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.

Loss of DDRGK1 modulates SOX9 ubiquitination in spondyloepimetaphyseal dysplasia.

Shohat-type spondyloepimetaphyseal dysplasia (SEMD) is a skeletal dysplasia that affects cartilage development. Similar skeletal disorders, such as spondyloepiphyseal dysplasias, are linked to mutations in type II collagen (COL2A1), but the causative gene in SEMD is not known. Here, we have performed whole-exome sequencing to identify a recurrent homozygous c.408+1G>A donor splice site loss-of-function mutation in DDRGK domain containing 1 (DDRGK1) in 4 families affected by SEMD. In zebrafish, ddrgk1 deficiency disrupted craniofacial cartilage development and led to decreased levels of the chondrogenic master transcription factor sox9 and its downstream target, col2a1. Overexpression of sox9 rescued the zebrafish chondrogenic and craniofacial phenotype generated by ddrgk1 knockdown, thus identifying DDRGK1 as a regulator of SOX9. Consistent with these results, Ddrgk1-/- mice displayed delayed limb bud chondrogenic condensation, decreased SOX9 protein expression and Col2a1 transcript levels, and increased apoptosis. Furthermore, we determined that DDRGK1 can directly bind to SOX9 to inhibit its ubiquitination and proteasomal degradation. Taken together, these data indicate that loss of DDRGK1 decreases SOX9 expression and causes a human skeletal dysplasia, identifying a mechanism that regulates chondrogenesis via modulation of SOX9 ubiquitination.

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.

Losartan increases bone mass and accelerates chondrocyte hypertrophy in developing skeleton.

Angiotensin receptor blockers (ARBs) are a group of anti-hypertensive drugs that are widely used to treat pediatric hypertension. Recent application of ARBs to treat diseases such as Marfan syndrome or Alport syndrome has shown positive outcomes in animal and human studies, suggesting a broader therapeutic potential for this class of drugs. Multiple studies have reported a benefit of ARBs on adult bone homeostasis; however, its effect on the growing skeleton in children is unknown. We investigated the effect of Losartan, an ARB, in regulating bone mass and cartilage during development in mice. Wild type mice were treated with Losartan from birth until 6 weeks of age, after which bones were collected for microCT and histomorphometric analyses. Losartan increased trabecular bone volume vs. tissue volume (a 98% increase) and cortical thickness (a 9% increase) in 6-weeks old wild type mice. The bone changes were attributed to decreased osteoclastogenesis as demonstrated by reduced osteoclast number per bone surface in vivo and suppressed osteoclast differentiation in vitro. At the molecular level, Angiotensin II-induced ERK1/2 phosphorylation in RAW cells was attenuated by Losartan. Similarly, RANKL-induced ERK1/2 phosphorylation was suppressed by Losartan, suggesting a convergence of RANKL and angiotensin signaling at the level of ERK1/2 regulation. To assess the effect of Losartan on cartilage development, we examined the cartilage phenotype of wild type mice treated with Losartan in utero from conception to 1 day of age. Growth plates of these mice showed an elongated hypertrophic chondrocyte zone and increased Col10a1 expression level, with minimal changes in chondrocyte proliferation. Altogether, inhibition of the angiotensin pathway by Losartan increases bone mass and accelerates chondrocyte hypertrophy in growth plate during skeletal development.

Notch activation as a driver of osteogenic sarcoma.

Osteogenic sarcoma (OS) is a deadly skeletal malignancy whose cause is unknown. We report here a mouse model of OS based on conditional expression of the intracellular domain of Notch1 (NICD). Expression of the NICD in immature osteoblasts was sufficient to drive the formation of bone tumors, including OS, with complete penetrance. These tumors display features of human OS; namely, histopathology, cytogenetic complexity, and metastatic potential. We show that Notch activation combined with loss of p53 synergistically accelerates OS development in mice, although p53-driven OS is not Rbpj dependent, which demonstrates a dual dominance of the Notch oncogene and p53 mutation in the development of OS. Using this model, we also reveal the osteoblasts as the potential sources of OS.

The swaying mouse as a model of osteogenesis imperfecta caused by WNT1 mutations.

