PRPF31-retinitis pigmentosa: Challenges and opportunities for clinical translation.

Mutations in pre-mRNA processing factor 31 cause autosomal dominant retinitis pigmentosa (PRPF31-RP), for which there is currently no efficient treatment, making this disease a prime target for the development of novel therapeutic strategies. PRPF31-RP exhibits incomplete penetrance due to haploinsufficiency, in which reduced levels of gene expression from the mutated allele result in disease. A variety of model systems have been used in the investigation of disease etiology and therapy development. In this review, we discuss recent advances in both in vivo and in vitro model systems, evaluating their advantages and limitations in the context of therapy development for PRPF31-RP. Additionally, we describe the latest approaches for treatment, including AAV-mediated gene augmentation, genome editing, and late-stage therapies such as optogenetics, cell transplantation, and retinal prostheses.

Adaptable three-pin skull clamp for large animal research.

Traditionally, surgical head immobilization for neurobiological research with large animals is achieved using stereotaxic frames. Despite their widespread use, these frames are bulky, expensive, and inflexible, ultimately limiting surgical access and preventing research groups from practicing surgical approaches used to treat humans. Here, we designed a mobile, low-cost, three-pin skull clamp for performing a variety of neurosurgical procedures on non-human primates. Modeled after skull clamps used to operate on humans, our system was designed with added adjustability to secure heads with small or irregular geometries for innovative surgical approaches. The system has six degrees of freedom with skull pins attached to setscrews for independent, fine-tuned depth adjustment. Unlike other conventional skull clamps which require additional mounting fixtures, our system has an integrated tray with mounting bracket for easy use on most operating room tables. Our system has successfully secured primate heads in the supine and lateral position, allowing surgeons to match surgical approaches currently practiced when operating on humans. The system also expands the opportunity for researchers to utilize imaged-guided robotic surgery techniques. Overall, we hope that our system can serve as an adaptable, affordable, and robust surgery platform for any laboratory performing neurobiological research with large animal models.

Developing New Vectors for Retinal Gene Therapy.

Since their discovery over 55 years ago, adeno-associated virus (AAV) vectors have become powerful tools for experimental and therapeutic in vivo gene delivery, particularly in the retina. Increasing knowledge of AAV structure and biology has propelled forward the development of engineered AAV vectors with improved abilities for gene delivery. However, major obstacles to safe and efficient therapeutic gene delivery remain, including tropism, inefficient and untargeted gene delivery, and limited carrying capacity. Additional improvements to AAV vectors will be required to achieve therapeutic benefit while avoiding safety issues. In this review, we provide an overview of recent methods for engineering-enhanced AAV capsids, as well as remaining challenges that must be overcome to achieve optimized therapeutic gene delivery in the eye.

Gene augmentation prevents retinal degeneration in a CRISPR/Cas9-based mouse model of PRPF31 retinitis pigmentosa.

Mutations in PRPF31 cause autosomal dominant retinitis pigmentosa, an untreatable form of blindness. Gene therapy is a promising treatment for PRPF31-retinitis pigmentosa, however, there are currently no suitable animal models in which to develop AAV-mediated gene augmentation. Here we establish Prpf31 mutant mouse models using AAV-mediated CRISPR/Cas9 knockout, and characterize the resulting retinal degeneration phenotype. Mouse models with early-onset morphological and functional impairments like those in patients were established, providing new platforms in which to investigate pathogenetic mechanisms and develop therapeutic methods. AAV-mediated PRPF31 gene augmentation restored the retinal structure and function in a rapidly degenerating mouse model, demonstrating the first in vivo proof-of-concept for AAV-mediated gene therapy to treat PRPF31-retinitis pigmentosa. AAV-CRISPR/Cas9-PRPF31 knockout constructs also mediated efficient PRPF31 knockout in human and non-human primate retinal explants, laying a foundation for establishing non-human primate models using the method developed here.

Modeling PRPF31 retinitis pigmentosa using retinal pigment epithelium and organoids combined with gene augmentation rescue.

Mutations in the ubiquitously expressed pre-mRNA processing factor (PRPF) 31 gene, one of the most common causes of dominant form of Retinitis Pigmentosa (RP), lead to a retina-specific phenotype. It is uncertain which retinal cell types are affected and animal models do not clearly present the RP phenotype observed in PRPF31 patients. Retinal organoids and retinal pigment epithelial (RPE) cells derived from human-induced pluripotent stem cells (iPSCs) provide potential opportunities for studying human PRPF31-related RP. We demonstrate here that RPE cells carrying PRPF31 mutations present important morphological and functional changes and that PRPF31-mutated retinal organoids recapitulate the human RP phenotype, with a rod photoreceptor cell death followed by a loss of cones. The low level of PRPF31 expression may explain the defective phenotypes of PRPF31-mutated RPE and photoreceptor cells, which were not observed in cells derived from asymptomatic patients or after correction of the pathogenic mutation by CRISPR/Cas9. Transcriptome profiles revealed differentially expressed and mis-spliced genes belonging to pathways in line with the observed defective phenotypes. The rescue of RPE and photoreceptor defective phenotypes by PRPF31 gene augmentation provide the proof of concept for future therapeutic strategies.

