Hepatic Gene Therapy Research Articles

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.

Improving Molecular Therapy in the Kidney.

Mutations in approximately 80 genes have been implicated as the cause of various genetic kidney diseases. However, gene delivery to kidney cells from the blood is inefficient because of the natural filtering functions of the glomerulus, and research into and development of gene therapy directed toward kidney disease has lagged behind as compared with hepatic, neuromuscular, and ocular gene therapy. This lack of progress is in spite of numerous genetic mouse models of human disease available to the research community and many vectors in existence that can theoretically deliver genes to kidney cells with high efficiency. In the past decade, several groups have begun to develop novel injection techniques in mice, such as retrograde ureter, renal vein, and direct subcapsular injections to help resolve the issue of gene delivery to the kidney through the blood. In addition, the ability to retarget vectors specifically toward kidney cells has been underutilized but shows promise. This review discusses how recent advances in gene delivery to the kidney and the field of gene therapy can leverage the wealth of knowledge of kidney genetics to work toward developing gene therapy products for patients with kidney disease.

Lipid nanoparticle technology for therapeutic gene regulation in the liver.

Hereditary genetic disorders, cancer, and infectious diseases of the liver affect millions of people around the globe and are a major public health burden. Most contemporary treatments offer limited relief as they generally aim to alleviate disease symptoms. Targeting the root cause of diseases originating in the liver by regulating malfunctioning genes with nucleic acid-based drugs holds great promise as a therapeutic approach. However, employing nucleic acid therapeutics in vivo is challenging due to their unfavorable characteristics. Lipid nanoparticle (LNP) delivery technology is a revolutionary development that has enabled clinical translation of gene therapies. LNPs can deliver siRNA, mRNA, DNA, or gene-editing complexes, providing opportunities to treat hepatic diseases by silencing pathogenic genes, expressing therapeutic proteins, or correcting genetic defects. Here we discuss the state-of-the-art LNP technology for hepatic gene therapy including formulation design parameters, production methods, preclinical development and clinical translation.

Rational Engineering and Preclinical Evaluation of Neddylation and SUMOylation Site Modified Adeno-Associated Virus Vectors in Murine Models of Hemophilia B and Leber Congenital Amaurosis.

Synthetic engineering of viral vectors such as adeno-associated virus (AAV) is crucial to overcome host transduction barriers observed during clinical gene therapy. We reasoned that exploring the role of cellular ubiquitin-like modifiers (UBLs) such as Neddylation or SUMOylation during AAV transduction could be beneficial. Using a combination of in silico biochemical and molecular engineering strategies, we have studied the impact of these UBLs during AAV2 infection and further developed Neddylation or SUMOylation site–modified AAV vectors and validated them in multiple disease models in vitro and in vivo. Hepatic gene transfer of two novel vectors developed, K105Q (SUMOylation-site mutant) and K665Q (Neddylation-site mutant), demonstrated a significantly improved human coagulation factor (F) IX expression (up to two-fold) in a murine model of hemophilia B. Furthermore, subretinal gene transfer of AAV2-K105Q vector expressing RPE65 gene demonstrated visual correction in a murine model of a retinal degenerative disease (rd12 mice). These vectors did not have any adverse immunogenic events in vivo. Taken together, we demonstrate that gene delivery vectors specifically engineered at UBLs can improve the therapeutic outcome during AAV-mediated ocular or hepatic gene therapy.

The Factor VIII Variant X5 Enhances Hemophilia a Gene Therapy Efficiency By Its Improved Secretion

