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Gene therapies using recombinant adeno-associated virus (AAV) vectors have demonstrated considerable clinical success in the treatment of genetic disorders. Improved vectors with favorable tropism profiles, decreased immunogenicity, and enhanced manufacturability are poised to further improve the state of gene therapies. Such vectors can be identified through directed evolution, a process of subjecting a diverse capsid library to a selection pressure to identify individual variants with a desired trait. Currently, libraries that involve changes distributed throughout the AAV capsid coding region, such as DNA family shuffled libraries, are largely characterized using low-throughput Sanger sequencing of individual clones. However, improvements in long-read sequencing technologies have increased their applicability to capsid libraries and evaluation of the selection process. Here, we explore the application of Oxford Nanopore Technologies refined by a concatemeric consensus method for initial library characterization and monitoring selection of a shuffled AAV capsid library. Furthermore, we present AAVolve, a bioinformatic pipeline for processing long-read data from AAV-directed evolution experiments. Our approach allows high-throughput characterization of AAV capsids in a streamlined manner, facilitating deeper insights into library composition through multiple rounds of selection, and generalization through training of machine learning models., Competing Interests: L.L. is a cofounder of LogicBio Therapeutics, S.S. and L.L. are cofounders of Sendatu Therapeutics, and L.L. and I.E.A. are co-founders of Exigen Biotherapeutics, companies that utilize technologies similar to those broadly discussed in this paper., (© 2024 The Author(s).)

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spf ash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types., Competing Interests: Declaration of interests All authors except for S.C.C., E.B.v.D., and I.E.A. are or were employees of Tessera Therapeutics and currently receive or previously received salary along with stock options as compensation for their employment. I.E.A. is a consultant of Tessera Therapeutics, and this work was performed under a Tessera Therapeutics-sponsored research agreement., (Copyright © 2024 The American Society of Gene and Cell Therapy. All rights reserved.)

To date, 3,900 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical activity from trial databases, official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our March 2023 update, we have entries on 3,900 trials undertaken in 46 countries. We have analyzed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and which genes have been transferred. Details of the analyses presented, and our searchable database are on The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at https://a873679.fmphost.com/fmi/webd/GTCT. We also provide an overview of the progress being made around the world, and discuss key trends since the previous review, namely the unprecedented increase in gene therapy clinical trial activity, including the implementation of genome editing technology with the potential to transform the field moving forward., (© 2024 John Wiley & Sons Ltd.)

Background: Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored., Methods: We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC)., Results: At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spf ash mouse following elimination of residual endogenous murine OTC., Conclusions: The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset., (© 2024 John Wiley & Sons, Ltd.)

Adiponectin (APN) is an adipokine which predominantly expresses in adipocytes with neuroprotective and anti-inflammatory effects. We have recently indicated that circulatory trimeric APN can enter the brain by crossing the blood-brain barrier (BBB) and modulate microglia-mediated neuroinflammation. Here, we found that the microglial NLR family pyrin domain containing 3 (NLRP3)-inflammasome activation was exacerbated in APN -/- 5xFAD mice in age-dependent manner. The focus of this study was to develop a new and tractable therapeutic approach for treating Alzheimer's disease (AD)-related pathology in 5xFAD mice using peripheral APN gene therapy. We have generated and transduced adeno-associated virus (AAV2/8) expressing the mouse mutated APN gene (APN C39S ) into the liver of 5xFAD mice that generated only low-molecular-weight trimeric APN (APN Tri ). Single dose of AAV2/8-APN C39S in the liver increased circulatory and cerebral APN levels indicating the overexpressed APN Tri was able to cross the BBB. Overexpression of APN Tri decreased both the soluble and fibrillar Aβ in the brains of 5xFAD mice. AAV2/8-APN Tri treatment reduced Aβ-induced IL-1β and IL-18 secretion by suppressing microglial NLRP3-inflammasome activation. The memory functions improved significantly in AAV-APN Tri -treated 5xFAD mice with reduction of dystrophic neurites. These findings demonstrate that peripheral gene delivery to overexpress trimeric APN can be a potential therapy for AD., (© 2024. The Author(s).)

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies., (© 2024. The Author(s).)

Despite the availability of life-saving corticosteroids for 70 years, treatment for adrenal insufficiency is not able to recapitulate physiological diurnal cortisol secretion and results in numerous complications. Gene therapy is an attractive possibility for monogenic adrenocortical disorders such as congenital adrenal hyperplasia; however, requires further development of gene transfer/editing technologies and knowledge of the target progenitor cell populations. Vectors based on adeno-associated virus are the leading system for direct in vivo gene delivery but have limitations in targeting replicating cell populations such as in the adrenal cortex. One strategy to overcome this technological limitation is to deliver the relevant adrenocortical gene to a currently targetable organ outside of the adrenal cortex. To explore this possibility, we developed a vector encoding human 21-hydroxylase and directed expression to the liver in a mouse model of congenital adrenal hyperplasia. This extra-adrenal expression resulted in reconstitution of the steroidogenic pathway. Aldosterone and renin levels normalized, and corticosterone levels improved sufficiently to reduce adrenal hyperplasia. This strategy could provide an alternative treatment option for monogenic adrenal disorders, particularly for mineralocorticoid defects. These findings also demonstrate, when targeting the adrenal gland, that inadvertent liver transduction should be precluded as it may confound data interpretation., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Gene therapies and associated technologies are transforming biomedical research and enabling novel therapeutic options for patients living with debilitating and incurable genetic disorders. The vector system based on recombinant adeno-associated viral vectors (AAVs) has shown great promise in recent clinical trials for genetic diseases of multiple organs, such as the liver and the nervous system. Despite recent successes toward the development of novel bioengineered AAV variants for improved transduction of primary human tissues and cells, vectors that can efficiently transduce human Schwann cells (hSCs) have yet to be identified. Here, we report the application of the functional transduction-RNA selection method in primary hSCs for the development of AAV variants for specific and efficient transgene delivery to hSCs. The two identified capsid variants, Pep2hSC1 and Pep2hSC2, show conserved potency for delivery across various in vitro , in vivo , and ex vivo models of hSCs. These novel AAV capsids will serve as valuable research tools, forming the basis for therapeutic solutions for both SC-related disorders or peripheral nervous system injury., Competing Interests: M.D., J.M., M.C.-C., A.W., S.L.G., I.E.A., and L.L. are inventors on patent applications filed by Children’s Medical Research Institute related to AAV capsid sequences, in vivo function of novel AAV variants and AAV selection platforms. L.L. is a co-founder and scientific advisor of LogicBio Therapeutics. L.L. and I.A.E. are co-founders of Exigen Biotherapeutics. L.L. and I.E.A. have consulted on broad technologies addressed in this paper. L.L. and I.A.E. have stock and/or equity in companies with technology broadly related to this paper., (© 2024 The Authors.)

Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood-brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuffled rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAV-BBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270-fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAV-PHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes in vivo. To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modifications based on rational design. Overall, our study highlights the potential of AAV1-like vectors for efficient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies., Competing Interests: Declaration of interests M.D., I.E.A., and L.L. are inventors on patent applications filed by CMRI related to AAV capsid sequences, in vivo function of novel AAV variants, and AAV selection platforms. L.L. and I.A.E. are cofounders of and own equity in Exigen Biotherapeutics. L.L. and I.E.A. have consulted on technologies addressed in this paper. L.L. and I.A.E. have stock and/or equity in companies with technology broadly related to this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Urea cycle defects (UCDs) are severe inherited metabolic diseases with high unmet needs which present a permanent risk of hyperammonaemic decompensation and subsequent acute death or neurological sequelae, when treated with conventional dietetic and medical therapies. Liver transplantation is currently the only curative option, but has the potential to be supplanted by highly effective gene therapy interventions without the attendant need for life-long immunosuppression or limitations imposed by donor liver supply. Over the last three decades, pioneering genetic technologies have been explored to circumvent the consequences of UCDs, improve quality of life and long-term outcomes: adenoviral vectors, adeno-associated viral vectors, gene editing, genome integration and non-viral technology with messenger RNA. In this review, we present a summarised view of this historical path, which includes some seminal milestones of the gene therapy's epic. We provide an update about the state of the art of gene therapy technologies for UCDs and the current advantages and pitfalls driving future directions for research and development., (© 2023 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.

Recombinant adeno-associated viruses (rAAVs) have emerged as one of the most promising gene therapy vectors that have been successfully used in pre-clinical models of heart disease. However, this has not translated well to humans due to species differences in rAAV transduction efficiency. As a result, the search for human cardiotropic capsids is a major contemporary challenge. We used a capsid-shuffled rAAV library to perform directed evolution in human iPSC-derived cardiomyocytes (hiPSC-CMs). Five candidates emerged, with four presenting high sequence identity to AAV6, while a fifth divergent variant was related to AAV3b. Functional analysis of the variants was performed in vitro using hiPSC-CMs, cardiac organoids, human cardiac slices, non-human primate and porcine cardiac slices, as well as mouse heart and liver in vivo . We showed that cell entry was not the best predictor of transgene expression efficiency. The novel variant rAAV.KK04 was the best-performing vector in human-based screening platforms, exceeding the benchmark rAAV6. None of the novel capsids demonstrate a significant transduction of liver in vivo . The range of experimental models used revealed the value of testing for tropism differences under the conditions of human specificity, bona fide, myocardium and cell type of interest., Competing Interests: C.Y.K., S.L.G., M.C.-C., I.E.A., L.L., and E.K. are inventors on patent applications filed by Children’s Medical Research Institute related to AAV capsid sequences and in vivo function of novel AAV variants. L.L. is a cofounder and a scientific advisor at LogicBio Therapeutics. L.L. and I.E.A. are co-founders of Exigen Biotherapeutics. L.L. has a sponsored research agreement with LogicBio Therapeutics. M.C.-C., I.E.A., and L.L. have IP licensed to biopharmaceutical companies. L.L. and I.A.E. have consulted on technologies addressed in this paper. L.L. and I.A.E. have stock and/or equity in companies with technology broadly related to this paper. J.E.H. is a co-founder, scientific advisor, and stockholder in Dynomics. All other authors declare no conflicts of interest., (© 2023 The Author(s).)

