Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.

Antibody inhibits defined stages in the pathogenesis of reovirus serotype 3 infection of the central nervous system.

The mammalian reoviruses provide a model for studying specific aspects of the immunopathogenesis of viral infection. We have used two serotype 3 reoviruses to define stages in the pathogenesis of central nervous system (CNS) infection at which a mAb specific for the reoviral cell attachment protein sigma 1 (sigma 1mAbG5) acts to protect mice against lethal disease. sigma 1mAbG5 administered either before or at the time of footpad inoculation with reovirus T3D prevented entry of T3D into the CNS. sigma 1mAbG5 also inhibited the spread of reovirus T3C9 from the gastrointestinal tract to the CNS after peroral inoculation with T3C9. These effects occurred in the absence of a significant effect of sigma 1mAbG5 on primary replication in skeletal muscle (T3D) or the gastrointestinal tract (T3C9). sigma 1mAbG5 administered after T3D had reached the spinal cord inhibited subsequent spread of infectious virus from spinal cord to brain. Even after direct intracerebral inoculation of T3D, sigma 1mAbG5 prevented both growth in the brain and spread of infectious virus from brain to eye, spinal cord, and muscle. Treatment with sigma 1mAbG5 after intracerebral inoculation with T3D prevented neuronal necrosis and resulted in a delayed and topographically restricted inflammatory response. We detected no antibody-resistant T3D variants in vivo after treatment with sigma 1mAbG5. We conclude that systemic IgG does not play a significant role at the primary site of infection with reoviruses, while it clearly acts to prevent infection of the CNS and extension of infection with the CNS. Further study will be directed to defining what components of the immune system do act at primary sites of infection, and to defining the mechanisms by which antibody acts at defined stages in pathogenesis.

Transient expression of a winged-helix protein, MNF-beta, during myogenesis.

A novel winged-helix transcription factor, MNF-beta, is expressed coincidentally with cell cycle withdrawal and differentiation of skeletal myogenic cells. MNF-beta is closely related to the myocyte nuclear factor (MNF) protein previously described (now termed MNF-alpha), but expression of the two isoforms is differentially regulated, and they exhibit distinctive functional properties with respect to DNA binding in vitro and transcriptional regulatory activity in transient-transfection assays. A DNA sequence motif binding MNF-beta with high affinity was selected from a library of random oligonucleotides and was found to be similar to but distinct from the cognate binding site for HNF-3beta, a more distantly related winged-helix protein. The temporal pattern of MNF-beta expression and the presence of MNF binding motifs within conserved promoter elements of several genes that modulate cell cycle progression support a working hypothesis that MNF proteins may modulate proliferation of myogenic precursor cells during development and muscle regeneration.

Synergistic interactions between heterologous upstream activation elements and specific TATA sequences in a muscle-specific promoter.

Previous investigations have defined three upstream activation elements--CCAC, A/T, and TATA sequences--necessary for muscle-specific transcription of the myoglobin gene. In the present study, we demonstrate that these three sequences elements, prepared as synthetic oligonucleotide cassettes, function synergistically to constitute a cell-type-specific transcription unit. Previously, cognate binding factors that recognize the CCAC and TATA elements were identified. In this study we determine that the A/T element binds two nuclear factors, including myocyte enhancer factor-2 (MEF-2) and an apparently unknown factor we provisionally termed ATF35 (A/T-binding factor, 35 kDa). Mutations that alter in vitro binding of either MEF-2 or ATF35 to this site diminish promoter function in vivo. Functional synergism between factors binding the CCAC and A/T elements is sensitive to subtle mutations in the TATA sequence, recapitulating the unusual preference for specific TATA variants exhibited by the native myoglobin promoter. These results provide new insights into mechanisms that underlie the distinctive pattern of myoglobin gene regulation in mammalian muscle development and lay a foundation for further studies to elucidate general principles of transcriptional control of complex mammalian promoters through combinatorial actions of heterologous transcriptional factors.

Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes.

A sequence motif (CCAC box) within an upstream enhancer region of the human myoglobin gene is essential for transcriptional activity in both cardiac and skeletal muscle. A cDNA clone, myocyte nuclear factor (MNF), was isolated from a murine expression library on the basis of sequence-specific binding to the myoglobin CCAC box motif and was found to encode a novel member of the winged-helix or HNF-3/fork head family of transcription factors. Probes based on this sequence identify two mRNA species that are upregulated during myocyte differentiation, and antibodies raised against recombinant MNF identify proteins of approximately 90, 68, and 65 kDa whose expression is regulated following differentiation of myogenic cells in culture. In addition, the 90-kDa form of MNF is phosphorylated and is upregulated in intact muscles subjected to chronic motor nerve stimulation, a potent stimulus to myoglobin gene regulation. Amino acid residues 280 to 389 of MNF demonstrate 35 to 89% sequence identity to the winged-helix domain from other known members of this family, but MNF is otherwise divergent. A proline-rich amino-terminal region (residues 1 to 206) of MNF functions as a transcriptional activation domain. These studies provide the first evidence that members of the winged-helix family of transcription factors have a role in myogenic differentiation and in remodeling processes of adult muscles that occur in response to physiological stimuli.