Machine learning sequence prioritization for cell type-specific enhancer design.

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations within heterogeneous tissue. Available approaches for engineering-targeted technologies for new neuron subtypes are low yield, involving intensive transgenic strain or virus screening. Here, we present Specific Nuclear-Anchored Independent Labeling (SNAIL), an improved virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and then make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV+) neurons. Nuclear isolation using SNAIL in wild-type mice is sufficient to capture characteristic open chromatin features of PV+ neurons in the cortex, striatum, and external globus pallidus. The SNAIL framework also has high utility for multispecies cell probe engineering; expression from a mouse PV+ SNAIL enhancer sequence was enriched in PV+ neurons of the macaque cortex. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

Quantitative single-cell transcriptome-based ranking of engineered AAVs in human retinal explants.

Gene therapy is a rapidly developing field, and adeno-associated viruses (AAVs) are a leading viral-vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wild-type and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA sequencing (RNA-seq) AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers except for a photoreceptor population with altered expression of photoreceptor-specific genes. The efficiency and tropism of AAVs in human explants were quantified with single-cell resolution. The top-performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.

Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness.

SignificanceCanine models of inherited retinal diseases have helped advance adeno-associated virus (AAV)-based gene therapies targeting specific cells in the outer retina for treating blinding diseases in patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the midretina remains underdeveloped. Using a leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) mutant canine model of CSNB exhibiting ON-BC dysfunction, we tested the ability of cell-specific AAV capsids and promotors to specifically target ON-BCs for gene delivery. Subretinal injection of one vector demonstrated safety and efficacy with robust and stable rescue of electroretinography signals and night vision up to 1 y, paving the way for clinical trials in patients.

Transcriptional and anatomical diversity of medium spiny neurons in the primate striatum.

Medium spiny neurons (MSNs) constitute the vast majority of striatal neurons and the principal interface between dopamine reward signals and functionally diverse cortico-basal ganglia circuits. Information processing in these circuits is dependent on distinct MSN types: cell types that are traditionally defined according to their projection targets or dopamine receptor expression. Single-cell transcriptional studies have revealed greater MSN heterogeneity than predicted by traditional circuit models, but the transcriptional landscape in the primate striatum remains unknown. Here, we set out to establish molecular definitions for MSN subtypes in Rhesus monkeys and to explore the relationships between transcriptionally defined subtypes and anatomical subdivisions of the striatum. Our results suggest at least nine MSN subtypes, including dorsal striatum subtypes associated with striosome and matrix compartments, ventral striatum subtypes associated with the nucleus accumbens shell and olfactory tubercle, and an MSN-like cell type restricted to µ-opioid receptor rich islands in the ventral striatum. Although each subtype was demarcated by discontinuities in gene expression, continuous variation within subtypes defined gradients corresponding to anatomical locations and, potentially, functional specializations. These results lay the foundation for achieving cell-type-specific transgenesis in the primate striatum and provide a blueprint for investigating circuit-specific information processing.

scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution.

Background: Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Methods: Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to simultaneously quantify and rank efficiency of competing AAV vectors across all cell types in the same animal. Results: To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant identified using this pipeline, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. scAAVengr was then used to identify top-performing AAV variants in mouse brain, heart, and liver following systemic injection. Conclusions: These results validate scAAVengr as a powerful method for development of AAV vectors. Funding: This work was supported by funding from the Ford Foundation, NEI/NIH, Research to Prevent Blindness, Foundation Fighting Blindness, UPMC Immune Transplant and Therapy Center, and the Van Sloun fund for canine genetic research.

Gene therapy is an experimental approach to treating disease that involves altering faulty genes or replacing them with new, working copies. Most often, the new genetic material is delivered into cells using a modified virus that no longer causes disease, called a viral vector. Virus-mediated gene therapies are currently being explored for degenerative eye diseases, such as retinitis pigmentosa, and neurological disorders, like Alzheimer's and Parkinson's disease. A number of gene therapies have also been approved for treating some rare cancers, blood disorders and a childhood form of motor neuron disease. Despite the promise of virus-mediated gene therapy, there are significant hurdles to its widespread success. Viral vectors need to deliver enough genetic material to the right cells without triggering an immune response or causing serious side effects. Selecting an optimal vector is key to achieving this. A type of viruses called adeno-associated viruses (AAV) are prime candidates, partly because they can be easily engineered. However, accurately comparing the safety and efficacy of newly engineered AAVs is difficult, due to variation between test subjects and the labor and cost involved in careful testing. Öztürk et al. addressed this issue by developing an experimental pipeline called scAAVengr for comparing gene therapy vectors head-to-head. The process involves tagging potential AAV vectors with unique genetic barcodes, which can then be detected and quantified in individual cells using a technique called single-cell RNA sequencing. This means that when several vectors are used to infect lab-grown cells or a test animal at the same time, they can be tracked. The vectors can then be ranked on their ability to infect specific cell types and deliver useful genetic material. Using scAAVengr, Öztürk et al. compared viral vectors designed to target the light-sensitive cells of the retina, which allow animals to see. First, a set of promising viral vectors were evaluated using the scAAVengr pipeline in the eyes of marmosets and macaques, two small primates. Precise levels and locations of gene delivery were quantified. The top-performing vector was then identified and used to deliver Cas9, a genome editing tool, to primate retinas. Öztürk et al. also used scAAVengr to compare viral vectors in mice, analysing the vectors' ability to deliver their genetic cargo to the brain, heart, and liver. These experiments demonstrated that scAAVengr can be used to evaluate vectors in multiple tissues and in different organisms. In summary, this work outlines a method for identifying and precisely quantifying the performance of top-performing viral vectors for gene therapy. By aiding the selection of optimal viral vectors, the scAAVengr pipeline could help to improve the success of preclinical studies and early clinical trials testing gene therapies.

