A phenylalanine-free recombinant nutritional protein for the dietary management of phenylketonuria.

Phenylketonuria (PKU) is a congenital metabolic disorder that causes the systemic elevation of phenylalanine (Phe), which is neurotoxic and teratogenic. PKU is currently incurable, and management involves lifelong adherence to an unpalatable protein-restricted diet based on Phe-free amino acid mixtures. Seeking a palatable dietary alternative, we identified a Bacillus subtilis protein (GSP16O) with a well-balanced but low-Phe amino acid profile. We optimized the sequence and expressed a modified Phe-free version (GSP105) in Pseudomonas fluorescens, achieving yields of 20 g/L. The purified GSP105 protein has a neutral taste and smell, is highly soluble, and remains stable up to 80°C. Homozygous enu2 mice, a model of human PKU, were fed with diets containing either GSP105 or normal protein. The GSP105 diet led to normalization of blood Phe levels and brain monoamine neurotransmitter metabolites, and prevented maternal PKU. The GSP105 diet thus provides an alternative and efficacious dietary management strategy for PKU.

Core-Shell Structured Chitosan-Polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency.

The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially nonviral gene delivery systems are of increasing interest. However, low transgene expression levels and limited tolerability of these systems remain a challenge. The improvement of nucleic acid delivery using depolymerized chitosan-polyethylenimine DNA complexes (dCS-PEI/DNA) is investigated. The secore complexes are further combined with chitosan-based shells and functionalized with polyethylene glycol (PEG) and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physicochemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-linear PEI/DNA complex with a shell consisting of dCS-PEG-COOH, which results in improved nucleic acid encapsulation, cellular uptake and transfection potency in human hepatoma HuH-7cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induced a high reporter gene signal for three days. It is concluded that ternary coreshell structured nanoparticles comprising functionalized chitosan can be used for in vitro andin vivo gene delivery.

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 ß-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.

In vivo prime editing of a metabolic liver disease in mice.

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 1014 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

Delivery of non-viral naked DNA vectors to liver in small weaned pigs by hydrodynamic retrograde intrabiliary injection.

Hepatic gene therapy by delivering non-integrating therapeutic vectors in newborns remains challenging due to the risk of dilution and loss of efficacy in the growing liver. Previously we reported on hepatocyte transfection in piglets by intraportal injection of naked DNA vectors. Here, we established delivery of naked DNA vectors to target periportal hepatocytes in weaned pigs by hydrodynamic retrograde intrabiliary injection (HRII). The surgical procedure involved laparotomy and transient isolation of the liver. For vector delivery, a catheter was placed within the common bile duct by enterotomy. Under optimal conditions, no histological abnormalities were observed in liver tissue upon pressurized injections. The transfection of hepatocytes in all tested liver samples was observed with vectors expressing luciferase from a liver-specific promoter. However, vector copy number and luciferase expression were low compared to hydrodynamic intraportal injection. A 10-fold higher number of vector genomes and luciferase expression was observed in pigs using a non-integrating naked DNA vector with the potential for replication. In summary, the HRII application was less efficient (i.e., lower luciferase activity and vector copy numbers) than the intraportal delivery method but was significantly less distressful for the piglets and has the potential for injection (or re-injection) of vector DNA by endoscopic retrograde cholangiopancreatography.

Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis, Characterization, and Evaluation.

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; MW 6-9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.

Improvement of DNA Vector Delivery of DOTAP Lipoplexes by Short-Chain Aminolipids.

Cellular delivery of DNA vectors for the expression of therapeutic proteins is a promising approach to treat monogenic disorders or cancer. Significant efforts in a preclinical and clinical setting have been made to develop potent nonviral gene delivery systems based on lipoplexes composed of permanently cationic lipids. However, transfection efficiency and tolerability of such systems are in most cases not satisfactory. Here, we present a one-pot combinatorial method based on double-reductive amination for the synthesis of short-chain aminolipids. These lipids can be used to maximize the DNA vector delivery when combined with the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). We incorporated various aminolipids into such lipoplexes to complex minicircle DNA and screened these systems in a human liver-derived cell line (HuH7) for gene expression and cytotoxicity. The lead aminolipid AL-A12 showed twofold enhanced gene delivery and reduced toxicity compared to the native DOTAP:cholesterol lipoplexes. Moreover, AL-A12-containing lipoplexes enabled enhanced transgene expression in vivo in the zebrafish embryo model.

