Viral Gene Therapy Research Articles

Rationally engineered novel AAV capsids for intra-articular gene delivery

Intra-articular adeno-associated virus (AAV) gene therapy has been explored as a potential strategy for joint diseases. However, concerns of low transduction efficacy, off-target expression, and neutralizing antibodies (Nabs) still need to be addressed. In this study, we demonstrated that AAV6 was the best serotype to transduce joints after screening serotypes 1 to 9. To develop a more effective AAV vector, a set of novel AAV capsids were rationally engineered. The mutant AAV62 created by swapping variable region I (VRI) of AAV2 into AAV6 induced a higher transduction efficiency per AAV genome copy number. To further investigate the roles of specific amino acids in the transduction of AAV62 and AAV6, we found out that AAV6D with the deletion of threonine at residue 265 induced a 2-fold higher transduction than AAV6, while the transduction efficiency from AAV6M with the mutation of alanine to glutamine at residue 263 was 10-fold lower. AAV6D efficiently transduced both synoviocytes and chondrocytes with low AAV genome copy numbers in other tissues and less Nab formation. This study demonstrates that novel AAV mutants with rational engineering may enhance joint transduction after intra-articular administration in mice, with the potential to evade AAV Nabs and minimize off-target effects in the liver.

Treatment of infantile-onset Pompe disease in a rat model with muscle-directed AAV gene therapy

ObjectivePompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy. MethodsPD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured. ResultsPD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate. ConclusionsThis gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.

AAV gene therapy in companion dogs with severe hemophilia: Real-world long-term data on immunogenicity, efficacy, and quality of life

The hemophilias are the most common severe inherited bleeding disorders and are caused by deficiency of clotting factor (F) VIII (hemophilia A) or FIX (hemophilia B). The resultant bleeding predisposition significantly increases morbidity and mortality. The ability to improve the bleeding phenotype with modest increases in clotting factor levels has enabled the development and regulatory approval of adeno-associated viral (AAV) vector gene therapies for people with hemophilia A and B. The canine hemophilia model has proven to be one of the best predictors of therapeutic response in humans. Here, we report long-term follow-up of 12 companion dogs with severe hemophilia that were treated in a real-world setting with AAV gene therapy. Despite more baseline bleeding than in research dogs, companion dogs demonstrated a 94% decrease in bleeding rates and 61% improvement in quality-of-life over a median of 4.1 years (range 2.6-8.9). No new anti-transgene immune responses were detected; 1 dog with a preexisting anti-FVIII inhibitor achieved immune tolerance with gene therapy. Two dogs expressing 1-5% FVIII post gene therapy experienced fatal bleeding events. These data suggest AAV liver-directed gene therapy is efficacious in a real-world setting, but should target expression > 5% and closely monitor those with levels in the 1-5% range.

Adeno-associated viral gene therapy for sialidosis using small and large animal models

Adeno-associated viral gene therapy for sialidosis using small and large animal models

Human Pluripotent Stem Cell-Derived Retinal Organoids: A Viable Platform for Investigating the Efficacy of Adeno-Associated Virus Gene Therapy.

With recent advances in adeno-associated virus (AAV)-based gene therapy, efficacy and toxicity screening have become essential for developing gene therapeutic drugs for retinal diseases. Retinal organoids from human pluripotent stem cells (hPSCs) offer a more accessible and reproducible human test platform for evaluating AAV-based gene therapy. In this study, hPSCs were differentiated into retinal organoids composed of various types of retinal cells. The transduction efficiencies of AAV2 and AAV8, which are widely used in clinical trials of inherited retinal diseases, were analyzed using retinal organoids. These results suggest that retinal organoids derived from hPSCs serve as suitable screening platforms owing to their diverse retinal cell types and similarity to the human retina. In summary, we propose an optimal stepwise protocol that includes the generation of retinal organoids and analysis of AAV transduction efficacy, providing a comprehensive approach for evaluating AAV-based gene therapy for retinal diseases.

Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Improving the safety of systemic viral gene therapy.

Improving the safety of systemic viral gene therapy.

Enkephalin Rescues Temporomandibular Joint Pain-Related Behavior in Rats.

