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Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice., (© 2024. The Author(s).)

Recent advancements have focused on enhancing factor VIII half-life and refining its delivery methods, despite the well-established knowledge that factor VIII deficiency is the main clotting protein lacking in hemophilia. Consequently, both viral and non-viral delivery systems play a crucial role in enhancing the quality of life for hemophilia patients. The utilization of viral vectors and the manipulation of non-viral vectors through targeted delivery are significant advancements in the field of cellular and molecular therapies for hemophilia. These developments contribute to the progression of treatment strategies and hold great promise for improving the overall well-being of individuals with hemophilia. This review study comprehensively explores the application of viral and non-viral vectors in cellular (specifically T cell) and molecular therapy approaches, such as RNA, monoclonal antibody (mAb), and CRISPR therapeutics, with the aim of addressing the challenges in hemophilia treatment. By examining these innovative strategies, the study aims to shed light on potential solutions to enhance the efficacy and outcomes of hemophilia therapy., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2024 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Haemophilia is at the dawn of a new era in therapeutic management, one that can generate greater protection from bleeding and a functional cure in some individuals. Prior advances in protein engineering and monoclonal antibody technology have facilitated therapeutic options to maintain decreased risk of bleeding and less burdensome treatment. The use of gene transfer, first proposed in 1971 for monogenic diseases, is emerging as an effective long-term treatment for a variety of diseases. Transfer of functional factor VIII (FVIII) and factor IX (FIX) genes has witnessed a series of advances and setbacks since the first non-clinical experiments in animals were initiated nearly 30 years ago. More recently, multiyear therapeutic levels of FVIII and FIX activity have been achieved in human clinical trials, translated into meaningful clinical benefit and a functional cure. While clinical progress has been definitive, many questions remain unanswered as prelicensure phase 3 clinical trials are underway. These unanswered questions translate into a state of uncertainty about the known unknowns and unknown unknowns intrinsic to any new therapeutic platform. Accepting this modality as a means to functionally cure haemophilia also means accepting the uncertainty regarding the biology of viral vector-mediated gene transfer, which remains inadequately understood. Gene therapy is a far more complex biological 'drug' than small molecule and protein drugs, where manufacturing processes and the drugs themselves are now well characterized. Extent of community acceptance of uncertainty and acknowledgement of the need for an uncompromising drive for answers to the unknowns will characterize the introduction of this first generation of gene therapy for haemophilia to the wider patient population in both resource-rich and resource-poor countries., (© 2020 John Wiley & Sons Ltd.)

Although therapeutic outcomes have been achieved in hemophilia patients after delivery of clotting factor genes to the liver using adeno-associated virus (AAV) vectors, it is well known that the preclinical results generated from hemophilia animal models have not been directly predictive of successful translation in humans. To address this discrepancy humanized mouse models have recently been used to predict AAV transduction efficiency for human hepatocytes. In this study we evaluated AAV vector transduction from several serotypes in human liver hepatocytes xenografted into chimeric mice. After systemic administration of AAV vectors encoding a GFP transgene in humanized mice, the liver was harvested for either immunohistochemistry staining or flow cytometry assay for AAV human hepatocyte transduction analysis. We observed that AAV7 consistently transduced human hepatocytes more efficiently than other serotypes in both immunohistochemistry assay and flow cytometry analysis. To better assess the future application of AAV7 for systemic administration in the treatment of hemophilia or other liver diseases, we analyzed the prevalence of neutralizing antibodies (NAbs) to AAV7 in sera from healthy subjects and patients with hemophilia. In the general population, the prevalence of NAbs to AAV7 was lower than that of AAV2 or AAV3B. However, a higher prevalence of AAV7 NAbs was found in patients with hemophilia. In summary, results from this study suggest that AAV7 vectors should be considered as an effective vehicle for human liver targeting in future clinical trials.

Gene therapy using an adeno-associated virus (AAV) vector offers a new treatment option for individuals with monogenetic disorders. The major bottleneck is the presence of pre-existing anti-AAV antibodies, which impacts its use. Even very low titers of neutralizing antibodies (NAb) to capsids from natural AAV infections have been reported to inhibit the transduction of intravenously administered AAV in animal models and are associated with limited efficacy in human trials. Assessing the level of pre-existing NAb is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Techniques used to screen AAV-antibodies include AAV capsid enzyme-linked immunosorbent assay (ELISA) and transduction inhibition assay (TIA) for detecting total capsid-binding (TAb) and Nab, respectively. In this study, we screened 521 individuals with hemophilia A from India for TAb and NAb using ELISA and TIA, respectively. The prevalence of TAb and NAb in hemophilia A patients from India were 96% and 77.5%, respectively. There was a significant increase in anti-AAV3 NAb prevalence with age in the hemophilia A patient group from India. There was a trend in anti-AAV3 TAb positivity between the pediatric age group (94.4%) and the adult age group (97.4%)., (© 2022 Wiley Periodicals LLC.)

Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC + rLC protein had a higher activity than the hHC + hLC protein at comparable expression levels. The specific activity of hHC + rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC + rLC treatment exhibited a visibly higher activity than those treated with hHC + hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC + rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy., (© 2021. Higher Education Press.)

