Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.

Sensory neurons promote immune homeostasis in the lung.

Cytokines employ downstream Janus kinases (JAKs) to promote chronic inflammatory diseases. JAK1-dependent type 2 cytokines drive allergic inflammation, and patients with JAK1 gain-of-function (GoF) variants develop atopic dermatitis (AD) and asthma. To explore tissue-specific functions, we inserted a human JAK1 GoF variant (JAK1GoF) into mice and observed the development of spontaneous AD-like skin disease but unexpected resistance to lung inflammation when JAK1GoF expression was restricted to the stroma. We identified a previously unrecognized role for JAK1 in vagal sensory neurons in suppressing airway inflammation. Additionally, expression of Calcb/CGRPß was dependent on JAK1 in the vagus nerve, and CGRPß suppressed group 2 innate lymphoid cell function and allergic airway inflammation. Our findings reveal evolutionarily conserved but distinct functions of JAK1 in sensory neurons across tissues. This biology raises the possibility that therapeutic JAK inhibitors may be further optimized for tissue-specific efficacy to enhance precision medicine in the future.

Circuit-specific gene therapy reverses core symptoms in a primate Parkinson's disease model.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.

An evolved AAV variant enables efficient genetic engineering of murine T cells.

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.

Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.

Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.

Revisiting astrocyte to neuron conversion with lineage tracing in vivo.

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.

Adeno-Associated Virus Toolkit to Target Diverse Brain Cells.

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types.

Computationally guided AAV engineering for enhanced gene delivery.

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.

Adeno-Associated Virus Delivery of Anti-HIV Monoclonal Antibodies Can Drive Long-Term Virologic Suppression.

Long-term delivery of anti-HIV monoclonal antibodies (mAbs) using adeno-associated virus (AAV) vectors holds promise for the prevention and treatment of HIV infection. We describe a therapy trial in which four rhesus monkeys were infected with SHIV-AD8 for 86 weeks before receiving the AAV-encoded mAbs 3BNC117, 10-1074, and 10E8. Although anti-drug antibody (ADA) responses restricted mAb delivery, one monkey successfully maintained 50-150 µg/mL of 3BNC117 and 10-1074 for over 2 years. Delivery of these two mAbs to this monkey resulted in an abrupt decline in plasma viremia, which remained undetectable for 38 successive measurements over 3 years. We generated two more examples of virologic suppression using AAV delivery of a cocktail of four mAbs in a 12-monkey study. Our results provide proof of concept for AAV-delivered mAbs to produce a "functional cure." However, they also serve as a warning that ADAs may be a problem for practical application of this approach in humans.

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4+ T-cells for the treatment of Hyper IgM1.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

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.

Comparison of different promoters to improve AAV vector-mediated gene therapy for neuronopathic Gaucher disease.

Gaucher Disease (GD) is an inherited metabolic disorder caused by mutations in the GBA1 gene. It can manifest with severe neurodegeneration and visceral pathology. The most acute neuronopathic form (nGD), for which there are no curative therapeutic options, is characterised by devastating neuropathology and death during infancy. In this study, we investigated the therapeutic benefit of systemically delivered AAV9 vectors expressing the human GBA1 gene at two different doses comparing a neuronal-selective promoter with ubiquitous promoters. Our results highlight the importance of a careful evaluation of the promoter sequence used in gene delivery vectors, suggesting a neuron-targeted therapy leading to high levels of enzymatic activity in the brain but lower GCase expression in the viscera, might be the optimal therapeutic strategy for nGD.