Introduction. Adeno-associated virus (AAV)-based factor VIII (FVIII) gene therapy holds great promise to provide clinical benefit in patients with hemophilia A. However, very high doses are currently needed to achieve therapeutic factor levels and the durability appears to be limited to a couple of years. Vector efficiency could be improved by employing more potent liver-specific promoters, but this might come at the price of overstraining the cellular protein folding capacity, causing FVIII to misfold in the lumen of the Endoplasmic Reticulum (ER). This event would in turn activate the unfolded protein response, cause oxidative stress, and if not resolved may even induce cell death. Aims. The objective of the presented study was to test whether the B-domain deleted (BDD)-FVIII-X5 variant can overcome the secretion challenge of high level FVIII expression in the context of hepatic gene therapy. Methods. The human FVIII variant BDD-FVIII-X5 harboring 5 amino acid exchanges in the A1 domain was previously isolated in a screen aimed at identifying those residues in porcine FVIII that are critical for efficient secretion. BDD-FVIII and BDD-FVIII-X5 were produced in Chinese Hamster Ovary (CHO) cells and purified to apparent homogeneity using standard procedures. The preparations were assayed for total protein by UV absorbance at 280 nm and FVIII activity by a chromogenic assay. Both FVIII variants were vectorized using AAV8 and tested in the human liver cell line HepG2 and FVIII knockout mice (E17) at various doses. Resulting samples were assayed for FVIII chromogenic activity. The potential immunogenic risk was evaluated in three hemophilic mouse strains (E17, human FVIII transgenic, humanized HLA-DRB1*1501). Results. A characterization of purified recombinant Refacto-like BDD-FVIII and the corresponding X5 variant revealed similarity of the two proteins and their specific activities in particular, indicating that introduction of the 5 amino acids from porcine FVIII did not alter functionality of human BDD-FVIII. In vitro expression of BDD-FVIII-X5 in a human liver cell line resulted in substantially increased FVIII activity levels in the supernatant compared with the non-modified BDD-FVIII, commensurate with enhanced secretion of the X5 variant. Intravenous delivery of liver-targeted AAV8 vectors carrying the BDD-FVIII-X5 transgene achieved substantial increases in plasma coagulation activity over BDD-FVIII in FVIII-deficient mice, even when highly efficient codon-optimized F8 nucleotide sequences were employed. Evaluation of the immunogenicity of the BDD-FVIII-X5 variant by an immunological risk assessment did not reveal any increased immunogenic risk compared to BDD-FVIII. Conclusions: The fully active BDD-FVIII-X5 variant demonstrated improved secretion in vitro and in vivo, resulting in substantially higher FVIII levels in a hemophilia A mouse model. No signs of enhanced immunogenicity were noted in a comparative immunogenicity study. The results obtained warrant further exploration of the BDD-FVIII-X5 variant for a next generation hemophilia A gene therapy. Disclosures Horling: Baxalta Innovations GmbH, a Takeda company: Employment. Lengler:Baxalta Innovations GmbH, a Takeda company: Employment. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. De La Rosa:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Hoellriegl:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Reipert:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Scheiflinger:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Xiao:Ivygen: Other: Patent application on FVIII-X5 has been submitted. Rottensteiner:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.

Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration.

Recombinant adeno-associated virus (AAV)-based gene therapy has been promising, but several host-related transduction or immune challenges remain. For this mode of therapy to be widely applicable, it is crucial to develop high transduction and permeating vectors that infect the target at significantly low doses. Because glycosylation of capsid proteins is known to be rate limiting in the life cycle of many viruses, we reasoned that perturbation of glycosylation sites in AAV2 capsid will enhance gene delivery. In our first set experiments, pharmacological modulation of the glycosylation status in host cells, modestly decreased (1-fold) AAV2 packaging efficacy while it improved their gene expression (~74%) in vitro. We then generated 24 mutant AAV2 vectors modified to potentially create or disrupt a glycosylation site in its capsid. Three of them demonstrated a 1.3–2.5-fold increase in transgene expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic gene transfer of these vectors in hemophilia B mice, resulted in a 2-fold increase in human coagulation factor (F)IX levels, while its T/B-cell immunogenic response was unaltered. Subsequently, intravitreal gene transfer of glycosylation site-modified vectors in C57BL6/J mice demonstrated an increase in green fluorescence protein expression (~2- to 4-fold) and enhanced permeation across retina. Subretinal administration of these modified vectors containing RPE65 gene further rescued the photoreceptor response in a murine model of Leber congenital amarousis. Our studies highlight the translational potential of glycosylation site-modified AAV2 vectors for hepatic and ocular gene therapy applications.

Hepatic arginase deficiency fosters dysmyelination during postnatal CNS development.

