Spf-ash Mice Research Articles

Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

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. 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). 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. 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.

Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency

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 invivo 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 Spfash 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.

Excretion of excess nitrogen and increased survival by loss of SLC6A19 in a mouse model of ornithine transcarbamylase deficiency.

Protein catabolism ultimately yields toxic ammonia, which must be converted to urea by the liver for renal excretion. In extrahepatic tissues, ammonia is temporarily converted primarily to glutamine for subsequent hepatic extraction. Urea cycle disorders (UCDs) are inborn errors of metabolism causing impaired ureagenesis, leading to neurotoxic accumulation of ammonia and brain glutamine. Treatment includes dietary protein restriction and oral "ammonia scavengers." These scavengers chemically combine with glutamine and glycine to yield excretable products, creating an alternate pathway of waste nitrogen disposal. The amino acid transporter SLC6A19 is responsible for >95% of absorption and reabsorption of free neutral amino acids in the small intestine and kidney, respectively. Genetic SLC6A19 deficiency causes massive neutral aminoaciduria but is typically benign. We hypothesized that inhibiting SLC6A19 would open a novel and effective alternate pathway of waste nitrogen disposal. To test this, we crossed SLC6A19 knockout (KO) mice with spfash mice, a model of ornithine transcarbamylase (OTC) deficiency. Loss of SLC6A19 in spfash mice normalized plasma ammonia and brain glutamine and increased median survival in response to a high protein diet from 7 to 97 days. While induced excretion of amino acid nitrogen is likely the primary therapeutic mechanism, reduced intestinal absorption of dietary free amino acids, and decreased muscle protein turnover due to loss of SLC6A19 may also play a role. In summary, the results suggest that SLC6A19 inhibition represents a promising approach to treating UCDs and related aminoacidopathies.

Intrabiliary infusion of naked DNA vectors targets periportal hepatocytes in mice

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 thepotential to treat defects restricted to periportal hepatocytes, which opens new doors for non-viral liver-directed gene therapy.

Restoring ornithine transcarbamylase (OTC) activity in an OTC‐deficient mouse model using LUNAR‐OTC mRNA

AbstractOrnithine transcarbamylase (OTC) catalyses the reaction from ornithine to citrulline in the urea cycle. Ornithine transcarbamylase deficiency (OTCD) results in episodes of hyperammonemia. Arcturus Therapeutics developed a lipid nanoparticle (LNP)‐encapsulated OTC‐mRNA (LUNAR‐OTC) that results in a replacement enzyme and is currently undergoing clinical trials. In this study, the efficacy of LUNAR OTC‐mRNA drug in the spfash mouse model was examined by measuring the OTC enzyme activity and protein expression in the liver and plasma of OTC‐mRNA‐treated mice. Using purified citrulline‐D4 as the substrate improved the sensitivity of the OTC activity assay and allowed us to quantify the ornithine‐D4 product from the mouse plasma samples. OTC activity in the liver showed a clear dose response: The lowest dose, 0.3 mg/kg, resulted in higher activity than that of the untreated group, and the highest dose, 3 mg/kg, resulted in completely restored OTC activity in the liver. OTC activity in plasma was also dose‐dependent. A clear positive correlation between the OTC activity in the liver and that in the plasma suggests that the plasma OTC activity assay may serve as a surrogate for measuring OTC activity in liver biopsy samples. In addition, the OTC protein expression levels correlated well with the OTC activity in liver samples, but there was no quantifiable OTC protein in the plasma samples. This finding suggests that the sensitivity of the OTC activity assay is superior to that of the protein expression assay. Overall, the results of this study suggest that the OTC activity assay described here can be used as a clinical pharmacodynamic endpoint to measure the effectiveness of OTCD treatment.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies

BackgroundAberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA.MethodsCreation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors.ResultsComparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects.Moreover, the interplay between the authentic and the adjacent cryptic 5′ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5′ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5′ss, was rescuable by engineered U1snRNA.ConclusionsSubtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Long-term correction of ornithine transcarbamylase deficiency in Spf-Ash mice with a translationally optimized AAV vector

Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTCSpf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic.

