Targeting STAT3 in tumor-associated antigen-presenting cells as a strategy for kidney and bladder cancer immunotherapy.

Introduction: Immune checkpoint blockade (ICB) improved clinical outcomes in renal and bladder cancer patients, but the response rates remain limited especially in metastatic disease. While STAT3 transcription factor is well-known master regulator of tumor immune evasion, little is known about the role of STAT3 in the resistance of renal or bladder cancers to immunotherapy. Methods: To better understand immune alterations associated with ICB resistance, we assessed blood biomarkers in renal cancer patients classified as responders or non-responders to first line nivolumab/ipilimumab immunotherapy. Results: We observed that non-responders showed elevated levels of proinflammatory mediators, such as IL-1RA, IL-6, IL-8 and to lesser extent IL-10, which are associated with STAT3 activation and tumor immunosuppression. In addition, we found STAT3 activation primarily in circulating myeloid immune cells such as tolerogenic MDSCs. To assess whether STAT3 inhibition within these cell subsets can promote antitumor immune responses and/or enhance sensitivity to ICB in vivo, we used an original antisense oligonucleotide (ASO) strategy for myeloid-cell selective STAT3 knockdown (CpG-STAT3ASO). Our results in syngeneic models of renal and bladder cancers in mice demonstrated potent antitumor activity of CpG-STAT3ASO alone in contrast to PD1 blockade alone in both models. The CpG-STAT3ASO/anti-PD1 combination improved therapeutic efficacy especially against bladder tumors. Therapeutic efficacy correlated with activation of dendritic cells (DCs) and M1 macrophages in the tumor microenvironment, reduced percentages of regulatory T cells (Tregs) and the expansion of CD8 T cells in both tumor models. Discussion/Conclusion: Our study underscores the potential of using myeloid-cell targeted CpG-STAT3 inhibitors for genitourinary cancer therapy to disrupt tolerogenic signaling, restore immune cell activity and sensitivity to immune checkpoint inhibitors and/or T cell-based immunotherapies.

Establishing a stable, repeatable platform for measuring changes in sperm DNA methylation.

BACKGROUND: Several independent research groups have shown that alterations in human sperm methylation profiles correlate with decreased fecundity and an increased risk of poor embryo development. Moving these initial findings from the lab into a clinical setting where they can be used to measure male infertility though requires a platform that is stable and robust against batch effects that can occur between sample runs. Operating parameters must be established, performance characteristics determined, and guidelines set to ensure repeatability and accuracy. The standard for technical validation of a lab developed test (LDT) in the USA comes from the Clinical Laboratory Improvement Amendments (CLIA). However, CLIA was introduced in 1988, before the advent of genome-wide profiling and associated computational analysis. This, coupled with its intentionally general nature, makes its interpretation for epigenetic assays non-trivial. RESULTS: Here, we present an interpretation of the CLIA technical validation requirements for profiling DNA methylation and calling aberrant methylation using the Illumina Infinium platform (e.g., the 450HM and MethylationEPIC). We describe an experimental design to meet these requirements, the experimental results obtained, and the operating parameters established. CONCLUSIONS: The CLIA guidelines, although not intended for high-throughput assays, can be interpreted in a way that is consistent with modern epigenetic assays. Based on such an interoperation, Illumina's Infinium platform is quite amenable to usage in a clinical setting for diagnostic work.

Aberrant sperm DNA methylation predicts male fertility status and embryo quality.

OBJECTIVE: To evaluate whether male fertility status and/or embryo quality during in vitro fertilization (IVF) therapy can be predicted based on genomewide sperm deoxyribonucleic acid (DNA) methylation patterns. DESIGN: Retrospective cohort study. SETTING: University-based fertility center. PATIENT(S): Participants were 127 men undergoing IVF treatment (where any major female factor cause of infertility had been ruled out), and 54 normozoospermic, fertile men. The IVF patients were stratified into 2 groups: patients who had generally good embryogenesis and a positive pregnancy (n = 55), and patients with generally poor embryogenesis (n = 72; 42 positive and 30 negative pregnancies) after IVF. INTERVENTION(S): Genomewide sperm DNA methylation analysis was performed to measure methylation at >485,000 sites across the genome. MAIN OUTCOME MEASURE(S): A comparison was made of DNA methylation patterns of IVF patients vs. normozoospermic, fertile men. RESULT(S): Predictive models proved to be highly accurate in classifying male fertility status (fertile or infertile), with 82% sensitivity, and 99% positive predictive value. Hierarchic clustering identified clusters enriched for IVF patient samples and for poor-quality-embryo samples. Models built to identify samples within these groups, from neat samples, achieved positive predictive value ≥ 94% while identifying >one fifth of all IVF patient and poor-quality-embryo samples in each case. Using density gradient prepared samples, the same approach recovered 46% of poor-quality-embryo samples with no false positives. CONCLUSION(S): Sperm DNA methylation patterns differ significantly and consistently for infertile vs. fertile, normozoospermic men. In addition, DNA methylation patterns may be predictive of embryo quality during IVF.

