Interim analyses of a first-in-human phase 1/2 mRNA trial for propionic acidaemia.

Propionic acidaemia is a rare disorder caused by defects in the propionyl-coenzyme A carboxylase α or ß (PCCA or PCCB) subunits that leads to an accumulation of toxic metabolites and to recurrent, life-threatening metabolic decompensation events. Here we report interim analyses of a first-in-human, phase 1/2, open-label, dose-optimization study and an extension study evaluating the safety and efficacy of mRNA-3927, a dual mRNA therapy encoding PCCA and PCCB. As of 31 May 2023, 16 participants were enrolled across 5 dose cohorts. Twelve of the 16 participants completed the dose-optimization study and enrolled in the extension study. A total of 346 intravenous doses of mRNA-3927 were administered over a total of 15.69 person-years of treatment. No dose-limiting toxicities occurred. Treatment-emergent adverse events were reported in 15 out of the 16 (93.8%) participants. Preliminary analysis suggests an increase in the exposure to mRNA-3927 with dose escalation, and a 70% reduction in the risk of metabolic decompensation events among 8 participants who reported them in the 12-month pretreatment period.

Transforming the Future of Surgeon-Scientists.

OBJECTIVE: To create a blueprint for surgical department leaders, academic institutions, and funding agencies to optimally support surgeon-scientists. BACKGROUND: Scientific contributions by surgeons have been transformative across many medical disciplines. Surgeon-scientists provide a distinct approach and mindset toward key scientific questions. However, lack of institutional support, pressure for increased clinical productivity, and growing administrative burden are major challenges for the surgeon-scientist, as is the time-consuming nature of surgical training and practice. METHODS: An American Surgical Association Research Sustainability Task Force was created to outline a blueprint for sustainable science in surgery. Leaders from top NIH-sponsored departments of surgery engaged in video and in-person meetings between January and April 2023. A strength, weakness, opportunities, threats analysis was performed, and workgroups focused on the roles of surgeons, the department and institutions, and funding agencies. RESULTS: Taskforce recommendations: (1) SURGEONS: Growth mindset : identifying research focus, long-term planning, patience/tenacity, team science, collaborations with disparate experts; Skill set : align skills and research, fill critical skill gaps, develop team leadership skills; DEPARTMENT OF SURGERY (DOS): (2) MENTORSHIP: Chair : mentor-mentee matching/regular meetings/accountability, review of junior faculty progress, mentorship training requirement, recognition of mentorship (eg, relative value unit equivalent, awards; Mentor: dedicated time, relevant scientific expertise, extramural funding, experience and/or trained as mentor, trusted advisor; Mentee : enthusiastic/eager, proactive, open to feedback, clear about goals; (3) FINANCIAL SUSTAINABILITY: diversification of research portfolio, identification of matching funding sources, departmental resource awards (eg, T-/P-grants), leveraging of institutional resources, negotiation of formalized/formulaic funds flow investment from academic medical center toward science, philanthropy; (4) STRUCTURAL/STRATEGIC SUPPORT: Structural: grants administrative support, biostats/bioinformatics support, clinical trial and research support, regulatory support, shared departmental laboratory space/equipment; Strategic: hiring diverse surgeon-scientist/scientists faculty across DOS, strategic faculty retention/ recruitment, philanthropy, career development support, progress tracking, grant writing support, DOS-wide research meetings, regular DOS strategic research planning; (5) COMMUNITY AND CULTURE: Community: right mix of faculty, connection surgeon with broad scientific community; Culture: building research infrastructure, financial support for research, projecting importance of research (awards, grand rounds, shoutouts); (6) THE ROLE OF INSTITUTIONS: Foundation: research space co-location, flexible start-up packages, courses/mock study section, awards, diverse institutional mentorship teams; Nurture: institutional infrastructure, funding (eg, endowed chairs), promotion friendly toward surgeon-scientists, surgeon-scientists in institutional leadership positions; Expectations: RVU target relief, salary gap funding, competitive starting salaries, longitudinal salary strategy; (7) THE ROLE OF FUNDING AGENCIES: change surgeon research training paradigm, offer alternate awards to K-awards, increasing salary cap to reflect market reality, time extension for surgeon early-stage investigator status, surgeon representation on study section, focused award strategies for professional societies/foundations. CONCLUSIONS: Authentic recommitment from surgeon leaders with intentional and ambitious actions from institutions, corporations, funders, and society is essential in order to reap the essential benefits of surgeon-scientists toward advancements of science.

