Viral Vector-mediated Gene Transfer Research Articles

Gene Therapy in Hemophilia A: Achievements, Challenges, and Perspectives.

Strides in advancements of care of persons with hemophilia include development of long-acting factor replacement therapies, novel substitution and hemostatic rebalancing agents, and most recently approved gene therapy. Several decades of preclinical and clinical trials have led to development of adeno-associated viral (AAV) vector-mediated gene transfer for endogenous production of factor VIII (FVIII) in hemophilia A (HA). Only one gene therapy product for HA (valoctocogene roxaparvovec) has been approved by regulatory authorities. Results of valoctocogene roxaparvovec trial show significant improvement in bleeding rates and use of factor replacement therapy; however, sustainability and duration of response show variability with overall decline in FVIII expression over time. Further challenges include untoward adverse effects involving liver toxicity requiring immunosuppression and development of neutralizing antibodies to AAV vector rendering future doses ineffective. Real-life applicability of gene therapy for HA will require appropriate patient screening, infrastructure setup, long-term monitoring including data collection of patient-reported outcomes and innovative payment schemes. This review article highlights the success and development of HA gene therapy trials, challenges including adverse outcomes and variability of response, and perspectives on approach to gene therapy including shared decision-making and need for future strategies to overcome the several unmet needs.

Gene Ex Vivo Transduction Effects on Flap Survival and MicroRNAs Expression Analysis in Rat Allotransplantation Model

The lack of noninvasive biomarkers for graft rejection remains a challenge for the accurate monitoring of vascularized composite allotransplants. Viral vector-mediated gene transfer is a promising method for preventing graft rejection. In this study, we aimed to establish the expression profile of microRNAs (miRNAs) in skin flap allotransplantation, with or without gene transfer, and determine the potential role of several miRNAs as biomarkers of acute rejection and immune tolerance. An abdominal epigastric flap was transplanted from SD (RT1a) to Wistar rats (RT1Au). The adenoviral interleukin 10 (vIL-10) gene was transferred to the experimental group via flap pedicle injection. Postoperatively, flap appearance, hematoxylin and eosin staining, immunohistochemical staining, and miRNA expression analyses were performed. The viral IL-10 gene-treated group showed improved flap survival and reduced acute rejection response compared with the control group. On postoperative day 7, IL-10 expression in the flap was identified using immunohistochemistry and real-time polymerase chain reaction. The expression of miR-191a, miR-31a, miR-16, and miR-3473 was upregulated in the skin tissue, and that of miR-484, miR-132, miR-139, miR-150, and miR-6216 was upregulated in the serum. AV IL-10 gene transfer could be an effective immunosuppressive strategy for the prevention of skin flap allograft rejection. Additionally, some miRNAs were upregulated in the experimental group, serving as potential biomarkers of immune tolerance.

18F]pFBC, a Covalent CLIP-Tag Radiotracer for Detection of Viral Reporter Gene Transfer in the Murine Brain.

Preclinical models of neurological diseases and gene therapy are essential for neurobiological research. However, the evaluation of such models lacks reliable reporter systems for use with noninvasive imaging methods. Here, we report the development of a reporter system based on the CLIP-tag enzyme and [18F]pFBC, an 18F-labeled covalent CLIP-tag-ligand synthesized via a DoE-optimized and fully automated process. We demonstrated its specificity using a subcutaneous xenograft model and a model of viral vector-mediated brain gene transfer by engineering HEK293 cells and striatal neurons to express membrane-tethered CLIP-tag protein. After in vitro characterization of the reporter, mice carrying either CLIP-tag expressing or control subcutaneous xenografts underwent dynamic [18F]pFBC PET imaging. The CLIP-tag expressing xenografts showed a significantly higher uptake than control xenografts (tumor-to-muscle ratio 5.0 vs 1.7, p = 0.0379). In vivo, metabolite analysis by radio-HPLC from plasma and brain homogenates showed only one radio-metabolite in plasma and none in the brain. In addition, [18F]pFBC showed fast uptake and rapid clearance from the brain in animals injected with adeno-associated virus (AAV)-CLIP in the right striatum but no right-to-left (R-L) uptake difference in the striata in the acquired PET data. In contrast, autoradiography showed a clear accumulation of radioactivity in the AAV-CLIP-injected right striatum compared to the sham-injected left striatum control. CLIP-tag expression and brain integrity were verified by immunofluorescence and light sheet microscopy. In conclusion, we established a novel reporter gene system for PET imaging of gene expression in the brain and periphery and demonstrated its potential for a wide range of applications, particularly for neurobiological research and gene therapy with viral vectors.

