Quantitative patterns of visual impairment and recovery in children with brain injury.

Brain injury can cause many distinct types of visual impairment in children, but these deficits are difficult to quantify due to co-morbid deficits in communication and cognition. Clinicians must instead rely on low-resolution, subjective judgements of simple reactions to handheld stimuli, which limits treatment potential. We have developed an interactive assessment program called the Visual Ladder, which uses gaze-based responses to intuitive, game-like tasks to address the lack of broad-spectrum quantified data on the visual abilities of children with brain injury. Here, we present detailed metrics on eye movements, field asymmetries, contrast sensitivity, and other critical visual abilities measured longitudinally using the Ladder in hospitalized children with varying types and degrees of brain injury, many of whom were previously considered untestable. Our findings show which abilities are most likely to exhibit recovery and reveal how distinct patterns of task outcomes defined unique diagnostic clusters of visual impairment.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

Illusory optical defocus generated by shaded surface texture.

The human visual system is tasked with the problem of extracting information about the world from images that contain a conflated mixture of environmental sources and optical artifacts generated by the focal properties of our eyes. In most contexts, our brains manage to distinguish these sources, but this is not always the case. Recent work showed that shading gradients generated by smooth three-dimensional (3D) surfaces can elicit strong illusory percepts of optical defocus1,2 - the perception of illusory blur is only eliminated when the surface appears attached to self-occluding contours3, surface discontinuities1, or sharp specular reflections1,2, which all generate sharp ('high spatial frequency') image structure. This suggests that it should also be possible to eliminate the illusory blur elicited by shaded surfaces by altering the surface geometry to include small-scale surface relief, which would also generate high-frequency image structure. We report the surprising result here that this manipulation fails to eliminate the perception of blur; the fine texture fails to perceptually 'bind' to the low-frequency image structure when there is a sufficient gap between the spatial scales of the fine and coarse surface structure. These findings suggest that discontinuous 'gaps' in the spatial scale of textures are a segmentation cue the visual system uses to extract multiple causes of image structure.

Why Do Canadians Travel Abroad for Cosmetic Surgery? A Qualitative Analysis on Motivations for Cosmetic Surgery Tourism.

Background: Canadians are increasingly engaging in medial tourism. The purpose of this study was to review Canadians' experiences with travelling abroad for cosmetic surgery, including primary motivations for seeking care outside of Canada. Methods: A qualitative analysis was conducted using semistructured interviews following a pre-determined topic guide. People who had undergone cosmetic surgery outside of Canada were interviewed. The interviews were transcribed and coded to determine motivational themes. Patients were recruited until thematic saturation was achieved. Results: Thematic saturation was achieved after recruitment of 11 patients. The most common motivational themes identified in this study for seeking cosmetic surgery outside of Canada included cost, post-operative care provided, marketing/customer service, and word-of-mouth. Member checking and theory triangulation were validation techniques used to verify identified themes. Mexico was the most common location for cosmetic tourism. The most common procedures were breast augmentation, mastopexy, and abdominoplasty. Participants gathered pre- and post-operative information primarily through pamphlets and contact with surgeons' offices. Follow-up was only available for half of the participants in this study, and only 5 of the participants felt that they had received informed consent. Conclusions: The majority of participants engaged in cosmetic tourism due to cost reasons and the level of post-operative care provided.

