Regulatory Cassette Research Articles

Experience-dependent, sexually dimorphic synaptic connectivity defined by sex-specific cadherin expression.

Early-life experience influences subsequent maturation and function of the adult brain, sometimes even in a sex-specific manner, but underlying molecular mechanisms are poorly understood. We describe here how juvenile experience defines sexually dimorphic synaptic connectivity in the adult Caenorhabditis elegans nervous system. Starvation of juvenile males disrupts serotonin-dependent activation of the CREB transcription factor in a nociceptive sensory neuron, PHB. CREB acts through a cascade of transcription factors to control expression of an atypical cadherin protein, FMI-1/Flamingo/CELSR. During postembryonic development, FMI-1 promotes and maintains synaptic connectivity of PHB to a command interneuron, AVA, in both sexes, but a serotonin-dependent transcriptional regulatory cassette antagonizes FMI-1 expression in males, thereby establishing sexually dimorphic connectivity between PHB and AVA. A critical regulatory node is the CREB-target LIN-29, a Zn finger transcription factor that integrates four layers of information: sexual specificity, past experience, time and cell-type specificity. Our findings provide the mechanistic details of how an early juvenile experience defines sexually dimorphic synaptic connectivity.

Have a Little Heart (or Not): Highly Minimized Skeletal Muscle Regulatory Cassettes with Low or No Activity in the Heart.

Adeno-associated virus-mediated gene therapies for certain muscle disorders require regulatory cassettes that provide high-level, striated muscle-specific activity. However, cardiotoxicity has emerged as a serious concern in clinical trials for Duchenne muscular dystrophy and X-linked myotubular myopathy. While this may be caused by systemic inflammatory effects of the treatment, high transgene expression in the heart may also play a role. Thus, certain muscle disorders may require a modulated level of therapeutic expression in the heart, while others may not require any cardiac expression at all. Additionally, the size of some cargos requires regulatory cassettes to be small enough that large cDNAs and other therapeutic payloads can be accommodated. Thus, we have performed enhancer/promoter optimization to develop highly minimized regulatory cassettes that are active in skeletal muscles, with either low or no detectable activity in cardiac muscle. Our No-heart (NH) cassette is active in most skeletal muscles, but exhibits only very low activity in extensor digitorum longus (EDL), soleus, and diaphragm, and no activity in the heart. By contrast, our Have a Little Heart (HLH) cassette displays high activity in most skeletal muscles, comparable to the ∼800-bp CK8 cassette, with increased activity in EDL, soleus, and diaphragm, and low activity in the heart. Due to their small size, these cassettes can be used in therapeutic strategies with both flexible (e.g., antisense) and stringent (e.g., CRISPR/Cas or bicistronic) size limitations. Thus, our new cassettes may be useful for gene therapies of muscle disorders in which the need for low or almost no expression in cardiac muscle would outweigh the need for high levels of therapeutic product in certain skeletal muscles.

SEM-2/SoxC regulates multiple aspects of C. elegans postembryonic mesoderm development.

Development of multicellular organisms requires well-orchestrated interplay between cell-intrinsic transcription factors and cell-cell signaling. One set of highly conserved transcription factors that plays diverse roles in development is the SoxC group. C. elegans contains a sole SoxC protein, SEM-2. SEM-2 is essential for embryonic development, and for specifying the sex myoblast (SM) fate in the postembryonic mesoderm, the M lineage. We have identified a novel partial loss-of-function sem-2 allele that has a proline to serine change in the C-terminal tail of the highly conserved DNA-binding domain. Detailed analyses of mutant animals harboring this point mutation uncovered new functions of SEM-2 in the M lineage. First, SEM-2 functions antagonistically with LET-381, the sole C. elegans FoxF/C forkhead transcription factor, to regulate dorsoventral patterning of the M lineage. Second, in addition to specifying the SM fate, SEM-2 is essential for the proliferation and diversification of the SM lineage. Finally, SEM-2 appears to directly regulate the expression of hlh-8, which encodes a basic helix-loop-helix Twist transcription factor and plays critical roles in proper patterning of the M lineage. Our data, along with previous studies, suggest an evolutionarily conserved relationship between SoxC and Twist proteins. Furthermore, our work identified new interactions in the gene regulatory network (GRN) underlying C. elegans postembryonic development and adds to the general understanding of the structure-function relationship of SoxC proteins.

