Molecular Therapy Methods & Clinical Development

Adeno-associated viruses (AAVs) are popular gene therapy vectors, but AAVs can cause toxicity. This is particularly evident following expression of some transgenes, e.g. GFP, in the retinal pigment epithelium (RPE), which leads to loss of RPE cells and photoreceptors. Here, we sought to unravel the toxicity mechanism(s). Several transgenes, self and non-self, were tested for toxicity, with no clear correlation for this variable. RPE RNA-sequencing revealed upregulation of translational processes, cell stress, cytokine release, antiviral responses, and leukocyte infiltration pathways. Toxicity-inducing pathways were explored for causality by injecting toxic AAVs into mice deficient for intrinsic, innate, or adaptive immune pathways. The CHOP KO partially alleviated toxicity for RPE but not photoreceptors, whereas the type I interferon receptor KO partially alleviated toxicity for photoreceptors but not RPE. In situ hybridization of interferon pathway transcripts (IFNB1, IFNAR1) revealed that the RPE and retina can produce and potentially respond to interferon. These data suggest that transgene-induced cell stress responses in the RPE lead to RPE cell death, while interferon signaling contributes to the death of photoreceptors.

A major challenge in gene therapy for GJB2-related hearing loss (DFNB1)--the most common form of hereditary deafness--is achieving efficient and precise connexin 26 delivery. Herein, we engineered two cell type-specific promoters (GJB2-1 and WFS1-2274) and developed an AAV capsid, AAV-MAS012, with enhanced transduction efficiency in mature cochlear cells. Our AAV-mediated gene therapy systems restored hearing of low-to-mid-frequencies in newborn Gjb2 cKO mice to wild-type levels and maintained for 45 weeks. Additionally, our therapeutic systems restored low-to-mid-frequencies hearing function to wild-type levels in adult Gjb2 cKO mice. A humanized version of the therapy, AAV-MAS012-WFS1-2274-hGJB2, rescued hearing function in two distinct Gjb2-deficient mouse models, and demonstrated a favorable safety profile in nonhuman primates. This study represents the first successful hearing restoration in adult Gjb2-deficient mice. The significant therapeutic efficacy of the humanized gene therapy system shows great potential for clinical translation in DFNB1 patients.

Mutations in the Eyes Shut Homolog (EYS) gene are a leading cause of autosomal recessive retinitis pigmentosa, a progressive retinal degenerative disease for which no effective treatment currently exists. However, the large size of the EYS coding sequence ([~]9.4 kb) exceeds the packaging limit of adeno-associated virus (AAV) vectors, posing a major barrier to gene replacement therapy. To address this challenge, we developed a tripartite AAV vector system that enables delivery and reconstitution of the full-length EYS gene using a Cre-lox-based unidirectional DNA recombination strategy, Uni-STAR (Uni-directional and Site-specific Transgene Assembly by Recombination). The system consists of three AAV constructs carrying discrete EYS segments flanked by engineered, non-compatible lox sites that drive ordered and unidirectional recombination in target cells. We validated this system in vitro by demonstrating successful reconstitution and expression of full-length EYS protein in HEK293T cells. In vivo, subretinal co-injection of the three AAV vectors into mouse eyes led to precise reconstitution and expression of full-length EYS protein in the retina. These findings establish the feasibility of using a tripartite AAV system to deliver the complete EYS gene and provide a foundation for future therapeutic development targeting EYS-associated retinal degenerations.

Recombinant adeno-associated virus (AAV) vectors are widely used for gene therapeutics, yet their early effects on host chromatin remodeling and nuclear organization remain insufficiently characterized. In contrast, several DNA viruses are known to remodel host chromatin accessibility, for example HSV-1 in mammalian cells and baculoviruses in insect systems. Here we evaluated genome accessibility and nuclear organization of cultured primary human cells at 24 and 48 hours after infection with wildtype AAV2, single-stranded (ss) and self-complementary (sc) recombinant AAV2. Genome-wide ATAC-seq showed no detectable change in host chromatin accessibility at either time point. A DNase I digestion assay at five candidate loci supported this observation. In contrast, immunofluorescence imaging revealed modest decreases in histone H3, H3K27me3, and RNA polymerase II signals, consistent with reduced polymerase engagement under stress-linked pathways. H3K4me3 deviated from this pattern in G1 cells upon scAAV2 infection, where signals increased. Nuclear geometry shifted in parallel with protein signals, with changes in area, perimeter, convexity, and eccentricity. Markers of nuclear condensates also changed, including reduced fibrillarin, SP100, and SRSF2 intensities, altered object shape metrics, and higher counts of promyelocytic leukemia (PML) bodies and nuclear splicing speckles (NSs). Collectively, at the tested doses and times, AAV2-based vectors did not remodel chromatin accessibility at scale and induced only small changes in polymerase associated readouts and nuclear architecture. These data align with a favorable safety profile while highlighting assay limits and suggesting indirect stress related mechanisms.