A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter.

To define transcriptional control elements responsible for muscle-specific expression of the human myoglobin gene, we performed mutational analysis of upstream sequences (nucleotide positions -373 to +7 relative to the transcriptional start site) linked to a firefly luciferase gene. Transient expression assays in avian and mammalian cells indicated that a CCCACCCCC (CCAC box) sequence (-223 to -204) is necessary for muscle-specific transcription directed either by the native myoglobin promoter or by a heterologous minimal promoter linked to the myoglobin upstream enhancer region. A putative MEF2-like site (-160 to -169) was likewise necessary for full transcriptional activity in myotubes. Mutations within either of two CANNTG (E-box) motifs (-176 to -148) had only minimal effects on promoter function. We identified and partially purified from nuclear extracts a 40-kDa protein (CBF40) that binds specifically to oligonucleotides containing the CCAC box sequence. A mutation of the CCAC box that disrupted promoter function in vivo also impaired binding of CBF40 in vitro. These data suggest that cooperative interactions between CBF40 and other factors including MEF-2 are required for expression of the human myoglobin gene in skeletal muscle.

ZBP-89, a Krüppel-like zinc finger protein, inhibits epidermal growth factor induction of the gastrin promoter.

We have shown previously that a GC-rich element (GGGGCGGGGTGGGGGG) conferring epidermal growth factor (EGF) responsiveness to the human gastrin promoter binds Sp1 and additional undefined complexes. A rat GH4 cell line expression library was screened by using a multimer of the gastrin EGF response element, and three overlapping cDNA clones were identified. The full-length rat cDNA encoded an 89-kDa zinc finger protein (ZBP-89) that was 89% identical to a 49-kDa human factor, ht(beta), that binds a GTGGG/CACCC element in T-cell receptor promoters. The conservation of amino acids between the zinc fingers indicates that ZBP-89 is a member of the C2H2 zinc finger family subclass typified by the Drosophila Krüppel protein. ZBP-89 is ubiquitously expressed in normal adult tissues. It binds specifically to the gastrin EGF response element and inhibits EGF induction of the gastrin promoter. Collectively, these results demonstrate that ZBP-89 functions as a repressor of basal and inducible expression of the gastrin gene.

Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles.

Calcineurin, a calcium/calmodulin-regulated protein phosphatase, modulates gene expression in cardiac and skeletal muscles during development and in remodeling responses such as cardiac hypertrophy that are evoked by environmental stresses or disease. Recently, we identified two genes encoding proteins (MCIP1 and MCIP2) that are enriched in striated muscles and that interact with calcineurin to inhibit its enzymatic activity. In the present study, we show that expression of MCIP1 is regulated by calcineurin activity in hearts of mice with cardiac hypertrophy, as well as in cultured skeletal myotubes. In contrast, expression of MCIP2 in the heart is not altered by activated calcineurin but responds to thyroid hormone, which has no effect on MCIP1. A approximately 900-bp intragenic segment located between exons 3 and 4 of the MCIP1 gene functions as an alternative promoter that responds to calcineurin. This region includes a dense cluster of 15 consensus binding sites for NF-AT transcription factors. Because MCIP proteins can inhibit calcineurin, these results suggest that MCIP1 participates in a negative feedback circuit to diminish potentially deleterious effects of unrestrained calcineurin activity in cardiac and skeletal myocytes. Inhibitory effects of MCIP2 on calcineurin activity may be pertinent to gene switching events driven by thyroid hormone in striated muscles. The full text of this article is available at http://www. circresaha.org.

Cardiac-specific LIM protein FHL2 modifies the hypertrophic response to beta-adrenergic stimulation.

BACKGROUND: A deficiency of muscle LIM protein results in dilated cardiomyopathy, but the function of other LIM proteins in the heart has not been assessed previously. We have characterized the expression and function of FHL2, a heart-specific member of the LIM domain gene family. METHODS AND RESULTS: Expression of FHL2 mRNA and protein was examined by Northern blot, in situ hybridization, and Western blot analyses of fetal and adult mice. FHL2 transcripts are present at embryonic day (E) 7.5 within the cardiac crescent in a pattern that resembles that of Nkx2.5 mRNA. During later stages of cardiac development and in adult animals, FHL2 expression is localized to the myocardium and absent from endocardium, cardiac cushion, outflow tract, or coronary vasculature. The gene encoding FHL2 was disrupted by homologous recombination, and knockout mice devoid of FHL2 were found to undergo normal cardiovascular development. In the absence of FHL2, however, cardiac hypertrophy resulting from chronic infusion of isoproterenol is exaggerated (59% versus 20% increase in heart weight/body weight in FHL null versus wild-type mice; P<0.01). CONCLUSIONS: FHL2 is an early marker of cardiogenic cells and a cardiac-specific LIM protein in the adult. FHL2 is not required for normal cardiac development but modifies the hypertrophic response to beta-adrenergic stimulation.