State-of-the-Art 2019 on Gene Therapy for Phenylketonuria.

Phenylketonuria (PKU) is considered to be a paradigm for a monogenic metabolic disorder but was never thought to be a primary application for human gene therapy due to established alternative treatment. However, somewhat unanticipated improvement in neuropsychiatric outcome upon long-term treatment of adults with PKU with enzyme substitution therapy might slowly change this assumption. In parallel, PKU was for a long time considered to be an excellent test system for experimental gene therapy of a Mendelian autosomal recessive defect of the liver due to an outstanding mouse model and the easy to analyze and well-defined therapeutic end point, that is, blood l-phenylalanine concentration. Lifelong treatment by targeting the mouse liver (or skeletal muscle) was achieved using different approaches, including (1) recombinant adeno-associated viral (rAAV) or nonviral naked DNA vector-based gene addition, (2) genome editing using base editors delivered by rAAV vectors, and (3) by delivering rAAVs for promoter-less insertion of the PAH-cDNA into the Pah locus. In this article we summarize the gene therapeutic attempts of correcting a mouse model for PKU and discuss the future implications for human gene therapy.

Treatment of Cystathionine ß-Synthase Deficiency in Mice Using a Minicircle-Based Naked DNA Vector.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by extremely elevated total homocysteine (tHcy) in the blood. Patients diagnosed with CBS deficiency have a variety of clinical problems, including dislocated lenses, osteoporosis, cognitive and behavioral issues, and a significantly increased risk of thrombosis. Current treatment strategies involve a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine. Here, a mouse model for CBS deficiency (Tg-I278T Cbs-/-) was used to evaluate the potential of minicircle-based naked DNA gene therapy to treat CBS deficiency. A 2.3 kb DNA-minicircle containing the liver-specific P3 promoter driving the human CBS cDNA (MC.P3-hCBS) was delivered into Tg-I278T Cbs-/- mice via a single hydrodynamic tail vein injection. Mean serum tHcy decreased from 351 µM before injection to 176 µM 7 days after injection (p = 0.0005), and remained decreased for at least 42 days. Western blot analysis reveals significant minicircle-directed CBS expression in the liver tissue. Liver CBS activity increased 34-fold (12.8 vs. 432 units; p = 0.0004) in MC.P3-hCBS-injected animals. Injection of MC.P3-hCBS in young mice, subsequently followed for 202 days, showed that the vector can ameliorate the mouse homocystinuria alopecia phenotype. The present findings show that minicircle-based gene therapy can lower tHcy in a mouse model of CBS deficiency.

Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads.

The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEG-lipid on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation also applies to LNP containing macromolecular payloads including mRNA, DNA vectors, and gold nanoparticles.

Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH4) treatment in Pahenu1/enu2 (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type. We found that brain L-tyrosine, L-tryptophan, BH4 cofactor and catecholamine concentrations, and brain tyrosine hydroxylase (TH) activity were normal in these mice but that brain serotonin, 5-hydroxyindolacetic acid (5HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) content, and brain TH protein, as well as tryptophan hydroxylase type 2 (TPH2) protein levels and activity were reduced in comparison to wild-type mice. Parenteral L-Phe loading conditions did not lead to significant changes in brain neurometabolite concentrations. Remarkably, enteral BH4 treatment, which normalized brain L-Phe levels in ENU1/2 mice, lead to only partial recovery of brain serotonin and 5HIAA concentrations. Furthermore, indirect evidence indicated that the GTP cyclohydrolase I (GTPCH) feedback regulatory protein (GFRP) complex may be a sensor for brain L-Phe elevation to ameliorate the toxic effects of HPA. We conclude that BH4 treatment of HPA toward systemic L-Phe lowering reverses elevated brain L-Phe content but the recovery of TPH2 protein and activity as well as serotonin levels is suboptimal, indicating that patients with mild HPA and mood problems (depression or anxiety) treated with the current diet may benefit from supplementation with BH4 and 5-OH-tryptophan.

Low-Dose Gene Therapy for Murine PKU Using Episomal Naked DNA Vectors Expressing PAH from Its Endogenous Liver Promoter.

Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of <1 vector genome per diploid hepatocyte genome and was achieved at a vector dose that was significantly lowered. Partial hepatectomy and subsequent liver regeneration was associated with >95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.

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

BACKGROUND: Clinical 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. METHODS: Here 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. RESULTS: Different 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. CONCLUSION: This 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.