Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it’s Insertion into genomic safe harbor AAVS1 site using CRISPR–Cas9

Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.

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Cloning of Human ABCB11 Gene in E. coli required the removal of an Intragenic Pribnow-Schaller Box before it’s Insertion into Genomic Safe Harbor AAVS1 Site using CRISPR Cas9

10.1101/2020.09.05.284125 ◽

2020 ◽

Author(s):

Nisha Vats ◽

Madhusudana Girija Sanal ◽

Senthil Kumar Venugopal ◽

Pankaj Taneja ◽

Shiv Kumar Sarin

Keyword(s):

Bile Salts ◽

Lipid A ◽

Safe Harbor ◽

Human Bile ◽

E Coli ◽

Transporter Protein ◽

Genomic Location ◽

A Genome ◽

Pribnow Box

AbstractBackgroundGenomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the site which is disrupted upon integration of Adeno Associated Virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in Progressive Familial Intra Hepatic Cholestasis Type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient.MethodsWe used CRISPR Cas9 a genome editing tool to insert ABCB11 gene at AAVS1 site in human cell-lines.ResultsWe found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli and the removal of the same was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that ABCB11 protein has similarity with E. coli Endotoxin (Lipid A) Transporter MsbA.ConclusionWe inserted ABCB11 at AAVS1 site using CRISPR-Cas9, however, the frequency of homologous recombination was very low for this approach to be successful in-vivo (Figure: pictorial abstract).Pictorial AbstractABCB11 gene (which codes the transporter of human bile salts) is targeted to AAVS1 site using a construct which has 5’ and 3’ overhangs which are homologous to the AAVS1 site. A Pribnow box was detected inside ABCB11 gene which allowed the gene to transcribe in E. Coli causing bacterial lysis probably through competitive replacement of a homologous transporter protein in E. Coli (E. coli Endotoxin (Lipid A) Transporter) MsbA, resulting in Lipid A (L) accumulation inside the bacteria.

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Induction of an Antitumoral Immune Response by Wild-Type Adeno-Associated Virus Type 2 in an In Vivo Model of Pancreatic Carcinoma

Pancreas ◽

10.1097/mpa.0b013e31804b4941 ◽

2007 ◽

Vol 35 (1) ◽

pp. 63-72 ◽

Cited By ~ 6

Author(s):

Sven Eisold ◽

Jan Schmidt ◽

Eduard Ryschich ◽

Michael Gock ◽

Ernst Klar ◽

...

Keyword(s):

Immune Response ◽

Pancreatic Carcinoma ◽

Virus Type ◽

In Vivo Model ◽

Wild Type ◽

Adeno Associated Virus

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Synergy in Polymicrobial Infections in a Mouse Model of Type 2 Diabetes

Infection and Immunity ◽

10.1128/iai.73.9.6055-6063.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 6055-6063 ◽

Cited By ~ 37

Author(s):

Matthew D. Mastropaolo ◽

Nicholas P. Evans ◽

Meghan K. Byrnes ◽

Ann M. Stevens ◽

John L. Robertson ◽

...

Keyword(s):

Type 2 Diabetes ◽

White Blood Cells ◽

In Vivo Model ◽

Diabetic Patients ◽

Diabetic Mice ◽

E Coli ◽

Polymicrobial Infections ◽

Young Mice

ABSTRACT Human diabetics frequently suffer delayed wound healing, increased susceptibility to localized and systemic infections, and limb amputations as a consequence of the disease. Lower-limb infections in diabetic patients are most often polymicrobial, involving mixtures of aerobic, facultative anaerobic, and anaerobic bacteria. The purpose of this study is to determine if these organisms contribute to synergy in polymicrobial infections by using diabetic mice as an in vivo model. The model was the obese diabetic mouse strain BKS.Cg-m +/+ Lepr db /J, a model of human type 2 diabetes. Young (5- to 6-week-old) prediabetic mice and aged (23- to 24-week-old) diabetic mice were compared. The mice were injected subcutaneously with mixed cultures containing Escherichia coli, Bacteroides fragilis, and Clostridium perfringens. Progression of the infection (usually abscess formation) was monitored by examining mice for bacterial populations and numbers of white blood cells at 1, 8, and 22 days postinfection. Synergy in the mixed infections was defined as a statistically significant increase in the number of bacteria at the site of injection when coinfected with a second bacterium, compared to when the bacterium was inoculated alone. E. coli provided strong synergy to B. fragilis but not to C. perfringens. C. perfringens and B. fragilis provided moderate synergy to each other but only in young mice. B. fragilis was anergistic (antagonistic) to E. coli in coinfections in young mice at 22 days postinfection. When age-matched nondiabetic mice (C57BLKS/J) were used as controls, the diabetic mice exhibited 5 to 35 times the number of CFU as did the nondiabetic mice, indicating that diabetes was a significant factor in the severity of the polymicrobial infections.