High-throughput transcriptome analyses from ASPIRO, a phase 1/2/3 study of gene replacement therapy for X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a severe congenital disease characterized by profound muscle weakness, respiratory failure, and early death. No approved therapy for XLMTM is currently available. Adeno-associated virus (AAV)-mediated gene replacement therapy has shown promise as an investigational therapeutic strategy. We aimed to characterize the transcriptomic changes in muscle biopsies of individuals with XLMTM who received resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) in the ASPIRO clinical trial and to identify potential biomarkers that correlate with therapeutic outcome. We leveraged RNA-sequencing data from the muscle biopsies of 15 study participants and applied differential expression analysis, gene co-expression analysis, and machine learning to characterize the transcriptomic changes at baseline (pre-dose) and at 24 and 48 weeks after resamirigene bilparvovec dosing. As expected, MTM1 expression levels were significantly increased after dosing (p < 0.0001). Differential expression analysis identified upregulated genes after dosing that were enriched in several pathways, including lipid metabolism and inflammatory response pathways, and downregulated genes were enriched in cell-cell adhesion and muscle development pathways. Genes involved in inflammatory and immune pathways were differentially expressed between participants exhibiting ventilator support reduction of either greater or less than 6 h/day after gene therapy compared to pre-dosing. Co-expression analysis identified similarly regulated genes, which were grouped into modules. Finally, the machine learning model identified five genes, including MTM1, as potential RNA biomarkers to monitor the progress of AAV gene replacement therapy. These findings further extend our understanding of AAV-mediated gene therapy in individuals with XLMTM at the transcriptomic level.

Viral Strategies for Targeting the Central and Peripheral Nervous Systems.

Recombinant viruses allow for targeted transgene expression in specific cell populations throughout the nervous system. The adeno-associated virus (AAV) is among the most commonly used viruses for neuroscience research. Recombinant AAVs (rAAVs) are highly versatile and can package most cargo composed of desired genes within the capsid's ∼5-kb carrying capacity. Numerous regulatory elements and intersectional strategies have been validated in rAAVs to enable cell type-specific expression. rAAVs can be delivered to specific neuronal populations or globally throughout the animal. The AAV capsids have natural cell type or tissue tropism and trafficking that can be modified for increased specificity. Here, we describe recently engineered AAV capsids and associated cargo that have extended the utility of AAVs in targeting molecularly defined neurons throughout the nervous system, which will further facilitate neuronal circuit interrogation and discovery.

Targeted evolution of adeno-associated virus capsids for systemic transgene delivery to microglia and tissue-resident macrophages.

Tissue macrophages, including microglia, are notoriously resistant to genetic manipulation. Here, we report the creation of Adeno-associated viruses (AAV) variants that efficiently and widely transduce microglia and tissue macrophages in vivo following intravenous delivery, with transgene expression of up to 80%. We use this technology to demonstrate manipulation of microglia gene expression and microglial ablation, thereby providing invaluable research tools for the study of these important cells.

Vectored immunoprophylaxis and treatment of SARS-CoV-2 infection in a preclinical model.

Vectored immunoprophylaxis was first developed as a means of establishing engineered immunity to HIV using an adenoassociated viral vector expressing a broadly neutralizing antibody. We applied this concept to establish long-term prophylaxis against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mouse model using adenoassociated virus and lentiviral vectors expressing a high-affinity angiotensin-converting enzyme 2 (ACE2) decoy. Administration of decoy-expressing (adenoassociated virus) AAV2.retro and AAV6.2 vectors by intranasal instillation or intramuscular injection protected mice against high-titered SARS-CoV-2 infection. AAV and lentiviral vectored immunoprophylaxis was durable and was active against SARS-CoV-2 Omicron subvariants. The AAV vectors were also effective therapeutically when administered postinfection. Vectored immunoprophylaxis could be of value for immunocompromised individuals for whom vaccination is not practical and as a means to rapidly establish protection from infection. Unlike monoclonal antibody therapy, the approach is expected to remain active despite continued evolution viral variants.

Populations of Hindbrain Glucagon-Like Peptide 1 (GLP1) Neurons That Innervate the Hypothalamic PVH, Thalamic PVT, or Limbic Forebrain BST Have Axon Collaterals That Reach All Central Regions Innervated by GLP1 Neurons.

