Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codonoptimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.

Background: The hepatitis C virus (HCV) is an attractive target for RNA interference (RNAi) based therapeutics because its genome consists of a single, positive stranded RNA and its replication occurs strictly within the cytoplasm. TT-034 is a DNA-directed RNAi (ddRNAi) based gene therapy product for the treatment of chronic HCV infection and is currently in a phase I/IIa clinical trial. TT-034 is comprised of a self-complementary recombinant adeno-associated virus (AAV) serotype 8 vector for transduction of hepatic tissues and is systemically administered as a single intravenous dose. Inside the hepatocyte, TT-034 uses the cell's own transcriptional machinery to continuously express three independent short hairpin RNAs (shRNAs) that simultaneously target the 5' UTR and NS5B regions of the HCV genome. Results: Following a single administration of up to 3.75E12 vg/kg of TT-034, biodistribution analyses demonstrated that greater than 90% of the quantified vector is found in the Cynomolgus monkey liver tissues. In situ hybridization analyses demonstrated almost 100% transduction of the hepatocytes when dosed at 1.25E12 vg/kg. Furthermore, qPCR data revealed that shRNA levels persisted for the duration of the 180-day experiment, demonstrating the durability of expression following a single injection. We examined the impact of continuous TT-034 expression on endogenous miRNAs in liver and cardiac tissues because previous reports have suggested that high expression of shRNA may cause global dysregulation of endogenous microRNA (miRNA) levels within cells. Non-human primates were dosed with saline control or either 1.00E11, 1.00E12, 1.00E13 vg/kg of TT-034. Expression profiles of mature miRNAs were generated from liver biopsies taken 15 days after TT-034 administration. Additionally, samples from liver and heart tissues were collected 60 and 180 days after administration. Approximately 260 different miRNAs were reliably detected in each sample type and used for the analyses. The day 15 liver samples showed significant differences in the expression levels of 27 miRNAs. When analyzing all 260 miRNAs, the day 60 and day 180 liver samples showed no significant statistical differences from the control group at any dose. In the heart tissues, no significant statistical differences between the TT-034 treated group and the control group were noted at any timepoint. Conclusions: These data suggest that a single, intravenous infusion of TT-034 results in almost complete transduction of Cynomolgus monkey hepatocytes. Furthermore, TT-034 results in durable expression of three anti-HCV shRNAs. These doses of TT-034 do not cause long-term perturbation of endogenous miRNA levels.

Background: TT-034 is a DNA-directed RNA interference (ddRNAi) agent designed for the treatment of chronic HCV infection and is currently being tested in a phase I/IIa clinical study. TT-034 is comprised of a vector that expresses three independent short hairpin RNAs (shRNAs) simultaneously targeting three well-conserved regions of the HCV genome. The recombinant genome is packaged in a self-complementary aden-associated virus serotype 8 (AAV8) capsid with tropism for hepatic tissues and delivered as a single dose intravenous (IV) infusion. Methods: Chimeric mouse models in which human hepatocytes replace the majority of mouse hepatocytes are used to study human hepatic function. In order to assess validity of dose/transduction relationships in this murine model to those observed in a human clinical study, chimeric mice were infected with identical doses of TT-034 used in a phase I/IIa study using the same clinical lot of TT-034. In the clinical study, eight subjects have received a single IV infusion of TT-034 at 4.00E10, 1.25E11, 4.00E11 or 1.25E12 vg/kg. At 21 days post dosing, a liver biopsy was collected to assess TT-034 DNA levels and shRNA expression by qPCR. PXB chimeric mice (Phoenix Bio) repopulated with a minimum of 80% hepatocytes were dosed identically with the TT-034 drug product (5 groups, n=4). After 21-28 days, the livers of two mice in each group were removed and hand curated to purify human hepatic tissues (>93% purity). The liver tissues of the other two mice were dissociated and human hepatocytes were enriched to >99% using mouse hepatocyte-capturing Dynabeads. TT-034 DNA and shRNA expression were assessed by qPCR. Results: In the human study, modest levels of TT-034 DNA copies were detected in the 3 subjects dosed at 1.25E11 vg/kg, yielding 0.48, 3.65 and 10.44 copies per cell respectively. Variability in transduction was noted at the higher dose of 4.00E11 vg/kg, with the two subjects yielding 17.74 and 1.01 copies per cell. qPCR analysis of the three anti-HCV shRNAs confirms concomitant, dose dependent expression. In the hand curated samples from the chimeric mouse model (>93% purity), a dose of 1.25E11 vg/kg yielded 1.1 or 1.3 DNA copies per cell while the 4.00E11 vg/kg dose resulted in 1.9 and 5.5 copies DNA per cell. Dynabead-enriched human hepatocytes (>99%) resulted in a considerable drop in DNA copy levels: the 1.25E11 vg/kg dose averaged 0.35 copies per cell while the 4.00E11 vg/kg resulted in 0.85 copies per cell. The lowered DNA levels in the chimeric mouse model led to a concomitant reduction in shRNA expressed from the hand curated tissues. Likewise, shRNA expression was reduced even further in enriched human hepatocytes. Conclusion: Our data suggests that residual mouse hepatocytes present in the chimeric livers are transduced with the scAAV8 vector more efficiently than human hepatocytes and results in lower overall transduction as compared to human clinical samples. Thus, while these models can serve as a surrogate to assess the activity of gene therapy constructs against functions of normal human liver, the doses required for optimal activity may be modestly higher than required in the human clinical setting.