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which model is the most relevant for the selection of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

To achieve cell type-specific gene expression, using target cell-tropic AAV capsids is advantageous. However, their tropism across brain cell types remains unexplored in non-human primates. We assessed the tropism of nine AAV serotype capsids (AAV1, 2, 5, 6, 7, 8, 9, rh.10 (rh10), and DJ) on marmoset cerebral cortical cell types. Marmoset cerebral cortex was injected with different serotype AAVs expressing enhanced GFP (EGFP) by the ubiquitous chicken {beta}-actin hybrid (CBh) promoter. After 4 weeks, all nine AAV capsid vectors, especially AAV9 and AAVrh10, caused highly neuron-selective EGFP expression. Some AAV capsids, including AAV5, caused EGFP expression in oligodendrocytes to a lesser extent, with minimal or no expression in astrocytes and microglia. Different ubiquitous CMV and CAG promoters showed similar neuron-predominant transduction. Conversely, all nine AAV capsid vectors with the astrocyte-specific hGFA(ABC1D) promoter selectively transduced astrocytes, except AAV5, which transduced oligodendrocytes modestly. Oligodendrocyte-specific mouse myeline basic protein (mMBP) promoter in AAV5 vectors transduced oligodendrocytes specifically and efficiently. Our results suggest optimal combinations of capsids and promoters for cell type-specific expression: AAV9 or AAVrh10 and ubiquitous CBh, CMV, or CAG promoter for neuron-specific transduction; AAV2 or 7 and hGFA(ABC1D) promoter for astrocyte-specific transduction; and AAV5 and mMBP promoter for oligodendrocyte-specific transduction.

CRISPR-Cas9 is a powerful gene-editing tool with great potential for treating genetic diseases, including inherited retinal disorders. However, its bacterial origin can induce immune responses that may eliminate transduced cells, threatening editing efficiency. A deeper understanding of CRISPR-Cas9 immunogenicity is therefore needed. Previous studies have shown that systemic delivery via Cas9 induces an immune response, but the detailed inflammation and the impact of the vector remain unclear, especially in immune-privileged organs like the eye. In this study, we found that Cas9 delivered to the retina using adeno-associated virus (AAV) induced persistent inflammation, whereas delivery as naked ribonucleoprotein (RNP) complexes resulted in acute inflammation that faded three weeks post-injection. Inflammation was more severe in the rd10 mouse model of inherited retinal degeneration, which exhibits basal inflammation. These findings provide new insights into vector-dependent immune responses to Cas9 in the eye and highlight potential risks associated with its clinical application. TEASERUnderstanding immune reactions to CRISPR-Cas9 and linking these to their delivery methodology increases their safety.

Cystic fibrosis (CF) is the most common monogenic lung disease and results from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). There have been over 2000 variants identified in patients that result in loss of function of the CFTR protein leading to systemic disease and respiratory failure in adolescence. While some variants encode proteins with residual activity that can be corrected or potentiated by CFTR modulators, at least 10% of CF individuals cannot tolerate the modulators or have nonsense mutations which fail to make any protein. For all people with CF, a mutation agnostic gene replacement strategy could provide a cure for CF lung disease. Here, we propose using a systemic route of administration to deliver a functional CFTR minigene cargo with a lung tropic AAV capsid. This would serve to reach multiple organs, most importantly the lung epithelium, and would provide a functional CFTR transgene that could be expressed in any cell type with a ubiquitous promoter. To achieve this, we generated the smallest CFTR minigene tested in an AAV delivery to date. We demonstrate its expression and function following transfection in cell-based assays and restoration of function in primary CF airway cells after viral delivery. Furthermore, we identify an AAV capsid that can transduce alveolar and airway epithelium with systemic delivery in non-human primates. These data provide tools for delivering a functional CFTR minigene that fits within the packaging capacity of an AAV and demonstrate lung transduction with an AAV following systemic delivery in a large animal model. This strategy first and foremost can reach target airway cells by circumventing the strong mucosal barrier in CF airways but may also provide a method by which to restore CFTR function in additional CF affected organs.