Temporomandibular joint disorders include a variety of clinical syndromes that are difficult to manage if associated with debilitating severe jaw pain. Thus, seeking additional experimental therapies for temporomandibular joint pain reduction is warranted. Targeted enkephalin gene therapy approaches provide clear promise for pain control. The studies detailed here indicate significant analgesia and protection of joint tissue are provided after injection of an overexpression viral vector gene therapy near the joint. The viral vector gene therapy described provides overexpression of naturally occurring opioid peptides after its uptake by trigeminal nerve endings. The viral vectors act as independent "minipump" sources for the opioid peptide synthesis in the neuronal cytoplasm producing the intended biological function, reduction of pain, and tissue repair. The antinociceptive effects provided with this delivery method of opioid expression persist for over 4weeks. This is coincident with the expected time frame for the duration of the transgene overproduction of the endogenous opioid peptide before its diminution due to dormancy of the virus. These experimental studies establish a basis for the use of replication-defective herpes simplex type 1-based gene therapy for severe chronic inflammatory temporomandibular joint destruction and pain. As innovative means of significantly reducing joint inflammation and preserving tissue architecture, gene therapies may extend their clinical usefulness for patients with temporomandibular joint disorders.

Gene therapy for neurotransmitter-related disorders.

Inborn errors of neurotransmitter (NT) metabolism are a group of rare, heterogenous diseases with predominant neurological features, such as movement disorders, autonomic dysfunction, and developmental delay. Clinical overlap with other disorders has led to delayed diagnosis and treatment, and some conditions are refractory to oral pharmacotherapies. Gene therapies have been developed and translated to clinics for paediatric inborn errors of metabolism, with 38 interventional clinical trials ongoing to date. Furthermore, efforts in restoring dopamine synthesis and neurotransmission through viral gene therapy have been developed for Parkinson's disease. Along with the recent European Medicines Agency (EMA)and Medicines and Healthcare Products Regulatory Agency (MHRA) approval of an AAV2 gene supplementation therapy for AADC deficiency, promising efficacy and safety profiles can be achieved in this group of diseases. In this review, we present preclinical and clinical advances to address NT-related diseases, and summarise potential challenges that require careful considerations for NT gene therapy studies.

Liver-directed gene therapy for inherited metabolic diseases.

Gene therapy clinical trials are rapidly expanding for inherited metabolic liver diseases whilst two gene therapy products have now been approved for liver based monogenic disorders. Liver-directed gene therapy has recently become an option for treatment of haemophilias and is likely to become one of the favoured therapeutic strategies for inherited metabolic liver diseases in the near future. In this review, we present the different gene therapy vectors and strategies for liver-targeting, including gene editing. We highlight the current development of viral and nonviral gene therapy for a number of inherited metabolic liver diseases including urea cycle defects, organic acidaemias, Crigler-Najjar disease, Wilson disease, glycogen storage disease Type Ia, phenylketonuria and maple syrup urine disease. We describe the main limitations and open questions for further gene therapy development: immunogenicity, inflammatory response, genotoxicity, gene therapy administration in a fibrotic liver. The follow-up of a constantly growing number of gene therapy treated patients allows better understanding of its benefits and limitations and provides strategies to design safer and more efficacious treatments. Undoubtedly, liver-targeting gene therapy offers a promising avenue for innovative therapies with an unprecedented potential to address the unmet needs of patients suffering from inherited metabolic diseases.

A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios.

The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild-type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2, and VP3, from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires a theoretical modification of the translational context. We have developed a Lac repressor-inducible system to empirically regulate the expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer, and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that the ratios of capsid proteins expressed were different than the ratios that ultimately were in purified capsids. Overexpressed VP1 did not become incorporated into capsids, while overexpressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling the production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.

Viral Vector-Based Gene Therapy for Epilepsy: What Does the Future Hold?

In recent years, many pre-clinical studies have tested gene therapy approaches as possible treatments for epilepsy, following the idea that they may provide an alternative to conventional pharmacological and surgical options. Multiple gene therapy approaches have been developed, including those based on anti-sense oligonucleotides, RNA interference, and viral vectors. In this opinion article, we focus on translational issues related to viral vector-mediated gene therapy for epilepsy. Research has advanced dramatically in addressing issues like viral vector optimization, target identification, strategies of gene expression, editing or regulation, and safety. Some of these pre-clinically validated potential gene therapies are now being tested in clinical trials, in patients with genetic or focal forms of drug-resistant epilepsy. Here, we discuss the ongoing translational research and the advancements that are needed and expected in the near future. We then describe the clinical trials in the pipeline and the further challenges that will need to be addressed at the clinical and economic levels. Our optimistic view is that all these issues and challenges can be overcome, and that gene therapy approaches for epilepsy will soon become a clinical reality.

Distributional comparison of different AAV vectors after unilateral cochlear administration.