Guanidinoacetate (GAA) is a potent GABAA receptor GABA mimetic: Implications for neurological disease pathology.

Impairment of excretion and enzymatic processing of nitrogen, for example, because of liver or kidney failure, or with urea cycle and creatine synthesis enzyme defects, surprisingly leads to primarily neurologic symptoms, yet the exact mechanisms remain largely mysterious. In guanidinoacetate N-methyltransferase (GAMT) deficiency, the guanidino compound guanidinoacetate (GAA) increases dramatically, including in the cerebrospinal fluid (CSF), and has been implicated in mediating the neurological symptoms in GAMT-deficient patients. GAA is synthesized by arginine-glycine amidinotransferase (AGAT), a promiscuous enzyme that not only transfers the amidino group from arginine to glycine, but also to primary amines in, for example, GABA and taurine to generate γ-guanidinobutyric acid (γ-GBA) and guanidinoethanesulfonic acid (GES), respectively. We show that GAA, γ-GBA, and GES share structural similarities with GABA, evoke GABAA receptor (GABAA R) mediated currents (whereas creatine [methylated GAA] and arginine failed to evoke discernible currents) in cerebellar granule cells in mouse brain slices and displace the high-affinity GABA-site radioligand [3 H]muscimol in total brain homogenate GABAA Rs. While γ-GBA and GES are GABA agonists and displace [3 H]muscimol (EC50 /IC50 between 10 and 40 µM), GAA stands out as particularly potent in both activating GABAA Rs (EC50 ~6 µM) and also displacing the GABAA R ligand [3 H]muscimol (IC50 ~3 µM) at pathophysiologically relevant concentrations. These findings stress the role of substantially elevated GAA as a primary neurotoxic agent in GAMT deficiency and we discuss the potential role of GAA in arginase (and creatine transporter) deficiency which show a much more modest increase in GAA concentrations yet share the unique hyperexcitability neuropathology with GAMT deficiency. We conclude that orthosteric activation of GABAA Rs by GAA, and potentially other GABAA R mimetic guanidino compounds (GCs) like γ-GBA and GES, interferes with normal inhibitory GABAergic neurotransmission which could mediate, and contribute to, neurotoxicity.

Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency.

Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.

Targeted deletion of PAC1 receptors in retinal neurons enhances neuron loss and axonopathy in a model of multiple sclerosis and optic neuritis.

