AAV Transduction Research Articles

Comparative analysis of six adeno-associated viral vector serotypes in mouse inferior colliculus and cerebellum.

AAVs have become ubiquitous gene expression tools in neuroscience research and are becoming more common in clinical settings. Naturally occurring and engineered serotypes have varying abilities to infect neurons and cause them to produce proteins of interest. The efficacy of AAV transduction in specific cell types depends on many factors and remains difficult to predict, so an empirical approach is often required to determine the best performing serotype in each population of cells. In the present study we show that AAV1 produces the highest expression in these two regions, labels the most axonal projections, and labels Purkinje cells and unipolar brush cells better than the other serotypes tested, while AAV2 labels granule cells most effectively.

Identification of SLC35A1 as an essential host factor for the transduction of multi-serotype recombinant adeno-associated virus (AAV) vectors.

rAAV is an essential tool for gene delivery in the treatment of genetic disorders, yet the mechanisms of rAAV transduction remain partially understood. GPR108 is vital for the transduction of most rAAV vectors, but not for rAAV5. We aimed to identify host factors that impact AAV5 transduction akin to GPR108. Using a genome-wide CRISPR/Cas9 screen in 293-F cells, we identified SLC35A1, a Golgi apparatus-localized CMP-sialic acid transporter that transports CMP-sialic acid from cytoplasm into the Golgi apparatus for sialylation, is essential to rAAV transduction. Further studies across various AAV serotypes showed SLC35A1 significantly affects vector nuclear import post-internalization. These results underscore the crucial role of SLC35A1 in intracellular trafficking beyond the initial cell attachment of rAAV.

Inhalation of SP-101 Followed by Inhaled Doxorubicin Results in Robust and Durable hCFTRΔR Transgene Expression in the Airways of Wild-Type and Cystic Fibrosis Ferrets.

Cystic fibrosis (CF) is a serious genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Approved small molecule therapies benefit the majority of people with CF (pwCF), but unfortunately not all. Gene addition offers a mutation agnostic treatment option for all pwCF. SP-101 is an adeno-associated virus gene therapy vector (AAV2.5T) that has been optimized for efficient human airway cell transduction, and that contains a functional and regulated shortened human CFTR minigene (hCFTRΔR) with a small synthetic promoter/enhancer. To understand SP-101 airway distribution, activity, and the associated immune response, invivo studies were performed in wild-type and CF ferrets. After single dose inhaled delivery of SP-101, followed by single dose inhaled doxorubicin (an AAV transduction augmenter) or saline, SP-101 vector genomes were detected throughout the respiratory tract. hCFTRΔR mRNA expression was highest in ferrets also receiving doxorubicin and persisted for the duration of the study (13 weeks). Pre-existing mucus in the CF ferrets did not present a barrier to effective transduction. Binding and neutralizing antibodies to the AAV2.5T capsid were observed regardless of doxorubicin exposure. Only a portion of ferrets exhibited a weak T-cell response to AAV2.5T and no T-cell response was seen against hCFTRΔR. These data strongly support the continued development of inhaled SP-101, followed by inhaled doxorubicin, for the treatment of CF.

Endothelial-to-mesenchymal transition enhances permissiveness to AAV vectors in cardiac endothelial cells

A major obstacle in inducing therapeutic angiogenesis in the heart is inefficient gene transfer to endothelial cells (ECs). Here, we identify compounds able to enhance the permissiveness of cardiac ECs to adeno-associated virus (AAV) vectors, which stand as ideal tools for invivo gene delivery. We screened a library of >1,500US Food and Drug Administration (FDA)-approved drugs, in combination with AAV vectors, in cardiac ECs. Among the top drugs increasing AAV-mediated transduction, we found vatalanib, an inhibitor of multiple tyrosine kinase receptors. The increased AAV transduction efficiency by vatalanib was paralleled by induction of the endothelial-to-mesenchymal transition, asdocumented by decreased endothelial and increased mesenchymal marker expression. Induction of the endothelial-to-mesenchymal transition by other strategies similarly increased EC permissiveness to AAV vectors. Invivo injection of AAV vectors in the heart after myocardial infarction resulted in the selective transduction of cells undergoing the endothelial-to-mesenchymal transition, which is known to happen transiently after cardiac ischemia. Collectively, these results point to the endothelial-to-mesenchymal transition as a mechanism for improving AAV transduction in cardiac ECs, with implications for both basic research and the induction of therapeutic angiogenesis in the heart.