Experience-dependent, sexually dimorphic synaptic connectivity defined by sex-specific cadherin expression.

We describe here the molecular mechanisms by which juvenile experience defines patterns of sexually dimorphic synaptic connectivity in the adult nervous system of the nematode C. elegans. We show that starvation of juvenile males disrupts serotonin-dependent activation of the CREB transcription factor in a nociceptive sensory neuron, PHB. CREB acts through a cascade of transcription factors to control expression of an atypical cadherin protein, FMI-1/Flamingo. During postembryonic development, FMI-1/Flamingo has the capacity to promote and maintain synaptic connectivity of the PHB nociceptive sensory to a command interneuron, AVA, in both sexes, but the serotonin transcriptional regulatory cassette antagonizes FMI-1/Flamingo expression in males, thereby establishing sexually dimorphic connectivity between PHB and AVA. A critical regulatory node in this process is the CREB-target LIN-29, a Zn finger transcription factor which integrates four different layers of information - sexual specificity, past feeding status, time and cell-type specificity. Our findings provide the mechanistic details of how an early juvenile experience defines sexually dimorphic synaptic connectivity.

Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium

Human induced pluripotent stem cells (hiPSCs) offer opportunities to study human biology where primary cell types are limited. CRISPR technology allows forward genetic screens using engineered Cas9-expressing cells. Here, we sought to generate a CRISPR activation (CRISPRa) hiPSC line to activate endogenous genes during pluripotency and differentiation. We first targeted catalytically inactive Cas9 fused to VP64, p65 and Rta activators (dCas9-VPR) regulated by the constitutive CAG promoter to the AAVS1 safe harbor site. These CRISPRa hiPSC lines effectively activate target genes in pluripotency, however the dCas9-VPR transgene expression is silenced after differentiation into cardiomyocytes and endothelial cells. To understand this silencing, we systematically tested different safe harbor sites and different promoters. Targeting to safe harbor sites hROSA26 and CLYBL loci also yielded hiPSCs that expressed dCas9-VPR in pluripotency but silenced during differentiation. Muscle-specific regulatory cassettes, derived from cardiac troponin T or muscle creatine kinase promoters, were also silent after differentiation when dCas9-VPR was introduced. In contrast, in cell lines where the dCas9-VPR sequence was replaced with cDNAs encoding fluorescent proteins, expression persisted during differentiation in all loci and with all promoters. Promoter DNA was hypermethylated in CRISPRa-engineered lines, and demethylation with 5-azacytidine enhanced dCas9-VPR gene expression. In summary, the dCas9-VPR cDNA is readily expressed from multiple loci during pluripotency but induces silencing in a locus- and promoter-independent manner during differentiation to mesoderm derivatives. Researchers intending to use this CRISPRa strategy during stem cell differentiation should pilot their system to ensure it remains active in their population of interest.

Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression.

Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5'-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5' end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits.

Mechanism of CCL21/CCL19-CCR7 as a Key Regulatory Signaling Cassette for Non-Small Cell Lung Cancer Patients with Brain Metastasis

