Retinal Explant Culture from Mouse, Human, and Nonhuman Primates and Its Applications in Vision Research.

The retinal explant culture system is a valuable tool for studying the pharmacological, toxicological, and developmental aspects of the retina. It is also used for translational studies such as gene therapy. While no photoreceptor-like cell lines are available for in vitro studies of photoreceptor cell biology, the retinal explant culture maintains the laminated retinal structure ex vivo for as long as a month. Human and nonhuman primate (NHP) postmortem retinal explants cut into small pieces offer the possibility of testing multiple conditions for safety and adeno-associated viral (AAV) vector optimization. In addition, the cone-enriched foveal area can be studied using the retinal explants. Here, we present a detailed working protocol for retinal explant isolation and culture from mouse, human, and NHP for testing drug efficacy and AAV transduction. Future applications of this protocol include combining live imaging and multiwell retinal explant culture for high-throughput drug screening systems in rodent and human retinal explants to identify new drugs against retinal degeneration.

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.

Pre-clinical development of AP4B1 gene replacement therapy for hereditary spastic paraplegia type 47.

Spastic paraplegia 47 (SPG47) is a neurological disorder caused by mutations in the adaptor protein complex 4 ß1 subunit (AP4B1) gene leading to AP-4 complex deficiency. SPG47 is characterised by progressive spastic paraplegia, global developmental delay, intellectual disability and epilepsy. Gene therapy aimed at restoring functional AP4B1 protein levels is a rational therapeutic strategy to ameliorate the disease phenotype. Here we report that a single delivery of adeno-associated virus serotype 9 expressing hAP4B1 (AAV9/hAP4B1) into the cisterna magna leads to widespread gene transfer and restoration of various hallmarks of disease, including AP-4 cargo (ATG9A) mislocalisation, calbindin-positive spheroids in the deep cerebellar nuclei, anatomical brain defects and motor dysfunction, in an SPG47 mouse model. Furthermore, AAV9/hAP4B1-based gene therapy demonstrated a restoration of plasma neurofilament light (NfL) levels of treated mice. Encouraged by these preclinical proof-of-concept data, we conducted IND-enabling studies, including immunogenicity and GLP non-human primate (NHP) toxicology studies. Importantly, NHP safety and biodistribution study revealed no significant adverse events associated with the therapeutic intervention. These findings provide evidence of both therapeutic efficacy and safety, establishing a robust basis for the pursuit of an IND application for clinical trials targeting SPG47 patients.

Preventing and treating neurotrophic keratopathy by a single intrastromal injection of AAV-mediated gene therapy.

PURPOSE: Neurotrophic keratopathy (NK) is a degenerative corneal condition resulting from corneal nerve injury. Current therapies, including the recombinant human nerve growth factor (rhNGF) therapy, requires continuous administration. This study aims to develop a novel and highly effective gene therapy strategy for the prevention and treatment of NK. METHODS: Adeno-associated virus (AAV) was transduced into corneal stromal cells by intrastromal injection. Three dimensional corneal wholemount imaging with co-immunostaining of ZO-1 and tubulin was utilized to assess the transduction of AAV.rh10. The efficacy of prevention and treatment of NK by a single intrastromal injection of AAV-Ngf was tested using capsaicin mouse model, herpes simplex keratitis (HSK) model, type Ⅱ diabetes model and alkali burn model. rhNGF eye drops served as the positive control. RESULTS: Intrastromal injection of AAV.rh10 efficiently transduced the subepithelial nerve plexus and retrogradely transported to the trigeminal ganglion (TG). A single injection of AAV.rh10-Ngf can significantly promote corneal nerve repair, accelerate corneal epithelial repair, reduce corneal stromal edema, and improve corneal sensitivity across the four NK models. The therapeutic effects were consistent with those achieved by continuous administration of rhNGF drops by 6 times daily. CONCLUSIONS: This proof-of-concept study demonstrates that AAV.rh10-Ngf gene therapy is a promising method for preventing and treating of NK. Our results underline the potential for developing clinical trials to further explore the safety and efficacy of such gene therapy.

A Novel and Simplified Anion Exchange Flow-Through Polishing Approach for the Separation of Full From Empty Adeno-Associated Virus Capsids.

