTF) by epitope mapping. Admantylated glycine was added to its C-terminal to increase its blood-brain barrier permeability and decrease its degradation by exopeptidases to make it druggable. Here, we report on the preventive effect of P021 in 3 × Tg-AD, a transgenic mouse model of AD. METHODS: P021 was administered in the diet at 3 months, i.e., 6-9 months before any overt amyloid beta (Aß) or tau pathology, and during the period of synaptic compensation, and was continued until 21 months in 3 × Tg-AD mice. The 3 × Tg-AD mice and wild-type (WT) mice were treated identically but with a vehicle-only diet serving as controls. The effects of P021 on neurogenesis, dendritic and synaptic markers, and cognitive performance were investigated. RESULTS: We found that P021 treatment was able to rescue dendritic and synaptic deficits, boost neurogenesis, and reverse cognitive impairment in 3 × Tg-AD mice. CONCLUSIONS: Availability of appropriate neurotrophic support during the period of synaptic compensation can prevent synaptic deficit and cognitive impairment, and P021 is a promising neurotrophic compound for this purpose.

Alleviative effect of ciliary neurotrophic factor analogue on high fat-induced hepatic steatosis is partially independent of the central regulation.

Ciliary neurotrophic factor (CNTF) analogues were reported to ameliorate fatty liver in db/db or high-fat diet-fed mice. It is generally thought that CNTF exerts its actions centrally. The aim of this study was to investigate whether peripheral effects of CNTF analogues are involved in the therapeutic effect on high fat-induced hepatic steatosis. The rat model of fatty liver was induced by a high-fat diet (HFD) for 12 weeks. In the next 2 weeks, rats were fed the HFD along with subcutaneous injection of vehicle or mutant recombinant human CNTF (rhmCNTF 0.05-0.2 mg/kg per day). Steatotic HepG2 cells were induced by 50% fetal bovine serum (FBS) for 48 hours, and then treated with rhmCNTF for 24 hours. The results showed that after rhmCNTF treatment, hepatic triglyceride (TG) accumulation was attenuated both in vivo and in vitro. RhmCNTF increased protein expression of CPT-1 and PPARα, and decreased SREBP-1c, FAS and SCD-1 in steatotic HepG2 cells. But the production of nitric oxide and 8-isoPGF2α in steatotic HepG2 cells was not affected by rhmCNTF. These results suggest that rhmCNTF has a peripheral effect that alleviates fat-induced hepatic steatosis.

AAV-mediated transfer of RhoA shRNA and CNTF promotes retinal ganglion cell survival and axon regeneration.

The aim of the present study was to determine whether adeno-associated viral vector (AAV) mediated transfer of ciliary neurotrophic factor (CNTF) and RhoA shRNA has additive effects on promoting the survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve crush (ONC). Silencing effects of AAV-RhoA shRNA were confirmed by examining neurite outgrowth in PC12 cells, and by quantifying RhoA expression levels with western blotting. Young adult Fischer rats received an intravitreal injection of (i) saline, (ii) AAV green fluorescent protein (GFP), (iii) AAV-CNTF, (iv) AAV-RhoA shRNA, or (v) a combination of both AAV-CNTF and AAV-RhoA shRNA. Two weeks later, the ON was completely crushed. Three weeks after ONC, RGC survival was estimated by counting ßIII-tubulin-positive neurons in retinal whole mounts. Axon regeneration was evaluated by counting GAP-43-positive axons in the crushed ON. It was found that AAV-RhoA shRNA decreased RhoA expression levels and promoted neurite outgrowth in vitro. In the ONC model, AAV-RhoA shRNA by itself had only weak beneficial effects on RGC axon regeneration. However, when combined with AAV-CNTF, AAV-RhoA shRNA significantly improved the therapeutic effect of AAV-CNTF on axon regeneration by nearly two fold, even though there was no significant change in RGC viability. In sum, this combination of vectors increases the regenerative response and can lead to more successful therapeutic outcomes following neurotrauma.