Deficiency of arginase is associated with hyperargininemia, and prominent features include spastic diplegia/tetraplegia, clonus, and hyperreflexia; loss of ambulation, intellectual disability and progressive neurological decline are other signs. To gain greater insight into the unique neuromotor features, we performed gene expression profiling of the motor cortex of a murine model of the disorder. Coexpression network analysis suggested an abnormality with myelination, which was supported by limited existing human data. Utilizing electron microscopy, marked dysmyelination was detected in 2-week-old homozygous Arg1-KO mice. The corticospinal tract was found to be adversely affected, supporting dysmyelination as the cause of the unique neuromotor features and implicating oligodendrocyte impairment in a deficiency of hepatic Arg1. Following neonatal hepatic gene therapy to express Arg1, the subcortical white matter, pyramidal tract, and corticospinal tract all showed a remarkable recovery in terms of myelinated axon density and ultrastructural integrity with active wrapping of axons by nearby oligodendrocyte processes. These findings support the following conclusions: arginase deficiency is a leukodystrophy affecting the brain and spinal cord while sparing the peripheral nervous system, and neonatal AAV hepatic gene therapy can rescue the defects associated with myelinated axons, strongly implicating the functional recovery of oligodendrocytes after restoration of hepatic arginase activity.

Tolerance induction in hemophilia: innovation and accomplishments.

Hemophilia is an X-linked blood coagulation genetic disorder, which can cause significant disability. Replacement therapy for coagulation factor VIII (hemophilia A) or factor IX (hemophilia B) may result in the development of high-affinity alloantibodies ('inhibitors') to the replacement therapy, thus making it ineffective. Therefore, there is interest in directing immunological responses towards tolerance to infused factors. In this review, we will discuss latest advancements in the development of potentially less immunogenic replacement clotting factors, optimization of current tolerance induction protocols (ITI), preclinical and clinical data of pharmacological immune modulation, hepatic gene therapy, and the rapidly advancing field of cell therapies. We will also evaluate publications reporting data from preclinical studies on oral tolerance induction using chloroplast-transgenic (transplastomic) plants. Until now, no clinical prophylactic immune modulatory protocol exists to prevent inhibitor formation to infused clotting factors. Recent innovative technologies provide hope for improved eradication and perhaps even prevention of inhibitors.

Anti-TCRβ mAb in Combination With Neurogenin3 Gene Therapy Reverses Established Overt Type 1 Diabetes in Female NOD Mice.

Insulin-producing β cells in patients with type 1 diabetes (T1D) are destroyed by T lymphocytes. We investigated whether targeting the T-cell receptor (TCR) with a monoclonal antibody (mAb) abrogates T-cell response against residual and newly formed islets in overtly diabetic nonobese diabetic (NOD) mice. NOD mice with blood glucose levels of 250 to 350 mg/dL or 350 to 450 mg/dL were considered as new-onset or established overt diabetes, respectively. These diabetic NOD mice were transiently treated with an anti-TCR β chain (TCRβ) mAb, H57-597, for 5 days. Two weeks later, some NOD mice with established overt diabetes further received hepatic gene therapy using the islet-lineage determining gene Neurogenin3 (Ngn3), in combination with the islet growth factor gene betacellulin (Btc). We found that anti-TCRβ mAb (50 µg/d) reversed >80% new-onset diabetes in NOD mice for >14 weeks by reducing the number of effector T cells in the pancreas. However, anti-TCRβ mAb therapy alone reversed only ∼20% established overt diabetes in these mice. Among those overtly diabetic NOD mice whose diabetes was resistant to anti-TCRβ mAb treatment, ∼60% no longer had diabetes when they also received Ngn3-Btc hepatic gene transfer 2 weeks after initial anti-TCRβ mAb treatment. This combination of Ngn3-Btc gene therapy and anti-TCRβ mAb treatment induced the sustained formation of periportal insulin-producing cells in the liver of overtly diabetic mice. Therefore, directly targeting TCRβ with a mAb potently reverses new-onset T1D in NOD mice and protects residual and newly formed gene therapy-induced hepatic neo-islets from T-cell‒mediated destruction in mice with established overt diabetes.

Optimized AAV rh.10 Vectors That Partially Evade Neutralizing Antibodies during Hepatic Gene Transfer.