Success in the treatment of infants with spinal muscular atrophy (SMA) underscores the potential of vectors based on adeno-associated virus (AAV). However, a major obstacle to the full realization of this potential is pre-existing natural and therapy-induced anti-capsid humoral immunity. Structure-guided capsid engineering is one possible approach to surmounting this challenge but necessitates an understanding of capsid-antibody interactions at high molecular resolution. Currently, only mouse-derived monoclonal antibodies (mAbs) are available to structurally map these interactions, which presupposes that mouse and human-derived antibodies are functionally equivalent. In this study, we have characterized the polyclonal antibody responses of infants following AAV9-mediated gene therapy for SMA and recovered 35 anti-capsid mAbs from the abundance of switched-memory B (smB) cells present in these infants. For 21 of these mAbs, seven from each of three infants, we have undertaken functional and structural analysis measuring neutralization, affinities, and binding patterns by cryoelectron microscopy (cryo-EM). Four distinct patterns were observed akin to those reported for mouse-derived mAbs, but with early evidence of differing binding pattern preference and underlying molecular interactions. This is the first human and largest series of anti-capsid mAbs to have been comprehensively characterized and will prove to be powerful tools for basic discovery and applied purposes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for hereditary diseases such as retinal dystrophy or spinal muscular atrophy 1 (SMA1). Many preclinical studies have used AAV vectors for gene therapy in models of cardiac disease with outcomes of varying translational potential. However, major barriers to effective and safe therapeutic gene delivery to the human heart remain to be overcome. These include tropism, efficient gene transfer, mitigating off-target gene delivery and avoidance of the host immune response. Developing such an enhanced AAV vector for cardiac gene therapy is of great interest to the field of advanced cardiac therapeutics. In this review, we provide an overview of the approaches currently being employed in the search for cardiac cell-specific AAV capsids, ranging from natural AAVs selected as a result of infection and latency in the heart, to the use of cutting-edge molecular techniques to engineer and select AAVs specific for cardiac cells with the use of high-throughput methods., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The clinical outcome for children and adolescents with homozygous familial hypercholesterolaemia (HoFH) can be devastating, and treatment options are limited in the presence of a null variant. In HoFH, atherosclerotic risk accumulates from birth. Gene therapy is an appealing treatment option as restoration of low-density lipoprotein receptor (LDLR) gene function could provide a cure for HoFH. A clinical trial using a recombinant adeno-associated vector (rAAV) to deliver LDLR DNA to adult patients with HoFH was recently completed; results have not yet been reported. However, this treatment strategy may face challenges when translating to the paediatric population. The paediatric liver undergoes substantial growth which is significant as rAAV vector DNA persists primarily as episomes (extra-chromosomal DNA) and are not replicated during cell division. Therefore, rAAV-based gene addition treatment administered in childhood would likely only have a transient effect. With over 2,000 unique variants in LDLR, a goal of genomic editing-based therapy development would be to treat most (if not all) mutations with a single set of reagents. For a robust, durable effect, LDLR must be repaired in the genome of hepatocytes, which could be achieved using genomic editing technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and a DNA repair strategy such as homology-independent targeted integration. This review discusses this issue in the context of the paediatric patient group with severe compound heterozygous or homozygous null variants which are associated with aggressive early-onset atherosclerosis and myocardial infarction, together with the important pre-clinical studies that use genomic editing strategies to treat HoFH in place of apheresis and liver transplantation., (Copyright © 2023 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). All rights reserved.)

Primary adrenal insufficiency (PAI) occurs in 1 in 5 to 7000 adults. Leading etiologies are autoimmune adrenalitis in adults and congenital adrenal hyperplasia (CAH) in children. Oral replacement of cortisol is lifesaving, but poor quality of life, repeated adrenal crises, and dosing uncertainty related to lack of a validated biomarker for glucocorticoid sufficiency persists. Adrenocortical cell therapy and gene therapy may obviate many of the shortcomings of adrenal hormone replacement. Physiological cortisol secretion regulated by pituitary adrenocorticotropin could be achieved through allogeneic adrenocortical cell transplantation, production of adrenal-like steroidogenic cells from either stem cells or lineage conversion of differentiated cells, or for CAH, gene therapy to replace or repair a defective gene. The adrenal cortex is a high-turnover organ and thus failure to incorporate progenitor cells within a transplant will ultimately result in graft exhaustion. Identification of adrenocortical progenitor cells is equally important in gene therapy, for which new genetic material must be specifically integrated into the genome of progenitors to ensure a durable effect. Delivery of gene-editing machinery and a donor template, allowing targeted correction of the 21-hydroxylase gene, has the potential to achieve this. This review describes advances in adrenal cell transplants and gene therapy that may allow physiological cortisol production for children and adults with PAI., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. In the present study, we first investigated the validity of a liver xenograft mouse model repopulated with primary hepatocytes using single-nucleus RNA sequencing (sn-RNA-seq) by studying the transcriptomic profile of human hepatocytes pre- and post-engraftment. Complementary immunofluorescence analyses performed in highly engrafted animals confirmed that the human hepatocytes organize and present appropriate patterns of zone-dependent enzyme expression in this model. Next, we tested a set of rationally designed HSPG de-targeted AAV-LK03 variants for relative transduction performance in human hepatocytes. We used immunofluorescence, next-generation sequencing, and single-nucleus transcriptomics data from highly engrafted FRG mice to demonstrate that the optimally HSPG de-targeted AAV-LK03 displayed a significantly improved lobular transduction profile in this model., Competing Interests: L.L. is a cofounder of LogicBio Therapeutics, and L.L. and I.E.A. are co-founders of Exigen Biotherapeutics, companies that utilize similar technologies broadly discussed in this paper., (© 2023 The Authors.)

