Gene therapy cargo delivery to specific cell types in the central nervous system (CNS) remains a major challenge for the development of adeno-associated virus (AAV) vectors as a therapeutic modality. Here, we leverage high-plex in situ transcriptomics (10× Genomics Xenium) to spatially map barcoded AAV payloads at subcellular resolution in the intact mouse brain while preserving anatomical context. Tropism profiling of 22 barcoded AAV variants including novel AAV9 derivatives and CNS-targeted capsids revealed that established vectors, AAV9-PHP.eB and AAV9-CAP-B10, demonstrated distinct neuronal and non-neuronal subtype preferences, recapitulating previous findings. Several novel AAV variants candidates predominantly transduced endothelial and vascular cells, suggesting limited blood-brain barrier (BBB) penetration. Notably, three novel variants (AAV9-BTX166, -BTX168, and -BTX175) exhibited enhanced endothelial and mural cell tropism, despite robust CNS activity in bulk assays. Intriguingly, the novel variants AAV9-BTX149 and -BTX001 displayed selective targeting of specific inhibitory neuron subtypes, with a single Trp503Arg substitution in the capsid being sufficient to redirect tropism. Our results establish in situ spatial transcriptomics as a powerful tool for resolving AAV biodistribution and BBB traversal capacities at high resolution, providing a blueprint for capsid engineering to achieve precise cell subtype targeting in the CNS.

Intravitreal (IVT) administration of adeno-associated virus (AAV) vectors is considered a promising strategy for retinal gene therapy. Random peptide-display-based directed evolution has yielded novel AAV capsids with potent retinal bioactivity. However, recent reports on safety risks following IVT AAV administration indicate that significant opportunities remain to further refine AAV vector technology. Here, we performed iterative in vivo NGS-guided screening of AAV2 peptide-display libraries in both mice and non-human primates (NHPs), integrating barcoded functional analytics, and single-nuclei RNA sequencing to identify retina-tropic AAV variants. The top-performing capsid in mice, AAV2-GAYPKSP robustly drove eGFP expression in a human retina-on-chip model but failed to drive gene expression in the NHP retina following IVT delivery. In NHPs, a barcoded evaluation of 70 retina-tropic variants revealed a complex correlation between viral genome enrichment and transgene transcriptional output, providing insights that enhance the mechanistic interpretation and methodological design of AAV library selection strategies. Nonetheless, several NHP variants, including AAV2-I.1 and AAV2-I.12, demonstrated broad retinal transduction and potent expression, outperforming benchmarks AAV2 and AAV2-7m8. Single-nucleus RNA sequencing confirmed broad cell-type tropism of the top performing variants. Collectively, these findings expand the AAV toolkit for retinal gene therapy and underscore the importance of optimized screening methodologies in vector discovery.

Designing enhancer-promoter combinations that enable potent and precise transgene expression is a promising strategy for advancing gene therapy mediated by adeno-associated virus (AAV). We introduce STARR-CRAAVT (self-transcribing active regulatory region sequencing of captured libraries for rAAV gene therapy), a STARR-Seq-based platform using in silico tailored sequence libraries to screen for enhancers in AAVs and assess relevant applications and critical screening parameters in an in vitro proof-of-concept study. Candidate libraries were generated by integrating epigenetic data, followed by the capture of candidate-containing genomic fragments and packaging into AAVs. Using STARR-CRAAVT, we identified enhancers driving cell type-specific expression and showed that the promoter type is a key determinant for the ability of candidates to act as enhancers. Notably, we also found that switching the candidate position in the AAV genome can significantly influence enhancer activity. Our study yields insights into enhancer function and describes a blueprint for the implementation of STARR-CRAAVT to identify cell type-specific enhancers for AAV-based gene therapy.