Genome-wide CRISPR screenings identified SMCHD1 as a host-restricting factor for AAV transduction.

AAV-mediated gene therapy typically requires a high dose of viral transduction, risking acute immune responses and patient safety, part of which is due to limited understanding of the host-viral interactions, especially post-transduction viral genome processing. Here, through a genome-wide CRISPR screen, we identified SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1), an epigenetic modifier, as a critical broad-spectrum restricting host factor for post-entry AAV transgene expression. SMCHD1 knock-down by RNAi and CRISPRi or knock-out by CRISPR all resulted in significantly enhanced transgene expression across multiple viral serotypes, as well as for both single-strand and self-complementary AAV genome types. Mechanistically, upon viral transduction, SMCHD1 effectively repressed AAV transcription by the formation of an LRIF1-HP1-containing protein complex and directly binding with the AAV genome to maintain a heterochromatin-like state. SMCHD1-KO or LRIF1-KD could disrupt such a complex and thus result in AAV transcriptional activation. Together, our results highlight the host factor-induced chromatin remodeling as a critical inhibitory mechanism for AAV transduction and may shed light on further improvement in AAV-based gene therapy.

Enhanced AAV transduction across preclinical CNS models: A comparative study in human brain organoids with cross-species evaluations

Viral vectors based on recombinant adeno-associated virus (rAAV) have become the most widely used system for therapeutic gene delivery in the central nervous system (CNS). Despite clinical safety and efficacy in neurological applications, a barrier to adoption of the current generation of vectors lies in their limited efficiency, resulting in limited transduction of CNS target cells. To address this limitation, researchers have bioengineered fit-for-purpose AAVs with improved CNS tropism and tissue penetration. While the preclinical assessment of these novel AAVs is primarily conducted in animal models, human induced pluripotent stem cell (hiPSC)-derived organoids offer a unique opportunity to functionally evaluate novel AAV variants in a human context. In this study, we performed a comprehensive and unbiased evaluation of a large number of wild-type and bioengineered AAV capsids for their transduction efficiency in hiPSC-derived brain organoids. We demonstrate that efficient AAV transduction observed in organoids was recapitulated in vivo in both mouse and non-human primate models following cerebrospinal fluid (CSF) delivery. In summary, our study showcases the utilization of brain organoid systems for the pre-screening of novel AAV vectors. Additionally, we report data for novel AAV variants that exhibit improved CNS transduction efficiency when delivered via the CSF in in vivo preclinical models.

Electric pulse exposure reduces AAV8 dosage required to transduce HepG2 cells.

We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.

Deep Learning for Cell Migration in Nonwoven Materials and Evaluating Gene Transfer Effects following AAV6-ND4 Transduction.

Studying cell settlement in the three-dimensional structure of synthetic biomaterials over time is of great interest in research and clinical translation for the development of artificial tissues and organs. Tracking cells as physical objects improves our understanding of the processes of migration, homing, and cell division during colonisation of the artificial environment. In this study, the 3D environment had a direct effect on the behaviour of biological objects. Recently, deep learning-based algorithms have shown significant benefits for cell segmentation tasks and, furthermore, for biomaterial design optimisation. We analysed the primary LHON fibroblasts in an artificial 3D environment after adeno-associated virus transduction. Application of these tools to model cell homing in biomaterials and to monitor cell morphology, migration and proliferation indirectly demonstrated restoration of the normal cell phenotype after gene manipulation by AAV transduction. Following the 3Rs principles of reducing the use of living organisms in research, modeling the formation of tissues and organs by reconstructing the behaviour of different cell types on artificial materials facilitates drug testing, the study of inherited and inflammatory diseases, and wound healing. These studies on the composition and algorithms for creating biomaterials to model the formation of cell layers were inspired by the principles of biomimicry.