Background: BB-HB-331 is a recombinant adeno-associated virus serotype 8 (AAV8) vector designed to treat chronic HBV infection using RNA interference. This self-complementary vector expresses three short hairpin RNAs (shRNAs) that simultaneously target three well-conserved sequences on the viral RNAs that correspond to regions that encode the Core, S-antigen and X-proteins. Using an identical capsid for delivery and an identical set of regulatory elements, BB-HB-331 was engineered as a mimic of TT-034, a ddRNAi therapeutic currently in phase I/IIa clinical studies for the treatment for hepatitis C virus (HCV) infection. The only significant difference between TT-034 and BB-HB-331 is that the anti-HCV shRNAs of TT-034 have been replaced with anti-HBV shRNAs in BB-HB-331. Methods: We first tested the efficacy of BB-HB-331 in an in vitro setting using primary hepatocytes (PHs) isolated from the PXB (Phoenix Bio) mice that are largely comprised of human hepatocytes. PHs were subjected to in vitro infection with HBV genotype C for 12 days prior to the treatment of BB-HB-331. Since AAV does not efficiently transduce PHs in vitro, we utilized adenovirus to deliver the recombinant BB-HB-331 DNA (Ad-BB-HB-331). Increasing doses of Ad-BB-HB-331 were applied to HBV infected primary hepatocytes for 16 days. Hepatocyte cultures treated with adenovirus Ad-TT-034 served as a control. For the in vivo study, PXB mice were first infected with HBV genotype C for 28 days to establish HBV baseline infection. This was followed by a single IV infusion of AAV8-BB-HB-331 at a dose of either 2.00E12 or 2.00E13 vg/kg. Untreated HBV infected PXB mice served as the negative control. Serum samples were collected on a weekly basis to assess HBeAg, HBsAg, and extracellular HBV DNA levels. Results: In vitro treatment of PXB primary hepatocytes with Ad-HB-BB-331 led to significant decreases in HBV parameters. PHs harvested at the conclusion of the experiment demonstrated dose dependent expression of the anti-HBV shRNAs and corresponding inhibition of the HBV viral RNAs. At an MOI of 3, the extracellular levels of HBsAg, HBeAg, and HBcrAg were reduced by 87% or more when compared to the control after 16 days. Although the total cellular DNA did not change, Ad-BB-HB-331 treated cells also demonstrated a 89% reduction of intracellular and extracellular HBV DNA quantities. The levels of cccDNA were correspondingly reduced by 70% in the same time frame. Similarly, effective suppression of HBV infection was observed following in vivo treatment of PXB mice with AAV8-BB-HB-331. Through the first 28 days of an ongoing 56-day experiment, treatment with the high dose resulted in decreases in extracellular levels of HBsAg and HBeAg by 90% and 84%, respectively, when compared to the untreated control. In addition, treatment with the same dose resulted in nearly a log reduction of extracellular HBV DNA at 28 days. Conclusion: Collectively, these data demonstrate suppression of HBV infection by HB-BB-331 in both a primary hepatocyte model and chimeric humanized mouse model.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a "single-shot" therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.