Historique: Les Canadiens font de plus en plus de tourisme médical. La présente étude vise à analyser les expériences des Canadiens qui se rendent à l'étranger pour recevoir des soins de chirurgie esthétique, y compris leur motivation primaire à faire ce choix. Méthodologie: Les chercheurs ont effectué une analyse qualitative au moyen d'entrevues semi-structurées selon un guide de sujets préétablis auprès de personnes ayant subi des chirurgies esthétiques hors du Canada. Les entrevues ont été transcrites et codées pour en tirer les thèmes. Des patients ont été recrutés jusqu'à ce que tous les thèmes aient été abordés. Résultats: Onze patients ont été recrutés pour parvenir au point de saturation des thèmes. Les principales motivations pour obtenir une chirurgie esthétique hors du Canada incluaient les coûts, les soins postopératoires reçus, les services de marketing et à la clientèle et le bouche-à-oreille. Les chercheurs ont utilisé la vérification des membres et la triangulation des théories pour vérifier les thèmes établis. Le tourisme esthétique avait surtout lieu au Mexique. Les interventions les plus courantes étaient l'augmentation mammaire, la mastopexie et l'abdominoplastie. Les participants accumulaient l'information préopératoire et postopératoire d'abord à l'aide de dépliants et de contacts au bureau des chirurgiens. Seulement la moitié des participants à l'étude ont eu accès au suivi, et seulement cinq ont eu l'impression d'avoir donné leur consentement éclairé. Conclusions: La majorité des participants faisaient du tourisme esthétique pour une question d'argent et pour le taux de soins postopératoires fournis.

Tracking-Based Interactive Assessment of Saccades, Pursuits, Visual Field, and Contrast Sensitivity in Children With Brain Injury.

Visual deficits in children that result from brain injury, including cerebral/cortical visual impairment (CVI), are difficult to assess through conventional methods due to their frequent co-occurrence with cognitive and communicative disabilities. Such impairments hence often go undiagnosed or are only determined through subjective evaluations of gaze-based reactions to different forms, colors, and movements, which limits any potential for remediation. Here, we describe a novel approach to grading visual health based on eye movements and evidence from gaze-based tracking behaviors. Our approach-the "Visual Ladder"-reduces reliance on the user's ability to attend and communicate. The Visual Ladder produces metrics that quantify spontaneous saccades and pursuits, assess visual field responsiveness, and grade spatial visual function from tracking responses to moving stimuli. We used the Ladder to assess fourteen hospitalized children aged 3 to 18 years with a diverse range of visual impairments and causes of brain injury. Four children were excluded from analysis due to incompatibility with the eye tracker (e.g., due to severe strabismus). The remaining ten children-including five non-verbal children-were tested multiple times over periods ranging from 2 weeks to 9 months, and all produced interpretable outcomes on at least three of the five visual tasks. The results suggest that our assessment tasks are viable in non-communicative children, provided their eyes can be tracked, and hence are promising tools for use in a larger clinical study. We highlight and discuss informative outcomes exhibited by each child, including directional biases in eye movements, pathological nystagmus, visual field asymmetries, and contrast sensitivity deficits. Our findings indicate that these methodologies will enable the rapid, objective classification and grading of visual impairments in children with CVI, including non-verbal children who are currently precluded from most vision assessments. This would provide a much-needed differential diagnostic and prognostic tool for CVI and other impairments of the visual system, both ocular and cerebral.

audiomath: A neuroscientist's sound toolkit.

In neuroscientific experiments and applications, working with auditory stimuli demands software tools for generation and acquisition of raw audio, for composition and tailoring of that material into finished stimuli, for precisely timed presentation of the stimuli, and for experimental session recording. Numerous programming tools exist to approach these tasks, but their differing specializations and conventions demand extra time and effort for integration. In particular, verifying stimulus timing requires extensive engineering effort when developing new applications. This paper has two purposes. The first is to present audiomath (https://pypi.org/project/audiomath), a sound software library for Python that prioritizes the needs of neuroscientists. It minimizes programming effort by providing a simple object-oriented interface that unifies functionality for audio generation, manipulation, visualization, decoding, encoding, recording, and playback. It also incorporates specialized tools for measuring and optimizing stimulus timing. The second purpose is to relay what we have learned, during development and application of the software, about the twin challenges of delivering stimuli precisely at a certain time, and of precisely measuring the time at which stimuli were delivered. We provide a primer on these problems and the possible approaches to them. We then report audio latency measurements across a range of hardware, operating systems and settings, to illustrate the ways in which hardware and software factors interact to affect stimulus presentation performance, and the resulting pitfalls for the programmer and experimenter. In particular, we highlight the potential conflict between demands for low latency, low variability in latency ("jitter"), cooperativeness, and robustness. We report the ways in which audiomath can help to map this territory and provide a simplified path toward each application's particular priority. By unifying audio-related functionality and providing specialized diagnostic tools, audiomath both simplifies and potentiates the development of neuroscientific applications in Python.

BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression.

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608.

Gradiate: A radial sweep approach to measuring detailed contrast sensitivity functions from eye movements.

The contrast sensitivity function (CSF) is an informative measure of visual health, but the practical difficulty of measuring it has impeded detailed analyses of its relationship to different visual disorders. Furthermore, most existing tasks cannot be used in populations with cognitive impairment. We analyzed detailed CSFs measured with a nonverbal procedure called "Gradiate," which efficiently infers visibility from eye movements and manipulates stimulus appearance in real time. Sixty observers of varying age (38 with refractive error) were presented with moving stimuli. Stimulus spatial frequency and contrast advanced along 15 radial sweeps through CSF space in response to stimulus-congruent eye movements. A point on the CSF was recorded when tracking ceased. Gradiate CSFs were reliable and in high agreement with independent low-contrast acuity thresholds. Overall CSF variation was largely captured by two orthogonal factors ("radius" and "slope") or two orthogonal shape factors when size was normalized ("aspect ratio" and "curvature"). CSF radius was highly predictive of LogMAR acuity, as were aspect ratio and curvature together, but only radius was predictive of observer age. Our findings suggest that Gradiate holds promise for assessing spatial vision in both verbal and nonverbal populations and indicate that variation between detailed CSFs can reveal useful information about visual health.

Shady: A software engine for real-time visual stimulus manipulation.

BACKGROUND: Precise definition, rendering and manipulation of visual stimuli are essential in neuroscience. Rather than implementing these tasks from scratch, scientists benefit greatly from using reusable software routines from freely available toolboxes. Existing toolboxes work well when the operating system and hardware are painstakingly optimized, but may be less suited to applications that require multi-tasking (for example, closed-loop systems that involve real-time acquisition and processing of signals). NEW METHOD: We introduce a new cross-platform visual stimulus toolbox called Shady (https://pypi.org/project/Shady)-so called because of its heavy reliance on a shader program to perform parallel pixel processing on a computer's graphics processor. It was designed with an emphasis on performance robustness in multi-tasking applications under unforgiving conditions. For optimal timing performance, the CPU drawing management commands are carried out by a compiled binary engine. For configuring stimuli and controlling their changes over time, Shady provides a programmer's interface in Python, a powerful, accessible and widely-used high-level programming language. RESULTS: Our timing benchmark results illustrate that Shady's hybrid compiled/interpreted architecture requires less time to complete drawing operations, exhibits smaller variability in frame-to-frame timing, and hence drops fewer frames, than pure-Python solutions under matched conditions of resource contention. This performance gain comes despite an expansion of functionality (e.g. "noisy-bit" dithering as standard on all pixels and all frames, to enhance effective dynamic range) relative to previous offerings. CONCLUSIONS: Shady simultaneously advances the functionality and performance available to scientists for rendering visual stimuli and manipulating them in real time.

Photogeometric Cues to Perceived Surface Shading.

The human visual system is remarkably adept at extracting the three-dimensional (3D) shape of surfaces from images of smoothly shaded surfaces (shape from shading). Most research into this remarkable perceptual ability has focused on understanding how the visual system derives a specific representation of 3D shape when it is known (or assumed) that shading and self-occluding contours are the sole causes of image structure [1-11]. But there is an even more fundamental problem that must be solved before any such analysis can take place: how does the visual system determine when it's viewing a shaded surface? Here, we present theoretical analyses showing that there is statistically reliable information generated along the bounding contours of smoothly curved surfaces that the visual system uses to identify surface shading. This information can be captured by two photogeometric constraints that link the shape of bounding contours to the distributions of shading intensity along the contours: one that links shading intensity to the local orientations along bounding contours and a second that links shading intensity to bounding contour curvature. We show that these constraints predict the perception of shading for surfaces with smooth self-occluding contours and a widely studied class of bounding contours (planar cuts). The results provide new insights into the information that the visual system exploits to distinguish surface shading from other sources of image structure and offer a coherent explanation of the influence of bounding contours on the perception of surface shading and 3D shape.