A profile of transcriptomic changes in the rd10 mouse model of retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is a photoreceptor disease that affects approximately 100,000 people in the United States. Treatment options are limited, and the prognosis for most patients is progressive vision loss. Unfortunately, understanding of the molecular underpinnings of RP initiation and progression is still limited. However, the development of animal models of RP, coupled with high-throughput sequencing, has provided an opportunity to study the underlying cellular and molecular changes in this disease. METHODS: Using RNA-Seq, we present the first retinal transcriptome analysis of the rd10 murine model of retinal degeneration. RESULTS: Our data confirm the loss of rod-specific transcripts and the increased relative expression of Müller-specific transcripts, emphasizing the important role of reactive gliosis and innate immune activation in RP. Moreover, we report substantial changes in relative isoform usage among neuronal differentiation and morphogenesis genes, including a marked shift to shorter transcripts. CONCLUSIONS: Our analyses implicate remodeling of the inner retina and possible Müller cell dedifferentiation.

Frequency and amplitude modulation have different effects on the percepts elicited by retinal stimulation.

PURPOSE: In an effort to restore functional form vision, epiretinal prostheses that elicit percepts by directly stimulating remaining retinal circuitry were implanted in human subjects with advanced retinitis pigmentosa RP). In this study, manipulating pulse train frequency and amplitude had different effects on the size and brightness of phosphene appearance. METHODS: Experiments were performed on a single subject with severe RP (implanted with a 16-channel epiretinal prosthesis in 2004) on nine individual electrodes. Psychophysical techniques were used to measure both the brightness and size of phosphenes when the biphasic pulse train was varied by either modulating the current amplitude (with constant frequency) or the stimulating frequency (with constant current amplitude). RESULTS: Increasing stimulation frequency always increased brightness, while having a smaller effect on the size of elicited phosphenes. In contrast, increasing stimulation amplitude generally increased both the size and brightness of phosphenes. These experimental findings can be explained by using a simple computational model based on previous psychophysical work and the expected spatial spread of current from a disc electrode. CONCLUSIONS: Given that amplitude and frequency have separable effects on percept size, these findings suggest that frequency modulation improves the encoding of a wide range of brightness levels without a loss of spatial resolution. Future retinal prosthesis designs could benefit from having the flexibility to manipulate pulse train amplitude and frequency independently (clinicaltrials.gov number, NCT00279500).

Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness.

Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness.

Temporal interactions during paired-electrode stimulation in two retinal prosthesis subjects.

PURPOSE: Since 2002, six blind patients have undergone implantation of an epiretinal 4 × 4 electrode array designed to directly stimulate the remaining cells of the retina after severe photoreceptor degeneration due to retinitis pigmentosa. This study was conducted to investigate how the brightness of percepts is affected by pulse timing across electrodes in two of these patients. METHODS: Subjects compared the perceived brightness of a standard stimulus (synchronous pulse trains presented across pairs of electrodes) to the perceived brightness of a test stimulus (pulse trains across the electrode pair phase shifted by 0.075, 0.375, 1.8, or 9 ms). The current amplitude necessary for each phase-shifted test stimulus to match the brightness of the standard was determined. RESULTS: Depending on the electrode pair, interactions between electrodes were either facilitatory (the perceived brightness produced by stimulating the pair of electrodes was greater than that produced by stimulating either electrode alone) or suppressive (the perceived brightness produced by stimulating the pair of electrodes was less than that produced by stimulating either electrode alone). The amount of interaction between electrodes decreased as a function of increased separation both in time (the phase-shift between pulse trains) and space (center-to-center distance between the electrode pair). CONCLUSIONS: For visual prostheses to represent visual scenes that are changing in both space and time requires the development of spatiotemporal models describing the effects of stimulation across multiple electrodes. During multielectrode stimulation, interactions between electrodes have a significant influence on subjective brightness that includes both facilitatory and suppressive effects, and these interactions can be described with a simple computational model. (ClinicalTrials.gov number, NCT00279500.).

Towards optogenetic sensory replacement.

Over the last several years we have developed a rapidly-expanding suite of genetically-encoded reagents (e.g., ChR2, Halo, Arch, Mac, and others) that, when expressed in specific neuron types in the nervous system, enable their activities to be powerfully and precisely activated and silenced in response to light. If the genes that encode for these reagents can be delivered to cells in the body using gene therapy methods, and if the resultant protein payloads operate safely and effectively over therapeutically important periods of time, these molecules could subserve a set of precise prosthetics that use light as the trigger of information entry into the nervous system, e.g. for sensory replacement. Here we discuss the use of ChR2 to make the photoreceptor-deprived retina, as found in diseases such as retinitis pigmentosa, sensitive to light, enabling restoration of functional vision in a mouse model of blindness. We also discuss arrays of light sources that could be useful for delivering patterned sensory information into the nervous system.

Spatiotemporal interactions in retinal prosthesis subjects.