Aromatic L-Amino-Acid Decarboxylase Deficiency Screening by Analysis of 3-O-Methyldopa in Dried Blood Spots: Results of a Multicentric Study in Neurodevelopmental Disorders.

Aromatic L-amino acid decarboxylase deficiency (AADCd) is a rare recessive metabolic disorder caused by pathogenic homozygous or compound heterozygous variants in the dopa decarboxylase (DDC) gene. Adeno-associated viral vector-mediated gene transfer of the human DDC gene injected into the putamen is available. The typical presentation is characterized by early-onset hypotonia, severe developmental delay, movement disorders, and dysautonomia. Recently, mild and even atypical phenotypes have been reported, increasing the diagnostic challenge. The aim of this multicentric study is to identify the prevalence of AADCd in a population of patients with phenotypic clusters characterized by neurodevelopmental disorders (developmental delay/intellectual disability, and/or autism) by 3-O-methyldopa (3-OMD) detection in dried blood spots (DBS). It is essential to identify AADCd promptly, especially within non-typical phenotypic clusters, because better results are obtained when therapy is quickly started in mild-moderate phenotypes. Between 2021 and 2023, 390 patients with non-specific phenotypes possibly associated with AADCd were tested; none resulted in a positive result. This result highlights that the population to be investigated for AADCd should have more defined clinical characteristics: association with common signs (hypotonia) and/or pathognomonic symptoms (oculogyric crisis and dysautonomia). It is necessary to continue to screen selected clusters for reaching diagnosis and improving long-term outcomes through treatment initiation. This underscores the role of newborn screening in identifying AADCd.

Considering gene therapy to protect from X-linked deafness DFNX2 and associated neurodevelopmental disorders.

Mutations and deletions in the gene or upstream of the gene encoding the POU3F4 transcription factor cause X-linked progressive deafness DFNX2 and additional neurodevelopmental disorders in humans. Hearing loss can be purely sensorineural or mixed, that is, with both conductive and sensorineural components. Affected males show anatomical abnormalities of the inner ear, which are jointly defined as incomplete partition type III. Current approaches to improve hearing and speech skills of DFNX2 patients do not seem to be fully effective. Owing to inner ear malformations, cochlear implantation is surgically difficult and may predispose towards severe complications. Even in cases where implantation is safely performed, hearing and speech outcomes remain highly variable among patients. Mouse models for DFNX2 deafness revealed that sensorineural loss could arise from a dysfunction of spiral ligament fibrocytes in the lateral wall of the cochlea, which leads to reduced endocochlear potential. Highly positive endocochlear potential is critical for sensory hair cell mechanotransduction and hearing. In this context, here, we propose to develop a therapeutic approach in male Pou3f4 -/y mice based on an adeno-associated viral (AAV) vector-mediated gene transfer in cochlear spiral ligament fibrocytes. Among a broad range of AAV vectors, AAV7 was found to show a strong tropism for the spiral ligament. Thus, we suggest that an AAV7-mediated delivery of Pou3f4 complementary DNA in the spiral ligament of Pou3f4 -/y mice could represent an attractive strategy to prevent fibrocyte degeneration and to restore normal cochlear functions and properties, including a positive endocochlear potential, before hearing loss progresses to profound deafness.

Recombinant Adeno-Associated Viral Vector-Mediated Gene Transfer of hTBX18 Generates Pacemaker Cells from Ventricular Cardiomyocytes.

