Chronic liver disease and impaired hepatic glycogen metabolism in argininosuccinate lyase deficiency.

BACKGROUNDLiver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear.METHODSWe estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTest to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the AslNeo/Neo mouse model of ASLD.RESULTSWe demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, 2 markers of disease severity, were significantly (P < 0.001 and P = 0.001, respectively) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTest revealed increased echogenicity and liver stiffness, even in individuals with ASLD and normal aminotransferases. The AslNeo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted before death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liver-specific (ApoE) promoter.CONCLUSIONOur results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov (NCT03721367, NCT00237315).FUNDINGFunding was provided by NIH, Burroughs Wellcome Fund, NUCDF, Genzyme/ACMG Foundation, and CPRIT.

Effects of Aspirin on Growth Factor Release From Freshly Isolated Leukocyte-Rich Platelet-Rich Plasma in Healthy Men: A Prospective Fixed-Sequence Controlled Laboratory Study.

BACKGROUND: The benefits of platelet-rich plasma (PRP) are believed to be in part dependent on growth factor release after platelet activation. Platelet activation is complex and involves multiple mechanisms. One important mechanism is driven by cyclooxygenase 1 (COX-1)-mediated conversion of arachidonic acid (AA) to precursor prostaglandins that then mediate proinflammatory responses that trigger growth factor release. Acetylsalicylic acid (ASA; also known as aspirin) is known to irreversibly inhibit COX-1, thereby blocking AA-mediated signaling; however, it is unclear whether ASA use alters growth factor release from freshly isolated PRP. PURPOSE: To assess the effects of low-dose ASA use on activation of growth factor release from freshly isolated human PRP via AA and thrombin (TBN). STUDY DESIGN: Controlled laboratory study. METHODS: Twelve healthy men underwent blood collection and leukocyte-rich PRP (LR-PRP) preparation through a double-spin protocol to obtain baseline whole blood and PRP counts the same day. PRP was aliquoted into 3 groups: nonactivated, AA activated, and TBN activated. Immediately after activation, the concentrations of transforming growth factor ß1 (TGF-ß1), vascular endothelial growth factor (VEGF), and platelet-derived growth factor AB (PDGF-AB) were measured using enzyme-linked immunosorbent assays (ELISAs). The same 12 participants were then placed on an 81-mg daily dose of oral ASA for 14 days. Repeat characterization of whole blood and PRP analyses was done on day 14, followed by repeat ELISAs of growth factors under the same nonactivated and activated settings as previously stated. RESULTS: Fourteen days of daily ASA had no effect on the number of platelets and leukocytes measured in whole blood and LR-PRP. Compared with nonactivated LR-PRP, AA- and TBN-mediated activation led to significant release of VEGF and PDGF-AB. In contrast, release of TGF-ß1 from LR-PRP was observed only with activation by AA, not with TBN. Consistent with its inhibitory role in AA signaling, ASA significantly inhibited AA-mediated release of all 3 growth factors measured in this study. Although ASA had no effect on TBN-mediated release of VEGF and TGF-ß1 from LR-PRP, ASA did partially block TBN-mediated release of PDGF-AB, although the mechanism remains unclear. CONCLUSION: Daily use of low-dose ASA reduces VEGF, PDGF-AB, and TGF-ß1 expression in freshly isolated human LR-PRP when activated with AA. CLINICAL RELEVANCE: Reduction in growth factor release attributed to daily use of low-dose ASA or other COX inhibitors can be mitigated when PRP samples are activated with TBN. Clinical studies are needed to determine whether activation before PRP injection is needed in all applications where ASA is in use and to what extent ASA may inhibit growth factor release in vivo at the site of injury.