Schwann cells develop from multipotent neural crest cells and form myelin sheaths around axons that allow rapid transmission of action potentials. Neuregulin signaling through the ErbB receptor regulates Schwann cell development; however, the downstream pathways are not fully defined. We find that mice lacking calcineurin B1 in the neural crest have defects in Schwann cell differentiation and myelination. Neuregulin addition to Schwann cell precursors initiates an increase in cytoplasmic Ca2+, which activates calcineurin and the downstream transcription factors NFATc3 and c4. Purification of NFAT protein complexes shows that Sox10 is an NFAT nuclear partner and synergizes with NFATc4 to activate Krox20, which regulates genes necessary for myelination. Our studies demonstrate that calcineurin and NFAT are essential for neuregulin and ErbB signaling, neural crest diversification, and differentiation of Schwann cells.

Congenital heart diseases are traditionally considered to be multifactorial in pathogenesis resulting from environmental and genetic interactions that determine penetrance and expressivity within a genetically predisposed family. Recent evidence suggests that genetic contributions have been significantly underestimated. However, single gene defects occur only in a minority of cases, and multigenetic causes of congenital heart diseases have not been fully demonstrated. Here, we show that interactions between alleles of 3 Pbx genes, which encode homeodomain transcription factors, are sufficient to determine the phenotypic presentation of congenital heart diseases in mice. A major role is served by Pbx1, whose inactivation results in persistent truncus arteriosus. Reduction or absence of Pbx2 or Pbx3 leads to Pbx1 haploinsufficiency and specific malformations that resemble tetralogy of Fallot, overriding aorta with ventricular septal defect, and bicuspid aortic valves. Disruption of Meis1, which encodes a Pbx DNA-binding partner, results in cardiac anomalies that resemble those caused by Pbx mutations. Each of the observed cardiac defects represents developmental abnormalities affecting distinct stages of cardiac outflow tract development and corresponds to specific types of human congenital heart disease. Thus, varied deficiencies in the Pbx gene family produce a full spectrum of cardiac defects involving the outflow tract, providing a framework for determining multigenetic causes of congenital heart anomalies.

The patterning of the cardiovascular system into systemic and pulmonic circulations is a complex morphogenetic process, the failure of which results in clinically important congenital defects. This process involves extensive vascular remodeling and coordinated division of the cardiac outflow tract ( OFT). We demonstrate that the homeodomain transcription factor Pbx1 orchestrates separate transcriptional pathways to control great-artery patterning and cardiac OFT septation in mice. Pbx1-null embryos display anomalous great arteries owing to a failure to establish the initial complement of branchial arch arteries in the caudal pharyngeal region. Pbx1 deficiency also results in the failure of cardiac OFT septation. Pbx1-null embryos lose a transient burst of Pax3 expression in premigratory cardiac neural crest cells (NCCs) that ultimately specifies cardiac NCC function for OFT development, but does not regulate NCC migration to the heart. We show that Pbx1 directly activates Pax3, leading to repression of its target gene Msx2 in NCCs. Compound Msx2/Pbx1-null embryos display significant rescue of cardiac septation, demonstrating that disruption of this Pbx1-Pax3-Msx2 regulatory pathway partially underlies the OFT defects in Pbx1-null mice. Conversely, the great-artery anomalies of compound Msx2/Pbx1-null embryos remain within the same spectrum as those of Pbx1-null embryos. Thus, Pbx1 makes a crucial contribution to distinct regulatory pathways in cardiovascular development.