A mutation-independent CRISPR-Cas9-mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality. Although a promising treatment for late-onset OTC deficiency, adeno-associated virus (AAV) neonatal gene therapy would only provide short-term therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation. CRISPR-Cas9-mediated homology-directed repair can correct a G-to-A mutation in 10% of OTC alleles in the livers of newborn OTC spfash mice. However, an editing vector able to correct one mutation would not be applicable for patients carrying different OTC mutations, plus expression would not be fast enough to treat a hyperammonemia crisis. Here, we describe a dual-AAV vector system that accomplishes rapid short-term expression from a non-integrated minigene and long-term expression from the site-specific integration of this minigene without any selective growth advantage for OTC-positive cells in newborns. This CRISPR-Cas9 gene-targeting approach may be applicable to all patients with OTC deficiency, irrespective of mutation and/or clinical state.

Comprehensive characterization of ureagenesis in the spfash mouse, a model of human ornithine transcarbamylase deficiency, reveals age-dependency of ammonia detoxification.

The most common ureagenesis defect is X-linked ornithine transcarbamylase (OTC) deficiency which is a main target for novel therapeutic interventions. The spf ash mouse model carries a variant (c.386G>A, p.Arg129His) that is also found in patients. Male spf ash mice have a mild biochemical phenotype with low OTC activity (5%-10% of wild-type), resulting in elevated urinary orotic acid but no hyperammonemia. We recently established a dried blood spot method for in vivo quantification of ureagenesis by Gas chromatography-mass spectrometry (GC-MS) using stable isotopes. Here, we applied this assay to wild-type and spf ash mice to assess ureagenesis at different ages. Unexpectedly, we found an age-dependency with a higher capacity for ammonia detoxification in young mice after weaning. A parallel pattern was observed for carbamoylphosphate synthetase 1 and OTC enzyme expression and activities, which may act as pacemaker of this ammonia detoxification pathway. Moreover, high ureagenesis in younger mice was accompanied by elevated periportal expression of hepatic glutamine synthetase, another main enzyme required for ammonia detoxification. These observations led us to perform a more extensive analysis of the spf ash mouse in comparison to the wild-type, including characterization of the corresponding metabolites, enzyme activities in the liver and plasma and the gut microbiota. In conclusion, the comprehensive enzymatic and metabolic analysis of ureagenesis performed in the presented depth was only possible in animals. Our findings suggest such analyses being essential when using the mouse as a model and revealed age-dependent activity of ammonia detoxification.

Ex Vivo Enteroids Recapitulate In Vivo Citrulline Production in Mice

BackgroundThe endogenous production of arginine relies on the synthesis of citrulline by enteral ornithine transcarbamylase (OTC). Mutations in the gene coding for this enzyme are the most frequent cause of urea cycle disorders. There is a lack of correlation between in vivo metabolic function and DNA sequence, transcript abundance, or in vitro enzyme activity. ObjectiveThe goal of the present work was to test the hypothesis that enteroids, a novel ex vivo model, are able to recapitulate the in vivo citrulline production of wild-type (WT) and mutant mice. MethodsSix-week-old male WT and OTC-deficient mice [sparse fur and abnormal skin (spf-ash) mutation] were studied. Urea and citrulline fluxes were determined in vivo, and OTC abundance was measured in liver and gut tissue. Intestinal crypts were isolated and cultured to develop enteroids. Ex vivo citrulline production and OTC abundance were determined in these enteroids. ResultsLiver OTC abundance was lower (mean ± SE: 0.16 ± 0.01 compared with 1.85 ± 0.18 arbitrary units; P < 0.001) in spf-ash mice than in WT mice, but there was no difference in urea production. In gut tissue, OTC was barely detectable in mutant mice; despite this, a lower but substantial citrulline production (67 ± 3 compared with 167 ± 8 µmol · kg−1 · h−1; P < 0.001) was shown in the mutant mice. Enteroids recapitulated the in vivo findings of a very low OTC content accompanied by a reduced citrulline production (1.07 ± 0.20 compared with 4.64 ± 0.44 nmol · µg DNA−1 · d−1; P < 0.001). ConclusionsEnteroids recapitulate in vivo citrulline production and offer the opportunity to study the regulation of citrulline production in a highly manipulable system.

Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency.

