Sheila Connelly - Academia.edu (original) (raw)

Papers by Sheila Connelly

John Wiley & Sons, Ltd eBooks, Apr 2, 2003

Journal of translational science, 2020

Ribaxamase is an orally delivered β-lactamase intended to be co-administered with intravenous β-l... more Ribaxamase is an orally delivered β-lactamase intended to be co-administered with intravenous β-lactam antibiotics (penicillins and cephalosporins) to protect the gut microbiome from excess antibiotics excreted into the gastrointestinal tract. In a placebo-controlled, multinational Phase 2b proof-of-concept clinical study, ribaxamase significantly reduced the incidence of Clostridium difficile infection in patients treated with ceftriaxone for a lower respiratory tract infection. Patients could also receive treatment with macrolides at the discretion of the clinician. During the clinical study, three sequential fecal samples were collected for analysis of the gut microbiome and microbiologic determination of intestinal colonization by certain pathogens. Changes in the gut microbiome were analyzed using 16S rRNA gene sequencing. Ribaxamase significantly ameliorated the loss of alpha and beta diversity as compared with the placebo group. During the clinical study, significantly more placebo patients became newly colonized with vancomycin resistant enterococci (VRE), and the present microbiome analysis determined that significantly more placebo patients also became monodominated by enterococci (15 vs. 4). Notably, VRE-colonized patients had a significant reduction in gut microbiome alpha diversity as compared with non-colonized patients. These data demonstrate that ribaxamase limited antibiotic-mediated damage to the gut microbiome in patients treated with ceftriaxone and support further clinical development. Ribaxamase is a unique gut microbiome protectant intended to reduce opportunistic infections like C. difficile in patients receiving IV β-lactam antibiotics. Kokai-Kun JF (2019) Ribaxamase, an orally administered β-lactamase, protects the gut microbiome in patients treated with ceftriaxone

PubMed, Oct 1, 1999

Adenoviral vectors currently represent one of the most efficient means of in vivo hepatocyte gene... more Adenoviral vectors currently represent one of the most efficient means of in vivo hepatocyte gene delivery. Consequently, liver-directed gene transfer has been increasingly explored as a promising approach for the treatment of a diverse range of genetic and acquired diseases. Numerous demonstrations of efficacious adenoviral vector-mediated delivery of a wide array of transgenes in several animal species and humans have been reported. In general, transgene expression was efficient, but transient, in many cases lasting < 1 month. Currently, efforts in the field are focused on the development of highly attenuated adenoviral vectors designed to prolong transgene expression by reducing vector immunogenicity and hepatoxicity. Vector optimization strategies include the development of vectors devoid of all viral coding regions, the generation of chimeric vectors engineered to capitalize on favorable aspects of the component viral systems, the development of tissue-specific regulated gene expression, and the development of strategies to circumvent the host immune system. The use of adenoviral vectors for gene therapy of hereditary, malignant and infectious diseases of the liver, and the vector optimization strategies outlined above are discussed in this work.

Pertussis remains a significant health problem, killing up to 200,000 infants annually. We are pu... more Pertussis remains a significant health problem, killing up to 200,000 infants annually. We are pursuing two complementary approaches to this problem, (1) engineering the adenylate cyclase toxin as an additional antigen for inclusion in the current accellular vaccine and (2) developing a neonatal antibody therapeutic to protect infants during the most vulnerable period before they are fully vaccinated.

Molecular and Cellular Biology, Oct 1, 1993

Formation of the 3' ends of RNA polymerase H (Pol H)-specific U small nuclear RNAs (U snRNAs) in ... more Formation of the 3' ends of RNA polymerase H (Pol H)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plwmbaginifolia. We found that the 3'-end-adjacent sequence CA (N)310AGTNNAA, conserved in plant Pol lI-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end

Molecular and Cellular Biology, Sep 1, 1994

RNA polymerase (Pol) IIand RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledon... more RNA polymerase (Pol) IIand RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the-30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol

Thrombosis and Haemostasis, 1997

Significant progress has been made on the development of gene therapy for the treatment of hemoph... more Significant progress has been made on the development of gene therapy for the treatment of hemophilia A, a common bleeding disorder caused by subnormal levels of blood coagulation factor VIII (FVIII). Recent advances in gene transfer technology have enabled the expression of therapeutic to physiological levels of human FVIII in normal animals as well as hemophiliac mice and dogs. However, the in vivo persistence of FVIII expression was variable, ranging from one day to over five months. Despite recent advances in the development of hemophilia A gene transfer vectors, each still faces limitations to its clinical utility. Current research is focused on improving gene transfer vehicles and delivery methods to enable sustained clotting factor expression, treatment readministration, and circumvention of the host immune response to the treatment.