Neurons in the caudal nucleus of the solitary tract (cNTS) and intermediate reticular nucleus (IRt) that express the glucagon gene (Gcg) give rise to glucagon-like peptide 1 (GLP1)-immunopositive axons in the spinal cord and many subcortical brain regions. Central GLP1 receptor signaling contributes to motivated behavior and stress responses in rats and mice, in which hindbrain GLP1 neurons are activated to express c-Fos in a metabolic state-dependent manner. The present study examined whether GLP1 inputs to distinct brain regions arise from distinct subsets of Gcg-expressing neurons, and mapped the distribution of axon collaterals arising from projection-defined GLP1 neural populations. Using our Gcg-Cre knock-in rat model, Cre-dependent adeno-associated virus (AAV) tracing was conducted in adult male and female rats to compare axonal projections of IRt versus cNTS GLP1 neurons. Overlapping projections were observed in all brain regions that receive GLP1 input, with the caveat that cNTS injections produced Cre-dependent labeling of some IRt neurons, and vice versa. In additional experiments, specific diencephalic or limbic forebrain nuclei were microinjected with Cre-dependent retrograde AAVs (AAVrg) that expressed reporters to fully label the axon collaterals of transduced GLP1 neurons. AAVrg injected into each forebrain site labeled Gcg-expressing neurons in both the cNTS and IRt. The collective axon collaterals of labeled neurons entered the spinal cord and every brain region previously reported to contain GLP1-positive axons. These results indicate that the axons of GLP1 neural populations that innervate the thalamic paraventricular nucleus, paraventricular nucleus of the hypothalamus, and/or bed nucleus of the stria terminalis collectively innervate all central regions that receive GLP1 axonal input.

AAV-mediated hepatic expression of SLC30A10 and the Thr95Ile variant attenuates manganese excess and other phenotypes in Slc30a10-deficient mice.

The manganese (Mn) export protein SLC30A10 is essential for Mn excretion via the liver and intestines. Patients with SLC30A10 deficiency develop Mn excess, dystonia, liver disease, and polycythemia. Recent genome-wide association studies revealed a link between the SLC30A10 variant T95I and markers of liver disease. The in vivo relevance of this variant has yet to be investigated. Using in vitro and in vivo models, we explore the impact of the T95I variant on SLC30A10 function. While SLC30A10 I95 expressed at lower levels than T95 in transfected cell lines, both T95 and I95 variants protected cells similarly from Mn-induced toxicity. Adeno-associated virus 8-mediated expression of T95 or I95 SLC30A10 using the liver-specific thyroxine binding globulin promoter normalized liver Mn levels in mice with hepatocyte Slc30a10 deficiency. Furthermore, Adeno-associated virus-mediated expression of T95 or I95 SLC30A10 normalized red blood cell parameters and body weights and attenuated Mn levels and differential gene expression in livers and brains of mice with whole body Slc30a10 deficiency. While our in vivo data do not indicate that the T95I variant significantly compromises SLC30A10 function, it does reinforce the notion that the liver is a key site of SLC30A10 function. It also supports the idea that restoration of hepatic SLC30A10 expression is sufficient to attenuate phenotypes in SLC30A10 deficiency.

Adeno-Associated Virus Gene Therapy for Hemophilia.

In vivo gene therapy is rapidly emerging as a new therapeutic paradigm for monogenic disorders. For almost three decades, hemophilia A (HA) and hemophilia B (HB) have served as model disorders for the development of gene therapy. This effort is soon to bear fruit with completed pivotal adeno-associated viral (AAV) vector gene addition trials reporting encouraging results and regulatory approval widely anticipated in the near future for the current generation of HA and HB AAV vectors. Here we review the clinical development of AAV gene therapy for HA and HB and examine outstanding questions that have recently emerged from AAV clinical trials for hemophilia and other monogenic disorders.