Long-term Follow-up of Patients With Retinitis Pigmentosa Receiving Intraocular Ciliary Neurotrophic Factor Implants.

PURPOSE: To evaluate the long-term efficacy of ciliary neurotrophic factor delivered via an intraocular encapsulated cell implant for the treatment of retinitis pigmentosa. DESIGN: Long-term follow-up of a multicenter, sham-controlled study. METHODS: Thirty-six patients at 3 CNTF4 sites were randomly assigned to receive a high- or low-dose implant in 1 eye and sham surgery in the fellow eye. The primary endpoint (change in visual field sensitivity at 12 months) had been reported previously. Here we measure long-term visual acuity, visual field, and optical coherence tomography (OCT) outcomes in 24 patients either retaining or explanting the device at 24 months relative to sham-treated eyes. RESULTS: Eyes retaining the implant showed significantly greater visual field loss from baseline than either explanted eyes or sham eyes through 42 months. By 60 months and continuing through 96 months, visual field loss was comparable among sham-treated eyes, eyes retaining the implant, and explanted eyes, as was visual acuity and OCT macular volume. CONCLUSIONS: Over the short term, ciliary neurotrophic factor released continuously from an intravitreal implant led to loss of total visual field sensitivity that was greater than the natural progression in the sham-treated eye. This additional loss of sensitivity related to the active implant was reversible when the implant was removed. Over the long term (60-96 months), there was no evidence of efficacy for visual acuity, visual field sensitivity, or OCT measures of retinal structure.

Intravitreal Ciliary Neurotrophic Factor Transiently Improves Cone-Mediated Function in a CNGB3-/- Mouse Model of Achromatopsia.

PURPOSE: Ciliary neurotrophic factor (CNTF) was recently shown to augment cone function in CNGB3 mutant achromat dogs. However, testing CNTF-releasing implant in human CNGB3 achromats failed to show benefit. We evaluated the effects of CNTF protein on the retinal function in an additional achromatopsia model, the CNGB3-/- mouse. METHODS: Fifty-nine CNGB3-/- mice (postnatal day [PD] ± SD = 30 ± 7) received a unilateral intravitreal injection of 1 or 2 µg CNTF protein, and 15 wild-type (WT) mice (PD = 34 ± 3) received 1 µg CNTF. Retinal function was evaluated by flash ERG and photopic flicker ERG (fERG) at 7 and 14 days after treatment. RESULTS: Seven days post CNTF, the photopic b-wave Vmax was significantly increased in CNGB3-/- mice (P < 0.01), whereas it was reduced in WT mice (P < 0.05). Ciliary neurotrophic factor significantly increased the amplitude of photopic fERG and the photopic oscillatory potentials (OPs) in CNGB3-/- mice. Ciliary neurotrophic factor did not alter the scotopic a-wave in either CNGB3-/- or WT mice, but it increased the scotopic b-wave k (P < 0.01) in CNGB3-/- mice, indicating diminished scotopic sensitivity, and reduced the scotopic b-wave Vmax in WT mice (P < 0.05). No difference was found in ERG parameters between 1 or 2 µg CNTF. Fourteen days after CNTF injection the ERG changes in CNGB3-/- mice were lost. CONCLUSIONS: Intravitreal bolus CNTF protein caused a small and transient improvement of cone-mediated function in CNGB3-/- mice, whereas it reduced rod-mediated function. The increase in photopic OPs and the lack of changes in scotopic a-wave suggest a CNTF effect on the inner retina.

Activation of transcription factors STAT1 and STAT5 in the mouse median eminence after systemic ciliary neurotrophic factor administration.