Adeno-associated viruses (AAV) are widely used viral vectors for in vivo gene therapy. The purification of AAV, particularly the separation of genome-containing from empty AAV capsids, is usually time-consuming and requires expensive equipment. In this study, we present a novel laboratory scale anion exchange flow-through polishing method designed to separate full and empty AAV. Once the appropriate conditions are defined, this method eliminates the need for a chromatography system. Determination of optimal polishing conditions using a chromatography system revealed that the divalent salt MgCl2 resulted in better separation of full and empty AAV than the monovalent salt NaCl. The efficacy of the method was demonstrated for three distinct AAV serotypes (AAV8, AAV5, and AAV2) on two different stationary phases: a membrane adsorber and a monolith, resulting in a 4- to 7.5-fold enrichment of full AAV particles. Moreover, the method was shown to preserve the AAV capsids' functional potency and structural integrity. Following the successful establishment of the flow-through polishing approach, it was adapted to a manual syringe-based system. Manual flow-through polishing using the monolith or membrane adsorber achieved 3.6- and 5.4-fold enrichment of full AAV, respectively. This study demonstrates the feasibility of separating full and empty AAV without complex linear or step gradient elution and the necessity of specialized equipment. Flow-through polishing provides a rapid and easy-to-perform platform for polishing multiple vector preparations, addressing a critical aspect in the research and development of novel gene therapies.

Schlemm's canal-selective Tie2/TEK knockdown induces sustained ocular hypertension in adult mice.

Deficient Angiopoietin-Tie2 signaling is linked to ocular hypertension in glaucoma. Receptor Tie2/TEK expression and signaling at Schlemm's canal (SC) is indispensable for canal integrity and homeostatic regulation of aqueous humor outflow (AHO) and intraocular pressure (IOP), as validated by conditional deletion of Tie2, its ligands (Angpt1, Angpt2 and Angpt3/4) or regulators (Tie1 and PTPRB/VE-PTP). However, these Tie2/TEK knockouts and conditional knockouts are global or endothelial, preventing separation of systemic and ocular vascular defects that impact retinal or renal integrity. To develop a more targeted model of ocular hypertension induced by selective knockdown of Tie2/TEK expressed in SC, we combined the use of viral vectors to target the canal, and two distinct gene-editing strategies to disrupt the Tie2 gene. Adeno-associated virus (AAV2) is known to transduce rodent SC when delivered into the anterior chamber by intracameral injection. First, delivery of Cre recombinase via AAV2.Cre into R26tdTomato/+ reporter mice confirmed preferential and stable transduction in SC endothelium. Next, to disrupt Tie2 expression in SC, we injected AAV2.Cre into homozygous floxed Tie2 (Tie2FL/FL) mice. This led to attenuated Tie2 protein expression along the SC inner wall, decreased SC area and reduced trabecular meshwork (TM) cellularity. Functionally, IOP was significantly and steadily elevated, whereas AHO facility was reduced. In contrast, hemizygous Tie2FL/+ mice responded to AAV2.Cre with inconsistent and low IOP elevation, corroborating the dose-dependency of ocular hypertension on Tie2 expression/activation. In a second model using CRISPR/SaCas9 genome editing, wild-type C57BL/6 J mice injected with AAV2.saCas9-sgTie2 showed similar selective SC transduction and comparable IOP elevation in course and magnitude to that induced by AAV2.Cre in Tie2FL/FL mice. Together, our findings, demonstrate that selective Tie2 knockdown in SC is a targeted strategy that reliably induces chronic ocular hypertension and reproduces glaucomatous damage to the conventional outflow pathway, providing novel models of SC-Tie2 signaling loss valuable for preclinical studies.

Generation, expansion, gene delivery, and single-cell profiling in rhesus macaque plasma B cells.

A key step in developing engineered B cells for therapeutic purposes is evaluation in immunocompetent, large-animal models. Therefore, we developed methods to purify, expand, and differentiate non-human primate (NHP; rhesus macaque) B cells. After 7 days in culture, B cells expanded 10-fold, differentiated into a plasma cell phenotype (CD38, CD138), and secreted immunoglobulin G. Using single-cell sequencing and flow cytometry, we verified the presence of plasma cell genes in differentiated NHP B cells and unearthed less-recognized markers, such as CD59 and CD79A. In contrast with human cells, we found that the immune checkpoint molecule CD274 (PD-L1) and major histocompatibility complex (MHC) class I molecules were upregulated in NHP plasma cells in the transcriptional data. Lastly, we established the conditions for efficient transduction of NHP B cells with adeno-associated virus (AAV) vectors, achieving a delivery rate of approximately 60%. We envision that this work will accelerate proof-of-concept studies using engineered B cells in NHPs.