Hydrodynamic tail vein injection (HTV) is the "gold standard" for delivering naked DNA vectors to mouse liver, thereby transfecting predominately perivenous hepatocytes. While HTV corrects metabolic liver defects such as phenylketonuria or cystathionine β-synthase deficiency, correction of spf ash mice with ornithine transcarbamylase (OTC) deficiency was not possible despite overexpression in the liver, as the OTC enzyme is primarily expressed in periportal hepatocytes. To target periportal hepatocytes, we established hydrodynamic retrograde intrabiliary injection (HRII) in mice and optimized minicircle (MC) vector delivery using luciferase as a marker gene. HRII resulted in a transfection efficiency below 1%, 100-fold lower than HTV. While HRII induced minimal liver toxicity compared with HTV, overexpression of luciferase by both methods, but not of a natural liver-specific enzyme, elicited an immune response that led to the elimination of luciferase expression. Further testing of MC vectors delivered via HRII in spf ash mice did not result in sufficient therapeutic efficacy and needs further optimization and/or selection of the corrected cells. This study reveals that luciferase expression is toxic for the liver. Furthermore, physical delivery of MC vectors via the bile duct has the potential to treat defects restricted to periportal hepatocytes, which opens new doors for non-viral liver-directed gene therapy., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Introduction: Gene therapy for spinal muscular atrophy (SMA) represents a significant milestone in the treatment of neurologic diseases. SMA is a neurodegenerative disease that results in motor neuron loss because of mutations of the survival motor neuron 1 gene, which directs survival motor neuron (SMN) protein production. Onasemnogene abeparvovec, a one-time gene replacement therapy, delivers a functional transgene to restore SMN protein expression. Onasemnogene abeparvovec has demonstrated improved survival and motor milestone achievements for presymptomatic infants and patients with SMA type 1., Areas Covered: This expert review describes the current state of gene therapy for SMA, reviews the mechanism of and clinical experience with onasemnogene abeparvovec, explains future efforts to expand applications of gene therapy for SMA, and provides context for developing gene therapy for other conditions., Expert Opinion: Onasemnogene abeparvovec has demonstrated efficacy in clinical trials and, because of this, is a valuable treatment option for patients with symptomatic infantile SMA and those identified by newborn screening. Gene therapy is still in its infancy, and challenges and uncertainties associated with transgene delivery must be addressed. With ongoing development of vector technology, more specific tissue tropism, reduced 'off-target' effects, and an enhanced safety profile will continue to evolve.

Gene therapy is making significant impact on a modest, yet growing, number of human diseases. Justifiably, the preferred viral vector for clinical use is that based on recombinant adeno-associated virus (rAAV). There is a need to scale up rAAV vector production with the transition from pre-clinical models to human application. Standard production methods based on the adherent cell type (HEK293) are limited in scalability and other methods, such as those based on the baculovirus and non-adherent insect cell (Sf9) system, have been pursued as an alternative to increase rAAV production. In this study, we compare these two production methods for cardiotropic rAAVs. Transduction efficiency for both production methods was assessed in primary cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and in mice following systemic delivery. We found that the rAAV produced by the traditional HEK293 method out-performed vector produced using the baculovirus/Sf9 system in vitro and in vivo. This finding provides a potential caveat for vector function when using the baculovirus/Sf9 production system and underscores the need for thorough assessment of vector performance when using diverse rAAV production methods.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7., (© 2022. The Author(s).)

Detecting viral and vector integration events is a key step when investigating interactions between viral and host genomes. This is relevant in several fields, including virology, cancer research and gene therapy. For example, investigating integrations of wild-type viruses such as human papillomavirus and hepatitis B virus has proven to be crucial for understanding the role of these integrations in cancer. Furthermore, identifying the extent of vector integration is vital for determining the potential for genotoxicity in gene therapies. To address these questions, we developed isling, the first tool specifically designed for identifying viral integrations in both wild-type and vector from next-generation sequencing data. Isling addresses complexities in integration behaviour including integration of fragmented genomes and integration junctions with ambiguous locations in a host or vector genome, and can also flag possible vector recombinations. We show that isling is up to 1.6-fold faster and up to 170% more accurate than other viral integration tools, and performs well on both simulated and real datasets. Isling is therefore an efficient and application-agnostic tool that will enable a broad range of investigations into viral and vector integration. These include comparisons between integrations of wild-type viruses and gene therapy vectors, as well as assessing the genotoxicity of vectors and understanding the role of viruses in cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an in vivo screen in a xenograft model of human hepatocytes, we identified the mRNA-based FT selection as the most optimal AAV selection method. Lastly, to gain insight into the mRNA-based selection mechanism driven by the native AAV-p40 promoter, we studied its activity in a range of in vitro and in vivo targets. We found AAV-p40 to be a ubiquitously active promoter that can be modified for cell-type-specific expression by incorporating binding sites for silencing transcription factors, allowing for cell-type-specific library selection.