Curveball: A tool for rapid measurement of contrast sensitivity based on smooth eye movements.

The contrast sensitivity function (CSF) is an informative measure of visual function, but current tools for assessing it are limited by the attentional, motor, and communicative abilities of the participant. Impairments in these abilities can prevent participants from engaging with tasks or following an experimenter's instructions. Here, we describe an efficient new tool for measuring contrast sensitivity, Curveball, and empirically validate it with a sample of healthy adults. The Curveball algorithm continuously infers stimulus visibility through smooth eye tracking instead of perceptual report, and rapidly lowers stimulus contrast in real time until a threshold is found. The procedure requires minimal instruction to administer and takes only five minutes to estimate a full CSF, which is comparable to the best existing methods available for healthy adults. Task repeatability was high: the coefficients of repeatability were 0.275 (in log10 units of RMS contrast) within the same session and 0.227 across different days. We also present evidence that the task is robust across illumination changes, well correlated with results from conventional psychophysical methods, and highly sensitive to improvements in visual acuity from refractive correction. Our findings indicate that Curveball is a promising means of accurately assessing contrast sensitivity in previously neglected populations.

Worldwide Esophageal Cancer Collaboration: pathologic staging data.

We report data-simple descriptions of patient characteristics, cancer categories, and non-risk-adjusted survival-for patients with pathologically staged cancer of the esophagus and esophagogastric junction after resection or ablation with no preoperative therapy from the Worldwide Esophageal Cancer Collaboration (WECC). Thirty-three institutions from six continents submitted de-identified data using standard definitions: demographics, comorbidities, clinical cancer categories, and all-cause mortality from first management decision. Of 13,300 patients, 5,631 had squamous cell carcinoma, 7,558 adenocarcinoma, 85 adenosquamous carcinoma, and 26 undifferentiated carcinoma. Patients were older (62 years) men (80%) with normal body mass index (51%), little weight loss (1.8 kg), 0-2 ECOG performance status (83%), and a history of smoking (70%). Cancers were pT1 (24%), pT2 (15%), pT3 (50%), pN0 (52%), pM0 (93%), and pG2-G3 (78%); most involved distal esophagus (71%). Non-risk-adjusted survival for both squamous cell carcinoma and adenocarcinoma was monotonic and distinctive across pTNM. Survival was more distinctive for adenocarcinoma than squamous cell carcinoma when pT was ordered by pN. Survival for pTis-1 adenocarcinoma was better than for squamous cell carcinoma, although monotonic and distinctive for both. WECC pathologic staging data is improved over that of the 7th edition, with more patients studied and patient and cancer variables collected. These data will be the basis for the 8th edition cancer staging manuals following risk adjustment for patient, cancer, and treatment characteristics, and should direct 9th edition data collection. However, the role of pure pathologic staging as the principal point of reference for esophageal cancer staging is waning.

Worldwide Esophageal Cancer Collaboration: clinical staging data.