Sinoatrial node dysfunction can manifest as bradycardia, leading to symptoms of syncope and sudden cardiac death. Electronic pacemakers are the current standard of care but are limited due to a lack of biological chronotropic control, cost of revision surgeries, and risk of lead- and device-related complications. We therefore aimed to develop a biological alternative to electronic devices by using a clinically relevant gene therapy vector to demonstrate conversion of cardiomyocytes into sinoatrial node-like cells in an in vitro context. Neonatal rat ventricular myocytes were transduced with recombinant adeno-associated virus vector 6 encoding either hTBX18 or green fluorescent protein and maintained for 3 weeks. At the endpoint, qPCR, Western blot analysis and immunocytochemistry were used to assess for reprogramming into pacemaker cells. Cell morphology and Arclight action potentials were imaged via confocal microscopy. Compared to GFP, hTBX18-transduced cells showed that hTBX18, HCN4 and Cx45 were upregulated. Cx43 was significantly downregulated, while sarcomeric α-actinin remained unchanged. Cardiomyocytes transduced with hTBX18 acquired the tapering morphology of native pacemaker cells, as compared to the block-like, striated appearance of ventricular cardiomyocytes. Analysis of the action potentials showed phase 4 depolarization and a significant decrease in the APD50 of the hTBX18-transduced cells. We have demonstrated that rAAV-hTBX18 gene transfer to ventricular myocytes results in morphological, molecular, physiological, and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. The generation of these induced pacemaker-like cells using a clinically relevant vector opens new prospects for biological pacemaker development.

The Effect of Rapamycin and Ibrutinib on Antibody Responses to Adeno-Associated Virus Vector-Mediated Gene Transfer.

Adeno-associated virus (AAV) vector-mediated gene transfer is lessening the impact of monogenetic disorders. Human AAV gene therapy recipients commonly mount immune responses to AAV or the encoded therapeutic protein, which requires transient immunosuppression. Most efforts to date have focused on blunting AAV capsid-specific T cell responses, which have been implicated in elimination of AAV-transduced cells. Here, we explore the use of immunosuppressants, rapamycin given alone or in combination with ibrutinib to inhibit AAV vector- or transgene product-specific antibody responses. Our results show that rapamycin or ibrutinib given alone reduces primary antibody responses against AAV capsid, but the combination of rapamycin and ibrutinib is more effective, blunts recall responses, and reduces numbers of circulating antibody-secreting plasma cells. The drugs fail to lower B cell memory formation or to reduce the inhibitory effects of pre-existing AAV capsid-specific antibodies on transduction efficiency.

Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells.

Despite their clinical success, chimeric antigen receptor (CAR)-T cell therapies for B-cell malignancies are limited by lengthy, costly and labor-intensive ex vivo manufacturing procedures that may lead to cell products with heterogeneous composition. Here we describe an implantable, multifunctional alginate scaffold for T cell engineering and release (MASTER) that streamlines in vivo CAR-T cell manufacturing and reduces processing time to a single day. When seeded with human peripheral blood mononuclear cells and CD19-encoding retroviral particles, MASTER provides the appropriate interface for viral vector-mediated gene transfer and, following subcutaneous implantation, mediates the release of functional CAR-T cells in mice. We further demonstrate that in vivo-generated CAR-T cells enter the bloodstream, and control distal tumor growth in a mouse xenograft model of lymphoma, showing greater persistence than conventional CAR-T cells. MASTER promises to transform CAR-T cell therapy by fast-tracking manufacture and potentially reducing the complexity and resources needed for provision of this type of therapy.

In vivo Gene Therapy to the Liver and Nervous System: Promises and Challenges.

In vivo genetic engineering has recently shown remarkable potential as a novel effective treatment for an ever-growing number of diseases, as also witnessed by the recent marketing authorization of several in vivo gene therapy products. In vivo genetic engineering comprises both viral vector-mediated gene transfer and the more recently developed genome/epigenome editing strategies, as long as they are directly administered to patients. Here we first review the most advanced in vivo gene therapies that are commercially available or in clinical development. We then highlight the major challenges to be overcome to fully and broadly exploit in vivo gene therapies as novel medicines, discussing some of the approaches that are being taken to address them, with a focus on the nervous system and liver taken as paradigmatic examples.