Factor VIII gene transfer with a single intravenous infusion of valoctocogene roxaparvovec (AAV5-hFVIII-SQ) has demonstrated clinical benefits lasting 5 years to date in people with severe hemophilia A. Molecular mechanisms underlying sustained AAV5-hFVIII-SQ-derived FVIII expression have not been studied in humans. In a substudy of the phase 1/2 clinical trial ( NCT02576795 ), liver biopsy samples were collected 2.6-4.1 years after gene transfer from five participants. Primary objectives were to examine effects on liver histopathology, determine the transduction pattern and percentage of hepatocytes transduced with AAV5-hFVIII-SQ genomes, characterize and quantify episomal forms of vector DNA and quantify transgene expression (hFVIII-SQ RNA and hFVIII-SQ protein). Histopathology revealed no dysplasia, architectural distortion, fibrosis or chronic inflammation, and no endoplasmic reticulum stress was detected in hepatocytes expressing hFVIII-SQ protein. Hepatocytes stained positive for vector genomes, showing a trend for more cells transduced with higher doses. Molecular analysis demonstrated the presence of full-length, inverted terminal repeat-fused, circular episomal genomes, which are associated with long-term expression. Interindividual differences in transgene expression were noted despite similar successful transduction, possibly influenced by host-mediated post-transduction mechanisms of vector transcription, hFVIII-SQ protein translation and secretion. Overall, these results demonstrate persistent episomal vector structures following AAV5-hFVIII-SQ administration and begin to elucidate potential mechanisms mediating interindividual variability., (© 2022. The Author(s).)

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

Hemophilia A is an inherited coagulation disorder resulting in the loss of functional clotting factor VIII (FVIII). Presently, the most effective treatment is prophylactic protein replacement therapy. However, this requires frequent life-long intravenous infusions of plasma derived or recombinant clotting factors and is not a cure. A major complication is the development of inhibitory antibodies that nullify the replacement factor. Immune tolerance induction (ITI) therapy to reverse inhibitors can last from months to years, requires daily or every other day infusions of supraphysiological levels of FVIII and is effective in only up to 70% of hemophilia A patients. Preclinical and recent clinical studies have shown that gene replacement therapy with AAV vectors can effectively cure hemophilia A patients. However, it is unclear how hemophilia patients with high risk inhibitor F8 mutations or with established inhibitors will respond to gene therapy, as these patients have been excluded from ongoing clinical trials. AAV8- coF8 gene transfer in naïve BALB/c- F8e16 −/Y mice (BALB/c-HA) results in anti-FVIII IgG1 inhibitors following gene transfer, which can be prevented by transient immune modulation with anti-mCD20 (18B12) and oral rapamycin. We investigated if we could improve ITI in inhibitor positive mice by combining anti-mCD20 and rapamycin with AAV8- coF8 gene therapy. Our hypothesis was that continuous expression of FVIII protein from gene transfer compared to transient FVIII from weekly protein therapy, would enhance regulatory T cell induction and promote deletion of FVIII reactive B cells, following reconstitution. Mice that received anti-CD20 had a sharp decline in inhibitors, which corresponded to FVIII memory B (Bmem) cell deletion. Importantly, only mice receiving both anti-mCD20 and rapamycin failed to increase inhibitors following rechallenge with intravenous FVIII protein therapy. Our data show that B and T cell immune modulation complements AAV8- coF8 gene therapy in naïve and inhibitor positive hemophilia A mice and suggest that such protocols should be considered for AAV gene therapy in high risk or inhibitor positive hemophilia patients. [ABSTRACT FROM AUTHOR]

Hemophilia A (HA) is an X-linked bleeding disorder due to deficiencies in coagulation factor VIII (FVIII). The major complication of current protein-based therapies is the development of neutralizing anti-FVIII antibodies, termed inhibitors, that block the hemostatic effect of therapeutic FVIII. Inhibitors develop in about 20–30% of people with severe HA, but the risk is dependent on the interaction between environmental and genetic factors, including the underlying F8 gene mutation. Recently, multiple clinical trials evaluating adeno-associated viral (AAV) vector liver-directed gene therapy for HA have reported promising results of therapeutically relevant to curative FVIII levels. The inclusion criteria for most trials prevented enrollment of subjects with a history of inhibitors. However, preclinical data from small and large animal models of HA with inhibitors suggests that liver-directed gene therapy can in fact eradicate pre-existing anti-FVIII antibodies, induce immune tolerance, and provide long-term therapeutic FVIII expression to prevent bleeding. Herein, we review the accumulating evidence that continuous uninterrupted expression of FVIII and other transgenes after liver-directed AAV gene therapy can bias the immune system toward immune tolerance induction, discuss the current understanding of the immunological mechanisms of this process, and outline questions that will need to be addressed to translate this strategy to clinical trials. [ABSTRACT FROM AUTHOR]

Neutralizing antibodies to factor VIII (fVIII), referred to as "inhibitors," remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product- and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liver-specific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pre-transplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors. [ABSTRACT FROM AUTHOR]

Hemophilia A (coagulation factor VIII deficiency) is a debilitating genetic disorder that is primarily treated with intravenous replacement therapy. Despite a variety of factor VIII protein formulations available, the risk of developing anti-dug antibodies ("inhibitors") remains. Overall, 20-30% of patients with severe disease develop inhibitors. Current clinical immune tolerance induction protocols to eliminate inhibitors are not effective in all patients, and there are no prophylactic protocols to prevent the immune response. New experimental therapies, such as gene and cell therapies, show promising results in pre-clinical studies in animal models of hemophilia. Examples include hepatic gene transfer with viral vectors, genetically engineered regulatory T cells (Treg), in vivo Treg induction using immune modulatory drugs, and maternal antigen transfer. Furthermore, an oral tolerance protocol is being developed based on transgenic lettuce plants, which suppressed inhibitor formation in hemophilic mice and dogs. Hopefully, some of these innovative approaches will reduce the risk of and/or more effectively eliminate inhibitor formation in future treatment of hemophilia A. [ABSTRACT FROM AUTHOR]