Adeno-associated virus (AAV) vectors are proving to be clinically transformative tools in the treatment of monogenic genetic disease. Rapid ongoing development of this technology promises to not only increase the number of monogenic disorders amenable to this approach but also to bring diseases with complex multigenic and nongenetic etiologies within therapeutic reach. In this study, we explore the broader paradigm of converting the liver into a biofactory for systemic output of therapeutic molecules using AAV-mediated delivery of the endonuclease DNaseI as an exemplar. DNaseI can clear neutrophil extracellular traps (NETs), which are nuclear-protein structures possessing antimicrobial action, also involved in the pathophysiology of clinically troubling immune-mediated diseases. However, a translational challenge is short half-life of the enzyme in vivo (<5 h). This study demonstrates that AAV-mediated liver-targeted gene transfer stably induces serum DNaseI activity to >190-fold above physiological levels. In lupus-prone mice (NZBWF1), the activity was maintained for longer than 6 months, the latest time point tested, and resulted in a clear functional effect with reduced renal presence of neutrophils, NETs, IgG, and complement C3. However, treatment in this complex disease model did not extend lifespan, improve serological endpoints, or preserve renal function, indicating there are elements of pathophysiology not accessible to DNaseI in the NZBWF1 model. We conclude that a translational solution to the challenge of short half-life of DNaseI is AAV-mediated gene delivery and that this may be efficacious in treating disease where NETs are a dominant pathological mechanism.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease and is frequently associated with type 2 diabetes. However, there is no specific medical therapy to treat this condition. Angiotensin-converting enzyme 2 (ACE2) of the protective renin angiotensin system generates the antifibrotic peptide angiotensin-(1-7) from profibrotic angiotensin II peptide. In this study, we investigated the therapeutic potential of ACE2 in diabetic NAFLD mice fed a high-fat (20%), high-cholesterol (2%) diet for 40 weeks. Mice were given a single intraperitoneal injection of ACE2 using an adeno-associated viral vector at 30 weeks of high-fat, high-cholesterol diet (15 weeks after induction of diabetes) and sacrificed 10 weeks later. ACE2 significantly reduced liver injury and fibrosis in diabetic NAFLD mice compared with the control vector injected mice. This was accompanied by reductions in proinflammatory cytokine expressions, hepatic stellate cell activation, and collagen 1 expression. Moreover, ACE2 therapy significantly increased islet numbers, leading to an increased insulin protein content in β-cells and plasma insulin levels with subsequent reduction in plasma glucose levels compared with controls. Conclusion: We conclude that ACE2 gene therapy reduces liver fibrosis and hyperglycemia in diabetic NAFLD mice and has potential as a therapy for patients with NAFLD with diabetes., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Viral integration is a complex biological process, and it is useful to have a reference integration dataset with known properties to compare experimental data against, or for comparing with the results from computational tools that detect integration. To generate these data, we developed a pipeline for simulating integrations of a viral or vector genome into a host genome. Our method reproduces more complex characteristics of vector and viral integration, including integration of sub-genomic fragments, structural variation of the integrated genomes, and deletions from the host genome at the integration site. Our method [1] takes the form of a snakemake [2] pipeline, consisting of a Python [3] script using the Biopython [4] module that simulates integrations of a viral reference into a host reference. This produces a reference containing integrations, from which sequencing reads are simulated using ART [5]. The IDs of the reads crossing integration junctions are then annotated using another python script to produce the final output, consisting of the simulated reads and a table of the locations of those integrations and the reads crossing each integration junction. To illustrate our method, we provide simulated reads, integration locations, as well as the code required to simulate integrations using any virus and host reference. This simulation method was used to investigate the performance of viral integration tools in our research [6]., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2022 Published by Elsevier Inc.)

Objective: To provide a greater understanding of the tolerability, safety and clinical outcomes of onasemnogene abeparvovec in real-world practice, in a broad population of infants with spinal muscular atrophy (SMA)., Methods: A prospective cohort study of children with SMA treated with onasemnogene abeparvovec at Sydney Children's Hospital Network, Australia was conducted from August 2019 to November 2021. Safety outcomes included clinical and laboratory evaluations. Efficacy assessments included World Health Organisation (WHO) motor milestones, oral and swallowing abilities, and requirements for respiratory support. The implementation of a model of care for onasemnogene abeparvovec administration in health practice is described., Results: 21 children were treated (age range, 0.65-24 months; body weight range, 2.5-12.5 kg) and 19/21 (90.4%) had previous nusinersen. Transient treatment-related side effects occurred in all children; vomiting (100%), transaminitis (57%) and thrombocytopaenia (33%). Incidence of moderate/severe transaminitis was significantly greater in infants weighing ≥8 kg compared with <8 kg (p < 0.05). Duration of prednisolone following treatment was prolonged (mean 87.5 days, range 57-274 days). 16/21 (76%) children gained at least one WHO motor milestone. Stabilisation or improvement in bulbar or respiratory function was observed in 20/21 (95.2%) patients. Implementation challenges were mitigated by developing standard operating procedures and facilitating exchange of knowledge., Interpretation: This study provides real-world evidence to inform treatment decisions and guide therapeutic expectations for onasemnogene abeparvovec and combination therapy for SMA in health practice, especially for children weighing ≥8 kg receiving higher vector loads. Proactive clinical and laboratory surveillance is essential to facilitate individualised management of risks., (© 2022 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. Here, we report the bioengineering of a set of next-generation AAV vectors, named AAV-SYDs (where "SYD" stands for Sydney, Australia), with increased human hepato-tropism in a liver xenograft mouse model repopulated with primary human hepatocytes. We followed a two-step process that staggered directed evolution and domain-swapping approaches. Using DNA-family shuffling, we first mapped key AAV capsid regions responsible for efficient human hepatocyte transduction in vivo . Focusing on these regions, we next applied domain-swapping strategies to identify and study key capsid residues that enhance primary human hepatocyte uptake and transgene expression. Our findings underscore the potential of AAV-SYDs as liver gene therapy vectors and provide insights into the mechanism responsible for their enhanced transduction profile., Competing Interests: M.C.-C., I.E.A., M.H. and L.L. are inventors on patent applications filed by Children's Medical Research Institute related to AAV capsid sequences and in vivo function of novel AAV variants. L.L. is a co-founder and a scientific advisor at LogicBio Therapeutics. M.H. is an employee of, and holds stocks in, LogicBio Therapeutics. L.L. and I.E.A. are co-founders of Exigen Biotherapeutics. L.L. has a sponsored research agreement with LogicBio Therapeutics. M.C.-C., I.E.A., and L.L. have IP licensed to biopharmaceutical companies. L.L. and I.A.E. have consulted on technologies addressed in this paper. L.L. and I.A.E. have stock and/or equity in companies with technology broadly related to this paper. A.J.T. is a cofounder and scientific consultant for Orchard Therapeutics, consultant for Rocket Pharmaceuticals, Generation bio, bluebird bio, 4Bio Capital Partners, and Sana Biotechnology. G.J.L. consults for Gyroscope Therapeutics. All other authors declare no conflicts of interest., (© 2021 The Authors.)