Gene therapy for fat-1 prevents obesity-induced metabolic dysfunction, cellular senescence, and osteoarthritis.

Obesity is one of the primary risk factors for osteoarthritis (OA), acting through cross talk among altered biomechanics, metabolism, adipokines, and dietary free fatty acid (FA) composition. Obesity and aging have been linked to cellular senescence in various tissues, resulting in increased local and systemic inflammation and immune dysfunction. We hypothesized that obesity and joint injury lead to cellular senescence that is typically associated with increased OA severity or with aging and that the ratio of omega-6 (ω-6) to omega-3 (ω-3) FAs regulates these pathologic effects. Mice were placed on an ω-6-rich high-fat diet or a lean control diet and underwent destabilization of the medial meniscus to induce OA. Obesity and joint injury significantly increased cellular senescence in subcutaneous and visceral fat as well as joint tissues such as synovium and cartilage. Using adeno-associated virus (AAV) gene therapy for fat-1, a fatty acid desaturase that converts ω-6 to ω-3 FAs, decreasing the serum ω-6:ω-3 FA ratio had a strong senomorphic and therapeutic effect, mitigating metabolic dysfunction, cellular senescence, and joint degeneration. In vitro coculture of bone marrow-derived macrophages and chondrocytes from control and AAV8-fat1-treated mice were used to examine the roles of various FA mediators in regulating chondrocyte senescence. Our results suggest that obesity and joint injury result in a premature "aging" of the joint as measured by senescence markers, and these changes can be ameliorated by altering FA composition using fat-1 gene therapy. These findings support the potential for fat-1 gene therapy to treat obesity- and/or injury-induced OA clinically.

Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system.

Adeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene transfer vector. Here, we describe a strategy to deliver large genes that exceed the AAV's packaging capacity using up to four AAV vectors and the CRE-lox DNA recombination system. We devised novel lox sites by combining non-compatible and reaction equilibrium-modifying lox site variants. These lox sites facilitate sequence-specific and near-unidirectional recombination of AAV vector genomes, enabling efficient reconstitution of up to 16 kb of therapeutic genes in a pre-determined configuration. Using this strategy, we have developed AAV gene therapy vectors to deliver IFT140, PCDH15, CEP290, and CDH23 and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, AAV-IFT140 gene therapy vectors ameliorated retinal degeneration and preserved visual functions in an IFT140-associated retinitis pigmentosa mouse model. The CRE-lox approach described here provides a simple, flexible, and effective platform for generating AAV gene therapy vectors beyond AAV's packaging capacity.

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 × 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

An improved high-resolution method for quantitative separation of empty and filled AAV8 capsids by strong anion exchange HPLC.

Cell and gene therapy (CGT) is a field of therapeutic medicine that aims to treat, prevent, and cure diseases using engineered cells (stem cells, immune cells, and differentiated adult or fetal cells), vectors [Adeno Associated Virus (AAV), Adeno Virus (AV), Herpes Simplex Virus (HSV), Baculo Virus (BV), Lenti Virus (LV), Retro Virus (RV), etc.], and other carriers [non-viral vectors, virus-like particles (VLP), Lipid Nano-Particles (LNP), etc.]. Among viral CGT vectors, adeno-associated viruses and lentiviruses (AAV and LV) are the most widely applied vector platforms. The presence of non-functional (empty or non-infectious) vectors that carry null or partial genes in the final drug product is classified as an impurity by the FDA. These impurities impair dosage accuracy and induce non-specific immunogenicity and variability in drug efficacy. These non-functional viral vectors in the drug product need to be elucidated following International Conference on Harmonization (ICH) guidelines for clinical manufacturing of the final drug product. This article showcases an ion-exchange chromatography (IEX) high-resolution method supporting ICH guidelines using commercially available AAV8 filled and empty capsids as reference standards. Our method successfully separated empty to full capsids with a resolution of 15 and sustained a linearity greater than 0.98 even under a wide range of empty or full viral particle concentrations (E+9 to E+13 vp/mL), which is an upgrade to other IEX capsid separation methods. The medium-throughput capacity and shorter sample processing time improve testing efficiency and save costs while delivering quality as value. The discussed method is a reliable and reproducible platform to precisely evaluate the presence of non-functional viral particles in AAV8 samples. Aligned with other orthogonal results, the method is a powerful tool to improve the quality of rAAV analytics.