Norrin, secreted by retinal Müller cells, activates canonical Wnt signaling via Frizzled-4 and co-receptors. Loss-of-function mutations abolish intraretinal capillary formation in mice. In humans, mutations in NDP, which encodes norrin, cause Norrie disease, characterized by retinal hypovascularization and congenital blindness, and X-linked familial exudative vitreoretinopathy (FEVR), resembling retinopathy of prematurity (ROP). We evaluated adeno-associated viral (AAV) vectors expressing norrin as gene therapy for Norrie disease, FEVR, and ROP. AAV2-7m8 and AAV-ShH10 were tested in juvenile wild-type and norrin-deficient (Ndp KO) mice via intravitreal injection at postnatal day 7, with some mice subjected to oxygen-induced retinopathy (OIR). AAV2-7m8 transduced Müller glia, while AAV-ShH10 targeted retinal ganglion cells. Both vectors fully restored intraretinal capillary growth in Ndp KO mice, normalizing vessel density and plexus organization, preserving the blood-retinal barrier, and rescuing visual function. In OIR, scAAV2-7m8-huNorrin reduced vaso-obliteration and neovascular tuft formation, increasing deep plexus coverage, and suppressed Vegfa164 and Ang-2 upregulation. The findings demonstrate that AAV-mediated norrin delivery efficiently targets retinal glia and neurons, restores vascular structure and function, stabilizes the blood-retinal barrier, and mitigates OIR-induced pathological angiogenesis, supporting its potential as a therapeutic strategy for Norrie-related retinopathies and ROP.

Previous studies have shown that altered AXL signaling is implicated in various diseases, with GAS6 recognized as its only relevant ligand to date. In this study, we show for the first-time a direct interaction between AXL and PROS1 using biochemical methods. Furthermore, we validate the biological significance of PROS1-AXL interaction through advanced quantitative and functional spatial imaging in both murine lung tissue, as well as human lung samples of idiopathic pulmonary fibrosis (IPF) patients. Our findings reveal the role of AXL-mediated biology in alveolar repair and the fibrotic response driven by GAS6 as well as PROS1. Notably, this effect involves PROS1 interacting with AXL to counteract GAS6 mediated effects. Together with the distinct temporal expression of profibrotic genes and the interplay between AXL and TGF-ß pathway, this emphasizes the potential of targeting AXL mediated biology for therapeutic intervention in IPF to allow alveolar restoration. ### Competing Interest Statement The authors have declared no competing interest.

Precise control of transgene expression through novel enhancer-promoter combinations is a promising strategy for advancing gene therapy mediated by adeno-associated virus (AAV). We present STARR-CRAAVT, a novel STARR-Seq-derived platform to screen for enhancers in the AAV context. Using HepG2 and HaCaT cells as screening models, we applied STARR-CRAAVT for the identification of cell type-specific enhancers. We integrated epigenetic datasets into an in-silico library of putative HepG2 enhancers and captured corresponding fragments from genomic DNA. The fragments were processed into AAV libraries and applied to HepG2 and HaCaT cells. STARR-CRAAVT analysis revealed a selective activity of the libraries confirming the HepG2-directed in-silico design and the specificity of single enhancer-promoter combinations could be validated using luciferase reporter assays. In addition, we scrutinized the impact of key experimental parameters on enhancer identification and found that the used promoter type had significant influence on the ability of candidates to act as enhancers. Furthermore, switching the location of identified enhancers in reporter assays revealed that the level of enhancer activity is highly dependent on the position in the AAV genome. Taken together, our study yields novel insights into enhancer function and demonstrates that STARR-CRAAVT can be employed to identify cell type-specific enhancers, highlighting promoter preference and enhancer positioning as key considerations for enhancer screening campaigns. ### Competing Interest Statement COMPETING INTEREST STATEMENT R.B., M.O., Y.F., B.S., S. Ketterer, J.P., S. Kreuz, U.M., S.M., and C.S. are employees of Boehringer Ingelheim. P.C. is currently an employee of Bayer. Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, DE