Combinatorial Prg4 and Il-1ra Gene Therapy Protects Against Hyperalgesia and Cartilage Degeneration in Post-Traumatic Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease of synovial joints characterized by progressive loss of articular cartilage, subchondral bone remodeling, and intra-articular inflammation with synovitis that results in chronic pain and motor impairment. Despite the economic and health impacts, current medical therapies are targeted at symptomatic relief of OA and fail to alter its progression. Given the complexity of OA pathogenesis, we hypothesized that a combinatorial gene therapy approach, designed to inhibit inflammation with interleukin-1 receptor antagonist (IL-1Ra) while promoting chondroprotection using lubricin (PRG4), would improve preservation of the joint compared to monotherapy alone. Employing two surgical techniques to model mild, moderate and severe posttraumatic OA, we found that combined delivery of helper-dependent adenoviruses (HDVs), expressing IL-1Ra and PRG4, preserved articular cartilage better than either monotherapy in both models as demonstrated by preservation of articular cartilage volume and surface area. This improved protection was associated with increased expression of proanabolic and cartilage matrix genes together with decreased expression of catabolic genes and inflammatory mediators. In addition to improvements in joint tissues, this combinatorial gene therapy prolonged protection against thermal hyperalgesia compared to either monotherapy. Taken together, our results show that a combinatorial strategy is superior to monotherapeutic approaches for treatment of posttraumatic OA.

Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes.

Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting.

Restoration of the serum level of SERPINF1 does not correct the bone phenotype in Serpinf1 null mice.

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by bone fragility and deformity. OI type VI is unique owing to the mineralization defects observed in patient biopsies. Furthermore, it has been reported to respond less well to standard therapy with bisphosphonates [1]. Others and we have previously identified SERPINF1 mutations in patients with OI type VI. SERPINF1 encodes pigment epithelium derived factor (PEDF), a secreted collagen-binding glycoprotein that is absent in the sera of patients with OI type VI. Serpinf1 null mice show increased osteoid and decreased bone mass, and thus recapitulate the OI type VI phenotype. We tested whether restoration of circulating PEDF in the blood could correct the phenotype of OI type VI in the context of protein replacement. To do so, we utilized a helper-dependent adenoviral vector (HDAd) to express human SERPINF1 in the mouse liver and assessed whether PEDF secreted from the liver was able to rescue the bone phenotype observed in Serpinf1(-/-) mice. We confirmed that expression of SERPINF1 in the liver restored the serum level of PEDF. We also demonstrated that PEDF secreted from the liver was biologically active by showing the expected metabolic effects of increased adiposity and impaired glucose tolerance in Serpinf1(-/-) mice. Interestingly, overexpression of PEDF in vitro increased mineralization with a concomitant increase in the expression of bone gamma-carboxyglutamate protein, alkaline phosphatase and collagen, type I, alpha I, but the increased serum PEDF level did not improve the bone phenotype of Serpinf1(-/-) mice. These results suggest that PEDF may function in a context-dependent and paracrine fashion in bone homeostasis.

Differential type I interferon-dependent transgene silencing of helper-dependent adenoviral vs. adeno-associated viral vectors in vivo.

We previously dissected the components of the innate immune response to Helper-dependent adenoviral vectors (HDAds) using genetic models, and demonstrated that multiple pattern recognition receptor signaling pathways contribute to this host response to HDAds in vivo. Based on analysis of cytokine expression profiles, type I interferon (IFN) mRNA is induced in host mouse livers at 1 hour post-injection. This type I IFN signaling amplifies cytokine expression in liver independent of the nature of vector DNA sequences after 3 hours post-injection. This type I IFN signaling in response to HDAds administration contributes to transcriptional silencing of both HDAd prokaryotic and eukaryotic DNA in liver. This silencing occurs early and is mediated by epigenetic modification as shown by in vivo chromatin immunoprecipitation (ChIP) with anti-histone deacetylase (HDAC) and promyelocytic leukemia protein (PML). In contrast, self-complementary adeno-associated viral vectors (scAAVs) showed significantly lower induction of type I IFN mRNA in liver compared to HDAds at both early and late time points. These results show that the type I IFN signaling dependent transgene silencing differs between AAV and HDAd vectors after liver-directed gene transfer.

Cytokine-conditioned dendritic cells induce humoral tolerance to protein therapy in mice.