Open Forum Infectious Diseases, 2017

• SYN-006 has a broad antibiotic degradation profile including penicillins, cephalosporins, and c... more • SYN-006 has a broad antibiotic degradation profile including penicillins, cephalosporins, and carbapenems • In dogs, oral SYN-006 resulted in degradation of intestinal meropenem and did not affect meropenem serum levels

Blood, May 1, 1998

Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been wide... more Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy. 1998 by The American Society of Hematology. MATERIALS AND METHODS Construction of recombinant adenoviruses. The recombinant adenovirus encoding human FVIII, Av1H8101 (previously named Av1ALAPH81) 19 has been described. 19 The vector was checked for the presence of replication-competent adenovirus contamination by polymer

Blood, Nov 15, 1996

The publication co.st.7 of this urticle were defrayed in part by page charge puynimr. This urticl... more The publication co.st.7 of this urticle were defrayed in part by page charge puynimr. This urticltr must therefbrr be hereby murked "advertisement" in accordance with I8 U.S.C. section I734 solely to indicate this fact.

Gastroenterology, 2019

Background: Despite progress in IBD genetics/microbiome, more effective genetic strategies are ne... more Background: Despite progress in IBD genetics/microbiome, more effective genetic strategies are needed to decipher the role of the genetic modifications in the gut microbiome. Methods: We used a forward genetics strategy in zebrafish to screen the entire genome and identify the microbiome alterations present in a new mutant line that was characterized by exhibiting a fluorescent intestinal phenotype, detectable in larvae after 5 days post fertilization (when the digestive system starts to function). Culture and 16S microbiome analysis of the gut content of two cohorts of mutant and WT sibling larvae were conducted to i) contextualize the relevance of the fish microbiome with respect to human/mouse microbiome, and ii) to identify the bacterial species altered in mutant fish. Results: Genetic analysis showed that the mutation associated with this fluorescent shift in the intestinal content is in the locus FG-9.6, located on Chromosome 20. Culture data revealed distinct microbial colonies enriched in mutant fish. Of relevance, antibiotic treatment of the water prevented the fluorescent phenotype, suggesting that the mutation caused changes in the gut luminal content and not the gut wall. Microscopic analysis revealed no anatomical changes in fish morphology. In context, a multi-experiment study with samples from 240 mice (1298 OTUs, 2.95 million reads 235±74 OTU/sample), showed that although the fish microbiome is distinct in multivariable statistics (a separate cluster), correlation analysis based on absolute presence at family level (n=117 taxa), showed that the fish and mouse microbiome have a mediumhigh level similarity (y = 0.413x + 0.502, R 2 = 0.599), making the fish model useful to study the mammalian microbiome. At species level, 67.3% of all bacteria in the fish-mouse analysis (n=70/104 taxa) were present in both systems, including Mucispirillum schaedleri, Weissella paramesenteroides, Helicobacter hepaticus, Parabacteroides distasonis, Ruminococcus gnavus, Faecalibacterium prausnitzii, Lactobacillus plantarum, Clostridium clostridioforme, and Akkermansia muciniphila. Quantitatively, the mutation resulted in a gut microbiome shift characterized by alterations in several classes (Deinococci, Saprospirae, Fusobacteriia, Clostridia, Deltaproteobacteria; p<0.079). At species level (365 OTUs, 111 taxa), the mutagenic effect was evidenced as changes in Lactobacillus reuteri, Brevundimonas vesicularis, Bacteroides acidifaciens, Clostridium celatum, Haemophilus parainfluenzae and Pseudomonas veronii (p<0.078). Conclusion: Forward genetics in zebra fish allowed the identification of a new genetic locus responsible for gut microbiota selection. Because most bacterial species detected in the study were present in both, fish and mice, forward genetics could expedite the discovery of host genetics-microbiome interactions relevant to mammalian IBD.