BACKGROUND & AIMS:Glucagon (GCG) analogues are gaining attention as promising components in incretin-based therapeutics for obesity and metabolic dysfunction-associated steatohepatitis. However, the biological effects of chronic GCG treatment, particularly the molecular underpinnings of GCG-induced energy expenditure and lipid metabolism, remain poorly defined. METHODS:We utilized a long-acting GCG analogue (LA-GCG) in conjunction with hepatic and adipose glucagon receptor (GCGR) knockout mouse models. Through an integrative approach that combined metabolic, biochemical, and omics techniques, we investigated the molecular mechanisms underlying GCG-induced energy expenditure and metabolic benefits. RESULTS:LA-GCG enhanced energy expenditure in diet-induced obese mice, with hepatic, but not adipose, GCGR signaling playing an essential role. Notably, this increase in energy expenditure was only observed in obese, not lean, mice. This preferential effect appeared to be mediated by sustained activation of cAMP/PKA signaling due to LA-GCG-induced downregulation of PDE4B/4D. In contrast, in lean mice, cAMP/PKA signaling is rapidly attenuated by PDE4B/4D activity. Interestingly, unlike energy expenditure, the lipid-clearing effects of LA-GCG were independent of the PDE4/cAMP/PKA axis. CONCLUSIONS:These findings elucidate the molecular basis of GCG-induced energy expenditure and metabolic benefits, and highlight a phenotypic segregation between cAMP/PKA-dependent and -independent effects. IMPACT AND IMPLICATIONS:This study provides fundamental mechanistic insights into GCG pharmacology, which has direct clinical relevance. The obesity-specific enhancement of energy expenditure by GCGR agonism supports the superior efficacy of GCGR/glucagon-like peptide-1 receptor (GLP-1R) dual agonists over GLP-1R mono-agonists in individuals with obesity. Importantly, differential expression patterns of PDE4 may underlie variability in weight loss responses to GCG-based therapies, identifying PDE4 inhibition as a potential strategy to restore efficacy in GCG non-responders. Moreover, a PDE4-overexpression model preserved the lipid-clearing effects of GCGR agonism while attenuating hyperglycemic risk, offering a translatable approach to optimize the safety-efficacy profile of GCG-based treatments for cardio-renal-metabolic diseases, including obesity and metabolic dysfunction-associated steatohepatitis.

Functional validation of therapeutic concepts-including understanding a target protein's mode of action in disease-relevant tissues-is a critical component of early drug discovery research. Tissue selectivity of recombinant adeno-associated viral vectors (AAVs) is an important property for both gene therapy and the modulation of target genes in model organisms, to investigate protein function and signaling pathways in specific cell types and tissues. However, while many target tissues can be transduced efficiently by selected capsids, achieving tissue specificity remains challenging in certain contexts. In this study, we compared different microRNA (miRNA)-based strategies to transcriptionally restrict AAV expression to either adipose tissue or skeletal muscle. Our findings demonstrate that liver-specific miR122 effectively suppressed AAV9-driven liver expression, while preserving adipose tissue targeting efficiency. Similarly, AAVMYO, in combination with a muscle-specific promoter and target sites for miR208a, confined transgene expression to skeletal muscle, while successfully de-targeting the liver and, importantly, the heart. Notably, both approaches outperformed conditionally self-silencing constructs that utilized artificial miRNAs (amiRs) driven by liver (LP1)- or heart (troponin T)-specific promoters. In summary, we expand the AAV toolbox by introducing expression cassette designs that enable specific expression in adipose tissue and skeletal muscle, thereby facilitating mechanistic studies in metabolic and muscle disease research.

The tissue transduction profiles and transgene expression efficiencies of adeno-associated viruses (AAVs) depend not only on the utilized capsid and dose but also on the administration route. Yet, despite the plethora of available natural and capsid-engineered variants, a comprehensive evaluation of the administration route dependency of AAV tropism has been lacking so far. Therefore, we here compared transduction and transgene expression profiles for 34 well-known AAV capsids following intravenous (i.v.) and intraperitoneal (i.p.) injection in male C57BL/6 mice by multiplexed biodistribution analyses based on AAV genome-barcoding. Readout on viral genome and transcript level confirmed pronounced liver targeting by most AAV variants after i.v. administration, as well as known tissue tropism for benchmark capsids (e.g., AAV-PHP.eB: brain and AAV2-ESGHGYF: lung). In contrast, i.p. administration generally decreased liver targeting, while concurrently increasing expression in other abdominal organs in a capsid-specific fashion. For example, AAV6.2 and AAV-DJ, which showed almost exclusive liver transduction after i.v. administration, displayed differential biodistribution profiles with enhanced expression in the diaphragm, adipose tissue, and pancreas when administered intraperitoneally. In summary, our data guide study design by enabling the selection of optimal vector and administration route combinations for refined tissue targeting approaches in preclinical in vivo experiments.

