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Phenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno-associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon-optimized human PAH gene, with or without an intron spacer, to the Pahenu2 mouse model of PKU. Dose-dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pahenu2 mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 × 1012 VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV-dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki-67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration.

The effectiveness of cell-based therapies to treat liver failure is often limited by the diseased liver environment. Here, we provide preclinical proof of concept for hepatocyte transplantation into lymph nodes as a cure for liver failure in a large-animal model with hereditary tyrosinemia type 1 (HT1), a metabolic liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) enzyme. Autologous porcine hepatocytes were transduced ex vivo with a lentiviral vector carrying the pig Fah gene and transplanted into mesenteric lymph nodes. Hepatocytes showed early (6 h) and durable (8 months) engraftment in lymph nodes, with reproduction of vascular and hepatic microarchitecture. Subsequently, hepatocytes migrated to and repopulated the native diseased liver. The corrected cells generated sufficient liver mass to clinically ameliorate the acute liver failure and HT1 disease as early as 97 days post-transplantation. Integration site analysis defined the corrected hepatocytes in the liver as a subpopulation of hepatocytes from lymph nodes, indicating that the lymph nodes served as a source for healthy hepatocytes to repopulate a diseased liver. Therefore, ectopic transplantation of healthy hepatocytes cures this pig model of liver failure and presents a promising approach for the development of cures for liver disease in patients., Graphical Abstract, Lillegard and colleagues demonstrate that ectopic hepatocyte transplantation into lymph nodes cures liver failure in a large-animal model of metabolic disease, hereditary tyrosinemia type-1 (HT1). Autologous hepatocytes were transduced ex vivo with a lentiviral vector carrying the Fah gene, missing in HT1. Transplanted hepatocytes generated sufficient liver mass to cure the disease.

Robert and Joyce Hogan introduced the concept of the dark side of personality to identify common counterproductive dispositions associated with ineffective leadership and career derailment. Dark-side traits are more complex and complicated than the bright-side traits assessed by most personality inventories. This chapter is about untangling the risks and rewards of the dark side for those who conduct executive assessments for (1) hiring, promotion, and succession planning as well as (2) leadership development and executive coaching. First, a brief history of the origins of the dark-side concept is reviewed. Next, distinctions are made between bright-side traits, dark-side traits, and personality disorders. Then a paradox is identified and addressed: Dark-side derailers are associated with both upside potential for effective leadership and downside risks for ineffective leadership. Finally, practical implications are provided with recommendations for executive selection and development.

CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'

General safety and toxicology assessments supporting in vivo lentiviral vector-based therapeutic development are sparse. We have previously demonstrated the efficacy of a lentiviral vector expressi...

In order to investigate inter-disciplinary differences in substance use-related attitudes, 651 trainees from counseling, dentistry, nursing, physician assistant, and psychology programs wer...

Introduction Human cadaver allograft (HCA) is the current standard of care for temporary wound closure of large burns, but a critical need for high-quality alternatives exists. Porcine skin shares many similarities with human skin, and hyperacute rejection is prevented via a single genetic modification. Like human skin, non-terminally sterilized porcine skin contains viable dermal and epidermal cells and intact vasculature that enables restoration of barrier function. These characteristics are distinct from those of traditional, terminally sterilized “xenografts” and may offer greater therapeutic capability. We report here key efficacy outcomes specific to severe burn care from a first-in-human clinical trial to assess the capability of genetically engineered porcine skin xenotransplants to provide temporary wound closure for severe and extensive deep-partial and full-thickness burn wounds, compared to HCA. Methods Split-thickness skin containing epidermal and dermal layers was harvested from Designated Pathogen Free, GalT-KO, porcine donors, cGMP processed to achieve USP< 71 > sterility and cryopreserved to retain >70% cell viability. These were transplanted side by side with HCA on debrided full-thickness burn wounds in six human subjects. Temporary wound closure, incidence of complete wound closure following autografting, and quality of healing, including scarring, contour, and feel of healed skin, normalization of skin markings or pigmentation, were independently assessed. Results Across all patients and assessment time points, adherence, vascularity, and overall appearance were indistinguishable between porcine skin xenotransplants and HCA control. After surgical removal, wound beds treated with each type of dressing were perfused and otherwise appeared equivalent and clinically suitable for autografting. Long-term outcomes were comparable between wound sites treated with porcine skin or HCA with no discernable differences in scarring or cosmesis. Conclusions Skin xenotransplants effectively provided temporary wound closure and restoration of barrier function via intact native vasculature, active cells, decreased antigenicity, and high-quality tissue architecture unimpacted by cryopreservation and thawing. These results show clinical promise as an interchangeable alternative to HCA in the treatment of severe burns. Expanded clinical evaluation is ongoing.

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)−1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate for the first time a cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy was well tolerated and provided stable long-term expression of FAH in pigs with HT1. Genomic integration displayed a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.