Exogenously administered ciliary neurotrophic factor (CNTF) causes weight loss in obese rodents and humans through leptin-like activation of the Jak-STAT3 signaling pathway in hypothalamic arcuate neurons. Here we report for the first time that 40min after acute systemic treatment, rat recombinant CNTF (intraperitoneal injection of 0.3mg/kg of body weight) induced nuclear translocation of the tyrosine-phosphorylated forms of STAT1 and STAT5 in the mouse median eminence and other circumventricular organs, including the vascular organ of the lamina terminalis and the subfornical organ. In the tuberal hypothalamus of treated mice, specific nuclear immunostaining for phospo-STAT1 and phospho-STAT5 was detected in ependymal cells bordering the third ventricle floor and lateral recesses, and in median eminence cells. Co-localization studies documented STAT1 and STAT5 activation in median eminence ß-tanycytes and underlying radial glia-like cells. A few astrocytes in the arcuate nucleus responded to CNTF by STAT5 activation. The vast majority of median eminence tanycytes and radial glia-like cells showing phospho-STAT1 and phospho-STAT5 immunoreactivity were also positive for phospho-STAT3. In contrast, STAT3 was the sole STAT isoform activated by CNTF in arcuate nucleus and median eminence neurons. Finally, immunohistochemical evaluation of STAT activation 20, 40, 80, and 120min from the injection demonstrated that cell activation was accompanied by c-Fos expression. Collectively, our findings show that CNTF activates STAT3, STAT1, and STAT5 in vivo. The distinctive activation pattern of these STAT isoforms in the median eminence may disclose novel targets and pathways through which CNTF regulates food intake.

Ciliary neurotrophic factor for macular telangiectasia type 2: results from a phase 1 safety trial.

PURPOSE: To evaluate the safety and tolerability of intraocular delivery of ciliary neurotrophic factor (CNTF) using an encapsulated cell implant for the treatment of macular telangiectasia type 2. DESIGN: An open-label safety trial conducted in 2 centers enrolling 7 participants with macular telangiectasia type 2. METHODS: The participant's more severely affected eye (worse baseline visual acuity) received the high-dose implant of CNTF. Patients were followed for a period of 36 months. The primary safety outcome was a change in the parameters of the electroretinogram (ERG). Secondary efficacy outcomes were changes in visual acuity, en face measurements of the optical coherence tomography of the disruption in the ellipsoid zone, and microperimetry when compared with baseline. RESULTS: The ERG findings demonstrated a reduction in the amplitude of the scotopic b-wave in 4 participants 3 months after implantation (month 3). All parameters returned to baseline values by month 12 and remained so at month 36 with no clinical impact on dark adaptation. There was no change in visual acuity compared with baseline. The area of the defect as measured functionally by microperimetry and structurally by the en face OCT imaging of the ellipsoid zone loss appeared unchanged from baseline. CONCLUSIONS: The intraocular delivery of CNTF in the encapsulated cell implant appeared to be safe and well tolerated in eyes with macular telangiectasia type 2. Further evaluation in a randomized controlled clinical trial is warranted to test for efficacy.

Neural stem cell-based intraocular administration of ciliary neurotrophic factor attenuates the loss of axotomized ganglion cells in adult mice.

PURPOSE: To analyze the neuroprotective effect of intravitreally grafted neural stem (NS) cells genetically modified to secrete ciliary neurotrophic factor (CNTF) on intraorbitally lesioned retinal ganglion cells (RGCs) in adult mice. METHODS: Adherently cultivated NS cells were genetically modified to express a secretable variant of mouse CNTF together with the fluorescent reporter protein Venus. Clonal CNTF-secreting NS cell lines were established using fluorescence activated cell sorting, and intravitreally grafted into adult mice 1 day after an intraorbital crush of the optic nerve. Brn-3a-positive RGCs were counted in flat-mounted retinas at different postlesion intervals to evaluate the neuroprotective effect of the CNTF-secreting NS cells on the axotomized RGCs. Anterograde axonal tracing experiments were performed to analyze the regrowth of the injured RGC axons in CNTF-treated retinas. RESULTS: Intravitreally grafted NS cells preferentially differentiated into astrocytes that survived in the host eyes, stably expressed CNTF, and significantly attenuated the loss of the axotomized RGCs over a period of at least 4 months, the latest postlesion time point analyzed. Depending on the postlesion interval analyzed, the number of RGCs in eyes with grafted CNTF-secreting NS cells was 2.8-fold to 6.4-fold higher than in eyes with grafted control NS cells. The CNTF-secreting NS cells additionally induced long-distance regrowth of the lesioned RGC axons. CONCLUSIONS: Genetically modified clonal NS cell lines may serve as a useful tool for preclinical studies aimed at evaluating the therapeutic potential of a sustained cell-based intravitreal administration of neuroprotective factors in mouse models of glaucoma.