Parallel functional testing identifies enhancers active in early postnatal mouse brain.

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type-specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

In vivo-directed evolution of adeno-associated virus in the primate retina.

Efficient adeno-associated virus-mediated (AAV-mediated) gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution - guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library - to create AAV viral capsids with the capability to deliver genes to the outer retina in primates. A replication-incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates - involving intravitreal library administration, recovery of genomes from outer retina, and extensive next-generation sequencing of each round - resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These viral vectors expand the toolbox of vectors available for primate retina, and they may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

Screening for Neutralizing Antibodies Against Natural and Engineered AAV Capsids in Nonhuman Primate Retinas.

Adeno-associated virus (AAV) has shown promise as a therapeutic gene delivery vector for inherited retinal degenerations in both preclinical disease models and human clinical trials. The retinas of nonhuman primates (NHPs) share many anatomical similarities to humans and are an important model for evaluating AAV gene delivery. Recent evidence has shown that preexisting immunity in the form of neutralizing antibodies (NABs) in NHPs strongly correlates with weak or lack of AAV transduction in the retina when administered intravitreally, work with translational implications. This necessitates prescreening of NHPs before intravitreal delivery of AAV. In this chapter, we describe a method for screening NHP serum for preexisting NABs.

In vivo genome editing improves motor function and extends survival in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord and brain. In particular, autosomal dominant mutations in the superoxide dismutase 1 (SOD1) gene are responsible for ~20% of all familial ALS cases. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas9) genome editing system holds the potential to treat autosomal dominant disorders by facilitating the introduction of frameshift-induced mutations that can disable mutant gene function. We demonstrate that CRISPR-Cas9 can be harnessed to disrupt mutant SOD1 expression in the G93A-SOD1 mouse model of ALS following in vivo delivery using an adeno-associated virus vector. Genome editing reduced mutant SOD1 protein by >2.5-fold in the lumbar and thoracic spinal cord, resulting in improved motor function and reduced muscle atrophy. Crucially, ALS mice treated by CRISPR-mediated genome editing had ~50% more motor neurons at end stage and displayed a ~37% delay in disease onset and a ~25% increase in survival compared to control animals. Thus, this study illustrates the potential for CRISPR-Cas9 to treat SOD1-linked forms of ALS and other central nervous system disorders caused by autosomal dominant mutations.

Anti-VEGF AAV2 injections: The fewer the better.

A single intravitreal injection of AAV2 provides sustained delivery of anti-VEGF protein for the treatment of neovascular AMD.

What's old is new again: Autologous stem cell transplant for AMD.

Transplanted RPE cells derived from induced pluripotent stem cells maintained vision and were well tolerated in a patient with age-related macular degeneration.

Massively parallel cis-regulatory analysis in the mammalian central nervous system.

Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell-derived organoids.

Rod-derived cone viability factor promotes cone survival by stimulating aerobic glycolysis.

Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.

CRALBP supports the mammalian retinal visual cycle and cone vision.

Mutations in the cellular retinaldehyde-binding protein (CRALBP, encoded by RLBP1) can lead to severe cone photoreceptor-mediated vision loss in patients. It is not known how CRALBP supports cone function or how altered CRALBP leads to cone dysfunction. Here, we determined that deletion of Rlbp1 in mice impairs the retinal visual cycle. Mice lacking CRALBP exhibited M-opsin mislocalization, M-cone loss, and impaired cone-driven visual behavior and light responses. Additionally, M-cone dark adaptation was largely suppressed in CRALBP-deficient animals. While rearing CRALBP-deficient mice in the dark prevented the deterioration of cone function, it did not rescue cone dark adaptation. Adeno-associated virus-mediated restoration of CRALBP expression specifically in Müller cells, but not retinal pigment epithelial (RPE) cells, rescued the retinal visual cycle and M-cone sensitivity in knockout mice. Our results identify Müller cell CRALBP as a key component of the retinal visual cycle and demonstrate that this pathway is important for maintaining normal cone-driven vision and accelerating cone dark adaptation.