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.

Safety of intravitreally delivered AAV2 vector-mediated multi-characteristic opsin genetic construct in wild type beagle dogs.

BACKGROUND: A novel adeno-associated virus 2 (AAV2)-carried multi-characteristic opsin (MCO) (MCO-010) is undergoing several clinical trials as a novel therapeutic modality for the treatment of degenerative retinal diseases including retinitis pigmentosa and Stargardt disease. The present study aimed to determine the ocular and systemic safety of MCO-010 and the AAV2 vehicle in adult Beagle dogs following intravitreal (IVT) injection. METHODS: The current safety/toxicology studies spanning 13 weeks described here utilized well-documented techniques to assess the effects of IVT injection of MCO-010 up to 2.2 × 1011 genome copies (gc) per eye, or the AAV2 capsid (vehicle control) on gross behavioral and immunogenic changes, alterations in body weights, blood biochemistry, hematology, blood coagulation, gross necropsy lesions, organ weight changes and histopathology in the dogs (n = 4 per group; two males and two females per group). Immunohistochemical and functional electroretinogram studies were also conducted to determine MCO expression in the retina and determine any retinal toxicity associated with MCO-010. RESULTS: There were no significant deleterious effects of the MCO-010 (or the AAV2 at the tested doses) on any of the examined parameters, including the absence of any severe ocular or systemic adverse events. However, as expected, inflammation after IVT delivery of AAV2 and MCO-010 was observed in the conjunctivae of all groups of animals, although this self-resolved within 1 week post-injection. Quantitative immunohistochemical analyses of MCO-010-associated mCherry revealed successful delivery of the gene therapy within the inner retina. CONCLUSIONS: In summary, MCO-010 demonstrated a favorable safety profile when administered to the eyes of adult Beagle dogs of both sexes at dose levels up to 2.2 × 1011 gc per eye, with no adverse effects observed. This dose was identified as the No Observed Adverse Effect Level (i.e. NOAEL) and guided selection of safe doses for human clinical trials.

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.

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.

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.

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.

Broad spectral excitation of opsin for enhanced stimulation of cells.

Optical stimulation of cells expressing light-sensitive proteins (opsins) has allowed targeted activation with cellular specificity. However, since narrow-band light has been used for excitation of these optogenetic probes, only active stimulation strategies are being attempted for clinical applications such as restoration of vision. Here, we report use of broad spectral excitation (white light) for optogenetic stimulation of opsin-sensitized cells. We found that ReaChR is optimally excited with white light offering significantly higher photocurrents compared to spectrally filtered narrow-band light stimulation. Our findings open up the possibility of passive stimulation strategy by use of natural sunlight for retinal stimulation, which could have benefits for ambient light stimulated vision restoration.

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.

Decoupling of geometric thickness and refractive index in quantitative phase microscopy.

In quantitative phase imaging, a priori knowledge of either refractive index or physical thickness is used to estimate the change in one of these parameters. Here, we report a method for decoupling geometric thickness from refractive index in quantitative phase microscopy.

Fiber-optic spanner.

Methods of controllable, noncontact rotation of optically trapped microscopic objects have garnered significant attention for tomographic imaging and microfluidic actuation. Here, we report development of a fiber-optic spanner and demonstrate controlled rotation of smooth muscle cells. The rotation is realized by introducing a transverse offset between two counterpropagating beams emanating from single-mode optical fibers. The rotation speed and surrounding microfluidic flow could be controlled by varying balanced laser beam powers. Further, we demonstrate simultaneous translation and rotation of the fiber-optically trapped cell by varying the laser power of one fiber-optic arm.

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.

Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells.

Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed.

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.

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.

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.

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.

Digital holographic microscopy for quantitative cell dynamic evaluation during laser microsurgery.

Digital holographic microscopy allows determination of dynamic changes in the optical thickness profile of a transparent object with sub-wavelength accuracy. Here, we report a quantitative phase laser microsurgery system for evaluation of cellular/ sub-cellular dynamic changes during laser micro-dissection. The proposed method takes advantage of the precise optical manipulation by the laser microbeam and quantitative phase imaging by digital holographic microscopy with high spatial and temporal resolution. This system will permit quantitative evaluation of the damage and/or the repair of the cell or cell organelles in real time.

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.

In-depth activation of channelrhodopsin 2-sensitized excitable cells with high spatial resolution using two-photon excitation with a near-infrared laser microbeam.

We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.

Manipulation of mammalian cells using a single-fiber optical microbeam.

The short working distance of microscope objectives has severely restricted the application of optical micromanipulation techniques at larger depths. We show the first use of fiber-optic tweezers toward controlled guidance of neuronal growth cones and stretching of neurons. Further, by mode locking, the fiber-optic tweezers beam was converted to fiber-optic scissors, enabling dissection of neuronal processes and thus allowing study of the subsequent response of neurons to localized injury. At high average powers, lysis of a three-dimensionally trapped cell was accomplished.