A major obstacle in the genetic therapy of inherited metabolic disease is host immune responses to the therapeutic protein. This is best exemplified by inhibitor formation in the protein therapy for hemophilia A. An approach to overcoming this is induction of immunological tolerance to the therapeutic protein. Tolerogenic dendritic cells (DCtols) have been reported to induce tolerance. In addition, cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-ß(1) are known to induce tolerance. To model protein therapy, we used ovalbumin (OVA) as antigen in BALB/c mice and their transgenic derivative, DO11.10 mice. In this study we show that adoptive transfer of antigen-pulsed dendritic cells (DCs) treated with a combination of IL-10 and TGF-ß(1) can suppress the antibody response in mice. Adoptive transfer of cytokine-conditioned DCs in preimmunized mice results in reduction of antibody response in the mice. Furthermore, the effect is antigen specific, as the recipient mice were able to mount a potent antibody response to the control antigen. Last, we show that TGF-ß(1) and IL-10-conditioned DCs are able to inhibit anti-FVIII antibody responses in FVIII knockout (KO) mice. Analysis of the contribution of IL-10 and TGF-ß(1) to the DCtol phenotype shows that IL-10 treatment of DCs is sufficient for inducing OVA-specific tolerance in BALB/c mice, but we observed a requirement for treatment with both human TGF-ß(1) and human IL-10 to significantly inhibit anti-FVIII antibody responses in FVIII KO mice. This paper demonstrates that autologous cell therapy for antigen-targeted immune suppression may be developed to facilitate long-term therapy.

NOD2 signaling contributes to the innate immune response against helper-dependent adenovirus vectors independently of MyD88 in vivo.

We previously demonstrated that Toll-like receptor/myeloid differentiation primary response gene 88 (MyD88) signaling is required for maximal innate and acquired [T helper cell type 1 (Th1)] immune responses following systemic administration of helper-dependent adenoviral vectors (HDAds). However, MyD88-deficient mice injected with HDAdLacZ exhibited only partial reduction of innate immune cytokine expression compared with wild-type mice, suggesting MyD88-independent pathways also respond to HDAds. We now show that NOD2, a nucleotide-binding and oligomerization domain (NOD)-like receptor known to detect muramyl dipeptides in bacterial peptidoglycans, also contributes to innate responses to HDAds, but not to humoral or Th1 immune responses. We established NOD2/MyD88 double-deficient mice that, when challenged with HDAds, showed a significant reduction of the innate response compared with mice deficient for either gene singly, suggesting that NOD2 signaling contributes to the innate response independently of MyD88 signaling following systemic administration of HDAds. In addition, NOD2-deficient mice exhibited significantly higher transgene expression than did wild-type mice at an early time point (before development of an acquired response), but not at a later time point (after development of an acquired response). These results indicate that the intracellular sensor NOD2 is required for innate responses to HDAds and can limit transgene expression during early phases of infection.

Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII.

Neonatal gene therapy is a promising strategy for treating a number of congenital diseases diagnosed shortly after birth as expression of therapeutic proteins during postnatal life may limit the pathologic consequences and result in a potential "cure." Hemophilia A is often complicated by the development of antibodies to recombinant protein resulting in treatment failure. Neonatal administration of vectors may avoid inhibitory antibody formation to factor VIII (FVIII) by taking advantage of immune immaturity. A helper-dependent adenoviral vector expressing human factor VIII was administered i.v. to neonatal hemophilia A knockout mice. Three days later, mice produced high levels of FVIII. Levels declined rapidly with animal growth to 5 wk of age with stable factor VIII expression thereafter to >1 y of age. Decline in factor VIII expression was not related to cell-mediated or humoral responses with lack of development of antibodies to capsid or human factor VIII proteins. Subsequent readministration and augmentation of expression was possible as operational tolerance was established to factor VIII without development of inhibitors; however, protective immunity to adenovirus remained.

Large-scale production of high-quality helper-dependent adenoviral vectors using adherent cells in cell factories.

The most efficient and widely used system for generating helper-dependent adenoviral vectors (HDAds) is the Cre/loxP system developed by Graham and co-workers (Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. [ 1996 ]. Proc. Natl. Acad. Sci. U. S. A. 93, 13565-13570). Alternative systems have been developed for HDAd production, but all are limited by the technical complexity of a three-component vector production system for reproducibly generating large quantities of adenovirus with high infectivity and low helper virus (HV) contamination. Recently, these problems were addressed by Ng and co-workers (Palmer, D., and Ng, P. [ 2003 ]. Mol Ther. 8, 846-852), who developed an improved system that combines the use of a suspension-adapted producer cell line expressing high levels of Cre recombinase, a HV resistant to mutation, and a refined purification protocol. With this system, >1 x 10(13) highly infectious vector particles are easily produced without vector genome rearrangements and having very low HV contamination levels. However, the Ng system incorporates a spinner flask culture system that involves considerable time, effort, and tissue culture medium to produce HDAds. We have an alternative system to obtain comparable quantities with equivalent quality to the spinner flask approach but requiring reduced labor and lower volumes of medium. This method utilizes a 10-chamber cell factory with adherent cells to produce high infectivity of HDAds with minimal HV contamination while improving yield and reducing technical complexity, effort, and medium requirements. This system is easily translatable to the production of clinical-grade HDAds for human trials.