The adeno-associated virus (AAV) gene therapy has been widely applied to mouse models for deafness. But, AAVs could transduce non-targeted organs after inner ear delivery due to their low cell-type specificity. This study compares transgene expression and biodistribution of AAV1, AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB after round window membrane (RWM) injection in neonatal mice. The highest virus concentration was detected in the injected cochlea. AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB transduced both inner hair cells (IHCs) and outer hair cells (OHCs) with high efficiency, while AAV1 transduced IHCs with high efficiency but OHCs with low efficiency. All AAV subtypes finitely transduced contralateral inner ear, brain, heart, and liver compared with the injected cochlea. In most brain regions, the enhanced green fluorescent protein (eGFP) expression of AAV1 and AAV2 was lower than that of other four subtypes. We suggested the cochlear aqueduct might be one of routes for vectors instantaneously infiltrating into the brain from the cochlea through a dye tracking test. In summary, our results provide available data for further investigating the biodistribution of vectors through local inner ear injection and afford a reference for selecting AAV serotypes for gene therapy toward deafness.

AAV capsid bioengineering in primary human retina models

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

Ataxia with giant axonopathy in Acbd5-deficient mice halted by adeno-associated virus gene therapy.

Acyl-CoA binding domain containing 5 (ACBD5) is a critical player in handling very long chain fatty acids (VLCFA) en route for peroxisomal β-oxidation. Mutations in ACBD5 lead to the accumulation of VLCFA and patients present retinal dystrophy, ataxia, psychomotor delay and a severe leukodystrophy. Using CRISPR/Cas9, we generated and characterized an Acbd5 Gly357* mutant allele. Gly357* mutant mice recapitulated key features of the human disorder, including reduced survival, impaired locomotion and reflexes, loss of photoreceptors, and demyelination. The ataxic presentation of Gly357* mice involved the loss of cerebellar Purkinje cells and a giant axonopathy throughout the CNS. Lipidomic studies provided evidence for the extensive lipid dysregulation caused by VLCFA accumulation. Following a proteomic survey, functional studies in neurons treated with VLCFA unravelled a deregulated cytoskeleton with reduced actin dynamics and increased neuronal filopodia. We also show that an adeno-associated virus-mediated gene delivery ameliorated the gait phenotypes and the giant axonopathy, also improving myelination and astrocyte reactivity. Collectively, we established a mouse model with significance for VLCFA-related disorders. The development of relevant neuropathological outcomes enabled the understanding of mechanisms modulated by VLCFA and the evaluation of the efficacy of preclinical therapeutic interventions.

Contributed Session III: AAV-mediated gene therapy for PDE6C achromatopsia: Progress and challenges.

To determine the clinical circumstances under which viral mediated gene therapy can rescue cone function in a nonhuman primate model of PDE6C achromatopsia. Infant rhesus macaques homozygous for the PDE6C R565Q mutation were generated through a breeding program at the California National Primate Research Center. Homozygotes were treated in the right eye with adeno-associated virus (AAV5) carrying rhesus PDE6C under the control of the PR1.7 cone-specific promoter. The left eye was used as a control. Animals were tested by full-field and multifocal electroretinography. A total of 7 animals have been treated. The virus was found to be safe, but with variable inflammatory response. There were no obvious alterations in retinal lamination in treated eyes. The virus was expressed specifically in cone photoreceptors. Pre- and post-treatment systemic steroids led to minimal to moderate inflammatory response. In general, the gene therapy partially restored the cone responses on ERG within one month of injection in infants, but not in the older animals. If restored, the rescued cone responses were sustained and durable for over a year. Chromatic ERG testing showed restoration of amplitudes in all three cone subtypes. AAV-mediated gene therapy partially restored cone function and was relatively durable. Inflammation and age of administration may be important to outcomes. Similar approaches in human patients may warrant investigation.