To elucidate the molecular mechanism underlying the high metastasis rate of brain for non-small cell lung cancer patients and to establish a comprehensive differential gene profile for non-small cell lung cancer brain metastasis. The differentially expressed genes for non-small cell lung cancer brain metastasis patients were systematically investigated. The enriched biological processes and signaling pathways were explored. The protein-protein interaction network was constructed. Compared with primary non-small cell lung cancer, the brain metastasis patients demonstrated 352 differentially expressed genes with the majority as down-regulated ones (342) and 10 up-regulated ones. Meanwhile, C-C motif chemokine ligand 21, C-C motif chemokine ligand 19, deleted in malignant brain tumors 1 gene, tryptase alpha/beta 1, lactotransferrin, complement component 4 binding protein alpha, C-C motif chemokine ligand 14, C-X-C motif chemokine ligand 9, cluster of differentiation 79A as well as membrane spanning 4-domains A1 were significant and differentially expressed (all down-regulated). The genes of cluster including C-C motif chemokine ligand 21, C-C motif chemokine ligand 19 played a pivotal role in non-small cell lung cancer brain metastasis. The cytokines C-C motif chemokine ligand 19 and C-C motif chemokine ligand 21 as well as their closely associated receptors, C-C chemokine receptor 7 could be detected as high expression levels in non-small cell lung cancer cell line (A549). Inhibition of C-C motif chemokine ligand 19 and C-C motif chemokine ligand 21 as well as C-C chemokine receptor 7 using small interfering ribonucleic acid obviously repressed the cell invasion and metastasis but not for cell proliferation and apoptosis. Meanwhile, the procedure was independent of micro ribonucleic acid lethal-7a regulation. This study hypothesized an innovative signaling axis (C-C motif chemokine ligand 21/C-C motif chemokine ligand 19-C-C chemokine receptor 7) for non-small cell lung cancer brain metastasis and provided a beneficial strategy for better understanding the lung metastases in clinic.

Antagonistic Control of Genetic Circuit Performance for Rapid Analysis of Targeted Enzyme Activity in Living Cells.

Genetic circuits have been developed for quantitative measurement of enzyme activity, metabolic engineering of strain development, and dynamic regulation of microbial cells. A genetic circuit consists of several bio-elements, including enzymes and regulatory cassettes, that can generate the desired output signal, which is then used as a precise criterion for enzyme screening and engineering. Antagonists and inhibitors are small molecules with inhibitory effects on regulators and enzymes, respectively. In this study, an antagonist and an inhibitor were applied to a genetic circuit for a dynamic detection range. We developed a genetic circuit relying on regulators and enzymes, allowing for straightforward control of its output signal without additional genetic modification. We used para-nitrophenol and alanine as an antagonist of DmpR and inhibitor of tyrosine phenol-lyase, respectively. We show that the antagonist resets the detection range of the genetic circuit similarly to a resistor in an electrical logic circuit. These biological resistors in genetic circuits can be used as a rapid and precise controller of variable outputs with minimal circuit configuration.

Targeted epigenetic repression by CRISPR/dSaCas9 suppresses pathogenic DUX4-fl expression in FSHD

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress DUX4 in FSHD myocytes. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. Thus, we re-engineered our platform to allow in vivo delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from Staphylococcus aureus fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the DUX4 promoter/exon 1 increased chromatin repression at the locus, specifically suppressing DUX4 and its target genes in FSHD myocytes and in a mouse model of the disease. Importantly, minimizing the regulatory cassette and using the smaller Cas9 ortholog allowed our therapeutic cassettes to be effectively packaged into adeno-associated virus (AAV) vectors for in vivo delivery. By engineering a muscle-specific epigenetic CRISPR platform compatible with AAV vectors for gene therapy, we have laid the groundwork for clinical use of dCas9-based chromatin effectors in skeletal muscle disorders.

Split AAV-Mediated Gene Therapy Restores Ureagenesis in a Murine Model of Carbamoyl Phosphate Synthetase 1 Deficiency

The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

Permutational analysis of Saccharomyces cerevisiae regulatory elements.

Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements—enhancers (upstream activating sequences), core promoters, 5′ untranslated regions (5′ UTRs) and transcription terminators/3′ untranslated regions (3′ UTRs). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remain under explored. We describe a simple and rapid approach to build a combinatorial library of regulatory elements and have used this library to study 26 different enhancers, core promoters, 5′ UTRs and transcription terminators/3′ UTRs to estimate the contribution of individual regulatory parts in gene expression. Our combinatorial analysis shows that while enhancers initiate gene expression, core promoters modulate the levels of enhancer-mediated expression and can positively or negatively affect expression from even the strongest enhancers. Principal component analysis (PCA) indicates that enhancer and promoter function can be explained by a single principal component while UTR function involves multiple functional components. The PCA also highlights outliers and suggest differences in mechanisms of regulation by individual elements. Our data also identify numerous regulatory cassettes composed of different individual regulatory elements that exhibit equivalent gene expression levels. These data thus provide a catalog of elements that could in future be used in the design of synthetic regulatory circuits.

Development of Novel Micro-dystrophins with Enhanced Functionality.

Gene therapies using adeno-associated viral (AAV) vectors have advanced into clinical trials for several diseases, including Duchenne muscular dystrophy (DMD). A limitation of AAV is the carrying capacity (∼5 kb) available for genes and regulatory cassettes (RCs). These size constraints are problematic for the 2.2-Mb dystrophin gene. We previously designed a variety of miniaturized micro-dystrophins (μDys) that displayed significant, albeit incomplete, function in striated muscles. To develop μDys proteins with improved performance, we explored structural modifications of the dystrophin central rod domain. Eight μDys variants were studied that carried unique combinations of between four and six of the 24 spectrin-like repeats present in the full-length protein, as well as various hinge domains. Expression of μDys was regulated by a strong but compact muscle-restricted RC (CK8e) or by the ubiquitously active cytomegalovirus (CMV) RC. Vectors were evaluated by intramuscular injection and systemic delivery to dystrophic mdx4cv mice, followed by analysis of skeletal muscle pathophysiology. Two μDys designs were identified that led to increased force generation compared with previous μDys while also localizing neuronal nitric oxide synthase to the sarcolemma. An AAV vector expressing the smaller of these (μDys5) from the CK8e RC is currently being evaluated in a DMD clinical trial.

TH.I.9 - Development of microdystrophins for gene therapy of DMD

Gene therapy represents a promising approach to treat DMD as it has the potential to circumvent the primary cause of the disorder, a mutant gene. Gene replacement therapies have focused on methods to deliver expression cassettes encoding the dystrophin protein. Challenges to this approach have included the enormous size of the dystrophin gene (2.2 MB), its muscle mRNA (14 kb) and difficulties in safely delivering vectors to muscles bodywide. We and others have shown that vectors derived from specific serotypes of adeno-associated virus (AAV) can systemically deliver genes to muscles after intravascular infusion. However, AAV vectors have a limited carrying capacity of approximately 5 kb, necessitating the design of miniaturized cDNAs and gene regulatory elements that enable expression of “microdystrophins”. Early generation microdystrophins displayed functional limitations due to their small size (<4 kb) and the absence of some protein interaction domains. To develop improved vectors we tested a variety of novel microdystrophins lacking the C-terminal domain but carrying 4–6 spectrin-like repeats, including the nitric oxide synthase localization domain. We also tested a set of powerful gene regulatory elements based on the muscle creatine kinase (MCK) enhancer plus promoter. These novel microdystrophins were evaluated by AAV-mediated delivery to dystrophic mdx4cv mice followed by extensive analysis of muscle pathophysiology. We identified a 5 repeat vector that when driven by the CK8 regulatory cassette leads to stable expression and profound reductions in muscle pathophysiology. This AAV/CK8-µDys5 vector performs better than previously tested microdystrophin cassettes and represents a promising clinical candidate. We have also been testing AAV vectors to deliver components of the CRISPR/Cas9 system as a method to potentially repair, rather than replace, the mutant dystrophin gene. While this gene editing approach shows promise, its efficiency remains low and as currently applied is less effective than direct delivery of microd

Cerberus–Nodal–Lefty–Pitx signaling cascade controls left–right asymmetry in amphioxus