Chronic inflammation drives synaptic loss in multiple sclerosis (MS) and is also commonly observed in other neurodegenerative diseases. Clinically approved treatments for MS provide symptomatic relief but fail to halt neurodegeneration and neurological decline. Studies in animal disease models have demonstrated that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) exhibits anti-inflammatory, neuroprotective and regenerative properties. Anti-inflammatory actions appear to be mediated primarily by two receptors, VPAC1 and VPAC2, which also bind vasoactive intestinal peptide (VIP). Pharmacological experiments indicate that another receptor, PAC1 (ADCYAP1R1), which is highly selective for PACAP, provides protection to neurons, although genetic evidence and other mechanistic information is lacking. To determine if PAC1 receptors protect neurons in a cell-autonomous manner, we used adeno-associated virus (AAV2) to deliver Cre recombinase to the retina of mice harboring floxed PAC1 alleles. Mice were then subjected to chronic experimental autoimmune encephalomyelitis (EAE), a disease model that recapitulates major clinical and pathological features of MS and associated optic neuritis. Unexpectedly, deletion of PAC1 in naïve mice resulted in a deficit of retinal ganglionic neurons (RGNs) and their dendrites, suggesting a homeostatic role of PAC1. Moreover, deletion of PAC1 resulted in increased EAE-induced loss of a subpopulation of RGNs purported to be vulnerable in animal models of glaucoma. Increased axonal pathology and increased secondary presence of microglia/macrophages was also prominently seen in the optic nerve. These findings demonstrate that neuronal PAC1 receptors play a homeostatic role in protecting RGNs and directly protects neurons and their axons against neuroinflammatory challenge. SIGNIFICANCE STATEMENT: Chronic inflammation is a major component of neurodegenerative diseases and plays a central role in multiple sclerosis (MS). Current treatments for MS do not prevent neurodegeneration and/or neurological decline. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to have anti-inflammatory, neuroprotective and regenerative properties but the cell type- and receptor-specific mechanisms are not clear. To test whether the protective effects of PACAP are direct on the PAC1 receptor subtype on neurons, we delete PAC1 receptors from neurons and investigate neuropathologigical changes in an animal model of MS. The findings demonstrate that PAC1 receptors on neurons play a homeostatic role in maintaining neuron health and can directly protect neurons and their axons during neuroinflammatory disease.

Split AAV-Mediated Gene Therapy Restores Ureagenesis in a Murine Model of Carbamoyl Phosphate Synthetase 1 Deficiency.

The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

Mesangial sclerosis in a patient with type 1 diabetes following simultaneous pancreas-kidney transplantation despite maintenance of normoglycemia: a case report.

BACKGROUND: Simultaneous pancreas-kidney transplantation is considered a curative treatment for type 1 diabetes complicated by end-stage kidney disease. We report herein a case of mesangial sclerosis in a patient who underwent successful kidney-pancreas transplantation despite well-controlled glucose and excellent pancreatic allograft function. CASE PRESENTATION: A 76-year-old type 1 diabetic man who underwent a simultaneous pancreas-kidney transplantation 19 years prior presented with persistent nephrotic range proteinuria although creatinine was at his baseline (normal) level. Hemoglobin A1c and fasting glucose were well controlled without the use of insulin or oral antihyperglycemic agents. Serum lipase and amylase were within the reference range and there was no evidence of donor-specific antibodies. Kidney allograft biopsy was performed to evaluate proteinuria and showed diffuse capillary loop thickening and diffuse moderate to severe mesangial sclerosis resembling diabetic nephropathy. CONCLUSIONS: This case demonstrates a case of mesangial sclerosis resembling diabetic nephropathy in a patient with good glucose control after simultaneous pancreas-kidney transplantation with excellent pancreatic allograft function.

CRISPR-Mediated Genomic Addition to CPS1 Deficient iPSCs is Insufficient to Restore Nitrogen Homeostasis.

CPS1 deficiency is an inborn error of metabolism caused by loss-of-function mutations in the CPS1 gene, catalyzing the initial reaction of the urea cycle. Deficiency typically leads to toxic levels of plasma ammonia, cerebral edema, coma, and death, with the only curative treatment being liver transplantation; due to limited donor availability and the invasiveness and complications of the procedure, however, alternative therapies are needed. Induced pluripotent stem cells offer an alternative cell source to partial or whole liver grafts that theoretically would not require immune suppression regimens and additionally are amenable to genetic modifications. Here, we genetically modified CPS1 deficient patient-derived stem cells to constitutively express human codon optimized CPS1 from the AAVS1 safe harbor site. While edited stem cells efficiently differentiated to hepatocyte-like cells, they failed to metabolize ammonia more efficiently than their unedited counterparts. This unexpected result appears to have arisen in part due to transgene promoter methylation, and thus transcriptional silencing, in undifferentiated cells, impacting their capacity to restore the complete urea cycle function upon differentiation. As pluripotent stem cell strategies are being expanded widely for potential cell therapies, these results highlight the need for strict quality control and functional analysis to ensure the integrity of cell products.

CPS1: Looking at an ancient enzyme in a modern light.