PURPOSE: Vision loss due to retinitis pigmentosa affects an estimated 15 million people worldwide. Through collaboration between Second Sight Medical Products, Inc., and the Doheny Eye Institute, six blind human subjects underwent implantation with epiretinal 4 x 4 electrode arrays designed to directly stimulate the remaining cells of the retina, with the goal of restoring functional vision by applying spatiotemporal patterns of stimulation. To better understand spatiotemporal interactions between electrodes during synchronous and asynchronous stimulation, the authors investigated how percepts changed as a function of pulse timing across the electrodes. METHODS: Pulse trains (20, 40, 80, and 160 Hz) were presented on groups of electrodes with 800, 1600, or 2400 microm center-to-center separation. Stimulation was either synchronous (pulses were presented simultaneously across electrodes) or asynchronous (pulses were phase shifted). Using a same-different discrimination task, the authors were able to evaluate how the perceptual quality of the stimuli changed as a function of phase shifts across multiple electrodes. RESULTS: Even after controlling for electric field interactions, subjects could discriminate between spatiotemporal pulse train patterns based on differences of phase across electrodes as small as 3 ms. These findings suggest that the quality of the percept is affected not only by electric field interactions but also by spatiotemporal interactions at the neural level. CONCLUSIONS: During multielectrode stimulation, interactions between electrodes have a significant influence on the quality of the percept. Understanding how these spatiotemporal interactions at the neural level influence percepts during multielectrode stimulation is fundamental to the successful design of a retinal prosthesis.

Brightness as a function of current amplitude in human retinal electrical stimulation.

PURPOSE: With the goal of eventually restoring functional vision in patients with retinal degenerative diseases, USC/Second Sight Medical Products, Inc. chronically implanted blind human subjects with a prototype epiretinal prosthesis consisting of a 4 x 4 array of 16 stimulating electrodes. To accurately represent a visual scene, a visual prosthesis must convey luminance information across a range of brightness levels. To achieve this, the brightness of phosphenes produced by an individual electrode should scale appropriately with luminance, and the same luminance should produce equivalently bright phosphenes across the entire electrode array. The goal was to examine how apparent brightness changes as a function of stimulation intensity across electrodes. METHODS: As described in previous studies, electrical stimulation of intact cells of the neural retina using this prosthetic device reliably elicits visual percepts in human subjects blinded by retinitis pigmentosa. Here, apparent brightness for a range of electrical amplitudes was measured using both subjective magnitude rating and brightness-matching procedures in chronically implanted human subjects. RESULTS: It was found that apparent brightness can be described as a power function of stimulation intensity. The same model can also predict brightness matching across electrodes. CONCLUSIONS: These results suggest that a relatively simple model for scaling current across electrodes may be capable of producing equivalently bright phosphenes across an entire array. (ClinicalTrials.gov number, NCT00279500.).

Predicting visual sensitivity in retinal prosthesis patients.

PURPOSE: With the long-term goal of restoring functional vision in patients with retinal degenerative diseases, the eyes of blind human subjects were implanted chronically with epiretinal prostheses consisting of two-dimensional electrode arrays that directly stimulated cells of the neural retina. METHODS: Psychophysical techniques were used to measure the brightness of electrically generated percepts on single electrodes using a variety of electrical stimulation patterns. RESULTS: It was possible to predict the sensitivity of the human visual system to a wide variety of retinal electrical stimulation patterns using a simple and biologically plausible model. CONCLUSIONS: This is the first study to demonstrate that, on the single-electrode level, retinal electrical stimulation in humans can produce visual qualia that are predictable using a quantitative model, a prerequisite for a successful retinal prosthesis. (ClinicalTrials.gov number, NCT00279500.).

Factors affecting perceptual thresholds in epiretinal prostheses.

PURPOSE: The goal was to evaluate how perceptual thresholds are related to electrode impedance, electrode size, the distance of electrodes from the retinal surface, and retinal thickness in six subjects blind as a result of retinitis pigmentosa, who received epiretinal prostheses implanted monocularly as part of a U.S. Food and Drug Administration (FDA)-approved clinical trial. METHODS: The implant consisted of an extraocular unit containing electronics for wireless data, power recovery, and generation of stimulus current, and an intraocular unit containing 16 platinum stimulating electrodes (260- or 520-microm diameter) arranged in a 4 x 4 pattern. The electrode array was held onto the retina by a small tack. Stimulation was controlled by a computer-based external system that allowed independent control over each electrode. Perceptual thresholds (the current necessary to see a percept on 79% of trials) and impedance were measured for each electrode on a biweekly basis. The distance of electrodes from the retinal surface and retinal thickness were measured by optical coherence tomography on a less regular basis. RESULTS: Stimulation thresholds for detecting phosphenes correlated with the distance of the electrodes from the retinal surface, but not with electrode size, electrode impedance, or retinal thickness. CONCLUSIONS: Maintaining close proximity between the electrode array and the retinal surface is critical in developing a successful retinal implant. With the development of chronic electrode arrays that are stable and flush on the retinal surface, it is likely that the influence of other factors such as electrode size, retinal degeneration, and subject age will become more apparent. (ClinicalTrials.gov number, NCT00279500.).