MyD88-dependent silencing of transgene expression during the innate and adaptive immune response to helper-dependent adenovirus.

Activation of the host innate immune response after systemic administration of adenoviral vectors constitutes a principal impediment to successful clinical gene replacement therapies. Although helper-dependent adenoviruses (HDAds) lack all viral functional genes, systemic administration of a high dose of HDAd still elicits a potent innate immune response in host animals. Toll-like receptors (TLRs) are innate receptors that sense microbial products and trigger the maturation of antigen-presenting cells and cytokine production via MyD88-dependent signaling (except TLR3). Here we show that mice lacking MyD88 exhibit a dramatic reduction in proinflammatory cytokines after intravenous injection of a high dose of HDAd, and show significantly reduced induction of the adaptive immune response when compared with wild-type and TLR2-deficient mice. Importantly, MyD88(-/-) mice also show significantly higher and longer sustained transgene expression than do wild-type mice. Chromatin immunoprecipitation studies using wild-type and MyD88-deficient primary mouse embryonic fibroblasts showed significant MyD88-dependent transcriptional silencing of the HDAd-encoded transgenes. Our results demonstrate that MyD88 signaling, activated by systemic delivery of HDAd, initiates an innate immune response that suppresses transgene expression at the transcriptional level before initiation of the adaptive immune response.

Correction of murine hemophilia A and immunological differences of factor VIII variants delivered by helper-dependent adenoviral vectors.

Bioengineering of the factor VIII (FVIII) molecule has led to the production of variants that overcome poor secretion and/or rapid inactivation. We tested six modified FVIII variants for in vivo efficacy by expressing them from helper-dependent adenoviral (HD-Ad) vectors. We constructed a wild-type (WT) variant, a B-domain-deleted (BDD) variant, a point mutant for improved secretion (F309S), a variant with a partial B-domain deletion for improved secretion (N6), a combination of the point mutant and partial BDD variant (F309N6), and an inactivation-resistant (IR8) FVIII variant. All the constructs expressed functional protein after injection of high-dose HD-Ad. Activity ranged from 20 to 50% with WT, to approximately 100% with the N6 and F309N6 variants. Interestingly, mice treated with N6 showed long-term FVIII activity and phenotypic correction for up to 74 weeks, with low anti-FVIII antibody titer. Importantly, the N6 variant was therapeutically efficacious even after a 50% reduction of viral dose, thereby indicating that transgene modification itself can improve the dose efficacy of HD-Ad. This finding is significant, because dose efficacy is a key factor in clinical application. In summary, bioengineering of the FVIII molecule may be an effective approach to improving the safety, immunogenicity, and efficacy of HD-Ad gene therapy in hemophilia A (HA).

Helper-dependent adenoviral vector-mediated long-term expression of human apolipoprotein A-I reduces atherosclerosis in apo E-deficient mice.

Apolipoprotein A-I (APOA-I) is the major protein component of high-density lipoproteins (HDL). It has been shown that over-expression of human APOA-I increases HDL cholesterol and decreases atherosclerosis. We constructed a helper-dependent adenoviral (HD-Ad) vector that contains the entire human APOA-I gene (hgAI). Intravenous delivery of 1x10(13) viral particles/kg of this vector was followed by high levels of human APOA-I expression (up to 200 mg/dl) in the absence of detectable hepatic toxicity. We treated apo E-deficient mice with the hgAI vector and fed them either with a high-fat diet or with regular chow. As a control, two groups of mice were treated with PBS. The apo E-deficient mice treated with the hgAI vector showed supraphysiological levels of expression of human APOA-I at week 4 and high levels of HDL cholesterol compared to the control groups. Analysis of aortic atherosclerotic lesions 20 weeks after treatment, showed a significant reduction of lesion size in the treated mice with both diets. In conclusion, liver-directed gene transfer of human APOA-I using a HD-Ad vector resulted in a reduction of the development of atherosclerosis with the absence of significant toxicity.