Adeno-associated virus (AAV) gene therapy for spinal muscular atrophy

Adeno-associated virus (AAV) gene therapy for spinal muscular atrophy

Adeno-associated virus (AAV) gene therapy in congenital myopathies

Adeno-associated virus (AAV) gene therapy in congenital myopathies

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

Adult Patients with Hemophilia B and with a History of Chronic HCV/HBV Infection Receiving Liver-Directed Gene Therapy Demonstrated Long-Term Bleeding Protection and Sustained FIX Activity: Efficacy and Safety Results from the HOPE-B Trial 3 Years after Administration of a Single Dose of Etranacogene Dezaparvovec

Introduction: Liver-targeted recombinant adeno-associated virus (rAAV) gene therapy for hemophilia B has recently become a real-world therapeutic option for an adult population burdened with prevalent co-morbid chronic hepatitis C virus (HCV) and hepatitis B virus (HBV). The pivotal phase 3 HOPE-B trial (NCT03489291) evaluated the efficacy and safety of etranacogene dezaparvovec (CSL222, formerly AMT-061), an AAV5 vector, containing a codon-optimized, highly active factor IX (FIX) Padua R338L transgene under the control of the liver-specific promoter LP-1. Here we evaluate the efficacy and safety of etranacogene dezaparvovec in the subset of HOPE-B participants with a history of chronic HCV and/or HBV. Methods: Adult male participants with hemophilia B (FIX ≤2%), were infused with a single dose of etranacogene dezaparvovec (2x10 13 gc/kg), following a ≥6-month lead-in period receiving their usual FIX prophylaxis. Relevant exclusion criteria included baseline liver chemistries &amp;gt; 2X the upper limit of normal (ULN); active HCV (HCV RNA detectable), HBV (HBV DNA detectable or HBV sAg reactive) or uncontrolled HIV infection; or advanced liver fibrosis (FibroScan™ score of ≥9 kPa). Regular liver ultrasound screening, serum chemistries, and alphafetoprotein (AFP) were collected along with FIX expression and bleeding data. During the first months post-treatment, alanine aminotransferase (ALT) increase to 2X the subject's baseline or &amp;gt;ULN was treated with a per-protocol tapering course of oral corticosteroids. Results: Among 54 HOPE-B trial participants, 31 (57.4%) had history of co-morbid chronic HCV, without active disease and with undetectable HCV RNA. Of these 31 subjects, 7 had a history of chronic HBV infection without active disease (HBV DNA undetectable; HBV sAg neg). Two subjects were HCV/HIV co-infected (HIV DNA neg; CD4+ T-cell count &amp;gt;200). Two subjects were HBV+ (HBV env AB reactive, HBV sAg non-reactive, HBV DNA undetectable)/HCV-/HIV-. The mean age in the HCV+ and/or HBV+ subgroup (HCBV n=33) was 50.0 years (range 31-75). All HCBV participants had central lab (CL) ALT &amp;lt;ULN (ULN=41 U/L) on the day of dosing except one subject who had a CL ALT of 48 U/L. The mean screening fibroscan (liver elastography) score for this subgroup was 5.2 kPa (range 2.8-8.0). In HCBV participants 16/33 (48.5%) had pre-existing AAV5 neutralizing antibodies (NAb), with titers ranging from 8.5-3232; all NAb+ HCBV patients had titer ≤678 except one participant who had a titer of 3212 and never demonstrated FIX expression; as a result his prophylactic FIX infusions were never discontinued and his efficacy outcomes reflect exogenous FIX therapy. One HCBV participant returned to continuous routine prophylaxis 30 months post treatment. In the HOPE-B trial 11/54 (20.4%) of participants had 12 adverse events of ALT elevation of 2X above baseline or &amp;gt;ULN within 12 weeks after etranacogene dezaparvovec treatment, which triggered immunosuppressive therapy with corticosteroids in 9/54 (16.7%) participants. In the HCBV subgroup, 5/33 (15.2%) participants had ALT elevations of which 4/33 (12.1%) participants received corticosteroids. As reported previously (Schmidt M, et al. Blood Advances. 2023), one HCBV subject maintained normal AFP levels however per-protocol screening ultrasound at one year after gene therapy this participant developed a hepatocellular carcinoma (HCC); molecular characterization demonstrated the HCC was not related to etranacogene dezaparvovec treatment. Factor IX expression was stably increased above baseline throughout 36 months after the single CSL222 infusion. History of chronic HCBV did not impact FIX expression (Table 1). After excluding the subject with the pre-existing AAV5 NAb titer 3212 whose continued prophylaxis use confounds analysis, the mean annualized bleeding rate (ABR) decreased after gene therapy compared to lead-in standard of care FIX prophylaxis. HCBV participants demonstrated an ABR rate ratio of 0.31 (95% CI 0.13, 0.72) indicating 69% reduction in all bleeding, sustained from months 7-36 following etranacogene dezaparvovec treatment. Conclusions: The majority of HOPE-B trial participants were adults with a history of chronic HCV and/or HBV infection without active viral disease or evident pre-existing liver fibrosis. Safety and efficacy are observed in the HCBV participants.