Reprogramming of Muller glial (MG) cells into retinal neurons has the potential to treat vision loss by regenerating the retina. Development of efficient gene delivery systems to target the MG cells is critical. Adeno-associated virus (AAV) serotypes and promoter specificity are important factors that influence AAV transduction profile in the retina. However, studies that optimize these parameters to specifically target MG cells are limited, in particular in rats which are commonly used for eye research. Here we tested 4 AAV serotypes and 14 promoters to optimize gene delivery to human MG cells in vitro and/or rat MG cells in vivo. We showed that the combinatorial use of MG-specific serotypes and promoters achieved high specificity for MG cell targeting, with ShH10Y serotype and the GFAP (gfaABC1D) promoter as the best performing tool to target rat MG cells in vivo. We developed new AAV vectors using known and novel MG-specific promoters and engineered short promoter variants to improve the cargo capacity of AAV delivery. Our results highlighted a number of promoters that can target MG cells in vitro or in vivo. This study further expands the AAV toolbox to target MG cells, which has important implications for retinal gene therapy development.

Adeno-associated viruses (AAVs) have emerged as powerful tools for delivery of genes to a variety of cell types including pancreatic endocrine cells. Currently, AAV serotype 8 (AAV8) is the main AAV vector employed for infecting pancreatic cells for transgene transfer. We aimed to address whether alternative serotypes (AAV2, AAV6, and AAV9) commonly used for gene transfer can be effective in transducing pancreatic cells efficiently. We also screened the additives heparin and neuraminidase to further understand the interaction between the individual AAV types included in this work and the cells for optimal infection. Murine pancreatic {beta}-cells and -cells as well as fibroblasts were infected with AAV serotypes 2, 6, and 9 carrying the transgene for enhanced green fluorescent protein (eGFP). AAV2 outperformed AAV9 in transducing pancreatic cells, while AAV6 induced cytotoxicity. Both AAV2 and AAV9 displayed slightly higher tropism for -cells than for {beta}-cells. Compared to the pancreatic cells, the fraction of GFP-expressing cells at various multiplicities of infection was consistently lower for fibroblasts. Incubation of AAV2 with heparin prior to transduction failed to induce any GFP expression in {beta}-cells, indicating that the primary site used for initial interaction with pancreatic cells are heparan sulfate proteoglycans. Treatment of {beta}-cells with neuraminidase prior to AAV9 infection appeared to improve the number of GFP-positive cells, but the increase was not statistically significant. These findings expand the repertoire of available serotypes for AAV-mediated delivery of transgenes to pancreatic endocrine cells and may contribute to gene therapy strategies for pancreas pathologies.

Adeno-associated viruses (AAVs) have been used in gene therapy, especially for inherited retinal diseases. Despite their effectiveness in gene transduction, immune responses to the AAV capsid and transgene products have been reported, which can compromise both the efficacy and safety of AAV-mediated therapies. The eye is regarded as an immune-privileged organ where immune activity is constitutively suppressed. Here, we highlight that immunomonitoring in an ocular gene transfer reveals variable immune responses, whatever the species (human clinical trial, non-human primates, mice), the site of injection, the cassette, and the dose. We further explored factors contributing to this variability, investigating the correlation among immune parameters in a controlled experimental setting. In a syngeneic murine model after an intraocular injection of AAV, our results highlight an inter-individual variability of immune parameters, emphasizing the importance of considering inherent variability among individuals while designing personalized therapies.

Recombinant adeno-associated virus (rAAV) vectors are pivotal for gene therapy; however, the encapsidation of residual DNA, particularly plasmid backbone sequences, pose significant safety risks. Recent studies have identified the p5 promoter, which contains a Rep-binding element and a terminal resolution site (TRS), as a cryptic origin of replication that facilitates packaging of upstream sequences. In this study, we investigated the effect of p5 TRS modifications on impurity DNA levels in a single-plasmid All-in-One (AiO) AAV production system. Wild-type p5 (p5wt) promoted significant packaging of upstream plasmid backbone DNA, especially when the backbone was positioned between p5wt and the inverted terminal repeat. Introducing mutations or deletions in the p5 TRS significantly reduced encapsidation of plasmid-derived sequences, including kanamycin resistance genes, and improved the ratio of full to partial particles, as seen with the p5{Delta}loop variant. Furthermore, the p5{Delta}loop-AiO system showed higher rAAV yields than both conventional triple-transfection methods and previously reported p5-spacer variants. Thus, our findings suggest a robust vector design strategy for minimizing DNA impurities, thereby enhancing the safety and efficacy of AAV-based gene therapy.

