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1.  Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle 
Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×1010 –1 × 1011 particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 109 AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.
doi:10.1038/mtna.2013.16
PMCID: PMC3650248  PMID: 23591809
AAV; eGFP; hrGFP; muscle toxicity
2.  Conditional over-expression of PITX1 causes skeletal muscle dystrophy in mice 
Biology open  2012;1(7):629-639.
Summary
Paired-like homeodomain transcription factor 1 (PITX1) was specifically up-regulated in patients with facioscapulohumeral muscular dystrophy (FSHD) by comparing the genome-wide mRNA expression profiles of 12 neuromuscular disorders. In addition, it is the only known direct transcriptional target of the double homeobox protein 4 (DUX4) of which aberrant expression has been shown to be the cause of FSHD. To test the hypothesis that up-regulation of PITX1 contributes to the skeletal muscle atrophy seen in patients with FSHD, we generated a tet-repressible muscle-specific Pitx1 transgenic mouse model in which expression of PITX1 in skeletal muscle can be controlled by oral administration of doxycycline. After PITX1 was over-expressed in the skeletal muscle for 5 weeks, the mice exhibited significant loss of body weight and muscle mass, decreased muscle strength, and reduction of muscle fiber diameters. Among the muscles examined, the tibialis anterior, gastrocnemius, quadricep, bicep, tricep and deltoid showed significant reduction of muscle mass, while the soleus, masseter and diaphragm muscles were not affected. The most prominent pathological change was the development of atrophic muscle fibers with mild necrosis and inflammatory infiltration. The affected myofibers stained heavily with NADH-TR with the strongest staining in angular-shaped atrophic fibers. Some of the atrophic fibers were also positive for embryonic myosin heavy chain using immunohistochemistry. Immunoblotting showed that the p53 was up-regulated in the muscles over-expressing PITX1. The results suggest that the up-regulation of PITX1 followed by activation of p53-dependent pathways may play a major role in the muscle atrophy developed in the mouse model.
PMCID: PMC3486706  PMID: 23125914
D4Z4; Grip strength; Rotarod; Tet-off; Cell death
3.  Conditional over-expression of PITX1 causes skeletal muscle dystrophy in mice 
Biology Open  2012;1(7):629-639.
Summary
Paired-like homeodomain transcription factor 1 (PITX1) was specifically up-regulated in patients with facioscapulohumeral muscular dystrophy (FSHD) by comparing the genome-wide mRNA expression profiles of 12 neuromuscular disorders. In addition, it is the only known direct transcriptional target of the double homeobox protein 4 (DUX4) of which aberrant expression has been shown to be the cause of FSHD. To test the hypothesis that up-regulation of PITX1 contributes to the skeletal muscle atrophy seen in patients with FSHD, we generated a tet-repressible muscle-specific Pitx1 transgenic mouse model in which expression of PITX1 in skeletal muscle can be controlled by oral administration of doxycycline. After PITX1 was over-expressed in the skeletal muscle for 5 weeks, the mice exhibited significant loss of body weight and muscle mass, decreased muscle strength, and reduction of muscle fiber diameters. Among the muscles examined, the tibialis anterior, gastrocnemius, quadricep, bicep, tricep and deltoid showed significant reduction of muscle mass, while the soleus, masseter and diaphragm muscles were not affected. The most prominent pathological change was the development of atrophic muscle fibers with mild necrosis and inflammatory infiltration. The affected myofibers stained heavily with NADH-TR with the strongest staining in angular-shaped atrophic fibers. Some of the atrophic fibers were also positive for embryonic myosin heavy chain using immunohistochemistry. Immunoblotting showed that the p53 was up-regulated in the muscles over-expressing PITX1. The results suggest that the up-regulation of PITX1 followed by activation of p53-dependent pathways may play a major role in the muscle atrophy developed in the mouse model.
doi:10.1242/bio.20121305
PMCID: PMC3486706  PMID: 23125914
D4Z4; Grip strength; Rotarod; Tet-off; Cell death
4.  Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases 
BMC Biotechnology  2011;11:107.
Background
RNA interference (RNAi) is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs).
Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes.
Results
As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system.