Many bilaterally symmetrical animals develop genetically programmed left-right asymmetries. In vertebrates, this process is under the control of Nodal signaling, which is restricted to the left side by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest diverging chordate lineage, has profound left-right asymmetry as a larva. We show that Cerberus, Nodal, Lefty, and their target transcription factor Pitx are sequentially activated in amphioxus embryos. We then address their function by transcription activator-like effector nucleases (TALEN)-based knockout and heat-shock promoter (HSP)-driven overexpression. Knockout of Cerberus leads to ectopic right-sided expression of Nodal, Lefty, and Pitx, whereas overexpression of Cerberus represses their left-sided expression. Overexpression of Nodal in turn represses Cerberus and activates Lefty and Pitx ectopically on the right side. We also show Lefty represses Nodal, whereas Pitx activates Nodal These data combine in a model in which Cerberus determines whether the left-sided gene expression cassette is activated or repressed. These regulatory steps are essential for normal left-right asymmetry to develop, as when they are disrupted embryos may instead form two phenotypic left sides or two phenotypic right sides. Our study shows the regulatory cassette controlling left-right asymmetry was in place in the ancestor of amphioxus and vertebrates. This includes the Nodal inhibitors Cerberus and Lefty, both of which operate in feedback loops with Nodal and combine to establish asymmetric Pitx expression. Cerberus and Lefty are missing from most invertebrate lineages, marking this mechanism as an innovation in the lineage leading to modern chordates.

Crinoid phylogeny: new interpretation of the main Permo-Triassic divergence, comparisons with echinoids and brachiopods, and EvoDevo interpretations of major morphological variations

An alignment of nuclear and mitochondrial gene sequences (1529 data columns from three genes: nuclear‐encoded 28S ribosomal DNA, mitochondrial 16S rDNA, and cytochrome oxidase subunit 1) from 120 specimens representing a wide selection of extant crinoids is used in Bayesian relaxed‐clock analyses to construct the maximum clade credibility tree and to locate the root. This tree, and its root position, largely agree with those previously published on the basis of mid‐point and outgroup rooting, and, using a likelihood ratio test, we find that our independently aligned data do not depart significantly from a strict molecular clock. Our tree, similar to those published previously, shows a basal split into two main clades but, unlike previous reports, we identify a phenotypic basis for the split and name the clades accordingly, ‘Stalked and quasi‐stalked’ (SqS) and ‘Stalked but facultatively unstalked’ (SfU), with the latter corresponding to the ‘comatulids’. There is some correspondence between clades and morphologically‐defined Linnean categories in the SqS clade (e.g. the family Hyocrinidae corresponds to a clade). However, there is almost no correspondence between them in the SfU clade, within which we abandon Linnean names and instead use the letters A to E, suggesting, as previously noted, that considerable morphology‐based taxonomic oversplitting has occurred, such that critical revision that takes account of molecular results is overdue. The distinctive, living crinoid Caledonicrinus is placed as sister to all other members of the SqS clade (i.e. its detailed relationships remain obscure). The results of the present study do not exclude the Articulata hypothesis under which all extant crinoids belong to a single radiation but, using the first appearance of fossil cyrtocrinid ossicles as a calibration datum, the root node of our time‐tree dates to before 258 Mya [i.e. in the Permian (Kungurian), rather than in the Triassic]. Simple comparisons are made between crinoids, echinoids, and selected articulate brachiopods using p‐distance networks and maximum likelihood pairwise patristic distances as proxies for molecular evolution, and the numbers of extant families as (the only available but potentially unreliable) proxy for morphological evolution. These analyses suggest that, over approximately the same period (Triassic to present), echinoids show approximately five‐fold lower rates of molecular evolution than crinoids or brachiopods, whereas both crinoids and echinoids show higher rates of morphological (taxonomic, family‐level) evolution than brachiopods. These differences are discussed briefly in terms of developmental constraint and character plasticity. The evolutionary history of extant crinoids is given a speculative interpretation in terms of evolutionary developmental gene expression (EvoDevo) on the basis of four hypothetical gene regulatory networks or cassettes (Stalk, Cirrus, Abscission, and Multiply‐or‐Divide) whose presence/absence and activity/inactivity could combine to account for the phenotypes of the main crinoid clades. The conventional division of living crinoids into ‘stalked’ and ‘unstalked’ categories is strongly deprecated.