Transfer of the longevity-associated variant of BPIFB4 gene rejuvenates immune system and vasculature by a reduction of CD38+ macrophages and NAD+ decline

As we age, our body experiences chronic, systemic inflammation contributing to the morbidity and mortality of the elderly. The senescent immune system has been described to have a causal role in driving systemic aging and therefore may represent a key therapeutic target to prevent pathological consequences associated with aging and extend a healthy lifespan. Previous studies from our group associated a polymorphic haplotype variant in the BPIFB4 gene (LAV-BPIFB4) with exceptional longevity. Transfer of the LAV-BPIFB4 in preclinical models halted the progression of cardiovascular diseases (CVDs) and frailty by counterbalancing chronic inflammation. In the present study, we aimed to delineate the action of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer (AAV-LAV-BPIFB4) on the deleterious age-related changes of the immune system and thereby the senescence-associated events occurring in C57BL/6J mice aged 26 months. Our in vivo data showed that 26-months-old mice had a higher frequency of CD45+SA-beta Gal+ immune cells in peripheral blood than young (4-months-old) C57BL/6J mice. Notably, AAV-LAV-BPIFB4 gene transfer in aged mice reduced the pool of peripheral immunosenescent cells that were shown to be enriched in the spleen. In addition, the proper tuning of the immune secretory phenotype (IL1βlow, IL6low, IL10high) associated with a significant reduction in SA-beta Gal-positive area of aorta from AAV-LAV treated mice. At the functional level, the reduction of senescence-associated inflammation ensured sustained NAD+ levels in the plasma of AAV-LAV-BPIFB4 old mice by preventing the NADase CD38 increase in F4/80+ tissue-resident macrophages and Ly6Chigh pro-inflammatory monocytes of the spleen and bone marrow. Finally, to validate the clinical implication of our findings, we showed that Long-living-individuals (LLIs, >95 years), which delay CVDs onset, especially if LAV-carriers, were characterized by high NAD+ levels. In conclusion, the new senotherapeutic action of LAV-BPIFB4 may offer a valuable therapeutic tool to control aging and reduce the burden of its pathophysiological disorders, such as CVDs.

Clozapine is my favorite color: Chemogenetic modulation of anxiety-related behavior in primates

The development of technologies to reversibly manipulate neural circuitry has given rise to the prospect of more precisely targeted therapies to normalize brain function in neuropsychiatric disorders. These approaches must be feasible using viral vector-mediated gene transfer to be practical in primates. Chemogenetic tools have great potential in fulfilling this goal. Chemogenetic neuromodulation changes neuronal activity via a systemically administered drug that triggers artificial receptors placed in anatomically defined neuronal populations via viral vectors.

Follistatin mediates learning and synaptic plasticity via regulation of Asic4 expression in the hippocampus

The biological mechanisms underpinning learning are unclear. Mounting evidence has suggested that adult hippocampal neurogenesis is involved although a causal relationship has not been well defined. Here, using high-resolution genetic mapping of adult neurogenesis, combined with sequencing information, we identify follistatin (Fst) and demonstrate its involvement in learning and adult neurogenesis. We confirmed that brain-specific Fst knockout (KO) mice exhibited decreased hippocampal neurogenesis and demonstrated that FST is critical for learning. Fst KO mice exhibit deficits in spatial learning, working memory, and long-term potentiation (LTP). In contrast, hippocampal overexpression of Fst in KO mice reversed these impairments. By utilizing RNA sequencing and chromatin immunoprecipitation, we identified Asic4 as a target gene regulated by FST and show that Asic4 plays a critical role in learning deficits caused by Fst deletion. Long-term overexpression of hippocampal Fst in C57BL/6 wild-type mice alleviates age-related decline in cognition, neurogenesis, and LTP. Collectively, our study reveals the functions for FST in adult neurogenesis and learning behaviors.

Expression and evaluation of porcine circovirus type 2 capsid protein mediated by recombinant adeno-associated virus 8.

BackgroundPorcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide.ObjectivesA suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy.MethodsIn the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer.ResultsThe obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2.ConclusionsThe injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.

Of rAAV and Men: From Genetic Neuromuscular Disorder Efficacy and Toxicity Preclinical Studies to Clinical Trials and Back.