Inhibitor-resistant tissue-type plasminogen activator: an improved thrombolytic agent in vitro.

Platelet-rich clots are inefficiently lysed by current fibrinolytic agents. Platelets contain a great deal of plasminogen activator inhibitor 1 (PAI-1), the principal endogenous inhibitor of tissue-type plasminogen activator (t-PA). We have tested whether PAI-1 resistant t-PAs would be more effective thrombolytic agents in an in vitro model of platelet-rich clots. Clots were formed with recalcified human plasma without or with the addition of platelets. The lysis of these clots was followed by the release of incorporated 125I-fibrinogen. Mutant and wild-type t-PA were almost equally effective against clots lacking platelets but the mutant was twice as effective at lysing platelet-rich clots. A mechanism for this effect is suggested by the demonstration that a complex between wild-type t-PA and extruded platelet contents resembles that between purified t-PA and PAI-1 and that the PAI-1 resistant t-PA does not interefer with formation of this adduct. Because of its enhanced ability to lyse platelet-rich clots in vitro, further in vivo work may find that PAI-1 resistant t-PA is a more efficacious therapeutic agent than wild-type t-PA in situations where platelets contribute to the failure of thrombolysis.

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity.

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.

Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation.

Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation.

Persistent expression of MNF identifies myogenic stem cells in postnatal muscles.

Skeletal muscles contain an undifferentiated myogenic stem cell pool (satellite cells) that can be mobilized to regenerate myofibers in response to injury. We have determined that the winged helix transcription factor MNF is expressed selectively in quiescent satellite cells, which do not express known regulators of the myogenic program. Following muscle injury, MNF is present transiently in proliferating satellite cells and in centralized nuclei of regenerating myofibers, but expression declines as these fibers mature, until only the residual stem cell pool continues to express detectable levels of MNF. MNF also is expressed selectively but transiently at embryonic stages of myogenesis in the developing myotome, limb bud precursors, and heart tube, but by late fetal stages of development, MNF is down-regulated within differentiated cardiac and skeletal myocytes, and persistently high expression is observed only in satellite cells. These data identify MNF as a marker of quiescent satellite cells and suggest that downstream genes controlled by MNF serve to modulate proliferative growth or differentiation in this unique cell population.

Identification of attenuating mutations on the reovirus type 3 S1 double-stranded RNA segment with a rapid sequencing technique.

Reovirus type 3 variants with mutations in the major neutralization domain of the sigma 1 protein have attenuated neurovirulence and restricted neurotropism. We devised a variation of the rapid RNA sequencing technique to facilitate the analysis of double-stranded RNA. We sequenced the S1 double-stranded RNA segment, which encodes the sigma 1 protein, of five attenuated reovirus type 3 variants. Four of the variants have changes in codon 419, and a fifth variant has a change at codon 340, all of which resulted in amino acid substitutions in the sigma 1 protein. We identified two sites on the reovirus type 3 sigma 1 protein that play a critical role in neurovirulence.

Persistent reovirus infections of L cells select mutations in viral attachment protein sigma1 that alter oligomer stability.

During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein sigma1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1 mutations in each S1 gene resulted in changes in the deduced amino acid sequence of sigma1 protein. Using electrophoresis conditions that favor migration of sigma1 oligomers, we found that sigma1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas sigma1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in sigma1 protein affecting stability of sigma1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 sigma1 protein, we used T1L X PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein sigma3. This finding suggests that in some cases, mutations in sigma3 protein in the absence of sigma1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L X PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the sigma1-encoding S1 gene. Given that the sigma1 and sigma3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.

Identification of a new polypeptide coded by reovirus gene S1.

The reovirus S1 gene has recently been shown potentially to encode two polypeptides (from two overlapping reading frames) having predicted molecular weights of 49,071 and 16,143 (Nagata et al., Nucleic Acids Res. 12:8699-8710, 1984; Bassel-Duby et al., Nature [London], in press). The larger polypeptide is reovirus protein sigma 1, but synthesis of the smaller polypeptide has not been described to date. A truncated clone of the S1 gene in which the first ATG is deleted was expressed in an in vitro protein synthesis system to yield a approximately 13-kilodalton polypeptide, as determined from migration on sodium dodecyl sulfate-polyacrylamide gels. A polypeptide with a similar migration pattern on sodium dodecyl sulfate-polyacrylamide gels was present in reovirus-infected cells and absent from mock-infected cells. Comparative tryptic peptide analysis of the 13-kilodalton polypeptides produced in vivo and in vitro showed them to be identical. Thus, the s1 mRNA of reovirus type 3 is apparently bicistronic, and we suggest that the approximately 13-kilodalton polypeptide be called sigma s (standing for sigma small).

