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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 Spfash 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive [CD8.sup.+] 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-2[K.sup.b]-associated peptides recognized by [CD8.sup.+] T cells from B10.BR (H-[2.sup.k]) mice, 13 of which were also recognized by BALB/c (H-[2.sup.d]) 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., Introduction Allorecognition may result in either graft rejection, graft versus host disease, or transplantation tolerance, depending on the context in which the recipient immune system encounters the allogeneic major histocompatibility [...]

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials (1-3). The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome (4-8). Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes invivo. As predicted from preclinical and clinical studies (4, 5, 8), rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly--approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications., rAAV clinical trials have been hampered by unanticipated immunological responses and lower than expected levels of transgene product (5-8). For a single serotype there is little correlation between in vitro [...]