380. Therapeutic Capacity of AAV-Delivered Muscle-Specific-Expressed Micro-Utrophin (ΔR4-R21/ΔCT) in mdX4cv Skeletal Muscles

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by dystrophin mutations. Utrophin is a dystrophin paralogue that can prevent necrosis in the mdx mouse DMD model. We designed an expression plasmid containing a miniaturized M-creatine kinase regulatory cassette (CK8e) and a rationally designed micro-utrophinΔR4-21/ΔCT (µUtrn) to accommodate the limited capacity of recombinant adeno-associated virus (rAAV). Both intramuscular (IM) and intravascular (IV) delivery of rAAV6-CK8-µUtrn at 2 weeks of age profoundly mitigated the dystrophic phenotype in skeletal muscles when examined at 3-6 months of age. These improvements were observed in many but not all skeletal muscle histological and functional parameters. At a pathological level µUtrn production was associated with significant reductions in centrally nucleated muscle fibers and with a greatly reduced prevalence of muscle regions containing inflammatory cells. µUtrn production also restored dystrophin-glycoprotein-complex (DGC) components β-Dystroglycan, Δ-Sarcoglycan, α-Dystrobrevin-2, and α1-Syntrophin to the sarcolemma. As expected, nNOS was not restored, presumably because CK8-µUtrn lacks direct nNOS binding sites. Analysis of µUtrn immunostaining intensities 3-months post-treatment suggested lower µUtrn levels in many of the 1, 2a and 2d/x fibers than in the 2b fibers. These differences would be consistent with the relative transcriptional activities of the CK8e regulatory cassette in different adult mouse fiber types: 2b > 2d/x > 2a > 1. Given the important structural role of costameres in connecting the sarcolemma to sarcomeres, we examined utrophin and µUtrn localization patterns and found endogenous utrophin localized in a costameric pattern adjacent to Z-line end points while abutting sarcolemma junctions. However, while µUtrn was also associated with a costameric lattice, its striations were unexpectedly only ~0.8 µm apart compared to ~2.2 µm for endogenous utrophin in untreated mdx4cv muscles. How the presence of two different costameric lattice patterns may affect skeletal muscle function is not known. Since mdx4cv mice are known to exhibit a fragmented synaptic phenotype, we also examined neuromuscular junctions (NMJs) in treated and untreated mice and found that µUtrn prevented synapse fragmentation and also restored the depth of synaptic folds. Interestingly, the NMJs in µUtrn-treated muscles exhibited a more highly branched architecture of the synaptic folds, and this may have compensated for the reduced number of fold openings observed in untreated mice. µUtrn treatment also provided partial restoration of the reduced Achilles myotendinous junction folds seen in mdx4cv mice, and did not lead to either the myotendinous strain injury or the ringed fiber formation associated with micro-dystrophinΔR4-R23/ΔCT mediated therapy. Importantly, physiological studies indicated that IM treated tibialis anterior muscles maintained peak force production and exhibited partially improved specific force production. Overall, rAAV6-CK8-µUtrn treatment of mdx4cv mice provided major improvements in many muscle parameters. However, it did not overcome all dystrophic deficits, and it modified the normal costameric lattice structure. Further improvements might be achieved by expressing µUtrn via regulatory cassettes with higher relative activity in 1, 2a and 2d/x fibers, and by further modifications of µUtrn (ΔR4-R21/ΔCT)'s design.