We describe a novel, two-nanoparticle mRNA delivery system and show that it is highly effective as a means of intracellular enzyme replacement therapy (i-ERT) using a murine model of ornithine transcarbamylase deficiency (OTCD). Our Hybrid mRNA Technology delivery system (HMT) comprises an inert lipid nanoparticle that protects the mRNA from nucleases in the blood as it distributes to the liver and a polymer micelle that targets hepatocytes and triggers endosomal release of mRNA. This results in high-level synthesis of the desired protein specifically in the liver. HMT delivery of human OTC mRNA normalizes plasma ammonia and urinary orotic acid levels, and leads to a prolonged survival benefit in the murine OTCD model. HMT represents a unique, non-viral mRNA delivery method that allows multi-dose, systemic administration for treatment of single-gene inherited metabolic diseases.

A simple dried blood spot-method for in vivo measurement of ureagenesis by gas chromatography–mass spectrometry using stable isotopes

BackgroundClinical management of inherited or acquired hyperammonemia depends mainly on the plasma ammonia level which is not a reliable indicator of urea cycle function as its concentrations largely fluctuate. The gold standard to assess ureagenesis in vivo is the use of stable isotopes. MethodsHere we developed and validated a simplified in vivo method with [15N]ammonium chloride ([15N]H4Cl) as a tracer. Non-labeled and [15N]urea were quantified by GC–MS after extraction and silylation. ResultsDifferent matrices were evaluated for suitability of analysis. Ureagenesis was assessed in ornithine transcarbamylase (OTC)-deficient spfash mice with compromised urea cycle function during fasted and non-fasted feeding states, and after rAAV2/8-vector delivery expressing the murine OTC-cDNA in liver. Blood (5μL) was collected through tail vein puncture before and after [15N]H4Cl intraperitoneal injections over a two hour period. The tested matrices, blood, plasma and dried blood spots, can be used to quantify ureagenesis. Upon [15N]H4Cl challenge, urea production in spfash mice was reduced compared to wild-type and normalized following rAAV2/8-mediated gene therapeutic correction. The most significant difference in ureagenesis was at 30min after injection in untreated spfash mice under fasting conditions (19% of wild-type). Five consecutive injections over a period of five weeks had no effect on body weight or ureagenesis. ConclusionThis method is simple, robust and with no apparent risk, offering a sensitive, minimal-invasive, and fast measurement of ureagenesis capacity using dried blood spots. The stable isotope-based quantification of ureagenesis can be applied for the efficacy-testing of novel molecular therapies.

481. CRISPR/Cas9-Mediated In Vivo Genome Editing to Correct the OTC spfash Mutation in Newborn Mice

Many genetic liver diseases, including OTC deficiency, present in newborns with repeated, often lethal, metabolic crises. Adeno-associated virus (AAV) neonatal gene therapy in this setting would require multiple vector administrations to maintain the therapeutic effects because the non-integrating genome is lost as developing hepatocytes proliferate. As such, we reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR/Cas9, a powerful genome-editing tool consisting of the Cas9 nuclease and a single-guide RNA (sgRNA). We developed a strategy using an AAV vector with high liver tropism (AAV8) to correct the point mutation in newborn spfash mice using Cas9 enzyme from Staphylococcus aureus (SaCas9). An animal model of OTC deficiency, the male sparse fur ash (spfash) mouse, has a G-to-A point mutation at the donor splice site at the end of exon 4 of the OTC gene, which leads to abnormal splicing and a 20-fold reduction in OTC mRNA and protein.We developed a two-vector approach to incorporate all 3 components of CRISPR/Cas9 into AAV. Vector 1 expresses the SaCas9 gene from a liver-specific TBG promoter, while vector 2 contains both the sgRNA1 sequence expressed from a U6 promoter and the 1.8 kb donor OTC DNA sequence. Spfash pups were injected intravenously on postnatal day 2 with mixtures of vector 1 and vector 2 and subsequently evaluated for indel (insertion and deletion) formation and functional correction of the spfash mutation. Following gene correction (3 and 8 weeks), indels were detected by deep sequencing in 31% of OTC alleles, and HDR-based correction of the G-to-A mutation was observed in 10% of OTC alleles. Liver sections were analyzed by immunohistochemistry for OTC expression, showing 15% OTC-positive cells at 3 weeks and 13% at 8 weeks. Direct measurements of OTC enzyme activity from liver homogenates and OTC mRNA from total cellular RNA from liver revealed similarly high levels of correction in treated animals. We further assessed the impact of gene correction on the clinical manifestations of OTC deficiency by evaluating the tolerance of spfash mice to a one-week course of high-protein diet. Spfash mice treated with the gene-editing vectors had significantly lower plasma ammonia levels and showed a survival improvement as compared with untreated spfash mice. This study provides convincing evidence for efficacy in an authentic animal model of a lethal human metabolic disease following in vivo genome editing.