CNGB3-achromatopsia clinical trial with CNTF: diminished rod pathway responses with no evidence of improvement in cone function.

PURPOSE: Ciliary neurotrophic factor (CNTF) protects rod photoreceptors from retinal degenerative disease in multiple nonhuman models. Thus far, CNTF has failed to demonstrate rod protection in trials for human retinitis pigmentosa. Recently, CNTF was found to improve cone photoreceptor function in a canine CNGB3 achromatopsia model. This study explores whether this finding translates to humans with CNGB3 achromatopsia. METHODS: A five-subject, open-label Phase I/II study was initiated by implanting intraocular microcapsules releasing CNTF (nominally 20 ng/d) into one eye each of CNGB3 achromat participants. Fellow eyes served as untreated controls. Subjects were followed for 1 year. RESULTS: Pupil constriction in treated eyes gave evidence of intraocular CNTF release. Additionally, scotopic ERG responses were reduced, and dark-adapted psychophysical absolute thresholds were increased, attributable to diminished rod or rod pathway activity. Optical coherence tomography revealed that the cone-rich fovea underwent structural changes as the foveal hyporeflective zone (HRZ) became diminished in CNTF-treated eyes. No objectively measurable enhancement of cone function was found by assessments of visual acuity, mesopic increment sensitivity threshold, or the photopic ERG. Careful measurements of color hue discrimination showed no change. Nonetheless, subjects reported beneficial changes of visual function in the treated eyes, including reduced light sensitivity and aversion to bright light, which may trace to decreased effective ambient light from the pupillary constriction; further they noted slowed adaptation to darkness, consistent with CNTF action on rod photoreceptors. CONCLUSIONS: Ciliary neurotrophic factor did not measurably enhance cone function, which reveals a species difference between human and canine CNGB3 cones in response to CNTF. (ClinicalTrials.gov number, NCT01648452.).

Disease modifying effect of chronic oral treatment with a neurotrophic peptidergic compound in a triple transgenic mouse model of Alzheimer's disease.

Besides the presence of amyloid beta (Aß) plaques and neurofibrillary tangles, neurogenesis and synaptic plasticity are markedly impaired in Alzheimer's disease (AD) possibly contributing to cognitive impairment. In this context, neurotrophic factors serve as a promising therapeutic approach via utilization of regenerative capacity of brain to shift the balance from neurodegeneration to neural regeneration. However, besides more conventional "bystander" effect, to what extent can neurotrophic compounds affect underlying AD pathology remains questionable. Here we investigated the effect of chronic oral treatment with a ciliary neurotrophic factor (CNTF) derived peptidergic compound, P021 (Ac-DGGL(A)G-NH2), on disease pathology both at moderate and severe stages in a transgenic mouse model of AD. 3xTg-AD and wild type female mice were treated for 12months with P021 or vehicle diet starting at 9-10months of age. A significant reduction in abnormal hyperphosphorylation and accumulation of tau at known major AD neurofibrillary pathology associated sites was observed. The effect of P021 on Aß pathology was limited to a significant decrease in soluble Aß levels and a trend towards reduction in Aß plaque load in CA1 region of hippocampus, consistent with reduction in Aß generation and not clearance. This disease modifying effect was probably via increased brain derived neurotrophic factor (BDNF) expression mediated decrease in glycogen synthase kinase-3-ß (GSK3ß) activity we found in P021 treated 3xTg-AD mice. P021 treatment also rescued deficits in cognition, neurogenesis, and synaptic plasticity in 3xTg-AD mice. These findings demonstrate the potential of the neurotrophic peptide mimetic as a disease modifying therapy for AD.