Conclusions
We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.
doi:10.1186/1472-6750-11-107
PMCID: PMC3252340  PMID: 22087765
5.  The bi-functional microRNA miR-9/miR-9* regulates REST and CoREST and is down-regulated in Huntington’s Disease 
The transcription factor REST silences neuronal gene expression in non-neuronal cells. In neurons, the protein is sequestered in the cytoplasm in part through binding to huntingtin. Polyglutamine expansions in huntingtin, which causes Huntington’s disease (HD), abrogates REST-huntingtin binding. Consequently, REST translocates to the nucleus, occupies RE1 repressor sequences and decreases neuronal gene expression. In this work, we found that levels of several microRNAs (miRNAs) with upstream RE1 sites are decreased in HD patient cortices relative to healthy controls. Interestingly, one of these, the bi-functional brain enriched miR-9/miR-9*, targets two components of the REST complex: miR-9 targets REST and miR-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates.
doi:10.1523/JNEUROSCI.2390-08.2008
PMCID: PMC3124002  PMID: 19118166
MIRNA; REST; HUNTINGTON’S DISEASE; Cortex; coordination; RNA interference
6.  In-vitro evidence for efficacy of antimicrobial mouthrinses 
Journal of dentistry  2010;38(Suppl 1):S16-S20.
SUMMARY
Objectives
The objective of this study was to compare the antimicrobial activity of commercially available antiseptic mouthrinses against saliva-derived plaque biofilms in static and flow-through biofilm systems in vitro.
Methods
Nine mouthrinses were tested in a recirculating flow-through biofilm model (RFTB) with viability assessment by ATP bioluminescence. In addition, five mouthrinses were evaluated in a batch chamber slide biofilm (BCSB) model, using live- dead staining and confocal laser scanning microscopy.
Results
In the RFTB model, essential oil (EO) and chlorhexidine (CHX)-containing rinses showed equivalent antimicrobial activity and were more effective than a range of cetyl pyridinium chloride (CPC1) formulations. In the BCSB model, twice-daily mouthrinse exposure demonstrated that the EO rinse was significantly more effective than rinses containing amine and stannous fluorides, a combination of CPC/CHX and CPC2. EO showed biofilm kill comparable to the CHX rinse.
Conclusions
The present studies have shown that mouthrinses vary significantly in their capability to kill plaque biofilm bacteria in BCSB and RFTB models. The EO mouthrinse demonstrated superior antiplaque biofilm activity to AFSF, CPC/CHX, and CPC rinses and comparable activity to CHX. The methods tested may be of value for the in-vitro screening of antiseptic rinses with different modes of antimicrobial action.
doi:10.1016/S0300-5712(10)70006-3
PMCID: PMC2954231  PMID: 20621239
biofilm; antiplaque; mouthrinse; antimicrobial; essential oils; chlorhexidine; cetylpyridinium chloride; amine fluoride; antiseptic; biocidal
7.  Microdystrophin Gene Therapy of Cardiomyopathy Restores Dystrophin-Glycoprotein Complex and Improves Sarcolemma Integrity in the Mdx Mouse Heart 
Circulation  2003;108(13):1626-1632.
Background
More than 90% of Duchenne muscular dystrophy (DMD) patients develop cardiomyopathy, and many die of cardiac failure. Despite tremendous progress in skeletal muscle gene therapy, few attempts have been made to treat cardiomyopathy. Microdystrophin genes are shown to correct skeletal muscle pathological lesions in the mdx mouse model for DMD. Here, we tested the therapeutic potential of adeno-associated virus (AAV)–mediated microdystrophin gene therapy in the mdx mouse heart.
Methods and Results
AAV was delivered to the newborn mdx mouse cardiac cavity. The procedure was rapid and well tolerated. Efficient expression was achieved in the inner and the outer layers of the myocardium. The ubiquitous cytomegalovirus promoter resulted in substantially higher expression than the muscle-specific CK6 promoter. The therapeutic effects of microdystrophin were evaluated at 10 months after infection. Immunostaining demonstrated extensive microdystrophin expression and successful restoration of the dystrophin-glycoprotein complex. Importantly, AAV-mediated microdystrophin expression improved the sarcolemma integrity in the mdx heart.