169. Expression Studies of Ornithine Transcarbamylase in Liver Using Minicircle Vectors for Gene Therapy: Flag-Tagging of a Mitochondrial Enzyme and Stable Expression Under Control of an Endogenous Otc Promoter-Enhancer

Ornithine transcarbamylase deficiency (OTCD) is the most common inherited defect of the urea cycle resulting in severe hyperammonemia and death if left untreated. We are aiming at correcting OTCD using naked-DNA minicircle (MC) vector mediated gene therapy in the spfash mouse model with low residual OTC activity. A critical parameter is delivery to periportal hepatocytes where the urea cycle is located because of metabolic zonation in the liver. To distinguish between MC-born and endogenous OTC enzyme, we generated an expression cassette with an internally Flag-tagged OTC enzyme. Internal epitope tagging is required because of the N-terminal mitochondrial import sequence. A corresponding Flag-tag sequence was introduced C-terminally of the mitochondrial import signal. The tagged protein performed similar to its non-tagged OTC enzyme upon hydrodynamic tail vein injection for liver targeting in spfash mice, indicating that the tag neither interferes with the mitochondrial import nor the formation of a functional OTC trimer. Furthermore we generated an endogenous Otc promoter-enhancer construct, termed PmO1, for potential specific or “natural” OTC transgene expression. According to others, the promoter (672 bp) sequence derived from mouse Otc was not sufficient for liver specificity (Veres et al, J Biol Chem 261: 7588-7591, 1986) and requires a corresponding enhancer sequence (Nishiyori et al, J Biol Chem 269: 1323-1331, 1994). An enhancer sequence (232 bp) originating from rat Otc was used that contains several binding sites for liver-selective transcription factors (Murakami et al, Mol Cell Biol 10: 1180-1191, 1990). The resulting MC-vector expressing OTC from this PmO1 promoter-enhancer construct was delivered to spfash mice via hydrodynamic tail vein injection, resulting in similar enzymatic activity in whole liver extracts compared to wild-type mice. Vectors designed to express Flag-tagged OTC driven from a natural Otc promoter might be useful to study biodistribution and/or long-term stable expression after MC-based gene therapy for OTCD.

150. Liver Directed Gene Therapy Using Non-Viral Minicircle-DNA Vector for Ornithine Transcarbamylase Deficiency (OTCD)

As an alternative to (AAV-) viral vector-transduction, we are using minicircle (MC) naked-DNA vectors devoid of any viral or bacterial sequences for delivery into the ornithine transcarbamylase (OTC) deficient murine model, Spfash. The MC-vectors which do not have a defined size limitation contained expression cassettes with a short (0.3 kb) synthetic liver-specific promoter or the natural (endogenous) Otc promoter with a length of up to 2 kb, the murine Otc-cDNA (mOtc) or the codon-optimized murine Otc (mcoOtc) with or without truncated 5’-intron, and a polyA signal. MC vectors were delivered to mouse liver by hydrodynamic tail vein injection. Upon sacrificing mice 2-3 weeks after vector infusion, transgenic OTC enzyme activity was found in liver extracts to be up to 2 or 3-fold higher than in normal wild-type controls. However, similar to what we have observed in our study to correct hyperphenylalaninemia in the phenylketonuria (PKU) mouse using hydrodynamic injection of MC vectors (Viecelli et al., Hepatology 2014), gene expression was mainly found at the perivenous area also in the OTC-treated mouse liver. Since the full active urea cycle is restricted to the periportal area and thus delivery of MC vectors to treat OTC deficiency needs to target these periportal hepatocytes, we have established delivery via hydrodynamic portal vein injection for gene transfer into Spfash mice. Preliminary analysis showed transgene expression in MC-treated mice however at a much lower level than via hydrodynamic tail vein injection. Analysis of urinary orotic acid and in vivo ureagenesis is ongoing for the evaluation of the therapeutic efficacy.