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Involvement of the Escherichia coli O157:H7(pO157) ecf Operon and Lipid A Myristoyl Transferase Activity in Bacterial Survival in the Bovine Gastrointestinal Tract and Bacterial Persistence in Farm Water Troughs

Infection and Immunity ◽

10.1128/iai.73.4.2367-2378.2005 ◽

2005 ◽

Vol 73 (4) ◽

pp. 2367-2378 ◽

Cited By ~ 23

Author(s):

Jang W. Yoon ◽

Ji Youn Lim ◽

Yong H. Park ◽

Carolyn J. Hovde

Keyword(s):

Escherichia Coli ◽

Membrane Structure ◽

Lipid A ◽

Transferase Activity ◽

Bacterial Membrane ◽

Dna Curvature ◽

Bacterial Survival ◽

Genetic Redundancy ◽

E Coli

ABSTRACT Escherichia coli O157:H7 is an important food-borne pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome in humans. Recently, we reported that the pO157 ecf (E. coli attaching and effacing gene-positive conserved fragments) operon is thermoregulated by an intrinsically curved DNA and contains the genes for bacterial surface-associated proteins, including a second copy of lipid A myristoyl transferase, whose chromosomal copy is the lpxM gene product. E. coli O157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tracts (GIT) to nutrient-dilute farm water troughs. Transcriptional regulation of the ecf operon by intrinsic DNA curvature and the genetic redundancy of lpxM that is associated with lipid A modification led us to hypothesize that the pO157 ecf operon and lpxM are associated with bacterial survival and persistence in various in vivo and ex vivo environments by optimizing bacterial membrane structure and/or integrity. To test this hypothesis, three isogenic ecf operon and/or lpxM deletion mutants of E. coli O157:H7 ATCC 43894 were constructed and analyzed in vitro and in vivo. The results showed that a double mutant carrying deletions in the ecf and lpxM genes had an altered lipid A structure and membrane fatty acid composition, did not survive passage through the bovine GIT, did not persist well in farm water troughs, had increased susceptibility to a broad spectrum of antibiotics and detergents, and had impaired motility. Electron microscopic analyses showed gross changes in bacterial membrane structure.

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Incorporation of Adeno-Associated Virus in a Calcium Phosphate Coprecipitate Improves Gene Transfer to Airway Epithelia In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.1.535-540.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 535-540 ◽

Cited By ~ 27

Author(s):

Robert W. Walters ◽

Dongsheng Duan ◽

John F. Engelhardt ◽

Michael J. Welsh

Keyword(s):

Calcium Phosphate ◽

Gene Transfer ◽

Mouse Lung ◽

Apical Surface ◽

Airway Epithelia ◽

Adeno Associated Virus ◽

Human Airway

ABSTRACT Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaPicoprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo.

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Conjugate-Based Targeting of Recombinant Adeno-Associated Virus Type 2 Vectors by Using Avidin-Linked Ligands

Journal of Virology ◽

10.1128/jvi.76.24.12900-12907.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12900-12907 ◽

Cited By ~ 84

Author(s):

Selvarangan Ponnazhagan ◽

Gandham Mahendra ◽

Sanjay Kumar ◽

John A. Thompson ◽

Mark Castillas,

Keyword(s):

Gene Therapy ◽

Growth Factor ◽

Virus Type ◽

Transgene Expression ◽

Future Application ◽

Adeno Associated Virus ◽

Tissue Specific ◽

Prokaryotic Expression System

ABSTRACT The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1α receptor (FGFR1α), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1α-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.

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Systematic identification of metabolites controlling gene expression in E. coli

Nature Communications ◽

10.1038/s41467-019-12474-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

Author(s):

Martin Lempp ◽

Niklas Farke ◽

Michelle Kuntz ◽

Sven Andreas Freibert ◽

Roland Lill ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

In Vitro Assays ◽

E Coli ◽

A Genome ◽

Concentration Changes ◽

Systematic Identification ◽

Genome Scale

Abstract Metabolism controls gene expression through allosteric interactions between metabolites and transcription factors. These interactions are usually measured with in vitro assays, but there are no methods to identify them at a genome-scale in vivo. Here we show that dynamic transcriptome and metabolome data identify metabolites that control transcription factors in E. coli. By switching an E. coli culture between starvation and growth, we induce strong metabolite concentration changes and gene expression changes. Using Network Component Analysis we calculate the activities of 209 transcriptional regulators and correlate them with metabolites. This approach captures, for instance, the in vivo kinetics of CRP regulation by cyclic-AMP. By testing correlations between all pairs of transcription factors and metabolites, we predict putative effectors of 71 transcription factors, and validate five interactions in vitro. These results show that combining transcriptomics and metabolomics generates hypotheses about metabolism-transcription interactions that drive transitions between physiological states.

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Adeno-Associated Virus Type 5 (AAV5) but Not AAV2 Binds to the Apical Surfaces of Airway Epithelia and Facilitates Gene Transfer

Journal of Virology ◽

10.1128/jvi.74.8.3852-3858.2000 ◽

2000 ◽

Vol 74 (8) ◽

pp. 3852-3858 ◽

Cited By ~ 238

Author(s):

Joseph Zabner ◽

Michael Seiler ◽

Robert Walters ◽

Robert M. Kotin ◽

Wendy Fulgeras ◽

...

Keyword(s):

Gene Transfer ◽

Virus Type ◽

Target Cells ◽

Apical Surface ◽

Airway Epithelia ◽

Adeno Associated Virus ◽

Human Airway ◽

Human Airway Epithelia

ABSTRACT In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring β-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.

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