Molecular and Cellular Biology, 1989

A region in the adenovirus major late promoter (MLP) containing a CCAAT consensus sequence can di... more A region in the adenovirus major late promoter (MLP) containing a CCAAT consensus sequence can direct transcription termination of RNA polymerase II, a mechanism that possibly prevents transcriptional interference from upstream genes. Using a chimeric plasmid template that contains the MLP directing expression of the simian virus 40 early region, we showed that an inserted oligonucleotide containing only 13 base pairs of MLP sequences, including the CCAAT box, is capable of inducing transcription termination in an orientation-dependent, position-independent manner. Point mutations within the CCAAT-specific protein-binding site abolished this effect, while a base substitution outside of this region did not affect termination. These data suggest that termination is mediated by a CCAAT box-binding protein. Several other transcription factor-binding sites do not, however, cause termination, suggesting that this may be a relatively specific property of a CCAAT-binding protein.

Molecular and Cellular Biology, 1985

Our previous studies of the 3'-end processing of simian virus 40 late mRNAs indicated the exi... more Our previous studies of the 3'-end processing of simian virus 40 late mRNAs indicated the existence of an essential element (or elements) downstream of the AAUAAA signal. We report here the use of transient expression analysis to study a functional element which we located within the sequence AGGUUUUUU, beginning 59 nucleotides downstream of the recognized signal AAUAAA. Deletion of this element resulted in (i) at least a 75% drop in 3'-end processing at the normal site and (ii) appearance of readthrough transcripts with alternate 3' ends. Some flexibility in the downstream position of this element relative to the AAUAAA was noted by deletion analysis. Using computer sequence comparison, we located homologous regions within downstream sequences of other genes, suggesting a generalized sequence element. In addition, specific complementarity is noted between the downstream element and U4 RNA. The possibility that this complementarity could participate in 3'-end site se...

Thrombosis and Haemostasis, 1999

SummaryAdenoviral vectors provide a promising gene therapy system for the treatment of hemophilia... more SummaryAdenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-spec...

Anaerobe, Jan 2, 2016

The gut microbiome, composed of the microflora that inhabit the gastrointestinal tract and their ... more The gut microbiome, composed of the microflora that inhabit the gastrointestinal tract and their genomes, make up a complex ecosystem that can be disrupted by antibiotic use. The ensuing dysbiosis is conducive to the emergence of opportunistic pathogens such as Clostridium difficile. A novel approach to protect the microbiome from antibiotic-mediated dysbiosis is the use of beta-lactamase enzymes to degrade residual antibiotics in the gastrointestinal tract before the microflora are harmed. Here we present the preclinical development and early clinical studies of the beta-lactamase enzymes, P3A, currently referred to as SYN-004, and its precursor, P1A. Both P1A and SYN-004 were designed as orally-delivered, non-systemically available therapeutics for use with intravenous beta-lactam antibiotics. SYN-004 was engineered from P1A, a beta-lactamase isolated from Bacillus licheniformis, to broaden its antibiotic degradation profile. SYN-004 efficiently hydrolyses penicillins and cephalos...

Investigative Ophthalmology & Visual Science, 2004

Increased expression of vascular endothelial cell growth factor (VEGF) in the retina is sufficien... more Increased expression of vascular endothelial cell growth factor (VEGF) in the retina is sufficient to stimulate sprouting of neovascularization from the deep capillary bed of the retina, but not the superficial retinal capillaries or the choriocapillaris. Coexpression of VEGF and angiopoietin 2 (Ang2) results in sprouting of neovascularization from superficial and deep retinal capillaries, but not the choriocapillaris. However, retina-derived VEGF and Ang2 may not reach the choriocapillaris, because of tight junctions between retinal pigmented epithelial (RPE) cells. To eliminate this possible confounding factor, we used the human vitelliform macular dystrophy 2 (VMD2) promoter, an RPE-specific promoter, combined with the tetracycline-inducible promoter system, to generate double transgenic mice with inducible expression of VEGF in RPE cells. Adult mice with increased expression of VEGF in RPE cells had normal retinas and choroids with no choroidal neovascularization (CNV), but when increased expression of VEGF in RPE cells was combined with subretinal injection of a gutless adenoviral vector containing an expression construct for Ang2 (AGVAng2), CNV consistently occurred. In contrast, triple transgenic mice with induced expression of Ang2 and VEGF in RPE cells, did not develop CNV. These data suggest that increased expression of VEGF and/or Ang2 in RPE cells is not sufficient to cause CNV unless it is combined with a subretinal injection of a gutless adenoviral vector, which is likely to perturb RPE cells. These data also suggest that the effects of angiogenic proteins may vary among vascular beds, even those that are closely related, and, therefore, generalizations should be avoided.