VEXAS syndrome as a mimicker of ANCA-associated vasculitis.

Objectives: Differentiating VEXAS syndrome from cases of canonical forms of primary vasculitis remains a significant clinical challenge, particularly for ANCA-associated vasculitis (AAV). We reviewed the clinical features of VEXAS as an AAV mimicker, while adding three new cases to the existing literature. Methods: We identified three cases of VEXAS with an AAV phenotype in our institution. We performed a comprehensive literature search of available similar cases and summarized and compared the findings. Inclusion criterion was a positive UBA1 mutation analysis. Results: Patient 1 was referred for evaluation of eosinophilic granulomatosis with polyangiitis (GPA), but had no active respiratory symptoms, despite CT imaging showing widespread ground-glass opacities. Patient 2 had no history of sinus disease, despite being referred under the diagnostic construct of limited GPA. Patient 3 developed a novel inflammatory syndrome suspected to represent GPA. Six other cases were identified upon literature review. In all the cases, the most common findings were pulmonary infiltrates (67%), skin involvement (55%) and ocular manifestations (44%). Additionally, 44% of cases had renal involvement, with half of them displaying kidney lesions resembling the typical AAV pattern. Conclusion: VEXAS can mimic different phenotypes of AAV and should be considered in atypical AAV presentations, especially when refractory to multiple treatments. Further studies are needed to explore the immunologic basis for an AAV phenotype within the spectrum of VEXAS.

Critically Ill Patients with Newly Diagnosed Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis: Case Series and Literature Review.

ANCA-associated vasculitides (AAVs) are rare diseases with a prevalence of less than 200 cases per million persons and an incidence of less than 25 cases per million person-years. Their presenting features can vary from prodromal and nonspecific symptoms to dramatic organ-specific symptoms such as respiratory failure due to diffuse alveolar hemorrhage (DAH) and acute kidney injury (AKI). The latter two are hallmark features of pulmonary-renal syndrome, a potentially fatal condition that necessitates early recognition and treatment in intensive care units (ICUs) and rapid induction of immunosuppressive therapy. Background and case summaries: We described three patients with newly diagnosed AAV during the treatment of critical illness. All patients had DAH and two had AKI. The initial disease severity was extremely high in patients with myeloperoxidase (MPO)-AAV, reaching Sequential Organ Failure Assessment (SOFA) scores of 15 and 14 with predicted mortality ≥ 95.2%. Both patients needed mechanical ventilation, one additional venovenous extracorporeal membrane oxygenation (VV-ECMO), and renal replacement therapy. The patient with proteinase 3 (PR3)-AAV had a less severe disease, SOFA 3, requiring only modest oxygen supplementation and exhibiting only hematuria with normal renal function parameters. Immunosuppressive therapy was initiated during the ICU stay. The patient with the most severe clinical presentation died during the ICU stay because of sepsis, and the other two patients were discharged home. Conclusions: Patients with AAV presenting with pulmonary-renal syndrome necessitate various degrees of organ support. Nevertheless, these patients can be successfully treated in the early, critical stages of the disease and achieve remission.

In Silico Structural Prediction for the Generation of Novel Performant Midi-Dystrophins Based on Intein-Mediated Dual AAV Approach.

Duchenne Muscular Dystrophy (DMD) is a pediatric disorder characterized by progressive muscle degeneration and premature death, and has no current cure. The current, most promising therapeutic avenue is based on gene replacement mediated by adeno-associated viruses (AAVs) using a shortened, but still functional, version of dystrophin, known as micro-dystrophin (µDys), to fit AAV capacity. The limited improvements observed in clinical trials suggest a sub-optimal performance of µDys in the human context that could be due to the lack of key domains in the protein. Therefore, expressing larger dystrophin proteins may be necessary for a more complete correction of the disease phenotype. In this study, we developed three novel midi-dystrophin constructs using a dual-AAV approach, leveraging split-intein-based protein trans-splicing. The midi-dystrophins include additional domains compared to µDys, such as the central cytoskeleton-binding domain, nNOS and Par1b interacting domains, and a complete C-terminal region. Given the limited capacity of each AAV vector, we strategically partially reduced hinge regions while ensuring that the structural stability of the protein remains intact. We predicted the interactions between the two halves of the split midi-Dys proteins thanks to the deep learning algorithm AphaFold3. We observed strong associations between the N- and C-termini in midi-Dys 1 and 2, while a weaker interaction in midi-Dys 3 was revealed. Our subsequent experiments confirmed the efficient protein trans-splicing both in vitro and in vivo in DBA2/mdx mice of the midi-Dys 1 and 2 and not in midi-Dys 3 as expected from the structural prediction. Additionally, we demonstrated that midi-Dys 1 and 2 exhibit significant therapeutic efficacy in DBA2/mdx mice, highlighting their potential as therapeutic agents for DMD. Overall, these findings highlight the potential of deep learning-based structural modeling for the generation of intein-based dystrophin versions and pose the basis for further investigation of these new midi-dystrophins versions for clinical studies.