A humanized mouse model for adeno-associated viral gene therapy

Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg−/−/Rag2−/−/Fah−/−/Aavr−/− (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.

A gene therapy targeting medium-chain acyl-CoA dehydrogenase (MCAD) did not protect against diabetes-induced cardiac pathology.

Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. KEY MESSAGES: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications.

Identification of host essential factors for recombinant AAV transduction of the polarized human airway epithelium.

The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

Casein kinase 2 activity is a host restriction factor for AAV transduction

So far, the mechanisms that impede AAV transduction, especially in the human heart, are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs), iPSC-derived cardiac fibroblasts (iPSC-CFs), and primary endothelial cells to model vector-host interactions. Through phosphoproteome analysis we established that casein kinase 2 (CK2) signaling is one of the most significantly affected pathways upon AAV exposure. Transient inhibition of CK2 activity substantially enhanced the transduction rate of AAV2, AAV6, and AAV9 in all tested cell types. In particular, CK2 inhibition improved the trafficking of AAVs through the cytoplasm, impaired DNA damage response through destabilization of MRE11, and altered the RNA processing pathways, which were also highly responsive to AAV transduction. Also, it augmented transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. In summary, the present study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, which may translate to improving the outcome of AAV-based therapies in the future.

MAGI2 ameliorates podocyte apoptosis of diabetic kidney disease through communication with TGF-β-Smad3/nephrin pathway.

Podocytes, the key component of the glomerular filtration barrier (GFB), are gradually lost during the progression of diabetic kidney disease (DKD), severely compromising kidney functionality. The molecular mechanisms regulating the survival of podocytes in DKD are incompletely understood. Here, we show that membrane-associated guanylate kinase inverted 2 (MAGI2) is specifically expressed in renal podocytes, and promotes podocyte survival in DKD. We found that MAGI2 expression was downregulated in podocytes cultured with high-glucose invitro, and in kidneys of db/db mice as well as DKD patients. Conversely, we found enforced expression of MAGI2 via AAV transduction protected podocytes from apoptosis, with concomitant improvement of renal functions. Mechanistically, we found that MAGI2 deficiency induced by high glucose levels activates TGF-β signaling to decrease the expression of anti-apoptotic proteins. These results indicate that MAGI2 protects podocytes from cell death, and can be harnessed therapeutically to improve renal function in diabetic kidney disease.

The circRNA MKLN1 regulates autophagy in the development of diabetic retinopathy

Diabetic retinopathy (DR) is a common complication in patients with diabetes and has become an important cause of blindness in working-age people. However, the mechanisms involved have not been fully elucidated. Circular RNAs (circRNAs) can play an important role in DR, and they can accurately regulate the expression of target genes through a new regulatory model: the competing endogenous RNA (ceRNA) model. We isolated total RNA from extracellular vesicles in the serum of healthy individuals (Con) and individuals with diabetes mellitus without DR (DM), nonproliferative DR (NPDR), or proliferative DR (PDR) and subjected them to deep sequencing. We found aberrantly high expression of circMKLN1. In a streptozotocin (STZ)-induced mice model of diabetes, the inhibition of circMKLN1 with AAV2 transduction markedly ameliorated retinal acellular vessels and vascular leakage, which was reversed by intravitreal injection of rapamycin, a potent autophagy inducer. In addition, circMKLN1 adsorbs miR-26a-5p as a molecular sponge and mediates high glucose (HG)/methylglyoxal (MG)-induced autophagy in hRMECs. CircMKLN1-silencing treatment reduces HG/MG-related reactive autophagy and inflammation. In addition, miR-26a-5p targeting by circMKLN1 plays an important role in the regulation of Rab11a expression. Thus, either new biomarkers or new therapeutic targets may be identified with the translation of these findings.