Recombinant Adeno-associated viruses (AAVs) are widely used for gene delivery in the central nervous system and have become central tools in both gene therapy and basic neuroscience research. However, although AAV serotypes have been extensively characterized in rodent models, their performance in human neurons, particularly those derived from induced pluripotent stem cells (iPSCs), remains poorly characterized. While human iPSC-derived neurons are increasingly used for disease modeling and drug screening, their susceptibility to viral transduction varies and remains difficult to predict. In this study, we systematically evaluated the transduction efficiency and toxicity profiles of 18 wild-type and engineered AAV serotypes across three distinct types of iPSC-derived neurons, relevant to disease modeling and drug discovery: cortical projection neurons, NGN2- induced forebrain-like neurons, and dopaminergic neurons and four doses (1E3, 1E4, 1E5 and 2E5 genome copies per cell). Using automated high-throughput confocal imaging and quantification of reporter gene expression, we identified several serotypes with robust and efficient transduction across all neuronal subtypes. Among these, three serotypes AAV6, AAV6.2 and AAV2.7m8 showed consistently high performance. To assess safety, we quantified cell number and neurite morphology, finding that while high transduction and gene expression correlate with toxicity, sensitivity varied across neuronal subtypes, with NGN2 neurons being most vulnerable and dopaminergic neurons most resilient. Finally, we validated our findings in a more complex 3D model by testing one of the best-performing serotypes, AAV2.7m8, in both whole and dissociated human cerebellar organoids. Together, our results establish a benchmark dataset for AAV performance in human iPSC- derived neurons and provide practical guidance for AAV based gene delivery in human in vitro neural models. This resource will be valuable for both basic research and preclinical applications aiming to manipulate gene expression in human neurons and understanding AAV tropism in disease-relevant cell types.

The efficacy of adeno-associated virus (AAV)-mediated systemic gene therapy for central nervous system (CNS) diseases is often limited by the blood-brain barrier (BBB). This study systematically evaluated the tissue distribution of three BBB-crossing AAV capsid variants (PHP.eB, CNSRCV300, and BI-hTFR1) following intravenous injection in mice, using either a constitutive promoter (CAG) or a neuron-specific promoter (hSyn) to drive EGFP reporter expression. Compared with AAV9, both PHP.eB and CNSRCV300 demonstrated significantly enhanced BBB penetration and brain transduction efficiency. While the use of the hSyn promoter led to reduced transgene expression in the brain compared with the CAG promoter, and substantially decreased visible reporter expression in peripheral organs, viral deposition in the liver could still be detected via immunohistochemistry. Overall, CNSRCV300 exhibited the most favorable balance between brain-targeting efficiency and biosafety, highlighting its potential as a promising delivery vector. In summary, both the capsid and promoter jointly influence AAV-mediated expression in vivo, and although cell type-specific promoters can reduce off-target expression, residual viral deposition in non-target tissues remains a potential safety concern.

Recombinant adeno-associated virus (rAAV) is a widely used viral vector for gene therapy. However, a limitation of AAV-mediated gene therapy is that patients are typically dosed only once. In this study, we investigated the possiblility to deliver multiple rounds of AAV through intracerebral injections in the mouse brain. We discovered a dose-dependent modulation of the second round AAV infection by the first round AAV injection in the brain-wide scales besides the injection region. High-dose AAV infection increases chemokines CXCL9 and CXCL10 to recruit the parenchymal infiltration of lymphocytes. Surprisingly, the blood-brain-barrier was relatively intact. Brain-wide dissection discovered the likely rountes of the infiltrated lymphocytes through perivascular space and ventricles. Further analysis using B-cell depleted mice revealed that B lymphocytes, but not T lymphocytes, played a critical role in inhibiting the second round AAV infection. Strategies against neutralizing antibodies had limited effects, while reducing the dosage for the first injection or switching the second AAV to a different serotype appeared to be more effective in antagonizing the first round AAV inhibition. Together, these results suggest that mammalian brains are not immunoprivileged for AAV infection, but multiple rounds of AAV gene therapy are still possible if designed carefully with proper doses and serotypes.