Adeno-associated virus (AAV) vectors currently represent the most attractive platform for therapeutic gene delivery. Ensuring efficient AAV production and vector integrity, defined by efficient packaging of full-size genomes, high full/empty ratios, and optimal bioactivity, therefore is of utmost importance. However, during AAV production, not only capsids that carry the full-size genome but also empty as well as capsids containing partial or oversized genomes are produced. To systematically investigate the impact of AAV genome size on packaging efficiency, genome integrity, (over)full/partial/empty ratio, and bioactivity, we designed single-stranded CMV-EGFP-poly(A) expression cassettes with successively increasing sizes (2-5 kb), by inserting non-coding stuffer sequences either upstream or downstream of the CMV-EGFP-poly(A) sequence. Our results demonstrate a systematic decrease of AAV yields and bioactivity with increasing expression cassette size. While significant overfilling was observed at expression cassette sizes <2.5 kb, an increase in partially filled capsids, due to genome truncation, was observed >4.5 kb. Surprisingly, the losses in productivity and bioactivity were not observed with a second stuffer sequence. Therefore, while the insertion of non-coding DNA is a straightforward approach to "right-size" the AAV genome and optimize CMC aspects, careful selection of the DNA sequence is required to assure AAV quality.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with substantial unmet medical needs. While aberrant epithelial remodeling is a key factor in IPF progression, the molecular mechanisms behind this process remain elusive. Harnessing a 3D patient-derived organoid model and multi-omics approach, the first inventory of the connection between metabolic alteration, chromatin accessibility, and transcriptional regulation in IPF aberrant epithelial remodeling is provided. This remodeling is characterized by an increase in chromatin accessibility, particularly at JUNB motif-enriched promoter regions proximal to transcription start sites of metabolic and pro-fibrotic genes. Mechanistically, JUNB undergoes O-linked β-N-acetylglucosamine modification (O-GlcNAcylation), a critical step in modulating pro-fibrotic responses to chronic injury. This modification is pivotal in fostering the emergence of aberrant epithelial basal cells in the alveolar niche, a proposed driver of IPF pathology. Specific deletion of O-GlcNAcylation sites on JUNB attenuates the metaplastic differentiation of basal cells, thereby aiding in the restoration of the alveolar lineage. Together, the findings reveal a novel link between metabolic dysregulation and cell fate regulation at the chromatin level in fibrosis, mediated by the O-GlcNAc-JUNB axis, suggesting avenues for the development of new therapeutic strategies in IPF.

Adeno-associated virus (AAV) vectors have become the leading platform for gene delivery in both preclinical research and therapeutic applications, making the production of high-titer AAV preparations essential. To date, most AAV-based studies use constitutive promoters (e.g., CMV, CAG), which are also active in human embryonic kidney (HEK)-293 producer cells, thus leading to the expression of the transgene already during production. Depending on the transgene's function, this might negatively impact producer cell performance and result in decreased AAV vector yields. Here, we evaluated a panel of diverse microRNA (miRNA)-based shRNA designs to identify a highly potent artificial miRNA for the transient suppression of transgenes during AAV production. Our results demonstrate that insertion of miRNA target sites into the 3' UTR of the transgene and simultaneous expression of the corresponding miRNA from the 3' UTR of conventional AAV production plasmids (rep/cap, pHelper) enabled efficient silencing of toxic transgene expression, thereby increasing AAV vector yields up to 240-fold. This strategy not only allows to maintain the traditional triple-transfection protocol, but also represents a universally applicable approach to suppress toxic transgenes, thereby boosting vector yields with so far unprecedented efficiency.

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.

