Chromatographic Purification and Polishing of AAV Particles.

The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.

In Vivo Imaging of Rodent Retina in Retinal Disease.

High-quality imaging of the retina is crucial to the diagnosis and monitoring of disease, as well as for evaluating the success of therapeutics in human patients and in preclinical animal models. Here, we describe the basic principles and methods for in vivo retinal imaging in rodents, including fundus imaging, fluorescein angiography, optical coherence tomography, fundus autofluorescence, and infrared imaging. After providing a concise overview of each method and detailing the retinal diseases and conditions that can be visualized through them, we will proceed to discuss the advantages and disadvantages of each approach.

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.

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.

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.

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.

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.

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.

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.

Targeting ON-bipolar cells by AAV gene therapy stably reverses -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 () 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.

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.

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.

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

Enhancers are -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.

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.

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.

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.

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.

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.

Viral-mediated RdCVF and RdCVFL expression protects cone and rod photoreceptors in retinal degeneration.

Alternative splicing of nucleoredoxin-like 1 (Nxnl1) results in 2 isoforms of the rod-derived cone viability factor. The truncated form (RdCVF) is a thioredoxin-like protein secreted by rods that promotes cone survival, while the full-length isoform (RdCVFL), which contains a thioredoxin fold, is involved in oxidative signaling and protection against hyperoxia. Here, we evaluated the effects of these different isoforms in 2 murine models of rod-cone dystrophy.

The expression pattern of systemically injected AAV9 in the developing mouse retina is determined by age.

Systemic delivery of AAV9 offers the potential for widespread and efficient gene delivery to the retina, and may thus be a useful approach for treatment of disease where intraocular injections are not possible, for syndromes affecting multiple organs, or where early intervention is required. The expression resulting from intravenous injection of AAV9 is more efficient in neonates than adults, and here we characterize the effect of age on retinal transduction of AAV9 in the mouse retina. We find that the pattern of expression in neonatal mice is correlated to the development of the retinal vasculature, and that the area of the retinal transduction as well as the cell types infected vary depending on the age at injection.