The complement system is a key component of innate immunity, but impaired regulation influences disease susceptibility, including age-related macular degeneration and some kidney diseases. While complete complement inhibition has been used successfully to treat acute kidney disease, key unresolved challenges include strategies to modulate rather than completely inhibit the system and to deliver therapy potentially over decades. Elevating concentrations of complement factor I (CFI) restricts complement activation in vitro and this approach was extended in the current study to modulate complement activation in vivo . Sustained increases in CFI levels were achieved using an adeno-associated virus (AAV) vector to target the liver, inducing a 4- to 5-fold increase in circulating CFI levels. This led to decreased activity of the alternative pathway as demonstrated by a reduction in the rate of inactive C3b (iC3b) deposition and more rapid formation of C3 degradation products. In addition, vector application in a mouse model of systemic lupus erythematosus (NZBWF1), where tissue injury is, in part, complement dependent, resulted in reduced complement C3 and IgG renal deposition. Collectively, these data demonstrate that sustained elevation of CFI reduces complement activation in vivo providing proof-of-principle support for the therapeutic application of AAV gene delivery to modulate complement activation.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

X-linked inherited ornithine transcarbamylase deficiency (OTCD) is the most common disorder affecting the liver-based urea cycle, a pathway enabling detoxification of nitrogen waste and endogenous arginine biosynthesis. Patients develop acute hyperammonemia leading to neurological sequelae or death despite the best-accepted therapy based on ammonia scavengers and protein-restricted diet. Liver transplantation is curative but associated with procedure-related complications and lifelong immunosuppression. Adeno-associated viral (AAV) vectors have demonstrated safety and clinical benefits in a rapidly growing number of clinical trials for inherited metabolic liver diseases. Engineered AAV capsids have shown promising enhanced liver tropism. Here, we conducted a good-laboratory practice-compliant investigational new drug-enabling study to assess the safety of intravenous liver-tropic AAVLK03 gene transfer of a human codon-optimized OTC gene. Juvenile cynomolgus monkeys received vehicle and a low and high dose of vector (2 × 10 12 and 2 × 10 13 vector genome (vg)/kg, respectively) and were monitored for 26 weeks for in-life safety with sequential liver biopsies at 1 and 13 weeks post-vector administration. Upon completion of monitoring, animals were euthanized to study vector biodistribution, immune responses, and histopathology. The product was well tolerated with no adverse clinical events, predominant hepatic biodistribution, and sustained supra-physiological OTC overexpression. This study supports the clinical deployment of intravenous AAVLK03 for severe OTCD., Competing Interests: I.E.A. and L.L. are inventors on patents related to the AAVLK03 capsid or the transgene cassette used in AAVLK03.hOTC. The other authors have no competing interests., (© 2021 The Author(s).)

Recent successes in clinical gene therapy applications have intensified the interest in using adeno-associated viruses (AAVs) as vectors for gene delivery into human liver. An inherent intriguing characteristic of AAVs is that vector variants vary substantially in their ability to transduce hepatocytes from different species. This has historically limited the value of preclinical studies using rodent models for predicting the efficiency of AAV vectors in liver-targeted gene therapy clinical studies. In this work, we aimed to investigate the key determinants of the observed differential interspecies transduction abilities among AAV variants. We took advantage of domain swapping strategies between AAV-KP1, a newly identified variant with enhanced murine liver tropism, and AAV3b, which functions poorly in mice. The systematic in vivo comparison of AAV3b/AAV-KP1 chimeric variants allowed us to identify a threonine insertion at position 265 within variable region I (VR-I) as the key residue that confers murine hepatic transduction to human-derived clade B (AAV2-like) and clade C (AAV3b-like) variants. We propose to use this insertion to generate phylogenetically related AAV surrogates in support of toxicology and dosing studies in the murine liver model., Competing Interests: M.C.-C., I.E.A., and L.L. are inventors on patent applications filed by Children’s Medical Research Institute related to AAV capsid sequences and in vivo function of novel AAV variants. L.L. is a co-founder and scientific advisor of LogicBio Therapeutics. L.L. and I.E.A. are co-founders of Exigen Biotherapeutics. L.L. has a sponsored research agreement with LogicBio Therapeutics. M.C.-C., I.E.A. and L.L. have intellectual property (IP) licensed to biopharmaceutical companies. L.L. and I.A.E. have consulted on technologies addressed in this paper. L.L. and I.A.E. have stock and/or equity in companies with technology broadly related to this paper. The remaining authors declare no competing interest., (© 2021 The Author(s).)

Genetic variation within the factor H-related (FHR) genes is associated with the complement-mediated kidney disease, C3 glomerulopathy (C3G). There is no definitive treatment for C3G, and a significant proportion of patients develop end-stage renal disease. The prototypical example is CFHR5 nephropathy, through which an internal duplication within a single CFHR5 gene generates a mutant FHR5 protein (FHR5mut) that leads to accumulation of complement C3 within glomeruli. To elucidate how abnormal FHR proteins cause C3G, we modeled CFHR5 nephropathy in mice. Animals lacking the murine factor H (FH) and FHR proteins, but coexpressing human FH and FHR5mut (hFH-FHR5mut), developed glomerular C3 deposition, whereas mice coexpressing human FH with the normal FHR5 protein (hFH-FHR5) did not. Like in patients, the FHR5mut had a dominant gain-of-function effect, and when administered in hFH-FHR5 mice, it triggered C3 deposition. Importantly, adeno-associated virus vector-delivered homodimeric mini-FH, a molecule with superior surface C3 binding compared to FH, reduced glomerular C3 deposition in the presence of the FHR5mut. Our data demonstrate that FHR5mut causes C3G by disrupting the homeostatic regulation of complement within the kidney and is directly pathogenic in C3G. These results support the use of FH-derived molecules with enhanced C3 binding for treating C3G associated with abnormal FHR proteins. They also suggest that targeting FHR5 represents a way to treat complement-mediated kidney injury., Competing Interests: Competing interest statement: M.C.P. has received consultancy fees for providing scientific advice for work for Alexion, ChemoCentryx, Achillion, Apellis, Gemini, Gyroscope, Ra, Silence Therapeutics, and Sobi Pharma. M.C.P. is a scientific advisory board member for Gemini and Gyroscope Pharma., (Copyright © 2021 the Author(s). Published by PNAS.)