For more than a decade, AAV-mediated gene transfer has been tested successfully in clinical trials to treat inherited retinal diseases. Despite the eyes immune-privileged status and the use of corticoids as an adjunct treatment, some patients display inflammatory events which led us to question the immune consequences of a subretinal AAV administration. We first characterized anti-transgene immune responses induced in the periphery by injecting increasing doses of AAV8 encoding reporter proteins fused with the HY male antigen into the subretinal space of female C57BL/6 and rd10 mice. Transgene expression was monitored over time with bioluminescence imaging and T-cell immune responses in the spleen were analyzed by IFN{gamma} ELISpot and cytokine multiplex assays. Our data show that an AAV8 injection causes proinflammatory T-cell immune response against the transgene product, correlated with the transgene expression level at 2.109 vg and above. Additionally, co-injection of immunodominant peptides from the transgene product, along with AAV8, modulates the immune response at all AAV doses tested. Taken together, our data suggest that injection of AAV8 in the subretinal space induces proinflammatory peripheral T-cell responses to the transgene product that can be modulated by the subretinal associated immune inhibition (SRAII) mechanism.

Cone photoreceptors mediate daylight vision and are the primary cells responsible for vision in humans. Cone dysfunction leads to poor quality daylight vision because rod photoreceptors become saturated and non-functional at high light levels. Here we demonstrate that in mice lacking cone function, AAV-mediated over-expression of Rgs9-anchor protein (R9AP), a critical component of the GTPase complex that mediates the deactivation of the phototransduction cascade, results in desensitization of rod function and a "photopic shift" of the rod-driven electroretinogram. This treatment enables rods to respond to brighter light (up to [~]2.0 log) with increased visually-evoked cortical responses to high intensity stimulation. These results suggest that AAV-mediated transfer of R9ap into rods might be used to improve daylight vision in humans visually handicapped by cone dysfunction.

Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal delivery of AAV vectors into the cerebral spinal fluid (CSF) can avoid many of the issues of systemic delivery, although achieving broad distribution of the vector and transgene expression throughout the spinal cord is challenging and vector entry to the periphery occurs, sometimes initiating hepatotoxicity. Here we performed two rounds of in vivo biopanning in non-human primates (NHPs) with an AAV9 peptide display library injected intrathecally and performed insert sequencing on DNA isolated from either whole tissue (conventional selection), isolated nuclei, or nuclei from transgene-expressing cells. A subsequent barcoded pool of candidates and AAV9 was compared at the DNA (biodistribution) and RNA (expression) level in spinal cord and liver of intrathecally injected NHPs. Most of the candidates displayed enhanced biodistribution compared to AAV9 at all levels of spinal cord ranging from 2 to 265-fold. Nuclear isolation or expression-based selection yielded 4 of 7 candidate capsids with enhanced transgene expression in spinal cord (up to 2.4-fold), while no capsid obtained by conventional selection achieved that level. Furthermore, several capsids displayed lower biodistribution to the liver of up to 1,250-fold, compared to AAV9, providing a remarkable on target/off target biodistribution ratio. These capsids may have potential for gene therapy programs directed at the spinal cord and the selection method described here should be useful in clinically relevant large animal models.

When producing Adeno-Associated Virus (AAV) gene therapies, a significant fraction of capsids can lack the desired DNA cargo. In AAV, Rep proteins mediate DNA packaging and virus assembly, suggesting that changes in Rep activity, expression, or DNA binding might affect genome packaging. To understand how mutations in the Rep gene affect activity, we selected a library of Rep mutants for their ability to produce active virions. By sequencing the Rep gene following the purification of viruses that package AAV genomes, we identified Rep mutants having non-synonymous mutations with a range of cellular activities. Surprisingly, synonymous mutations within the p19 promoter were enriched to the greatest extent, increasing in abundance by 102 to 104-fold. When the most highly enriched mutant was used to package a synthetic DNA cargo into the AAV capsid, the packaging efficiency could not be differentiated from native Rep. These findings suggest that these synonymous mutations enhance AAV genome packaging into capsids by affecting Rep-DNA interactions. They also suggest that silent sequence changes in the DNA cargo packaged by Rep can be used to tune packaging DNA packaging efficiency. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/617207v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1ba20d0org.highwire.dtl.DTLVardef@c05a6corg.highwire.dtl.DTLVardef@70a248org.highwire.dtl.DTLVardef@ae587c_HPS_FORMAT_FIGEXP M_FIG C_FIG

AbstractsPatients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10 for whom cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knock-in mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-hTMPRSS3 injection in the adult knock-in mouse inner ears results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. A single AAV2-hTMPRSS3 injection in aged Tmprss3A306T/A306T mice leads to sustained rescue of the auditory function, to a level similar to the wildtype mice. AAV2-hTMPRSS3 delivery rescues the hair cells and the spiral ganglions. This is the first study to demonstrate successful gene therapy in an aged mouse model of human genetic deafness. This study lays the foundation to develop AAV2-hTMPRSS3 gene therapy to treat DFNB8 patients, as a standalone therapy or in combination with cochlear implantation.