Conclusions
We established a simple gene transfer method for efficient and persistent transduction of the mdx mouse heart. AAV-mediated microdystrophin expression restored the critical dystrophin-glycoprotein complex and improved sarcolemma integrity of the mdx heart. Our results revealed the promise of AAV-microdystrophin gene therapy for cardiomyopathy in DMD.
doi:10.1161/01.CIR.0000089371.11664.27
PMCID: PMC2581719  PMID: 12952841
muscular dystrophy; genes; viruses; gene therapy; microdystrophin
8.  Adeno-Associated Virus-Mediated Microdystrophin Expression Protects Young mdx Muscle from Contraction-Induced Injury 
Duchenne muscular dystrophy (DMD) is the most common inherited lethal muscle degenerative disease. Currently there is no cure. Highly abbreviated microdystrophin cDNAs were developed recently for adeno-associated virus (AAV)-mediated DMD gene therapy. Among these, a C-terminal-truncated ΔR4-R23/ΔC microgene (ΔR4/ΔC) has been considered as a very promising therapeutic candidate gene. In this study, we packaged a CMV.ΔR4/ΔC cassette in AAV-5 and evaluated the transduction and muscle contractile profiles in the extensor digitorum longus muscles of young (7-week-old) and adult (9-month-old) mdx mice. At ∼3 months post-gene transfer, 50–60% of the total myofibers were transduced in young mdx muscle and the percentage of centrally nucleated myofibers was reduced from ∼70% in untreated mdx muscle to ∼22% in microdystrophin-treated muscle. Importantly, this level of transduction protected mdx muscle from eccentric contraction-induced damage. In contrast, adult mdx muscle was more resistant to AAV-5 transduction, as only ∼30% of the myofibers were transduced at 3 months postinfection. This transduction yielded marginal protection against eccentric contraction-induced injury. The extent of central nucleation was also more difficult to reverse in adult mdx muscle (from ∼83% in untreated to ∼58% in treated). Finally, we determined that the ΔR4/ΔC microdystrophin did not significantly alter the expression pattern of the endogenous full-length dystrophin in normal muscle. Neither did it have any adverse effects on normal muscle morphology or contractility. Taken together, our results suggest that AAV-mediated ΔR4/ΔC microdystrophin expression represents a promising approach to rescue muscular dystrophy in young mdx skeletal muscle.
doi:10.1016/j.ymthe.2004.09.013
PMCID: PMC2581717  PMID: 15668136
Duchenne muscular dystrophy; mdx; adeno-associated virus; microdystrophin; muscle contraction
9.  Optimization of Feline Immunodeficiency Virus Vectors for RNA Interference 
Journal of Virology  2006;80(19):9371-9380.
RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3′ untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.
doi:10.1128/JVI.00958-06
PMCID: PMC1617215  PMID: 16973543
10.  The Public Health Response and Epidemiologic Investigation Related to the Opening of a Bacillus anthracis–Containing Envelope, Capitol Hill, Washington, D.C. 
Emerging Infectious Diseases  2002;8(10):1039-1043.
On October 15, 2001, a U.S. Senate staff member opened an envelope containing Bacillus anthracis spores. Chemoprophylaxis was promptly initiated and nasal swabs obtained for all persons in the immediate area. An epidemiologic investigation was conducted to define exposure areas and identify persons who should receive prolonged chemoprophylaxis, based on their exposure risk. Persons immediately exposed to B. anthracis spores were interviewed; records were reviewed to identify additional persons in this area. Persons with positive nasal swabs had repeat swabs and serial serologic evaluation to measure antibodies to B. anthracis protective antigen (anti-PA). A total of 625 persons were identified as requiring prolonged chemoprophylaxis; 28 had positive nasal swabs. Repeat nasal swabs were negative at 7 days; none had developed anti-PA antibodies by 42 days after exposure. Early nasal swab testing is a useful epidemiologic tool to assess risk of exposure to aerosolized B. anthracis. Early, wide chemoprophylaxis may have averted an outbreak of anthrax in this population.
doi:10.3201/eid0810.020332
PMCID: PMC2730304  PMID: 12396912
Bacillus anthracis; nasal swabs; epidemiology; bioterrorism; postexposure prophylaxis
11.  Absence of HIV Antibody Among Dental Professionals Exposed to Infected Patients 
Western Journal of Medicine  1987;146(4):439-442.
Dental professionals have relatively frequent skin contact with saliva and small amounts of blood of patients infected with the human immunodeficiency virus (HIV). Despite this exposure, none of 255 dentists, hygienists and chairside assistants had the antibody to HIV following an estimated 189 or more exposures. These data provide further evidence that casual contact with the saliva of HIV-infected persons, such as may occur in households, the workplace or in public places, is unlikely to result in transmission of HIV to uninfected persons. Because of the small sample size in this study, however, and the relatively high frequency of exposure of HIV-infected patients that we found, we recommend that dental care professionals increase their use of disposable gloves and adhere to the Centers for Disease Control's guidelines for infection control practices for dentistry until more is known about the transmission of this virus.
PMCID: PMC1307332  PMID: 3646818

Results 1-11 (11)