Patients with Down syndrome have characteristic heart valve lesions resulting from endocardial cushion defects. The Down syndrome critical region 1 ( DSCR1) gene, identified at the conserved trisomic 21 region in those patients, encodes a calcineurin inhibitor that inactivates nuclear factor of activated T cells ( NFATc) activity. Here, we identify a regulatory sequence in the promoter region of human DSCR1 that dictates specific expression of a reporter gene in the endocardium, defined by the temporal and spatial expression of Nfatc1 during heart valve development. Activation of this evolutionally conserved DSCR1 regulatory sequence requires calcineurin and NFATc1 signaling in the endocardium. NFATc1 proteins bind to the regulatory sequence and trigger its enhancer activity. NFATc1 is sufficient to induce the expression of Dscr1 in cells that normally have undetectable or minimal NFATc1 or DSCR1. Pharmacologic inhibition of calcineurin or genetic Nfatc1 null mutation in mice abolishes the endocardial activity of this DSCR1 enhancer. Furthermore, in mice lacking endocardial NFATc1, the endogenous Dscr1 expression is specifically inhibited in the endocardium but not in the myocardium. Thus, our studies indicate that the DSCR1 gene is a direct transcriptional target of NFATc1 proteins within the endocardium during a critical window of heart valve formation.

Several hundred microRNAs (miRNAs) have been cloned from a wide range of organisms across phylogeny. miRNAs are 19–23 nucleotide transcripts with characteristic 3′ hydroxyl and 5′ phosphate termini cleaved from a ∼70-nt hairpin precursor by Dicer Ribonuclease III (Hütvagner et al., 2001; Ketting et al., 2001). Many miRNAs, often with highly conserved sequences, have been mapped in the genomes of C. elegans, Drosophila, rodents and humans (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001; Lagos-Quintana et al., 2002; Mourelatos et al., 2002; Dostie et al., 2003). Based on the length, hairpin structure and conservation the total number of miRNAs in the Drosophila genome has been estimated to be 110 (Lai et al., 2003) and in the human genome to be 255 (Lim et al., 2003). Some miRNAs are organized in the genome as clusters which can be separated by intervals as short as a few nucleotides (Lagos-Quintana et al., 2001; Lau et al., 2001). Despite the high degree of conservation of miRNAs, their functions in general, and in mammals particularly, have not been well defined. The first two miRNAs discovered, lin-4 and let-7, were found in C. elegans where they control developmental timing. These short transcripts form imperfect base pairing with elements within the 3′ UTR of target mRNAs and attenuate their translation (Lee et al., 1993; Wightman et al., 1993; Olsen and Ambros, 1999; Reinhart et al., 2000; Slack et al., 2000).

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.

A rat polyclonal anti-M-line protein antiserum and three mouse monoclonal anti-titin antibodies (E2, F3, and A12) were used to study the spatiotemporal relationship between M-line proteins and titin during myofibril assembly in cultured chicken cardiomyocytes by immunofluorescence microscopy. In day 2 cultures, M-line proteins and titin were detected as punctate staining in most cardiomyocytes, which possessed many nonstriated fibrils. At a late stage (day 3 cultures), M-line proteins were incorporated into dot-like structures along nonstriated fibrils, while titin staining was continuous on these structures. As development progressed, M-line proteins were registered in periodic pattern in the mid-A band. In cardiomyocytes from day 5 cultures, the titin bands were separated by an unstained region, and achieved their adult doublet pattern. Thus, the organization of titin in the sarcomere appears to occur later than that of M-line proteins in the M-line. Our morphological data indicate that the early registration of M-line proteins in primitive myofibrils may guide titin filament alignment via interaction between M-line proteins and titin. In order to investigate the role of M-line proteins in the assembly of titin filaments, anti-M-line protein or anti-titin antibodies were introduced into cultured cardiomyocytes by electroporation to functionally bind the respective proteins, and the profile of myofibril assembly was examined. Cardiomyocytes from day 2-3 cultures with incorporated anti-M-line protein antibodies became shrunk, and exhibited defective myofibrillar assembly, as shown by the failure of titin to assemble into a typical sarcomeric pattern. Incorporation of anti-titin antibody E2, which recognizes the M-line end domain of titin, resulted in the failure of M-line proteins organized into the M-line structure, as shown by random, sporadic staining with anti-M-line protein antibody. These studies confirm the essential role of M-line proteins in the organization of titin filaments in the sarcomere and that the interaction between titin and M-line proteins is crucial to the formation of the M-line structure.