Neuromuscular disorders are a large group of rare pathologies characterised by skeletal muscle atrophy and weakness, with the common involvement of respiratory and/or cardiac muscles. These diseases lead to life-long motor deficiencies and specific organ failures, and are, in their worst-case scenarios, life threatening. Amongst other causes, they can be genetically inherited through mutations in more than 500 different genes. In the last 20 years, specific pharmacological treatments have been approved for human usage. However, these “à-la-carte” therapies cover only a very small portion of the clinical needs and are often partially efficient in alleviating the symptoms of the disease, even less so in curing it. Recombinant adeno-associated virus vector-mediated gene transfer is a more general strategy that could be adapted for a large majority of these diseases and has proved very efficient in rescuing the symptoms in many neuropathological animal models. On this solid ground, several clinical trials are currently being conducted with the whole-body delivery of the therapeutic vectors. This review recapitulates the state-of-the-art tools for neuron and muscle-targeted gene therapy, and summarises the main findings of the spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD) and X-linked myotubular myopathy (XLMTM) trials. Despite promising efficacy results, serious adverse events of various severities were observed in these trials. Possible leads for second-generation products are also discussed.

A Single Injection of an Optimized Adeno-Associated Viral Vector into Cerebrospinal Fluid Corrects Neurological Disease in a Murine Model of GM1 Gangliosidosis.

GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (β-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore β-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human β-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased β-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.

Murine transfer of a human gene variant associated with exceptional longevity displays senolytic effects both in immune compartment and endothelium of aged mice

Abstract The persistence and accumulation of senescent cells has been shown to potentially play a role in the pathophysiology of age-related cardiovascular diseases. Indeed with time, a decline in immune efficacy, termed immunesenescence, and a deleterious secretory phenotype of senescent cells (SASP) generate that inflammatory background mainly mediating the elderly cardiovascular phenotype. Long Living Individuals (LLI) which delay aging, represent a model of positive biology and an exceptional resource to study and find a way to improve general public health. Previous studies from our group have shown that a human gene associated with exceptional longevity (LAV-BPIFB4) was able to block the atherosclerotic process in ApoE−/− mice by conferring the animals with a pro-resolving M2 macrophages profile. Furthermore, LAV-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization and frailty reduction. Here we sought to underpinn the role of LAV-BPIFB4 in counteracting the age-related remodeling of the immune responses. The effects of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the immune dynamics in old mice have been investigated by an extensive flow cytometric approach in lymphoid tissues (bone marrow, spleen and peripheral blood). C57BL/6J mice were assigned to two age-matched experimental groups: a treatment group (AAV-LAV-BPIFB4; N=6 mice, aged 18–23 months and a control group (AAV-GFP; N=6 mice, aged 18–23 months. 30th and 60th day since the beginning of the infection, SA-beta Gal substrate has been used to identify CD45+ senescent cells in freshly isolated PBMC, splenocytes, bone marrow (BM)-derived cells. As expected, we monitored an increase in SA-betaGal activity in blood. This increase is most significant in CD11b+ myeloid cells, whithout affecting neither CD3+T neither NK1.1+Natural Killer (NK) cell compartment. Notably 30 days AAV-LAV-BPIFB4 infection and to a more the 60 days-treatment, resulted in a significant decrease in senescent pool of peripheral immune cells and a concomitant enrichment of senescent cells in spleen. Concomitantly, aorta from AAV-LAV treated mice showed significant reduction in SA-beta Gal positive area. Furthermore a LAV-BPIFB4 induction of pro-resolving M2 macrophages compared to control group was documented in the main haemocateretic organ. As consequence the senolytic effect of LAV-BPIFB4 gene-therapy well correlated with the rescue of proliferative index of splenocytes upon PHA stimulation, and their functional protective response to lipopolysaccharide (LPS) in term of IL-6 and TNF-alpha secretion. The restoration of a protective and balanced immune response finally reflected the reduction of senescent phenopype acquired by mouse aortic endothelial cells during the aging process in vivo. A better underpinning of the senolytic action of LAV-BPIFB4 may offer a valuable therapeutic tool to reverse aging phenotype causing most of cardiac diseases Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Cariplo Foundation (n.2016-0874) to AAP and CV