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue characterized by bone fragility and low bone mass. Recently, our group and others reported that WNT1 recessive mutations cause OI, whereas WNT1 heterozygous mutations cause early onset osteoporosis. These findings support the hypothesis that WNT1 is an important WNT ligand regulating bone formation and bone homeostasis. While these studies provided strong human genetic and in vitro functional data, an in vivo animal model to study the mechanism of WNT1 function in bone is lacking. Here, we show that Swaying (Wnt1(sw/sw)) mice previously reported to carry a spontaneous mutation in Wnt1 share major features of OI including propensity to fractures and severe osteopenia. In addition, biomechanical and biochemical analyses showed that Wnt1(sw/sw) mice exhibit reduced bone strength with altered levels of mineral and collagen in the bone matrix that is also distinct from the type I collagen-related form of OI. Further histomorphometric analyses and gene expression studies demonstrate that the bone phenotype is associated with defects in osteoblast activity and function. Our study thus provides in vivo evidence that WNT1 mutations contribute to bone fragility in OI patients and demonstrates that the Wnt1(sw/sw) mouse is a murine model of OI caused by WNT1 mutations.

Combinatorial treatment with oncolytic adenovirus and helper-dependent adenovirus augments adenoviral cancer gene therapy.

Oncolytic adenoviruses (Onc.Ads) produce significant antitumor effects but as single agents they rarely eliminate tumors. Investigators have therefore incorporated sequences into these vectors that encode immunomodulatory molecules to enhance antitumor immunity. Successful implementation of this strategy requires multiple tumor immune inhibitory mechanisms to be overcome, and insertion of the corresponding multiple functional genes reduces the titer and replication of Onc.Ads, compromising their direct ant-tumor effects. By contrast, helper-dependent (HD) Ads are devoid of viral coding sequences, allowing inclusion of multiple transgenes. HDAds, however, lack replicative capacity. Since HDAds encode the adenoviral packaging signal, we hypothesized that the coadministration of Onc.Ad with HDAd would allow to be amplified and packaged during replication of Onc.Ad in transduced cancer cells. This combination could provide immunostimulation without losing oncolytic activity. We now show that coinfection of Onc.Ad with HDAd subsequently replicates HDAd vector DNA in trans in human cancer cell lines in vitro and in vivo, amplifying the transgenes the HDAd encode. This combinatorial treatment significantly suppresses the tumor growth compared to treatment with a single agent in an immunocompetent mouse model. Hence, combinatorial treatment of Onc.Ad with HDAd should overcome the inherent limitations of each agent and provide a highly immunogenic oncolytic therapy.

E-selectin ligand 1 regulates bone remodeling by limiting bioactive TGF-ß in the bone microenvironment.

TGF-ß is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-ß bioavailability by attenuating maturation of pro-TGF-ß during cartilage homeostasis. However, whether regulation of intracellular TGF-ß maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1(-/-) mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1(-/-) osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1(-/-) osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast-osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1(-/-) calvaria exhibit an elevated mature TGF-ß/pro-TGF-ß ratio, with increased expression of TGF-ß downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan-TGF-ß antibody, significantly improved the low bone mass of Esl-1(-/-) mice, suggesting that elevated TGF-ß signaling is the major cause of osteopenia in Esl-1(-/-) mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-ß maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast-osteoclast coupling.

Proteoglycan 4 expression protects against the development of osteoarthritis.

Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.

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.

SR-A and SREC-I are Kupffer and endothelial cell receptors for helper-dependent adenoviral vectors.

Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity. However, a toxic acute response with potentially lethal consequences has hindered their clinical applications. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells are major barriers to efficient hepatocyte transduction. Understanding the mechanisms of adenoviral vector uptake by non-parenchymal cells may allow the development of strategies aimed at overcoming these important barriers and to achieve preferential hepatocyte gene transfer with reduced toxicity. Scavenger receptors on Kupffer cells bind adenoviral particles and remove them from the circulation, thus preventing hepatocyte transduction. In the present study, we show that HDAd particles interact in vitro and in vivo with scavenger receptor-A (SR-A) and with scavenger receptor expressed on endothelial cells-I (SREC-I) and we exploited this knowledge to increase the efficiency of hepatocyte transduction by HDAd vectors in vivo through blocking of SR-A and SREC-I with specific fragments antigen-binding (Fabs).

Capsid-modified adenoviral vectors for improved muscle-directed gene therapy.

Skeletal muscle represents an attractive target tissue for adenoviral gene therapy to treat muscle disorders and as a production platform for systemic expression of therapeutic proteins. However, adenovirus serotype 5 vectors do not efficiently transduce adult muscle tissue. Here we evaluated whether capsid modifications on adenoviral vectors could improve transduction in mature murine muscle tissue. First-generation and helper-dependent serotype 5 adenoviral vectors featuring the serotype 3 knob (5/3) showed significantly increased transduction of skeletal muscle after intramuscular injection in adult mice. Furthermore, we showed that full-length dystrophin could be more efficiently transferred to muscles of mdx mice using a 5/3-modified helper-dependent adenoviral vector. In contrast to first-generation vectors, helper-dependent adenoviral vectors mediated stable marker gene expression for at least 1 year after intramuscular injection. In conclusion, 5/3 capsid-modified helper-dependent adenoviral vectors show enhanced transduction in adult murine muscle tissue and mediate long-term gene expression, suggesting the suitability of these vectors for muscle-directed gene therapy.

miRNA-34c regulates Notch signaling during bone development.