335. Toward a Cellular Therapy for Metabolic Liver Disease: Gene Targeting of the Ornithine Transcarbamylase (Otc) Locus in Bipotential Murine Oval Liver Cells (BMOL) Using Adeno-Associated Virus

Liver progenitor cells (LPC) possess significant potential as a cellular therapy as they are easily isolated, can differentiate into hepatocytes and cholangiocytes and maintain stable phenotype in long-term culture. However, their use to correct recessive genetic disease in an autologous context will require repair of mutant loci. Adeno-associated virus induced homologous recombination (AAV-HR) is a highly effective tool for this task as it is >1000-fold more efficient than alternative approaches such as plasmid DNA and adenovirus. As with other HR systems, the frequency of AAV-HR is increased by DNA strand breaks (DSB) at the target locus. To develop a model of gene targeting in LPC, an AAV vector was constructed to target the Otc locus in Spfash mice which would both correct the mutant base and insert Otc exons 5-10 as well as a polyadenylation signal to support transcription and translation of the targeted gene. A neomycin phosphotransferase expression cassette was also included to permit G418-selection of transduced cells. The capacity of the construct (OTCe4-neo) to correctly target was confirmed by restoration of OTC expression in hepatocytes of male Spfash mice injected as neonates with 2.2 × 1011 vg packaged in hepatotropic serotype 8 capsids. Using an enzyme activity assay, clusters of OTC positive cells were observed in liver tissue sections consistent with locus repair and subsequent replication of corrected cells. The vector was also used to target the Otc locus in BMOL-TAT-LacZ, a murine LPC line derived from a transgenic mouse after three weeks on a choline-deficient, ethionine-supplemented (CDE) diet to increase LPC frequency. In vitro targeting was detected by nested PCR analysis of G418 selected populations. Based on this promising data, cell lines were isolated from male Spfash mice maintained on the CDE diet and validated to be BMOL by (i) expression of LPC markers (E-Cad, A6, EpCAM, CD24 and CK19) using immuno-histochemistry and (ii) their differentiation into hepatoblasts or ductal epithelial cells when maintained in appropriate in vitro conditions. Ongoing experiments will test the capacity of OTCe4-neo to target the Otc locus in the BMOL-Spfash lines. We have also developed CRISPR technology to induce DSB in exon 4 of Otc and functionally validated the constructs in BMOL-Spfash. While not essential for success of the project, application of CRISPR should increase the basal frequency of AAV-HR in BMOL- Spfash and expedite downstream experiments designed to test the therapeutic efficacy of LPC.

700. Stable Correction of Severe Metabolic Liver Disease Phenotypes in the Growing Murine Liver Using a Hybrid rAAV/piggyBac Transposon Gene Delivery System

Liver-targeted gene therapy using adeno-associated virus-based vectors (rAAV) shows therapeutic promise in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver where rapid clearance of rAAV episomal genomes occurs in concert with hepatocellular proliferation. This phenomenon has been shown to underpin progressive loss of therapeutic efficacy, initially achieved in the neonatal period, in mouse models of early-onset urea cycle defects (UCDs). We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an EGFP expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks post-treatment compared to traditional rAAV gene delivery. We then modeled the therapeutic potential of the system in the context of severe early onset UCDs. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase (OTC) deficient Spfash mouse and neonatal lethal argininosuccinate synthetase (ASS) knock-out mouse. The system was next utilized in a murine model of chronic liver disease, progressive familial intrahepatic cholestasis type 3 (PFIC3), in which intervention prior to disease onset is necessary due to the development of chronic liver pathology which impedes vector delivery and hepatocyte transduction. Stable persistent expression extending into adulthood was achieved with increased mean biliary phosphatidylcholine concentration and dramatically reduced liver fibrosis. Finally, transposon integration patterns were analysed, revealing 127,286 unique integration sites which conformed to previously published piggyBac data. Conclusion: Using a hybrid rAAV/piggyBac transposon vector system we achieved stable therapeutic protection in two UCD mouse models and a murine model with chronic liver pathology. This strategy has the potential for treating a wide array of inherited liver diseases with early onset. A clinically conceivable early application of this technology in the management of severe UCDs could be as a bridging therapy while awaiting liver transplantation. Further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.