Background & Aims: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored., Methods: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC., Results: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5 + intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis., Conclusions: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver., (Copyright © 2021 AGA Institute. All rights reserved.)

Liver-targeted genome editing offers the prospect of life-long therapeutic benefit following a single treatment and is set to rapidly supplant conventional gene addition approaches. Combining progress in liver-targeted gene delivery with genome editing technology, makes this not only feasible but realistically achievable in the near term. However, important challenges remain to be addressed. These include achieving therapeutic levels of editing, particularly in vivo, avoidance of off-target effects on the genome and the potential impact of pre-existing immunity to bacteria-derived nucleases, when used to improve editing rates. In this chapter, we outline the unique features of the liver that make it an attractive target for genome editing, the impact of liver biology on therapeutic efficacy, and disease specific challenges, including whether the approach targets a cell autonomous or non-cell autonomous disease. We also discuss strategies that have been used successfully to achieve genome editing outcomes in the liver and address translational considerations as genome editing technology moves into the clinic., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte-derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah -/- /Rag2 -/- /Il2rg -/- (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Gene therapy (GT) has tremendous potential for the treatment of neurological disorders to transform patient care. The successful application of virus-mediated GT to treat spinal muscular atrophy is a significant milestone, serving to accelerate similar progress in a spectrum of neurological conditions, with more than 50 clinical trials currently underway, across neurodevelopmental, neurodegenerative, muscular dystrophy, epilepsy, chronic pain and neoplastic diseases. This review provides an overview of the key features of virus-mediated GT, paradigms of delivery and dosing, potential risks and highlights ongoing research to optimise safe and effective delivery of vectors into the nervous system. Examples of the application of GT in various neurological diseases alongside clinical development challenges will be presented. As the development and translation of GTs gain pace, success can only ultimately be realised for patients following implementation in the health system. The challenges and controversies of daunting costs, ethics, early diagnosis and health system readiness will require innovative pricing schemes, regulatory policies, education and organisation of a skilled workforce to deliver of high-quality care in clinical practice as we prepare for advanced therapeutics in neurology., Competing Interests: Competing interests: MF has received compensation as a member of the scientific advisory board for Biogen, Roche and AveXis. DK has received reimbursement for attending symposia, sponsored by Biogen. IA has received compensation for the cost of participation on a Novartis advisory panel.These funding bodies had no role in the manuscript design, preparation of the manuscript or decision to publish., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: The utility of genome sequencing (GS) in the diagnosis of suspected pediatric mitochondrial disease (MD) was investigated., Methods: An Australian cohort of 40 pediatric patients with clinical features suggestive of MD were classified using the modified Nijmegen mitochondrial disease severity scoring into definite (17), probable (17), and possible (6) MD groups. Trio GS was performed using DNA extracted from patient and parent blood. Data were analyzed for single-nucleotide variants, indels, mitochondrial DNA variants, and structural variants., Results: A definitive MD gene molecular diagnosis was made in 15 cases and a likely MD molecular diagnosis in a further five cases. Causative mitochondrial DNA (mtDNA) variants were identified in four of these cases. Three potential novel MD genes were identified. In seven cases, causative variants were identified in known disease genes with no previous evidence of causing a primary MD. Diagnostic rates were higher in patients classified as having definite MD., Conclusion: GS efficiently identifies variants in MD genes of both nuclear and mitochondrial origin. A likely molecular diagnosis was identified in 67% of cases and a definitive molecular diagnosis achieved in 55% of cases. This study highlights the value of GS for a phenotypically and genetically heterogeneous disorder like MD.

Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo , an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo . Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo . The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs., (© 2020 The Author(s).)

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro , ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.

Purpose: To evaluate the implementation of the first statewide newborn screening (NBS) program for spinal muscular atrophy (SMA) in Australia. Processes that hinder and support clinical development, translation, and sustainability of the first primary genetic screening program in Australia are appraised., Methods: The study prospectively describes the course (timelines, health processes, and preliminary clinical outcomes) for SMA screen-positive newborns from 1 August 2018 to 31 July 2019 in New South Wales and Australian Capital Territory, Australia., Results: In the first year of the program, 103,903 newborns were screened. Ten newborns screened positive for SMA. Genetic confirmation of SMA occurred in 9/10 (90%) of infants. Clinical signs of SMA evolved in 4/9 (44%) within 4 weeks of life, heralded by hypotonia and weakness initially recognized in the neck. Median time to implementing a care plan (including commencement of disease-modifying therapies) was 26.5 days (16-37 days) from birth., Conclusion: NBS is essential for early and equitable identification of patients with SMA. Expedient diagnosis and management are vital, as disease latency appears brief in some cases. NBS shows significant clinical utility to support early parental decision making, improve access to specialist neuromuscular expertise, and facilitate initiation of personalized therapeutic strategies.