AAV-mouse DNase I sustains long-term DNase I expression in vivo and suppresses breast cancer metastasis.

Neutrophil extracellular traps (NETs) have been implicated in the pathology of various inflammatory conditions. In cancer, NETs have been demonstrated to induce systemic inflammation, impair peripheral vessel and organ function and promote metastasis. Here we show that the plasma level of NETs is significantly higher in patients with metastatic breast cancer compared to those with local disease, or those that were considered cured at a 5-year follow-up, confirming NETs as interesting therapeutic targets in metastatic breast cancer. Administration of DNase I is one strategy to eliminate NETs but long-term treatment requires repeated injections and species-specific versions of the enzyme. To enhance administration and therapeutic efficacy, we have developed an adeno-associated virus (AAV) vector system for delivery of murine DNase I and addressed its potential to counteract cancer-associated pathology in the murine MMTV-PyMT model for metastatic mammary carcinoma. The AAV vector is comprised of capsid KP1 and an expression cassette encoding hyperactive murine DNase I (AAV-mDNase I) under the control of a liver-specific promotor. This AAV-mDNase I vector could support elevated expression and serum activity of murine DNase I over at least 8 months. Neutrophil Gelatinase-Associated Lipocalin (NGAL), a biomarker for kidney hypoperfusion that is upregulated in urine from MMTV-PyMT mice, was suppressed in mice receiving AAV-mDNase I compared to an AAV-null control group. Furthermore, the proportion of mice that developed lung metastasis was reduced in the AAV-mDNase I group. Altogether, our data indicate that AAV-mDNase I has the potential to reduce cancer-associated impairment of renal function and development of metastasis. We conclude that AAV-mDNase I could represent a promising therapeutic strategy in metastatic breast cancer.

Seven cases illustrating difficulties in the treatment of MPO-ANCA-positive refractory otitis media.

There are increasing reports of patients with refractory otitis media caused by anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), especially myeloperoxidase (MPO)-ANCA-positive middle ear disease.　However, making a definitive diagnosis can be difficult, which can adversely affect the outcome of treatment.　We reviewed the diagnostic features of MPO-ANCA-positive middle ear disease and here discuss the difficulties of timely diagnosis and treatment.　Seven cases were eligible (6 women, 1 man;aged 57-83 years), and all were MPO-ANCA positive and proteinase 3 (PR3)-ANCA negative.　The patients were referred to our institution for management of intractable otitis media (2/7), progressive hearing loss (7/7) with facial palsy (1/7), and/or a high MPO-ANCA titer (5/7).　All patients underwent tapering steroid therapy and their MPO-ANCA titer was monitored.　Refractory MPO-ANCA-positive otitis media was noted:5 of 7 cases showed improvement with tapering steroid therapy but cure was not achieved in the remaining 2 cases.　This study demonstrates the difficulties in the diagnosis and treatment of localized AAV.　Early diagnosis and treatment can improve the prognosis of patients with AAV but global diagnostic criteria for ear disease have not been established.　Additional cases should be prospectively examined to establish a treatment for MPO-ANCA-positive middle ear disease.

Enhanced interaction between genome-edited mesenchymal stem cells and platelets improves wound healing in mice.