A comparison of the impact on neuronal transcriptome and cognition of rAAV5 transduction with three different doses in the mouse hippocampus.

Recombinant adeno-associated viruses (rAAVs) are widely used in genetic therapeutics. AAV5 has shown superior transduction efficiency, targeting neurons and glial cells in primate brains. Nonetheless, the comprehensive impact of AAV5 transduction on molecular and behavioral alterations remains unexplored. This study focuses on evaluating the effects of AAV5 transduction in the hippocampus, a critical region for memory formation and emotional processes. In this experiment, fluorescence-activated cell sorting (FACS) was utilized to isolate the mCherry-labeled pyramidal neurons in the hippocampus of CaMkIIα-cre mice following three different doses rAAV5-mCherry infusion after 3 weeks, which were then subjected to RNA sequencing (RNA-seq) to assess gene expression profiles. The cytokines concentration, mRNA expression, and glial response in hippocampi were confirmed by ELASA, digital droplet PCR and immunohistochemistry respectively. Locomotion and anxiety-like behaviors were elevated by Open Field Test and Elevated Plus Maze Test, while the Y-Maze were used to assessed spatial working memory. Recognition memory and fear responses were examined by the Novel Object Recognition Test and Fear Conditioning Test, respectively. We found that 2.88 × 1010 v.g rAAV5 transduction significantly upregulated genes related to the immune response and apoptosis, and downregulated genes associated with mitochondrial function and synaptic plasticity in hippocampal pyramidal neurons, while did not induce neuronal loss and gliosis compared with 2.88 × 109 v.g and 2.88 × 108 v.g. Furthermore, the same doses impaired working memory and contextual fear memory, without effects on locomotion and anxiety-related behaviors. Our findings highlight the detrimental impact of high-dose administration compared to median-dose or low-dose, resulting in increased neural vulnerability and impaired memory. Therefore, when considering the expression effectiveness of exogenous genes, it is crucial to also take potential side effects into account in clinical settings. However, the precise molecular mechanisms underlying these drawbacks of high-dose rAAV5-mCherry still require further investigation in future studies.

Semi-automated workflows to quantify AAV transduction in various brain areas and predict gene editing outcome for neurological disorders

One obstacle to the development of gene therapies for the central nervous system is the lack of workflows for quantifying transduction efficiency in affected neural networks and ultimately predicting therapeutic potential. We integrated data from a brain cell atlas with 3D or 2D semi-automated quantification of transduced cells in segmented images to predict AAV transduction efficiency in multiple brain regions. We used this workflow to estimate the transduction efficiency of AAV2/rh.10 and AAV2.retro co-injection in the corticostriatal network affected in Huntington's disease. We then validated our pipeline in gene editing experiments targeting both human and mouse huntingtin genes in transgenic and wild-type mice, respectively. Our analysis predicted that 54% of striatal cells and 7% of cortical cells would be edited in highly transduced areas. Remarkably, in the treated animals, huntingtin gene inactivation reached 54.5% and 9.6%, respectively. These results demonstrate the power of this workflow to predict transduction efficiency and the therapeutic potential of gene therapies in the central nervous system.