McArdle disease is a disorder of carbohydrate metabolism that causes painful skeletal muscle cramps and skeletal muscle damage leading to transient myoglobinuria and increased risk of kidney failure. McArdle disease is caused by recessive mutations in the muscle glycogen phosphorylase (PYGM) gene leading to absence of PYGM enzyme in skeletal muscle and preventing access to energy from muscle glycogen stores. There is currently no cure for McArdle disease. Using a preclinical animal model, we aimed to identify a clinically translatable and relevant therapy for McArdle disease. We evaluated the safety and efficacy of recombinant adeno-associated virus serotype 8 (rAAV8) to treat a murine model of McArdle disease via delivery of a functional copy of the disease-causing gene, Pygm. Intraperitoneal injection of rAAV8-Pygm at post-natal day 1-3 resulted in Pygm expression at 8 weeks of age, accompanied by improved skeletal muscle architecture, reduced accumulation of glycogen and restoration of voluntary running wheel activity to wild-type levels. We did not observe any adverse reaction to the treatment at 8 weeks post-injection. Thus, we have investigated a highly promising gene therapy for McArdle disease with a clear path to the ovine large animal model endemic to Western Australia and subsequently to patients., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background & Aims: Genome editing technology has immense therapeutic potential and is likely to rapidly supplant contemporary gene addition approaches. Key advantages include the capacity to directly repair mutant loci with resultant recovery of physiological gene expression and maintenance of durable therapeutic effects in replicating cells. In this study, we aimed to repair a disease-causing point mutation in the ornithine transcarbamylase ( OTC ) locus in patient-derived primary human hepatocytes in vivo at therapeutically relevant levels., Methods: Editing reagents for precise CRISPR/SaCas9-mediated cleavage and homology-directed repair (HDR) of the human OTC locus were first evaluated against an OTC minigene cassette transposed into the mouse liver. The editing efficacy of these reagents was then tested on the native OTC locus in patient-derived primary human hepatocytes xenografted into the FRG ( Fah -/- Rag2 -/- Il2rg -/- ) mouse liver. A highly human hepatotropic capsid (NP59) was used for adeno-associated virus (AAV)-mediated gene transfer. Editing events were characterised using next-generation sequencing and restoration of OTC expression was evaluated using immunofluorescence., Results: Following AAV-mediated delivery of editing reagents to patient-derived primary human hepatocytes in vivo , OTC locus-specific cleavage was achieved at efficiencies of up to 72%. Importantly, successful editing was observed in up to 29% of OTC alleles at clinically relevant vector doses. No off-target editing events were observed at the top 10 in silico -predicted sites in the genome., Conclusions: We report efficient single-nucleotide correction of a disease-causing mutation in the OTC locus in patient-derived primary human hepatocytes in vivo at levels that, if recapitulated in the clinic, would provide benefit for even the most therapeutically challenging liver disorders. Key challenges for clinical translation include the cell cycle dependence of classical HDR and mitigation of unintended on- and off-target editing events., Lay Summary: The ability to efficiently and safely correct disease-causing mutations remains the holy grail of gene therapy. Herein, we demonstrate, for the first time, efficient in vivo correction of a patient-specific disease-causing mutation in the OTC gene in primary human hepatocytes, using therapeutically relevant vector doses. We also highlight the challenges that need to be overcome for this technology to be translated into clinical practice., (© 2019 The Authors.)

Recombinant adeno-associated viral (rAAV) vectors are highly promising vehicles for liver-targeted gene transfer, with therapeutic efficacy demonstrated in preclinical models and clinical trials. Progressive familial intrahepatic cholestasis type 3 (PFIC3), an inherited juvenile-onset, cholestatic liver disease caused by homozygous mutation of the ABCB4 gene, may be a promising candidate for rAAV-mediated liver-targeted gene therapy. The Abcb4 -/- mice model of PFIC3, with juvenile mice developing progressive cholestatic liver injury due to impaired biliary phosphatidylcholine excretion, resulted in cirrhosis and liver malignancy. Using a conventional rAAV strategy, we observed markedly blunted rAAV transduction in adult Abcb4 -/- mice with established liver disease, but not in disease-free, wild-type adults or in homozygous juveniles prior to liver disease onset. However, delivery of predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression due to hepatocyte proliferation in the growing liver. Conclusion: A hybrid vector system, combining the high transduction efficiency of rAAV with piggyBac transposase-mediated somatic integration, was developed to facilitate stable human ABCB4 expression in vivo and to correct juvenile-onset chronic liver disease in a murine model of PFIC3. A single dose of hybrid vector at birth led to life-long restoration of bile composition, prevention of biliary cirrhosis, and a substantial reduction in tumorigenesis. This powerful hybrid rAAV-piggyBac transposon vector strategy has the capacity to mediate lifelong phenotype correction and reduce the tumorigenicity of progressive familial intrahepatic cholestasis type 3 and, with further refinement, the potential for human clinical translation., (© 2019 by the American Association for the Study of Liver Diseases.)

Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and genome editing applications. While conceptually simple, the system harbors considerable complexity that presents many potential pitfalls for the inexperienced user. The short inverted terminal repeats (ITRs) can prove to be particularly problematic during vector engineering due to inherent instability necessitating diligent quality control measures during vector manufacture. This is especially important from a clinical standpoint when consistent purity and potency are paramount, and all components of the system are rigorously scrutinized by regulatory agencies. Despite the discovery over 30 years ago that the AAV ITRs are the only cis -acting elements of the virus required for vector production, there is a scarcity of reviews specifically focused on these complex elements. This review provides an overview of the ITR with the dual purpose of acting as a user's guide in the application of AAV vector technology and as a roadmap for ongoing vector development and optimization.