Of the 12 common serotypes used for gene delivery applications, Adeno-associated virus (AAV)rh.10 serotype has shown sustained hepatic transduction and has the lowest seropositivity in humans. We have evaluated if further modifications to AAVrh.10 at its phosphodegron like regions or predicted immunogenic epitopes could improve its hepatic gene transfer and immune evasion potential. Mutant AAVrh.10 vectors were generated by site directed mutagenesis of the predicted targets. These mutant vectors were first tested for their transduction efficiency in HeLa and HEK293T cells. The optimal vector was further evaluated for their cellular uptake, entry, and intracellular trafficking by quantitative PCR and time-lapse confocal microscopy. To evaluate their potential during hepatic gene therapy, C57BL/6 mice were administered with wild-type or optimal mutant AAVrh.10 and the luciferase transgene expression was documented by serial bioluminescence imaging at 14, 30, 45, and 72 days post-gene transfer. Their hepatic transduction was further verified by a quantitative PCR analysis of AAV copy number in the liver tissue. The optimal AAVrh.10 vector was further evaluated for their immune escape potential, in animals pre-immunized with human intravenous immunoglobulin. Our results demonstrate that a modified AAVrh.10 S671A vector had enhanced cellular entry (3.6 fold), migrate rapidly to the perinuclear region (1 vs. >2 h for wild type vectors) in vitro, which further translates to modest increase in hepatic gene transfer efficiency in vivo. More importantly, the mutant AAVrh.10 vector was able to partially evade neutralizing antibodies (~27–64 fold) in pre-immunized animals. The development of an AAV vector system that can escape the circulating neutralizing antibodies in the host will substantially widen the scope of gene therapy applications in humans.

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.

Hepatic gene therapy rescues high-fat diet responses in circadian Clock mutant mice

ObjectiveCircadian Clock gene mutant mice show dampened 24-h feeding rhythms and an increased sensitivity to high-fat diet (HFD) feeding. Restricting HFD access to the dark phase counteracts its obesogenic effect in wild-type mice. The extent to which altered feeding rhythms are causative for the obesogenic phenotype of Clock mutant mice, however, remains unknown. MethodsMetabolic parameters of wild-type (WT) and ClockΔ19 mutant mice (MT) were investigated under ad libitum and nighttime restricted HFD feeding. Liver circadian clock function was partially rescued by hydrodynamic tail vein delivery of WT-Clock DNA vectors in mutant mice and transcriptional, metabolic, endocrine and behavioral rhythms studied. ResultsNighttime-restricted feeding restored food intake, but not body weight regulation in MT mice under HFD, suggesting Clock-dependent metabolic dysregulation downstream of circadian appetite control. Liver-directed Clock gene therapy partially restored liver circadian oscillator function and transcriptome regulation without affecting centrally controlled circadian behaviors. Under HFD, MT mice with partially restored liver clock function (MT-LR) showed normalized body weight gain, rescued 24-h food intake rhythms, and WT-like energy expenditure. This was associated with decreased nighttime leptin and daytime ghrelin levels, reduced hepatic lipid accumulation, and improved glucose tolerance. Transcriptome analysis revealed that hepatic Clock rescue in MT mice affected a range of metabolic pathways. ConclusionLiver Clock gene therapy improves resistance against HFD-induced metabolic impairments in mice with circadian clock disruption. Restoring or stabilizing liver clock function might be a promising target for therapeutic interventions in obesity and metabolic disorders.

MRNA destabilization improves glycemic responsiveness of transcriptionally regulated hepatic insulin gene therapy in vitro and in vivo.

Hepatic insulin gene therapy (HIGT) employing a glucose and insulin sensitive promoter to direct insulin transcription can lower blood sugars within 2h of an intraperitoneal glucose challenge. However, post-challenge blood sugars frequently decline to below baseline. We hypothesize that this 'over-shoot' hypoglycemia results from sustained translation of long-lived transgene message, and that reducing pro-insulin message half-life will ameliorate post-challenge hypoglycemia. We compared pro-insulin message content and insulin secretion from primary rat hepatocytes expressing insulin from either a standard construct (2xfur), or a construct producing a destabilized pro-insulin message (InsTail), following exposure to stimulating or inhibitory conditions. Hepatocytes transduced with a 2xfur construct accumulated pro-insulin message, and exhibited increased insulin secretion, under conditions that both inhibit or stimulate transcription. By contrast, pro-insulin message content remained stable in InsTail expressing cells, and insulin secretion increased less than 2xfur during prolonged stimulation. During transitions from stimulatory to inhibitory conditions, or vice versa, amounts of pro-insulin message changed more rapidly in InsTail expressing cells than 2xfur expressing cells. Importantly, insulin secretion increased during the transition from stimulation to inhibition in 2xfur expressing cells, although it remained unchanged in InsTail expressing cells. Use of the InsTail destabilized insulin message tended to more rapidly reduce glucose induced glycemic excursions, and limit post-load hypoglycemia in STZ-diabetic mice in vivo. The data obtained in the present study suggest that combining transcriptional and post-transcriptional regulatory strategies may reduce undesirable glycemic excursion in models of HIGT.