Fundamental and State-of-the-Art Technologies In Retinal Studies and Disease Repair

ARTICLES DISCUSSED: Ma, Y., Li., T. Monitoring dynamic growth of retinal vessels in oxygen-induced retinopathy mouse model. Journal of Visualized Experiments. (170), e62410 (2021). Guan, Y., Xie, B. Zhong, X. Retinal organoid induction system for derivation of 3D retinal tissues from human pluripotent stem cells. Journal of Visualized Experiments. (170), e62435 (2021). Rose, K., Lokappa, S. Chen, J. Two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis. Journal of Visualized Experiments. (178), e62977 (2021). Ye., Q. et al. In vivo methods to assess retinal ganglion cell and optic nerve function and structure in large animals. Journal of Visualized Experiments. (180), e62879 (2022). Abbas, F., Vinberg, F., Becker, S. Optimizing the setup and conditions for ex vivo electroretinogram to study retina function in small and large eyes. Journal of Visualized Experiments. (184), e62763 (2022). Okan, I., Ahmadian, M., Tutuncu, Y., Altay, H., Agca, C. Digital droplet PCR method for the quantification of AAV transduction efficiency in murine retina. Journal of Visualized Experiments. (178), e63038 (2021).

Long non-coding RNA rhabdomyosarcoma 2-associated transcript contributes to neuropathic pain by recruiting HuR to stabilize DNA methyltransferase 3 alpha mRNA expression in dorsal root ganglion neuron.

Long non-coding RNAs (lncRNAs) act as key regulators in multiple human diseases. In particular, the dysfunction of lncRNAs in dorsal root ganglion (DRG) contributes to the pathogenesis of neuropathic pain (NP). Nevertheless, the role and mechanism of most lncRNAs in NP remain unclear. Two classic chronic NP models, including L4 spinal nerve ligation (SNL) model and chronic constriction injury (CCI) of the sciatic nerve, were performed. Mechanical allodynia and heat hyperalgesia were used to evaluate neuropathic pain. DRG microinjection was used to deliver agents into DRG. qRT-PCR, immunofluorescence, immunoprecipitation, western blotting, siRNA transfection, AAV transduction were performed to investigate the phenotypes and molecular basis. Here, we discovered that Rmst as a lncRNA was specifically expressed in Atf3 + injured DRG neurons and significantly upregulated following peripheral nerve damage. Rmst overexpression by direct DRG injection of AAV5-Rmst causes neuropathic symptoms in the absence of nerve damage. Conversely, blocking Rmst expression in injured DRGs alleviated nerve injury-induced pain hypersensitivities and downregulated Dnmt3a expression. Furthermore, we found peripheral nerve damage induced Rmst increase could interact with RNA-binding protein HuR to stabilize the Dnmt3a mRNA. Our findings reveal a crucial role of Rmst in damaged DRG neurons under NP condition and provide a novel target for drug development against NP.

AAV- based vector improvements unrelated to capsid protein modification.

Recombinant adeno-associated virus (rAAV) is the leading platform for delivering genetic constructs in vivo. To date, three AAV-based gene therapeutic agents have been approved by the FDA and are used in clinical practice. Despite the distinct advantages of gene therapy development, it is clear that AAV vectors need to be improved. Enhancements in viral vectors are mainly associated with capsid protein modifications. However, there are other structures that significantly affect the AAV life cycle and transduction. The Rep proteins, in combination with inverted terminal repeats (ITRs), determine viral genome replication, encapsidation, etc. Moreover, transgene cassette expression in recombinant variants is directly related to AAV production and transduction efficiency. This review discusses the ways to improve AAV vectors by modifying ITRs, a transgene cassette, and the Rep proteins.

Durability of Bleeding Protection and Factor IX Activity Levels Are Demonstrated in Individuals with and without Adeno-Associated Virus Serotype 5 Neutralizing Antibodies (Titers