760. Optimized AAV-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice and Cynomolgus Macaques

In an effort to optimize expression of human coagulation VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed combining liver-specific promoter and enhancer elements with a codon-optimized human B-domain-deleted hFVIII transgene. Due to the large size of the FVIII coding sequence, there is a strong requirement for gene expression control elements to be as short as possible while retaining hepatocyte-restricted transcription. Several strong liver-specific promoters were shortened and combined, with combinations of up to three liver-specific enhancer sequences, to generate 42 enhancer/promoter combinations.These 42 liver regulatory gene cassettes were packaged into the AAVrh10 capsid and were tested in FVIII KO mice. Following intravenous (IV) administration of 1010 genome copies (GC), mice were bled every two weeks to follow hFVIII activity and antibody generation to the transgene. At week 2 post-injection, mice showed a range in hFVIII activity from 0.12-2.12 IU/ml. FVIII KO mice developed antibodies to hFVIII at week 4, and by week 8, mice in most of the 42 vector groups had detectable anti-hFVIII IgG levels.Based on the FVIII KO mouse studies and a small pilot rhesus macaque study, two of the original 42 enhancer/promoter combinations were selected for further evaluation in cynomolgus macaques, using two different Clade E capsids for expression. Each of the four vector combinations were administered IV at a dose of 1.2×1013 GC/kg into five macaques per group. With one capsid plus enhancer/promoter combination, peak expression of 37% of normal FVIII levels was seen at week 2 post-vector administration, which then plateaued at 20% of normal. While antibodies to the hFVIII were detected in the majority of macaques by week 8, antibodies remained undetectable in two animals at week 30 post-vector administration.

532. Stable Transcriptional Repression, Gene Excision, and Parastism of HIV by Conditionally Replicating Lentiviral Vectors

Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV, as they have the potential for sustained viral inhibition via reduced treatment interventions. One innovative approach developed here involves using conditionally replicating vectors (CR-Vectors), as these vectors utilize HIV-expressed proteins to replicate. These vector payloads can spread along with HIV into the budding viral particles, and co-infect target cells, essentially disseminating to HIV infected cells. We have generated and characterized CR-Vectors carrying therapeutic payloads consisting of various non-coding RNA regulatory expression cassettes, which modulate HIV both transcriptionally and post-transcriptionally, as well as CRISPR directed excision machinery. Notably, we have followed both virus and vector expression in T-cells and in vivo in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-Vectors functionally suppress HIV expression in a long-term stable and potent manner in both the presence and absence of MPA; and that transcriptional targeting is more potent at modulating stable suppression of HIV than post-transcriptional targeting or CRISPR directed excision of HIV. This suppression may be physiologically relevant, as it appears to drive HIV to a sustained non-pathogenic set point. Our findings suggest that CR-Vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term stable suppression of HIV leading ultimately to a functional cure.

AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility.

Impaired systolic function, resulting from acute injury or congenital defects, leads to cardiac complications and heart failure. Current therapies slow disease progression but do not rescue cardiac function. We previously reported that elevating the cellular 2 deoxy-ATP (dATP) pool in transgenic mice via increased expression of ribonucleotide reductase (RNR), the enzyme that catalyzes deoxy-nucleotide production, increases myosin-actin interaction and enhances cardiac muscle contractility. For the current studies, we initially injected wild-type mice retro-orbitally with a mixture of adeno-associated virus serotype-6 (rAAV6) containing a miniaturized cardiac-specific regulatory cassette (cTnT(455)) composed of enhancer and promotor portions of the human cardiac troponin T gene (TNNT2) ligated to rat cDNAs encoding either the Rrm1 or Rrm2 subunit. Subsequent studies optimized the system by creating a tandem human RRM1-RRM2 cDNA with a P2A self-cleaving peptide site between the subunits. Both rat and human Rrm1/Rrm2 cDNAs resulted in RNR enzyme overexpression exclusively in the heart and led to a significant elevation of left ventricular (LV) function in normal mice and infarcted rats, measured by echocardiography or isolated heart perfusions, without adverse cardiac remodeling. Our study suggests that increasing RNR levels via rAAV-mediated cardiac-specific expression provide a novel gene therapy approach to potentially enhance cardiac systolic function in animal models and patients with heart failure.