Mechanisms of haem toxicity in haemolysis and protection by the haem‐binding protein, haemopexin

Haem (iron‐protoporphyrin IX) is vital to aerobic life. However, haemolysis generates massive amounts of free haem that can be maintained for several days. Haem is toxic because the chemical reactions of haem that occur in the presence of oxygen can destroy most biological molecules and haem activates cells of the immune system to drive inflammation. Haemopexin captures haem and disarms its chemical activity, thus protecting cells and molecules in biological fluids from haem toxicity. A surprisingly large number of disease states characterized by haemolysis or complicated by haemolysis (e.g. sickle cell disease, sepsis, haemolytic anaemias, viral haemorrhagic fevers) lack targeted therapies. Novel treatments from proof of principle studies in mice models of sickle cell disease and sepsis include replenishment therapies with haemopexin infusions or hepatic gene therapy to express haemopexin. Here, we characterize the key protective mechanisms that haemopexin instigates, explain how methaemoglobin and methemalbumin produce haem toxicity and evaluate the clinical importance of the haemopexin system in protecting against extracellular haem‐activated pathology in intravascular haemolysis. The main findings are that endocytosis of haem–haemopexin co‐regulates the safe trafficking of three redox‐active metals through subcellular compartments and, compared with the response to ‘free’ haem, uniquely activates several signalling pathways and transcription factors that induce the production of antioxidant enzymes and molecules known to maintain the intracellular redox state. Mounting evidence from patient longitudinal studies supports that impairments of haem and haemoglobin clearance are often unrecognized. Clinical data suggest that haemopexin is likely the best plasma biomarker for assessing a haem load as, unlike haptoglobin, it responds to heme and is not an acute‐phase reactant. Knowledge of the biology of haemopexin has yielded a potential novel therapeutic and also provided an impetus and means for improving diagnosis and longitudinal monitoring of patients with haemolysis.

Fah Knockout Animals as Models for Therapeutic Liver Repopulation.

Several animal models of Fah deficiency have been developed, including mice, pigs and most recently rats. Initially, the murine models were developed with the intent to mirror the human disease for pathophysiologic and therapeutic studies. However, it soon became apparent that Fah-positive hepatocytes have a potent selective growth advantage in mutant liver and can extensively repopulate the diseased organ. For this reason, Fah mutant mice have become a workhorse for liver biology and are widely used in liver stem cell and hepatic gene therapy research. Immune deficient Fah-knockout mice can be repopulated with human hepatocytes, creating "mice with human livers". These chimeric animals have become an important preclinical model for infectious diseases, metabolism and gene therapy. The potent expansion of human hepatocytes in Fah knockout mice has given rise to the concept of using Fah mutants as living bioreactors to produce large quantities of fully mature hepatocytes. As a consequence, larger animal models of Fah deficiency have recently been developed.

Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy.

Long-term glycemic control with hepatic insulin gene therapy in streptozotocin-diabetic mice.

Insulin self-administration is burdensome and can produce dangerous hypoglycemia. Insulin gene therapy may improve and simplify the treatment of diabetes mellitus. In rats, metabolically responsive hepatic insulin gene therapy (HIGT) delivered by adenovirus normalizes random blood sugars but with a limited duration. To prolong glycemic control, we delivered a metabolically regulated insulin transgene by adeno-associated virus (AAV). We administered increasing doses of self-complementary (SC), pseudotyped AAV8 expressing the (GlRE)3 BP1-2xfur insulin transgene to streptozotocin-diabetic CD-1 mice, and monitored blood sugar and body weight. We also compared responses to intraperitoneal glucose and chow withdrawal, assessed for viral genomes in liver by Southern blotting, and measured hepatic glycogen. Glucose lowering required the combination of SC genomes and AAV capsid pseudotyping. HIGT controlled glycemia in diabetic mice (DM) for > 1 year. However, glycemic responses were variable. Approximately 30% of mice succumbed to hypoglycemia, and approximately 30% of mice again became hyperglycemic. During an intraperitoneal glucose tolerance test, blood sugars declined to normal within 180 min in HIGT-treated DM compared to 90 min in control mice. Hypoglycemia was common among HIGT-treated mice during a 24-h fast. However, HIGT mice lost less weight than either diabetic or nondiabetic controls as a result of increased water intake. HIGT treatment reduced the hepatic glycogen content of fed mice. Our studies demonstrate the possibility for long-term glycemic correction following AAV-mediated HIGT in mice. However, the dose-response relationship is irregular, and metabolic responsiveness may be less than that observed in rats.