Transduction of Salivary Gland Acinar Cells with a Novel AAV Vector 44.9

The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren’s syndrome is a serious condition that affects a patient’s quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector–mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction. Previous studies with adeno-associated virus (AAV) focused on the submandibular gland and reported mostly ductal cell transduction. In this study, we have screened AAV vectors for acinar cell tropism in the parotid gland utilizing membrane-tomato floxed membrane-GFP transgenic mice to screen CRE recombinase encoding AAV vectors of different clades to rapidly identify capsid isolates able to transduce salivary gland acinar cells. We determined that AAVRh10 and a novel isolate found as a contaminant of a laboratory stock of simian adenovirus SV15, AAV44.9, are both able to transduce parotid and sublingual acinar cells. Persistence and localization of transduction of these AAVs were tested using vectors encoding firefly luciferase, which was detected 6 months after vector administration. Most luciferase expression was localized to the salivary gland compared to that of distal organs. Transduction resulted in robust secretion of recombinant protein in both blood and saliva. Transduction was species specific, with AAVRh10 having stronger transduction activity in rats compared with AAV44.9 or AAV2 but weaker in human primary salivary gland cells. This work demonstrates efficient transduction of parotid acinar cells by AAV that resulted in secretion of recombinant protein in both serum and saliva.

Retrograde gene transfer into neural pathways mediated by adeno-associated virus (AAV)-AAV receptor interaction

BackgroundViral vector systems delivering transgenes in the retrograde direction through axons to neural cell bodies are powerful experimental tools for the functional analysis of specific neural pathways. Generally, the efficiency of viral vector-mediated retrograde gene transfer depends on the expression of requisite viral receptors in neural pathways projecting to the viral vector-injected regions. This is known as viral tropism and can limit the utility of retrograde viral vectors. The adeno-associated virus (AAV) vector has become an increasingly popular platform for gene delivery to neural cells in vivo, and it is therefore meaningful to develop a new type of retrograde gene transfer approach based on a tropism-free AAV vector system. New MethodThe wild-type or mutant receptor gene of AAV was expressed to mitigate AAV tropism. ResultsEfficient AAV vector-mediated retrograde gene transfer was observed in diverse neural pathways by expression of the AAV receptor (AAVR) gene. Moreover, the expression of a minimal mutant of AAVR (miniAAVR), which maintains binding potential to AAV, demonstrated efficient retrograde gene expression comparable to that of AAVR. Comparison with Existing MethodsThe utility of existing AAV vector-mediated retrograde gene delivery methods is sometimes limited by tropism. Our newly developed AAV-AAVR and AAV-miniAAVR interaction approaches enabled efficient retrograde gene transfer into various neural pathways by mitigating tropism. ConclusionsAAV-AAVR and AAV-miniAAVR interaction approaches enabled us to induce efficient retrograde gene expression in targeted neural pathways and provide a powerful tool for analyzing specific neural pathways.

Hemophilia gene therapy-New country initiatives.

Gene therapy is an opportunity for haemophilia patients to receive a one-time treatment and have lasting factor levels for years or decades instead of dependence on repeated administration within short intervals and on sustained supply of drug. Great strides have been made in the development of gene therapy for haemophilia in the last decade. Adeno-associated virus (AAV) vector-mediated gene transfer in haemophilia A and B has entered the phase III trial stage. Gene transfer by lentiviral vector or gene editing technologies using factor VIII (FVIII) or IX (FIX) genes are now entering clinical evaluation. It is expected that the first FVIII and FIX gene therapy products will soon be approved and distributed in major markets. Global access to gene therapy is a critical goal. This review presents new and ongoing efforts towards this goal in countries other than North America and Europe. In Japan, researchers, regulators and funders have established a promising gene therapy development platform for multiple diseases including haemophilia. Decades of scientific and clinical research in haemophilia gene therapy in China have led to a recently registered clinical trial of AAV-mediated gene therapy for haemophilia B. Other countries are in earlier phases of building gene therapy programmes or participate in international trials. A phase 2 feasibility trial of AAV-mediated FIX gene therapy in low- and middle-income countries aims to demonstrate that gene therapy could become available in resource-constrained socio-economic settings. The different strategies for establishing gene therapy provide opportunities for closing the global gap in haemophilia care.

Uncertainty in an era of transformative therapy for haemophilia: Addressing the unknowns.

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 30years 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.