To address uncertainty of whether clinical stage groupings (cTNM) for esophageal cancer share prognostic implications with pathologic groupings after esophagectomy alone (pTNM), we report data-simple descriptions of patient characteristics, cancer categories, and non-risk-adjusted survival-for clinically staged patients from the Worldwide Esophageal Cancer Collaboration (WECC). Thirty-three institutions from six continents submitted data using variables with standard definitions: demographics, comorbidities, clinical cancer categories, and all-cause mortality from first management decision. Of 22,123 clinically staged patients, 8,156 had squamous cell carcinoma, 13,814 adenocarcinoma, 116 adenosquamous carcinoma, and 37 undifferentiated carcinoma. Patients were older (62 years) men (80%) with normal body mass index (18.5-25 mg/kg2 , 47%), little weight loss (2.4 ± 7.8 kg), 0-1 ECOG performance status (67%), and history of smoking (67%). Cancers were cT1 (12%), cT2 (22%), cT3 (56%), cN0 (44%), cM0 (95%), and cG2-G3 (89%); most involved the distal esophagus (73%). Non-risk-adjusted survival for squamous cell carcinoma was not distinctive for early cT or cN; for adenocarcinoma, it was distinctive for early versus advanced cT and for cN0 versus cN+. Patients with early cancers had worse survival and those with advanced cancers better survival than expected from equivalent pathologic categories based on prior WECC pathologic data. Thus, clinical and pathologic categories do not share prognostic implications. This makes clinically based treatment decisions difficult and pre-treatment prognostication inaccurate. These data will be the basis for the 8th edition cancer staging manuals following risk adjustment for patient characteristics, cancer categories, and treatment characteristics and should direct 9th edition data collection.

Worldwide Esophageal Cancer Collaboration: neoadjuvant pathologic staging data.

To address uncertainty of whether pathologic stage groupings after neoadjuvant therapy (ypTNM) for esophageal cancer share prognostic implications with pathologic groupings after esophagectomy alone (pTNM), we report data-simple descriptions of patient characteristics, cancer categories, and non-risk-adjusted survival-for pathologically staged cancers after neoadjuvant therapy from the Worldwide Esophageal Cancer Collaboration (WECC). Thirty-three institutions from six continents submitted data using variables with standard definitions: demographics, comorbidities, clinical cancer categories, and all-cause mortality from first management decision. Of 7,773 pathologically staged neoadjuvant patients, 2,045 had squamous cell carcinoma, 5,686 adenocarcinoma, 31 adenosquamous carcinoma, and 11 undifferentiated carcinoma. Patients were older (61 years) men (83%) with normal (40%) or overweight (35%) body mass index, 0-1 Eastern Cooperative Oncology Group performance status (96%), and a history of smoking (69%). Cancers were ypT0 (20%), ypT1 (13%), ypT2 (18%), ypT3 (44%), ypN0 (55%), ypM0 (94%), and G2-G3 (72%); most involved the distal esophagus (80%). Non-risk-adjusted survival for yp categories was unequally depressed, more for earlier categories than later, compared with equivalent categories from prior WECC data for esophagectomy-alone patients. Thus, survival of patients with ypT0-2N0M0 cancers was intermediate and similar regardless of ypT; survival for ypN+ cancers was poor. Because prognoses for ypTNM and pTNM categories are dissimilar, prognostication should be based on separate ypTNM categories and groupings. These data will be the basis for the 8th edition cancer staging manuals following risk adjustment for patient, cancer, and treatment characteristics and should direct 9th edition data collection.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100-500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).

Specular image structure modulates the perception of three-dimensional shape.

Retinal images are produced by interactions between a surface's 3D shape, material properties, and surrounding light field. In order to recover the 3D geometry of a surface, the visual system must somehow separate aspects of image structure generated by a surface's shape from structure generated by its material properties or the light field in which it is embedded. Attributing image structure to the wrong physical source would cause the visual system to interpret changes in one physical property (such as reflectance) as changes in another (such as shape). Many previous studies have shown that the visual system does not conflate image structure generated by specular reflectance with 3D shape, but they did not assess the physical conditions where it would be computationally most difficult to disentangle these different sources of image structure. Here, we show that varying the specular roughness and curvature of surfaces embedded in natural light fields can strongly modulate perceived shape. Despite the complexity of these interactions, we show how an image's gradient structure mediates its interpretation as a specular reflection or a change in 3D shape. Our findings provide a coherent explanation of when and why specular reflections impact perceived shape and reveal how the static surface properties, simplified light fields, and experimental methods used in previous studies may explain their inconsistent results.