Hepatic insulin gene therapy prevents diabetic enteropathy in STZ-treated CD-1 mice

Depending on the population examined, from 6 to 83% of people with diabetes mellitus exhibit symptoms of altered gut motility, manifesting as dysphagia, reflux, early satiety, nausea, abdominal pain, diarrhea, or constipation. Hyperglycemia-induced cell loss within the enteric nervous system has been demonstrated in both diabetic rodents and patients with diabetes. Glycemic control is recommended to prevent diabetic gastroenteropathy but is often difficult to achieve with current treatment modalities. We asked if hepatic insulin gene therapy (HIGT) could inhibit the development of diabetic gastroenteropathy in mice. Bowel length, bowel transit, colonic muscle relaxation, and the numbers of both stimulatory and inhibitory neurons in the colonic myenteric plexus were compared in groups of diabetic mice (DM), control nondiabetic mice (Con), and diabetic mice treated with HIGT (HIGT). Delivery of a metabolically responsive insulin transgene to the liver of STZ-diabetic mice with an adeno-associated virus, sero-type 8 (AAV8) produced near-normal blood sugars for over 1 month and prevented anatomic, functional, and neurohistologic changes observed in diabetic mice. We conclude that in addition to normalizing oxidative metabolism in diabetic rodents, HIGT is sufficient to prevent the development of diabetic gastroenteropathy.

Expression and purification of recombinant circumsporozoite proteins of Plasmodium falciparum and evaluation of their binding activities to hepatocytes

Objective To amplify four fragments of circumsporozoite （ CSP） protein gene from Plasmodium falciparum FCC1/HN strain and express recombinant CSP proteins in prokaryotic vector pET28 a/EGFP for further evaluation of their binding activities to hepatic cells .Methods Four pairs of primers were designed according to the cDNA sequence of CSP protein from Plasmodium falciparum FCC1/HN strain and used to amplify the gene fragments by PCR .The cloned gene fragments were inserted into pET28a/EGFP to construct the recombinant expression plasmids .The transformed E.coli BL21 strains carry-ing expression plasmids were induced by IPTG to express CPS proteins .The recombinant CSP proteins were purified with Ni2＋chelating HiTrap HP column and detected by SDS-PAGE.The binding activities of the ex-pressed proteins to different tissues were also detected .Results Four gene fragments encoding CSP protein were successfully amplified and expressed in E.coil BL21 strain as fusion protein CSR1a-EGFP, CSR1b-EGFP, CSR2a-EGFP and CSR2b-EGFP with a relative molecular mass of about 39×103, 31×103, 33×103 and 30 ×10 3 , respectively .The purified fusion proteins reacted specifically with Plasmodium falciparum-posi-tive serum samples.Moreover, the recombinant protein CSR2a-EGFP could bind to the hepatic cells specif-ically rather than other cells.Conclusion The purified recombinant CSR2a-EGFP protein has a strong binding activity to hepatocytes , indicating its potential value as a targeting molecule for hepatic gene therapy . Key words: Plasmodium falciparum; Circumsporozoite protein; Hepatic cells; Targeting

Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy

AbstractThe development of the next-generation gene therapy vectors for hemophilia requires using lower and thus potentially safer vector doses and augmenting their therapeutic efficacy. We have identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs) by using a computational strategy that increased factor IX (FIX) levels 11- to 15-fold. Vector efficacy could be enhanced by combining these hepatocyte-specific CRMs with a synthetic codon-optimized hyperfunctional FIX-R338LPadua transgene. This Padua mutation boosted FIX activity up to sevenfold, with no apparent increase in thrombotic risk. We then validated this combination approach using self-complementary adenoassociated virus serotype 9 (scAAV9) vectors in hemophilia B mice. This resulted in sustained supraphysiologic FIX activity (400%), correction of the bleeding diathesis at clinically relevant, low vector doses (5 × 1010 vector genomes [vg]/kg) that are considered safe in patients undergoing gene therapy. Moreover, immune tolerance could be induced that precluded induction of inhibitory antibodies to FIX upon immunization with recombinant FIX protein.