Expression of reovirus type 3 (Dearing) sigma 1 and sigma s polypeptides in Escherichia coli.

The reovirus S1 gene codes for two polypeptides: sigma 1 and sigma s. In order to characterize the structure and function of the sigma 1 polypeptide, we have expressed the sigma 1 protein in Escherichia coli. The S1 gene from mammalian reovirus type 3 (Dearing strain) and the variant K strain were subcloned into an expression vector containing the tac (trp-lac) promoter designed to express foreign gene products in E. coli efficiently. The hybrid plasmids, upon induction with isopropyl-beta-D-thiogalactopyranoside, expressed two polypeptides that were detected by [35S]methionine labelling. One of the induced proteins had a relative molecular mass (Mr) of approx. 46,000 and corresponded to sigma 1, as shown by immunoprecipitation with goat anti-reovirus antibody and a monoclonal antibody against sigma 1. The second induced protein had a Mr of approx. 12,000 and was very similar to sigma s as judged by comparative tryptic peptide map analysis. Protein sigma 1 produced in E. coli was shown to be functional as judged from its ability to bind to mouse L fibroblasts.

Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing).

The mammalian reoviruses have provided a valuable model for studying the pathogenesis of viral infections of the central nervous system (CNS). We have used this model to study the effect of antibody on disease produced by the neurally spreading reovirus type 3 (Dearing) (T3). Polyclonal and monoclonal antibodies protect mice from fatal infection with T3 after either footpad or intracerebral virus challenge. Protection occurs with monoclonal antibodies directed against the viral cell attachment protein sigma 1, and with polyclonal antisera without T3 sigma 1 binding activity. In vivo protection occurs with both neutralizing and nonneutralizing monoclonal antibodies. Antibody-mediated protection does not require serum complement and, under specific circumstances, can occur via Fc-independent mechanisms. Antibody can protect mice when transferred up to 5 days after intracerebral challenge and up to 7 days after footpad challenge, times when high titers of virus are present in the CNS. Thus, antibody mediated protection against this neurally spreading virus does not require neutralizing antibody or serum complement and occurs even in the face of established CNS infection.

Regulatory elements governing transcription in specialized myofiber subtypes.

Skeletal myofibers of vertebrates acquire specialized metabolic and physiological properties as a consequence of developmental cues in the embryo and different patterns of contractile activity in the adult. The myoglobin gene is regulated stringently in muscle fibers, such that high myoglobin expression is observed in mitochondria-rich, oxidative myofibers (Types I and IIa) compared with glycolytic fibers (Type IIb). Using germ-line transgenesis and somatic cell gene transfer methods, we defined discrete regions of the murine and human genes encoding myoglobin that are sufficient to confer muscle- and fiber type-specific expression to reporter genes. Mutational analysis confirms the importance of A/T-rich, MEF2-binding motifs in myoglobin gene regulation, as suggested by previous studies using different experimental approaches. In addition, we demonstrated a previously unsuspected role for an intragenic E-box motif as a negative regulatory element contributing to the tightly regulated variation in myoglobin gene expression among particular myofiber subtypes.

Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains.

The S1 gene nucleotide sequences of 10 type 3 (T3) reovirus strains were determined and compared with the T3 prototype Dearing strain in order to study sequence diversity in strains of a single reovirus serotype and to learn more about structure-function relationships of the two S1 translation products, sigma 1 and sigma 1s. Analysis of phylogenetic trees constructed from variation in the sigma 1-encoding S1 nucleotide sequences indicated that there is no pattern of S1 gene relatedness in these strains based on host species, geographic site, or date of isolation. This suggests that reovirus strains are transmitted rapidly between host species and that T3 strains with markedly different S1 sequences circulate simultaneously. Comparison of the deduced sigma 1 amino acid sequences of the 11 T3 strains was notable for the identification of conserved and variable regions of sequence that correlate with the proposed domain organization of sigma 1 (M.L. Nibert, T.S. Dermody, and B. N. Fields, J. Virol. 64:2976-2989, 1990). Repeat patterns of apolar residues thought to be important for sigma 1 structure were conserved in all strains examined. The deduced sigma 1s amino acid sequences of the strains were more heterogeneous than the sigma 1 sequences; however, a cluster of basic residues near the amino terminus of sigma 1s was conserved. This analysis has allowed us to investigate molecular epidemiology of T3 reovirus strains and to identify conserved and variable sequence motifs in the S1 translation products, sigma 1 or sigma 1s.