The mammalian urea cycle (UC) is responsible for siphoning catabolic waste nitrogen into urea for excretion. Disruptions of the functions of any of the enzymes or transporters lead to elevated ammonia and neurological injury. Carbamoyl phosphate synthetase 1 (CPS1) is the first and rate-limiting UC enzyme responsible for the direct incorporation of ammonia into UC intermediates. Symptoms in CPS1 deficiency are typically the most severe of all UC disorders, and current clinical management is insufficient to prevent the associated morbidities and high mortality. With recent advances in basic and translational studies of CPS1, appreciation for this enzyme's essential role in the UC has been broadened to include systemic metabolic regulation during homeostasis and disease. Here, we review recent advances in CPS1 biology and contextualize them around the role of CPS1 in health and disease.

A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia.

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.

Hepatic glutamine synthetase augmentation enhances ammonia detoxification.

The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.

Factors predicting kidney delayed graft function among recipients of simultaneous liver-kidney transplantation: A single-center experience.

BACKGROUND: Kidney delayed graft function (kDGF) remains a challenging problem following simultaneous liver and kidney transplantation (SLKT) with a reported incidence up to 40%. Given the scarcity of renal allografts, it is crucial to minimize the development of kDGF among SLKT recipients to improve patient and graft outcomes. We sought to assess the role of preoperative recipient and donor/graft factors on developing kDGF among recipients of SLKT. METHODS: A retrospective review of 194 patients who received SLKT in the period from January 2004 to March 2017 in a single center was performed to assess the effect of preoperative factors on the development of kDGF. RESULTS: Kidney delayed graft function was observed in 95 patients (49%). Multivariate analysis revealed that donor history of hypertension, cold static preservation of kidney grafts [versus using hypothermic pulsatile machine perfusion (HPMP)], donor final creatinine, physiologic MELD, and duration of delay of kidney transplantation after liver transplantation were significant independent predictors for kDGF. kDGF is associated with worse graft function and patient and graft survival. CONCLUSIONS: Kidney delayed graft function has detrimental effects on graft function and graft survival. Understanding the risks and combining careful perioperative patient management, proper recipient selection and donor matching, and graft preservation using HPMP would decrease kDGF among SLKT recipients.

Human hepatocyte transplantation corrects the inherited metabolic liver disorder arginase deficiency in mice.

The transplantation, engraftment, and expansion of primary hepatocytes have the potential to be an effective therapy for metabolic disorders of the liver including those of nitrogen metabolism. To date, such methods for the treatment of urea cycle disorders in murine models has only been minimally explored. Arginase deficiency, an inherited disorder of nitrogen metabolism that presents in the first two years of life, has the potential to be treated by such methods. To explore the potential of this approach, we mated the conditional arginase deficient mouse with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations to give a selective advantage to transplanted (normal) human hepatocytes. On day -1, a uroplasminogen-expressing adenoviral vector was administered intravenously followed the next day with the transplantation of 1 × 106 human hepatocytes (or vehicle alone) by intrasplenic injection. As the initial number of administered hepatocytes would be too low to prevent hepatotoxicity-induced mortality, NTBC cycling was performed to allow for hepatocyte expansion and repopulation. While all control mice died, all except one human hepatocyte transplanted mice survived. Four months after hepatocyte transplantation, 2 × 1011 genome copies of AAV-TBG-Cre recombinase was administered IV to disrupt endogenous hepatic arginase expression. While all control mice died within the first month, human hepatocyte transplanted mice did well. Ammonia and amino acids, analyzed in both groups before and after disruption of endogenous arginase expression, while well-controlled in the transplanted group, were markedly abnormal in the controls. Ammonium challenging further demonstrated the durability and functionality of the human repopulated liver. In conclusion, these studies demonstrate that human hepatocyte repopulation in the murine liver can result in effective treatment of arginase deficiency.

Conditional disruption of hepatic carbamoyl phosphate synthetase 1 in mice results in hyperammonemia without orotic aciduria and can be corrected by liver-directed gene therapy.

Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.

Rescue of the Functional Alterations of Motor Cortical Circuits in Arginase Deficiency by Neonatal Gene Therapy.

UNLABELLED: Arginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome. SIGNIFICANCE STATEMENT: These studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.

A High-Throughput Platform for Formulating and Screening Multifunctional Nanoparticles Capable of Simultaneous Delivery of Genes and Transcription Factors.

Simultaneous delivery of multiple genes and proteins (e.g., transcription factors; TFs) is an emerging issue surrounding therapeutic research due to their ability to regulate cellular circuitry. Current gene and protein delivery strategies, however, are based on slow batch synthesis, which is ineffective, poorly controlled, and incapable of simultaneous delivery of both genes and proteins with synergistic functions. Consequently, advances in this field have been limited to in vitro studies. Here, by integrating microfluidic technologies with a supramolecular synthetic strategy, we present a high-throughput approach for formulating and screening multifunctional supramolecular nanoparticles (MFSNPs) self-assembled from a collection of functional modules to achieve simultaneous delivery of one gene and TF with unprecedented efficiency both in vitro and in vivo. We envision that this new approach could open a new avenue for immunotherapy, stem cell reprogramming, and other therapeutic applications.

Augmentation of transgene-encoded protein after neonatal injection of adeno-associated virus improves hepatic copy number without immune responses.

BACKGROUND: Achieving persistent expression is a prerequisite for genetic therapies for inherited metabolic enzymopathies. Such disorders potentially could be treated with gene therapy shortly after birth to prevent pathology. However, rapid cell turnover leads to hepatic episomal vector loss, which diminishes effectiveness. The current studies assessed whether tolerance to transgene proteins expressed in the neonatal period is durable and if the expression may be augmented with subsequent adeno-associated virus (AAV) administration. METHODS: AAV was administered to mice on day 2 with reinjection at 14 or at 14 and 42 d with examination of changes in hepatic copies and B and T cell-mediated immune responses. RESULTS: Immune responses to the transgene protein and AAV were absent after neonatal administration. Reinjection at 14 or at 14 and 42 d resulted in augmented expression with greater hepatic genome copies. Unlike controls, immune responses to transgene proteins were not detected in animals injected as neonates and subsequently. However, while no immune response developed after neonatal administration, anticapsid immune responses developed with further injections suggesting immunological ignorance was the initial mechanism of unresponsiveness. CONCLUSIONS: Persistence of transgene protein allows for tolerance induction permitting readministration of AAV to re-establish protein levels that decline with growth.

Optimizing HLA matching in a highly sensitized pediatric patient using ABO-incompatible and paired exchange kidney transplantation.

BACKGROUND: Kidney transplantation is the treatment of choice for end-stage renal disease. However, since pediatric patients have long projected life-years, it is also optimal for them to get well-matched transplants to minimize long-term sensitization. In North America, pediatric kidney transplantation is largely dependent upon the use of deceased donor organs, making it challenging to identify timely, well-matched transplants. Pediatric recipients may have willing living donors who are either HLA- or ABO-incompatible (ABOi); therefore, one solution is to utilize ABOi transplants and paired exchange programs to enhance HLA matching and living donation. CASE-DIAGNOSIS/TREATMENT: We adopted this approach for a highly sensitized patient with cPRA 90%, who received a successful ABOi paired exchange transplant. The recipient received pre-transplant immunomodulation until an acceptable isohemagglutinin titer <1:8 was reached before transplantation. The patient was induced with anti-thymocyte globulin and maintained on steroid-based triple immunosuppression. Eighteen-month allograft function is excellent with an estimated glomerular filtration rate (eGFR) of 83.53 ml/min/1.73 m(2). The patient did not develop de novo donor-specific HLA antibodies or have any episodes of acute rejection CONCLUSIONS: This case highlights the safety and efficacy of using paired exchange in combination with ABOi transplants in pediatric kidney transplantation to optimize HLA matching, minimize wait times, and enhance allograft survival.