The translation of findings from animal models to human disease is a fundamental part in the field of drug development. However, only a small proportion of promising preclinical results in animals translate to human pathophysiology. This underscores the necessity for novel data analysis strategies to accurately evaluate the most suitable animal model for a specific purpose, ensuring cross-species translatability. To address this need, we present In Silico Treatment (IST), a computational method to assess translation of disease-related molecular expression patterns between animal models and humans. By simulating changes observed in animals onto humans, IST provides a holistic picture of how well animal models recapitulate key aspects of human disease, or how treatments transform pathogenic expression patterns to healthy ones. Furthermore, IST highlights particular genes that influence molecular features of pathogenesis or drug mode of action. We demonstrate the potential of IST with three applications using bulk transcriptomics data. First, we assessed two mouse models for idiopathic pulmonary fibrosis (IPF): one involving injury with intra-tubular Bleomycin exposure, and the other Adeno-associated-virus-induced, TGFβ1-mediated tissue transformation (AAV6.2-TGFβ1). Both models exhibited gene expression patterns resembling extracellular matrix derangement in human IPF, whereas differences in VEGF-driven vascularization were observed. Second, we confirmed known features of non-alcoholic steatohepatitis (NASH) mouse models, including choline-deficient, l-amino acid-defined diet (CDAA), carbon tetrachloride hepatotoxicity injury (CCl4) and bile duct ligation surgery (BDL). Overall, the three mouse models recapitulated expression changes related to fibrosis in human NASH, whereas model-specific differences were found in lipid metabolism, inflammation, and apoptosis. Third, we reproduced the strong anti-fibrotic signature and induction of the PPARα signaling observed in the Elafibranor experimental treatment for NASH in the CDAA model. We validated the contribution of known disease-related genes to the findings made with IST in the IPF and NASH applications. The complete data integration IST framework, including an interactive app to integrate and compare datasets, is made available as an open-source R package. Author summary Preclinical testing plays a pivotal role in the drug development process, serving as a crucial evaluation phase before a new drug can be tested on humans in clinical trials. The drug must undergo a rigorous evaluation in in vivo and in vitro preclinical studies to assess its safety and efficacy. However, positive outcomes in preclinical animal models do not always translate to positive results in humans, mainly due to biological differences. Therefore, selecting an animal model that closely mirrors human disease traits and detecting and accounting for model limitations is of paramount importance. Over the last decade, the availability of gene expression data in both animals and humans has substantially increased. Gene expression states and perturbations are routinely employed as a proxy to predict and understand changes in disease states. Here, we developed In Silico Treatment, a computational method designed to overlay the gene expression changes observed in animals onto humans, quantifying the change in human disease status. We applied this method to mouse models for idiopathic pulmonary fibrosis and non-alcoholic steatohepatitis, two severe fibrotic diseases. We successfully identified known features of the disease models and provide a granular gene-level rationale behind our predictions. Consequently, our method shows promise as an effective approach to improve animal model selection and thus clinical translation. ### Competing Interest Statement All the authors were paid employees by Boehringer Ingelheim Pharma GmbH & Co.KG

Transgenic animals with increased or abrogated target gene expression are powerful tools for drug discovery research. Here, we developed a CRISPR-based Rosa26-LSL-dCas9-VPR mouse model for targeted induction of endogenous gene expression using different Adeno-associated virus (AAV) capsid variants for tissue-specific gRNAs delivery. To show applicability of the model, we targeted low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), either individually or together. We induced up to ninefold higher expression of hepatocellular proteins. In consequence of LDLR upregulation, plasma LDL levels almost abolished, whereas upregulation of PCSK9 led to increased plasma LDL and cholesterol levels. Strikingly, simultaneous upregulation of both LDLR and PCSK9 resulted in almost unaltered LDL levels. Additionally, we used our model to achieve expression of all α 1 -Antitrypsin (AAT) gene paralogues simultaneously. These results show the potential of our model as a versatile tool for optimized targeted gene expression, alone or in combination.

Adeno-associated virus (AAV) vector applications are often limited by capsid-directed humoral immune responses, mainly through neutralizing antibodies (NAbs), which are present throughout the human population due to natural AAV infections. Currently, antibody levels are often quantified via ELISA-based protocols or by cellular NAb assays and less frequently by in vivo NAb assays in mice. These methods need optimization for each serotype and are often not applicable to AAV variants with poor in vitro transduction. To tackle these limitations, we have established Meso Scale Discovery (MSD)-based assays for the quantification of binding antibodies (BAbs) and NAbs against the three most commonly used AAV serotypes, AAV2, AAV8, and AAV9. Both assays detect antiAAV-IgG(1-3) with high sensitivity and consistency as shown in a screen of sera from 40 healthy human donors. Subsequently, BAb and NAb titers were determined for identification of seronegative animals in a non-human primate (NHP) cohort. Moreover, the MSD -based BAb assay protocol was extended to a panel of 14 different AAV serotypes. In summary, our platform allows a rapid and quantitative assessment of the immunological properties of any natural or engineered AAV variant irrespective of transduction efficiency and enables high-throughput screens.