AAV-encoded OTC activity persisting to adulthood following delivery to newborn spfash mice is insufficient to prevent shRNA-induced hyperammonaemia

Urea cycle defects presenting in the neonatal period with hyperammonaemia are associated with high morbidity and mortality, and necessitate liver transplantation for long-term management. Gene therapy is therefore an attractive possibility, with vectors based on adeno-associated virus (rAAV) currently showing exciting promise in liver-targeted clinical trials in adults. Successful use of rAAV vectors in infants, however, is more challenging as episomal rAAV genomes will be lost from proliferating hepatocytes during liver growth, leaving stable transgene expression dependent on the subset of vector genomes that undergo genomic integration. To explore this challenge, we exploited the partially ornithine transcarbamylase (OTC)-deficient spf(ash) mouse model and small hairpin RNA-mediated knockdown of residual endogenous OTC enzyme activity in adult mice that had received neonatal treatment with an OTC-encoding rAAV. This leaves mice reliant on vector-encoded OTC activity that has persisted from the newborn period. Despite stable transduction in approximately 8% of hepatocytes and residual vector-encoded OTC activity of up to 33% of wild-type, well above endogenous spf(ash) levels (5-7%), mice were not protected from hyperammonaemia. These data show that the distribution of OTC activity within the liver is critical and that rAAV vector re-delivery after early neonatal treatment is likely to be necessary for stable control of hyperammonaemia into adulthood.

Acute metabolic decompensation due to influenza in a mouse model of ornithine transcarbamylase deficiency

ABSTRACTThe urea cycle functions to incorporate ammonia, generated by normal metabolism, into urea. Urea cycle disorders (UCDs) are caused by loss of function in any of the enzymes responsible for ureagenesis, and are characterized by life-threatening episodes of acute metabolic decompensation with hyperammonemia (HA). A prospective analysis of interim HA events in a cohort of individuals with ornithine transcarbamylase (OTC) deficiency, the most common UCD, revealed that intercurrent infection was the most common precipitant of acute HA and was associated with markers of increased morbidity when compared with other precipitants. To further understand these clinical observations, we developed a model system of metabolic decompensation with HA triggered by viral infection (PR8 influenza) using spf-ash mice, a model of OTC deficiency. Both wild-type (WT) and spf-ash mice displayed similar cytokine profiles and lung viral titers in response to PR8 influenza infection. During infection, spf-ash mice displayed an increase in liver transaminases, suggesting a hepatic sensitivity to the inflammatory response and an altered hepatic immune response. Despite having no visible pathological changes by histology, WT and spf-ash mice had reduced CPS1 and OTC enzyme activities, and, unlike WT, spf-ash mice failed to increase ureagenesis. Depression of urea cycle function was seen in liver amino acid analysis, with reductions seen in aspartate, ornithine and arginine during infection. In conclusion, we developed a model system of acute metabolic decompensation due to infection in a mouse model of a UCD. In addition, we have identified metabolic perturbations during infection in the spf-ash mice, including a reduction of urea cycle intermediates. This model of acute metabolic decompensation with HA due to infection in UCD serves as a platform for exploring biochemical perturbations and the efficacy of treatments, and could be adapted to explore acute decompensation in other types of inborn errors of metabolism.

Preclinical evaluation of a clinical candidate AAV8 vector for ornithine transcarbamylase (OTC) deficiency reveals functional enzyme from each persisting vector genome

Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No curative therapy exists except for liver transplantation which is limited by available donors and carries significant risk of mortality and morbidity. Adeno-associated virus 8 (AAV8) has been shown to be the most efficient vector for liver-directed gene transfer and is currently being evaluated in a clinical trial for treating hemophilia B. In this study, we generated a clinical candidate vector for a proposed OTC gene therapy trial in humans based on a self-complementary AAV8 vector expressing codon-optimized human OTC (hOTCco) under the control of a liver-specific promoter. Codon-optimization dramatically improved the efficacy of OTC gene therapy. Supraphysiological expression levels and activity of hOTC were achieved in adult spf ash mice following a single intravenous injection of hOTCco vector. Vector doses as low as 1 × 10 10 genome copies (GC) achieved robust and sustained correction of the OTCD biomarker orotic aciduria and clinical protection against an ammonia challenge. Functional expression of hOTC in 40% of liver areas was found in mice treated with a low vector dose of 1 × 10 9 GC. We suggest that the clinical candidate vector we have developed has the potential to achieve therapeutic effects in OTCD patients.