Long-term follow-up after gene therapy for canavan disease.

Canavan disease is a hereditary leukodystrophy caused by mutations in the aspartoacylase gene (ASPA), leading to loss of enzyme activity and increased concentrations of the substrate N-acetyl-aspartate (NAA) in the brain. Accumulation of NAA results in spongiform degeneration of white matter and severe impairment of psychomotor development. The goal of this prospective cohort study was to assess long-term safety and preliminary efficacy measures after gene therapy with an adeno-associated viral vector carrying the ASPA gene (AAV2-ASPA). Using noninvasive magnetic resonance imaging and standardized clinical rating scales, we observed Canavan disease in 28 patients, with a subset of 13 patients being treated with AAV2-ASPA. Each patient received 9 × 10(11) vector genomes via intraparenchymal delivery at six brain infusion sites. Safety data collected over a minimum 5-year follow-up period showed a lack of long-term adverse events related to the AAV2 vector. Posttreatment effects were analyzed using a generalized linear mixed model, which showed changes in predefined surrogate markers of disease progression and clinical assessment subscores. AAV2-ASPA gene therapy resulted in a decrease in elevated NAA in the brain and slowed progression of brain atrophy, with some improvement in seizure frequency and with stabilization of overall clinical status.

Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector.

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

Assessment of hippocampal adeno-associated viral vector gene delivery via frameless stereotaxis in a nonhuman primate.

BACKGROUND/AIMS: Expression of the neuropeptide galanin in hippocampal neurons reduces seizures in the kainic acid rodent model of epilepsy. In order to translate these findings into a human clinical trial, the safety and feasibility of hippocampal adeno-associated viral (AAV) vector expression must be demonstrated in a nonhuman primate model. METHODS: The Stealth Frameless Stereotactic System and Navigus Biopsy Appliance (Medtronic) were used to inject self-complementary AAV2 carrying the gene for green fluorescent protein (GFP) into monkey hippocampi. Using a single occipital trajectory per side (n = 8 trajectories), multiple injections spaced by 5 mm were delivered to each hippocampus. RESULTS: GFP was expressed in both neuronal and glial cells. Injections led to nonhomogeneous gene expression, suggesting closer spacing of injections may lead to more gene expression. Increasing injection volumes entailed a general increase in volume of expression, but there was no overlap of expression within the 5-mm injection interval. Efforts to avoid the occipital horn failed to prevent leaking of vector into the ventricle, and resulted in deviation of the trajectory at proximal points from the hippocampus. CONCLUSION: Using the occipital approach, adequate cannulation of the monkey hippocampus will require transventricular trajectories.

Adeno-associated virus for the treatment of muscle diseases: toward clinical trials.

Muscle diseases include muscular dystrophies, cardiomyopathies, neuromuscular and metabolic disorders. The loss of normal muscle structure and function is associated with significant morbidity and mortality. Patients with Duchenne muscular dystrophy usually lose ambulation in their teenage years, and frequently experience severe respiratory problems and heart failure in later stages of life. These unmet medical needs have encouraged the development of genetic strategies targeting the underlying muscle disease processes. Adeno-associated virus (AAV) vectors have been identified as promising gene delivery candidates because of their ability to transduce muscle tissue efficiently while transporting a genetic payload. There is currently significant momentum in the research of AAV-mediated delivery of muscle genes. Various AAV-based therapeutic strategies are undergoing preclinical and clinical testing, including the use of miniaturized and codon-optimized transgenes, exon skipping expression cassettes, novel tissue-specific promoters, AAV capsid mutants and chimeras, and localized intravascular administration procedures. These advancements in gene delivery have led to the generation of AAV vectors with targeted transgene expression, tissue-selective tropism and minimal off-target effects. This review describes advances in AAV gene therapy that are specific to the treatment of muscle diseases, and discusses the implications of their clinical application.