Introduction: Pre-existing adeno-associated virus (AAV) neutralizing antibodies (NAbs) have limited the efficacy of AAV-based gene therapy in prior clinical applications, including in hemophilia. A unique aspect of the Phase 3 HOPE-B clinical trial (NCT03569891) assessing etranacogene dezaparvovec gene therapy, an AAV serotype 5 vector expressing Padua factor IX (FIX), was the enrollment of participants regardless of their baseline AAV5 NAb status. The HOPE-B clinical trial met its primary efficacy endpoint, providing hemostatic protection superior to standard of care FIX prophylaxis over 52 weeks of follow-up after stable FIX Padua expression (defined as Months 7-18). Aim: Assess efficacy and safety of etranacogene dezaparvovec in HOPE-B participants with (NAb+) and without (NAb-) pre-existing AAV5 NAbs over 18 and 24 months of follow-up. Methods: Adult male participants with severe or moderately severe hemophilia B (FIX ≤2%), NAb+ or NAb-, were treated in the Phase 3, open-label, single-arm, HOPE-B trial with a single intravenous infusion of etranacogene dezaparvovec (2x1013 gc/kg), following a ≥6-month lead-in period receiving FIX prophylaxis. FIX activity, annualized bleed rate (ABR), and use of infused replacement FIX concentrates were assessed regularly during the lead-in and the first 12 months after receiving etranacogene dezaparvovec, then every 6 months during the long-term follow-up (Years 2-5). Adverse events were recorded continuously. Although not an exclusion criterion, AAV5 NAbs were assessed on the day of intravenous dosing (baseline), using a custom-developed, cell-based in vitro AAV5 transduction inhibition assay (sensitivity 10 ng/mL antibody, titer 1:7, intra and inter-run coefficient of variation [CV] <30%). Results: Of the 54 participants who received etranacogene dezaparvovec, at baseline 33 participants were NAb- and 21 were NAb+. The median (Q1-Q3) titer among NAb+ participants was 56.9 (23.3-198.9) and 20/21 (95%) NAb+ participants had titers of <1:700. One participant with a markedly high NAb titer of 3212 prior to vector dosing and one participant who only received a partial dose (due to an infusion-related reaction; NAb titer: 198.9), did not express FIX Padua and did not discontinue FIX prophylaxis. All other participants (52/54) discontinued FIX prophylaxis. At 18- and 24-months post-dose, no clinically meaningful correlation between an individual's baseline AAV5 NAb titer and FIX activity levels was identified, up to a NAb titer of <1:700 (24-month Pearson coefficient: -0.29; Spearman coefficient: -0.25; R2: 0.086). Sustained FIX activity levels (median [min-max]) were demonstrated at 18 months post-dose in NAb+ <1:700 (32.0% [10.3-57.9]) and NAb- (35.0% [4.5-122.9]) participants and at 24 months post-dose in NAb+ <1:700 (33.5% [9.1-88.3]) and the NAb- (35.4% [4.7-99.2]) participants (Table 1). At both 18- and 24-months post-dose, NAb+ <1:700 and NAb- participants demonstrated a low ABR (Table 2). The ABR achieved by the NAb+ <1:700 and NAb- subgroups at 18 months was 1.30 and 0.93, and at 24 months was 1.65 and 0.80, respectively. These ABRs were significantly improved compared with the respective ABRs of 4.29 (p=0.0005 and p=0.0065 at 18 and 24 months, respectively) and 3.80 (p<0.0001 at 18 and 24 months) observed during the ≥6-month lead-in period of continuous FIX prophylaxis. The safety profile of etranacogene dezaparvovec was similar between NAb subgroups. At 24 months, corticosteroid-treated transaminase elevations occurred in 6/33 NAb- (18.2%) and 3/21 NAb+ (14.3%) participants. Infusion-related reactions occurred in 2/33 NAb- (6.1%) and 5/21 NAb+ (23.8%) participants. There was no statistically significant association between infusion-related reactions and NAb status (p=0.0956). Conclusions: Throughout 24 months of follow-up, AAV5 NAb- and NAb+ (<700 titer) HOPE-B participants receiving etranacogene dezaparvovec demonstrated significant reductions in ABR, freedom from continuous FIX prophylaxis and a comparable and acceptable safety profile, regardless of NAb status. FIX activity levels were stable, with no association between baseline NAb status (up to titer <1:700) and the long-term durability of FIX expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal