Response of heterologously expressed pressure sensor-actuator-modulator macromolecule to external mechanical stress.

Cells from different organs in the body experience a range of mechanical and osmotic pressures that change in various diseases, including neurological, cardiovascular, ophthalmological, and renal diseases. Here, we demonstrate the use of an engineered Sensor-Actuator-Modulator (SAM) of microbial origin derived from a mechanosensitive channel of large conductance (MscL) for sensing external mechanical stress and modulating activities of mammalian cells. SAM is reliably expressed in the mammalian cell membrane and acts as a tension-activated pressure release valve. Further, the activities of heterologously expressed SAM in mammalian cells could be modulated by osmotic pressure. A comparison of the mechanosensitive activities of SAM-variants from different microbial origins shows differential inward current and dye uptake in response to mechanical stress exerted by hypo-osmotic shock. The use of SAM channels as mechanical stress-activated modulators in mammalian cells could provide new therapeutic approaches for treating disorders related to mechanical or osmotic pressure.

OCT guided micro-focal ERG system with multiple stimulation wavelengths for characterization of ocular health.

Inherited retinal disorders and dry age-related macular degeneration are characterized by the degeneration and death of different types of photoreceptors at different rate and locations. Advancement of new therapeutic interventions such as optogenetics gene therapy and cell replacement therapies are dependent on electrophysiological measurements at cellular resolution. Here, we report the development of an optical coherence tomography (OCT) guided micro-focal multi-color laser stimulation and electroretinogram (ERG) platform for highly localized monitoring of retina function. Functional evaluation of wild type and transgenic pigs affected by retinal degeneration was carried out using OCT guided micro-focal ERG (µfERG) with selected stimulation wavelengths for S, M and L cones as well as rod photoreceptors. In wild type pigs, µfERG allowed functional recording from rods and each type of cone photoreceptor cells separately. Furthermore, functional deficits in P23H transgenic pigs consistent with their retinal degeneration phenotype were observed, including decrease in the S and M cone function and lack of rod photoreceptor function. OCT guided µfERG based monitoring of physiological function will enable characterization of animal models of retinal degenerative diseases and evaluation of therapeutic interventions at the cellular level.

Bioluminescent Multi-Characteristic Opsin for Simultaneous Optical Stimulation and Continuous Monitoring of Cortical Activities.

Stimulation and continuous monitoring of neural activities at cellular resolution are required for the understanding of the sensory processing of stimuli and development of effective neuromodulation therapies. We present bioluminescence multi-characteristic opsin (bMCOII), a hybrid optogenetic actuator, and a bioluminescence Ca2+ sensor for excitation-free, continuous monitoring of neural activities in the visual cortex, with high spatiotemporal resolution. An exceptionally low intensity (10 µW/mm2) of light could elicit neural activation that could be detected by Ca2+ bioluminescence imaging. An uninterrupted (>14 h) recording of visually evoked neural activities in the cortex of mice enabled the determination of strength of sensory activation. Furthermore, an artificial intelligence-based neural activation parameter transformed Ca2+ bioluminescence signals to network activity patterns. During continuous Ca2+-bioluminescence recordings, visual cortical activity peaked at the seventh to eighth hour of anesthesia, coinciding with circadian rhythm. For both direct optogenetic stimulation in cortical slices and visually evoked activities in the visual cortex, we observed secondary delayed Ca2+-bioluminescence responses, suggesting the involvement of neuron-astrocyte-neuron pathway. Our approach will enable the development of a modular and scalable interface system capable of serving a multiplicity of applications to modulate and monitor large-scale activities in the brain.

Monitoring Visual Cortical Activities During Progressive Retinal Degeneration Using Functional Bioluminescence Imaging.

Mouse models of inherited retinal degenerative diseases such as retinitis pigmentosa are characterized by degeneration of photoreceptors, which hinders the generation of signal to be transmitted to the visual cortex. By monitoring Ca2+-bioluminescence neural activity, we quantified changes in visual cortical activities in response to visual stimuli in RD10 mice during progression of retinal degeneration, which correlated with progressive deteriorations of electro-retinography signal from the eyes. The number of active neurons in the visual cortex, the intensity of Ca2+-bioluminescence response, and neural activation parameter showed progressive deterioration during aging. Further, we correlated the thinning of retina as measured by Optical Coherence Tomography with the decrease in visual cortical activities as retinal degeneration progressed. The present study establishes Ca2+-bioluminescence monitoring as a longitudinal imaging modality to characterize activities in visual cortex of retinal degenerative disease models and therapeutic interventions.

Biodistribution of adeno-associated virus type 2 carrying multi-characteristic opsin in dogs following intravitreal injection.

Gene therapy of retinal diseases using recombinant adeno-associated virus (rAAV) vector-based delivery has shown clinical success, and clinical trials based on rAAV-based optogenetic therapies are currently in progress. Recently, we have developed multi-characteristic opsin (MCO), which has been shown to effectively re-photosensitize photoreceptor-degenerated retina in mice leading to vision restoration at ambient light environment. Here, we report the biodistribution of the rAAV2 carried MCO (vMCO-I) in live samples and post-mortem organs following intraocular delivery in wild-type dogs. Immunohistochemistry showed that the intravitreal injection of vMCO-I resulted in gene transduction in the inner nuclear layer (INL) but did not induce detectable inflammatory or immune reaction in the dog retina. Vector DNA analysis of live body wastes and body fluids such as saliva and nasal secretions using quantitative polymerase chain reaction (qPCR) showed no correlative increase of vector copy in nasal secretions or saliva, minimal increase of vector copy in urine in the low-dose group 13 weeks after injection and in the faeces of the high-dose group at 3-13 weeks after injection suggesting clearance of the virus vector via urine and faeces. Further analysis of vector DNA extracted from faeces using PCR showed no transgene after 3 weeks post-injection. Intravitreal injection of vMCO-I resulted in few sporadic off-target presences of the vector in the mesenteric lymph node, liver, spleen and testis. This study showed that intravitreal rAAV2-based delivery of MCO-I for retinal gene therapy is safe.

Layer-specific nanophotonic delivery of therapeutic opsin-encoding genes into retina.

In recent time, gene therapy has proven to be a promising remedial approach for treating visual disorders either by replacement of nonfunctioning gene(s) or by introduction of light sensitive proteins (opsins) as artificial photoreceptors in retinal cells. Conventional viral vector-based gene delivery method is often confronted with limitations due to immunogenetic reaction, unintended non-targeted delivery, non-feasibility of repeated re-dosing due to immunorejection, and complicated manufacturing process, leading to significant roadblock in translational success. In this regard, non-viral delivery provides a safer, simpler and cost-effective alternative. However, most of the non-viral approaches lack spatial and/or cellular specificity and limited by low transfection efficacy and cytotoxicity. Here, we present a minimally invasive, non-viral and clinically translatable safe targeted gene delivery method utilizing functionalized plasmonic gold nanorods (fGNRs, targeted to attach to specific cell types of the organ of interest) and spatially targeted controlled light irradiation. Targeted in-vivo delivery and expression of opsin-encoding gene in bipolar and ganglion cell layers were achieved by use of cell specific fGNRs concurrent with light irradiation. Evaluation of safety and toxicity associated with the transduction of opsin-encoding genes by use of fGNRs and light irradiation were examined by electrophysiology, Optical coherence tomography, intra-ocular pressure and other analytical methods (confocal microscopy, immunohistochemistry). The non-viral light-based opsin-gene delivery provides a safe and effective alternative to viral-vector based gene delivery and holds promise for corrective cell-specific gene therapies for retinal degenerative diseases.

Sensitization of ON-bipolar cells with ambient light activatable multi-characteristic opsin rescues vision in mice.

Gene therapy-based treatment such as optogenetics offers a potentially powerful way to bypass damaged photoreceptors in retinal degenerative diseases and use the remaining retinal cells for functionalization to achieve photosensitivity. However, current approaches of optogenetic treatment rely on opsins that require high intensity light for activation thus adding to the challenge for use as part of a wearable device. Here, we report AAV2 assisted delivery of highly photosensitive multi-characteristic opsin (MCO1) into ON-bipolar cells of mice with retinal degeneration to allow activation by ambient light. Rigorous characterization of delivery efficacy by different doses of AAV2 carrying MCO1 (vMCO1) into targeted cells showed durable expression over 6 months after delivery as measured by reporter expression. The enduring MCO1 expression was correlated with the significantly improved behavioral outcome, that was longitudinally measured by visual water-maze and optomotor assays. The pro/anti-inflammatory cytokine levels in plasma and vitreous humor of the vMCO1-injected group did not change significantly from baseline or control group. Furthermore, biodistribution studies at various time points after injection in animal groups injected with different doses of vMCO1 showed non-detectable vector copies in non-targeted tissues. Immunohistochemistry of vMCO1 transfected retinal tissues showed bipolar specific expression of MCO1 and the absence of immune/inflammatory response. Furthermore, ocular imaging using SD-OCT showed no change in the structural architecture of vMCO1-injected eyes. Induction of ambient light responsiveness to remaining healthy bipolar cells in subjects with retinal degeneration will allow the retinal circuitry to gain visual acuity without requiring an active stimulation device.

Laser-assisted targeted gene delivery to degenerated retina improves retinal function.

Delivery of therapeutic genes into retina is proving to reverse degeneration and restore vision, however, viral vector-based gene delivery is prone to immunorejection, inflammatory/immune-response and nontargeted. Here, we report nonviral gene delivery and expression of opsin encoding genes in mouse retina in-vitro and in-vivo by use of pulsed femtosecond laser microbeam. In-vitro patch-clamp recording of the opsin-sensitized retinal cells and visually evoked in-vivo electrical recording from laser-transfected eye of mouse with degenerated retina showed functional response. The ultrafast laser-based naked gene delivery showed minimal damage and reliable expression of therapeutic opsin in cell membrane of the selected cells and in targeted retinal region. Laser-based "naked DNA gene therapy" in a spatially targeted manner will pave the way for treatment of inherited retinal diseases.

Near-Infrared Laser-Based Spatially Targeted Nano-enhanced Optical Delivery of Therapeutic Genes to Degenerated Retina.

Non-viral delivery of therapeutic genes into targeted areas of retina is essential for re-functionalizing the retinal circuitry. While a focused ultrafast laser beam has been recently used for intra-ocular delivery of molecules, it poses the significant technical challenge of overcoming aberrations of the eye and maintaining a tightly focused spot on the retinal cell membrane. Furthermore, to minimize collateral damage and increase the throughput of gene delivery, we introduced a weakly focused near-infrared (NIR) continuous wave (CW) or pulsed laser beam on to the cells wherein the intensity is locally enhanced by gold nanorods bound to the cell membranes to permit gene insertion. Parametric optimization of nano-enhanced optical delivery (NOD) was carried out by varying the exposure time, as well as the power of the CW NIR beam or the energy of the pulsed NIR beam. Using this NOD method, therapeutic genes encoding for multi-characteristic opsins (MCOs) were delivered to spatially targeted regions of degenerated retina ex vivo as well as in vivo. NOD-mediated cell membrane-specific expression of MCOs in targeted retinal regions with photoreceptor degeneration will allow functional recovery in an ambient light environment.

Pharmacologic fibroblast reprogramming into photoreceptors restores vision.

Photoreceptor loss is the final common endpoint in most retinopathies that lead to irreversible blindness, and there are no effective treatments to restore vision1,2. Chemical reprogramming of fibroblasts offers an opportunity to reverse vision loss; however, the generation of sensory neuronal subtypes such as photoreceptors remains a challenge. Here we report that the administration of a set of five small molecules can chemically induce the transformation of fibroblasts into rod photoreceptor-like cells. The transplantation of these chemically induced photoreceptor-like cells (CiPCs) into the subretinal space of rod degeneration mice (homozygous for rd1, also known as Pde6b) leads to partial restoration of the pupil reflex and visual function. We show that mitonuclear communication is a key determining factor for the reprogramming of fibroblasts into CiPCs. Specifically, treatment with these five compounds leads to the translocation of AXIN2 to the mitochondria, which results in the production of reactive oxygen species, the activation of NF-κB and the upregulation of Ascl1. We anticipate that CiPCs could have therapeutic potential for restoring vision.

Nano-enhanced Optical Gene Delivery to Retinal Degenerated Mice.

BACKGROUND: The efficient and targeted delivery of genes and other impermeable therapeutic molecules into retinal cells is of immense importance for the therapy of various visual disorders. Traditional methods for gene delivery require viral transfection, or chemical methods that suffer from one or many drawbacks, such as low efficiency, lack of spatially targeted delivery, and can generally have deleterious effects, such as unexpected inflammatory responses and immunological reactions. METHODS: We aim to develop a continuous wave near-infrared laser-based Nano-enhanced Optical Delivery (NOD) method for spatially controlled delivery of ambient-light-activatable Muti-Characteristic opsin-encoding genes into retina in-vivo and ex-vivo. In this method, the optical field enhancement by gold nanorods is utilized to transiently permeabilize cell membrane, enabling delivery of exogenous impermeable molecules to nanorod-binding cells in laser-irradiated regions. RESULTS AND DISCUSSION: With viral or other non-viral (e.g. electroporation, lipofection) methods, gene is delivered everywhere, causing uncontrolled expression over the whole retina. This will cause complications in the functioning of non-degenerated areas of the retina. In the NOD method, the contrast in temperature rise in laser-irradiated nanorod-attached cells at nano-hotspots is significant enough to allow site-specific delivery of large genes. The in-vitro and in-vivo results using NOD, clearly demonstrate in-vivo gene delivery and functional cellular expression in targeted retinal regions without compromising the structural integrity of the eye or causing immune response. CONCLUSION: The successful delivery and expression of MCO in the targeted retina after in-vivo NOD in the mice models of retinal degeneration opens a new vista for re-photosensitizing retina with geographic atrophies, such as in dry age-related macular degeneration.

Erratum: Publisher's note: Restoring vision in mice with retinal degeneration using multicharacteristic opsin.

[This corrects the article DOI: 10.1117/1.NPh.4.4.041412.].

Fe Core-Carbon Shell Nanoparticles as Advanced MRI Contrast Enhancer.

The aim of this study is to fabricate a hybrid composite of iron (Fe) core-carbon (C) shell nanoparticles with enhanced magnetic properties for contrast enhancement in magnetic resonance imaging (MRI). These new classes of magnetic core-shell nanoparticles are synthesized using a one-step top-down approach through the electric plasma discharge generated in the cavitation field in organic solvents by an ultrasonic horn. Transmission electron microscopy (TEM) observations revealed the core-shell nanoparticles with 10-85 nm in diameter with excellent dispersibility in water without any agglomeration. TEM showed the structural confirmation of Fe nanoparticles with body centered cubic (bcc) crystal structure. Magnetic multi-functional hybrid composites of Fe core-C shell nanoparticles were then evaluated as negative MRI contrast agents, displaying remarkably high transverse relaxivity (r2) of 70 mM-1·S-1 at 7 T. This simple one-step synthesis procedure is highly versatile and produces desired nanoparticles with high efficacy as MRI contrast agents and potential utility in other biomedical applications.

Label-free optical detection of action potential in mammalian neurons.

We describe an optical technique for label-free detection of the action potential in cultured mammalian neurons. Induced morphological changes due to action potential propagation in neurons are optically interrogated with a phase sensitive interferometric technique. Optical recordings composed of signal pulses mirror the electrical spike train activity of individual neurons in a network. The optical pulses are transient nanoscale oscillatory changes in the optical path length of varying peak magnitude and temporal width. Exogenous application of glutamate to cortical neuronal cultures produced coincident increase in the electrical and optical activity; both were blocked by application of a Na-channel blocker, Tetrodotoxin. The observed transient change in optical path length in a single optical pulse is primarily due to physical fluctuations of the neuronal cell membrane mediated by a yet unknown electromechanical transduction phenomenon. Our analysis suggests a traveling surface wave in the neuronal cell membrane is responsible for the measured optical signal pulses.

Restoring vision in mice with retinal degeneration using multicharacteristic opsin.

Retinal degenerative diseases, such as retinitis pigmentosa (RP) and dry age-related macular degeneration, have led to loss of vision in millions of individuals. Currently, no surgical or medical treatment is available, although optogenetic therapies are in clinical development. We demonstrate vision restoration using multicharacteristics opsin (MCO1) in animal models with degenerated retina. MCO1 is reliably delivered to specific retinal cells via intravitreal injection of adeno-associated virus (vMCO1), leading to significant improvement in visually guided behavior conducted using a radial arm water maze. The time to reach the platform and the number of error arms decreased significantly after delivery of MCO1. Notably, the improvement in visually guided behavior was observed even at light intensity levels orders of magnitude lower than that required for channelrhodopsin-2 opsin. Viability of vMCO1-treated retina is not compromised by chronic light exposure. Safe virus-mediated MCO1 delivery has potential for effective gene therapy of diverse retinal degenerations in patients.

Restoring vision in mice with retinal degeneration using multicharacteristic opsin.

Retinal degenerative diseases, such as retinitis pigmentosa (RP) and dry age-related macular degeneration, have led to loss of vision in millions of individuals. Currently, no surgical or medical treatment is available, although optogenetic therapies are in clinical development. We demonstrate vision restoration using multicharacteristics opsin (MCO1) in animal models with degenerated retina. MCO1 is reliably delivered to specific retinal cells via intravitreal injection of adeno-associated virus (vMCO1), leading to significant improvement in visually guided behavior conducted using a radial arm water maze. The time to reach the platform and the number of error arms decreased significantly after delivery of MCO1. Notably, the improvement in visually guided behavior was observed even at light intensity levels orders of magnitude lower than that required for channelrhodopsin-2 opsin. Viability of vMCO1-treated retina is not compromised by chronic light exposure. Safe virus-mediated MCO1 delivery has potential for effective gene therapy of diverse retinal degenerations in patients.

Ultrafast laser-assisted spatially targeted optoporation into cortical axons and retinal cells in the eye.

Visualization and assessment of the cellular structure and function require localized delivery of the molecules into specific cells in restricted spatial regions of the tissue and may necessitate subcellular delivery and localization. Earlier, we have shown ultrafast near-infrared laser beam-assisted optoporation of actin-staining molecules into cortical neurons with single-cell resolution and high efficiency. However, diffusion of optoporated molecules in soma degrades toward the growth cone, leading to difficulties in visualization of the actin network in the growth cone in cases of long axons. Here, we demonstrate optoporation of impermeable molecules to functional cortical neurons by precise laser subaxotomy near the growth cone, leading to visualization of the actin network in the growth cone. Further, we demonstrate patterned delivery of impermeable molecules into targeted retinal cells in the rat eye. The development of optoporation as a minimally invasive approach to reliably deliver exogenous molecules into targeted axons and soma of retinal neurons in vivo will enable enhanced visualization of the structure and function of the retina.

Quantitative analysis of nanoscale intranuclear structural alterations in hippocampal cells in chronic alcoholism via transmission electron microscopy imaging.

Chronic alcoholism is known to alter the morphology of the hippocampus, an important region of cognitive function in the brain. Therefore, to understand the effect of chronic alcoholism on hippocampal neural cells, we employed a mouse model of chronic alcoholism and quantified intranuclear nanoscale structural alterations in these cells. Transmission electron microscopy (TEM) images of hippocampal neurons were obtained, and the degree of structural alteration in terms of mass density fluctuation was determined using the light-localization properties of optical media generated from TEM imaging. The results, which were obtained at length scales ranging from ~30 to 200 nm, show that 10-12 week-old mice fed a Lieber-DeCarli liquid (alcoholic) diet had a higher degree of structural alteration than control mice fed a normal diet without alcohol. The degree of structural alteration became significantly distinguishable at a sample length of ~100 nm, which is the typical length scale of the building blocks of cells, such as DNA, RNA, proteins and lipids. Interestingly, different degrees of structural alteration at such length scales suggest possible structural rearrangement of chromatin inside the nuclei in chronic alcoholism.

Spatial temperature gradients guide axonal outgrowth.

Formation of neural networks during development and regeneration after injury depends on accuracy of axonal pathfinding, which is primarily believed to be influenced by chemical cues. Recently, there is growing evidence that physical cues can play crucial role in axonal guidance. However, detailed mechanism involved in such guidance cues is lacking. By using weakly-focused near-infrared continuous wave (CW) laser microbeam in the path of an advancing axon, we discovered that the beam acts as a repulsive guidance cue. Here, we report that this highly-effective at-a-distance guidance is the result of a temperature field produced by the near-infrared laser light absorption. Since light absorption by extracellular medium increases when the laser wavelength was red shifted, the threshold laser power for reliable guidance was significantly lower in the near-infrared as compared to the visible spectrum. The spatial temperature gradient caused by the near-infrared laser beam at-a-distance was found to activate temperature-sensitive membrane receptors, resulting in an influx of calcium. The repulsive guidance effect was significantly reduced when extracellular calcium was depleted or in the presence of TRPV1-antagonist. Further, direct heating using micro-heater confirmed that the axonal guidance is caused by shallow temperature-gradient, eliminating the role of any non-photothermal effects.