Impaired wound healing poses a significant burden on the healthcare system and patients. Stem cell therapy has demonstrated promising potential in the treatment of wounds. However, its clinical application is hindered by the low efficiency of cell homing. In this study, we successfully integrated P-selectin glycoprotein ligand-1 (PSGL-1) into the genome of human adipose-derived mesenchymal stem cells (ADSCs) using a Cas9-AAV6-based genome editing tool platform. Our findings revealed that PSGL-1 knock-in enhanced the binding of ADSCs to platelets and their adhesion to the injured site. Moreover, the intravenous infusion of PSGL-1 -engineered ADSCs (KI-ADSCs) significantly improved the homing efficiency and residence rate at the site of skin lesions in mice. Mechanistically, PSGL-1 knock-in promotes the release of some therapeutic cytokines by activating the canonical WNT/ß-catenin signaling pathway and accelerates the healing of wounds by promoting angiogenesis, re-epithelialization, and granulation tissue formation at the wound site. This study provides a novel strategy to simultaneously address the problem of poor migration and adhesion of mesenchymal stem cells (MSCs).

A Novel Method for Separating Full and Empty Adeno-Associated Viral Capsids Using Ultrafiltration.

Adeno-associated viral vectors (AAVs) are the predominant viral vectors used for gene therapy applications. A significant challenge in obtaining effective doses is removing non-therapeutic empty viral capsids lacking DNA cargo. Current methods for separating full (gene-containing) and empty capsids are challenging to scale, produce low product yields, are slow, and are difficult to operationalize for continuous biomanufacturing. This communication demonstrates the feasibility of separating full and empty capsids by ultrafiltration. Separation performance was quantified by measuring the sieving coefficients for full and empty capsids using ELISA, qPCR, and an infectivity assay based on the live cell imaging of green fluorescent protein expression. We demonstrated that polycarbonate track-etched membranes with a pore size of 30 nm selectively permeated empty capsids to full capsids, with a high recovery yield (89%) for full capsids. The average sieving coefficients of full and empty capsids obtained through ELISA/qPCR were calculated as 0.25 and 0.49, indicating that empty capsids were about twice as permeable as full capsids. Establishing ultrafiltration as a viable unit operation for separating full and empty AAV capsids has implications for developing the scale-free continuous purification of AAVs.

Viral vectors for gene delivery to the central nervous system.

Brain diseases with a known or suspected genetic basis represent an important frontier for advanced therapeutics. The central nervous system (CNS) is an intricate network in which diverse cell types with multiple functions communicate via complex signaling pathways, making therapeutic intervention in brain-related diseases challenging. Nevertheless, as more information on the molecular genetics of brain-related diseases becomes available, genetic intervention using gene therapeutic strategies should become more feasible. There remain, however, several significant hurdles to overcome that relate to (i) the development of appropriate gene vectors and (ii) methods to achieve local or broad vector delivery. Clearly, gene delivery tools must be engineered for distribution to the correct cell type in a specific brain region and to accomplish therapeutic transgene expression at an appropriate level and duration. They also must avoid all toxicity, including the induction of inflammatory responses. Over the last 40 years, various types of viral vectors have been developed as tools to introduce therapeutic genes into the brain, primarily targeting neurons. This review describes the most prominent vector systems currently approaching clinical application for CNS disorders and highlights both remaining challenges as well as improvements in vector designs that achieve greater safety, defined tropism, and therapeutic gene expression.

Evaluating Methods for the Prediction of Cell Type-Specific Enhancers in the Mammalian Cortex.

Identifying cell type-specific enhancers in the brain is critical to building genetic tools for investigating the mammalian brain. Computational methods for functional enhancer prediction have been proposed and validated in the fruit fly and not yet the mammalian brain. We organized the 'Brain Initiative Cell Census Network (BICCN) Challenge: Predicting Functional Cell Type-Specific Enhancers from Cross-Species Multi-Omics' to assess machine learning and feature-based methods designed to nominate enhancer DNA sequences to target cell types in the mouse cortex. Methods were evaluated based on in vivo validation data from hundreds of cortical cell type-specific enhancers that were previously packaged into individual AAV vectors and retro-orbitally injected into mice. We find that open chromatin was a key predictor of functional enhancers, and sequence models improved prediction of non-functional enhancers that can be deprioritized as opposed to pursued for in vivo testing. Sequence models also identified cell type-specific transcription factor codes that can guide designs of in silico enhancers. This community challenge establishes a benchmark for enhancer prioritization algorithms and reveals computational approaches and molecular information that are crucial for the identification of functional enhancers for mammalian cortical cell types. The results of this challenge bring us closer to understanding the complex gene regulatory landscape of the mammalian brain and help us design more efficient genetic tools and potential gene therapies for human neurological diseases.