During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling.

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.

E-selectin ligand-1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-beta.

The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-beta signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-beta signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-beta/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-beta during skeletal development and homeostasis.

Vasoactive intestinal peptide increases hepatic transduction and reduces innate immune response following administration of helper-dependent Ad.

Helper-dependent adenoviral vectors (HDAd) are effective tools for liver-directed gene therapy because they can mediate long-term transgene expression in the absence of chronic toxicity. However, high vector doses required for efficient hepatocyte transduction by intravascular delivery result in systemic vector dissemination and dose-dependent activation of the innate immunity. Therefore, strategies to achieve high-efficiency hepatocyte transduction using low vector doses and/or to reduce the acute elevations of proinflammatory cytokines and chemokines may have significant clinical potential. Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide involved in the regulation of hepatic blood flow and plays an important role as modulator of immune functions. Here, we show that VIP pretreatment in mice is able to increase hepatocyte transduction by HDAd, decrease vector uptake by the spleen, reduce elevation of proinflammatory serum cytokines interleukin (IL)-6 and IL-12, and reduce serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) following intravenous HDAd injection. VIP pretreatment also resulted in a reduction in the expression of the chemokines macrophage-inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1 (MCP-1), and regulated on activation normal T-cell expressed and secreted (RANTES) in the livers of mice injected with HDAd. These results suggest that VIP can improve the therapeutic index of HDAd by increasing hepatocyte transduction efficiency while reducing cytokine and chemokine expression following intravascular delivery of HDAd.

Osteosclerosis owing to Notch gain of function is solely Rbpj-dependent.

Osteosclerosis is a pathologic bone disease characterized by an increase in bone formation over bone resorption. Genetic factors that contribute to the pathogenesis of this disease are poorly understood. Dysregulation or mutation in many components of the Notch signaling pathway results in a wide range of human developmental disorders and cancers, including bone diseases. Our previous study found that activation of the Notch signaling in osteoblasts promotes cell proliferation and inhibits differentiation, leading to an osteosclerotic phenotype in transgenic mice. In this study we report a longer-lived mouse model that also develops osteosclerosis and a genetic manipulation that completely rescues the phenotype. Conditionally cre-activated expression of Notch1 intracellular domain (NICD) in vivo exclusively in committed osteoblasts caused massive osteosclerosis with growth retardation and abnormal vertebrae. Importantly, selective deletion of a Notch nuclear effector--Rbpj--in osteoblasts completely suppressed the osteosclerotic and growth-retardation phenotypes. Furthermore, cellular and molecular analyses of bones from the rescued mice confirmed that NICD-dependent molecular alterations in osteoblasts were completely reversed by removal of the Rbpj pathway. Together, our observations show that the osteosclerosis owing to activation of Notch signaling in osteoblasts is canonical in nature because it depends solely on Rbpj signaling. As such, it identifies Rbpj as a specific target for manipulating Notch signaling in a cell-autonomous fashion in osteoblasts in bone diseases where Notch may be dysregulated.

Ascorbate synthesis pathway: dual role of ascorbate in bone homeostasis.

Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of D-glucuronate to L-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) mice show that the two enzymes, GR and AR, provide approximately 85 and approximately 15% of L-gulonate, respectively. GRKO/ARKO double knock-out mice are unable to synthesize ASC (>95% ASC deficit) and develop scurvy. The GRKO mice ( approximately 85% ASC deficit) develop and grow normally when fed regular mouse chow (ASC content = 0) but suffer severe osteopenia and spontaneous fractures with stresses that increase ASC requirements, such as pregnancy or castration. Castration greatly increases osteoclast numbers and activity in GRKO mice and promotes increased bone loss as compared with wild-type controls and additionally induces proliferation of immature dysplastic osteoblasts likely because of an ASC-sensitive block(s) in early differentiation. ASC and the antioxidants pycnogenol and resveratrol block osteoclast proliferation and bone loss, but only ASC feeding restores osteoblast differentiation and prevents their dysplastic proliferation. This is the first in vivo demonstration of two independent roles for ASC as an antioxidant suppressing osteoclast activity and number as well as a cofactor promoting osteoblast differentiation. Although humans have lost the ability to synthesize ASC, our mouse models suggest the mechanisms by which suboptimal ASC availability facilitates the development of osteoporosis, which has important implications for human osteoporosis.

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.

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.