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Treating an N-Glycosylation Abnormality in Airways with a Cystic Fibrosis Transmembrane Conductance Regulator Protein De...

Permanent Link: http://ufdc.ufl.edu/UFE0021557/00001

Material Information

Title: Treating an N-Glycosylation Abnormality in Airways with a Cystic Fibrosis Transmembrane Conductance Regulator Protein Deficiency Reduces Pseudomonas aeruginosa Colonization; A Hallmark Symptom of Cystic Fibrosis
Physical Description: 1 online resource (134 p.)
Language: english
Creator: Martino, Ashley T
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aav, abnormal, adeno, aeruginosa, associated, bacterial, chronic, clearance, colonzation, cystic, deficiency, desquamation, fibrosis, gene, genetics, glycosylation, mannose, mpi, pmi, pseudomonas, therapy, virus
Genetics (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Chronic colonization of Pseudomonas aeruginosa is a hallmark symptom in cystic fibrosis (CF) patients. Abnormal glycosylation has been implicated as a contributing factor for this condition. A recent study reported that Mannose-6-Phosphate isomerase (MPI), a glycosylation enzyme involved N-glycosylation, is down regulated in a CFTR deficient airway epithelial cell line, suggesting MPI as a potential contributor to this pathogenesis. I observed a 40% decrease in N-glycosylation on the surface of CFTR deficient cells (IB3) compared to CFTR corrected cells (S9) along with a 2-fold lower attachment of P. aeruginosa laboratory strain PAO1 to IB3 cells compared to S9 cells. Blocking N-glycosylation in S9 cells prior to PAO1 binding significantly decreased the bacterial attachment, revealing a role of N-glycosylation in cell adhesion. Further analysis of the PAO1 ingestion by IB3 and S9 cells revealed a 2-fold lower uptake by IB3 cells. I also discovered an ensuing bacterial clearance deficiency in IB3 cells with a reduction in programmed cell death response as a mechanism for clearance. Transfecting IB3 cells with a MPI or CFTR over-expressing plasmid reversed these IB3 deficiencies. Additionally, as mannose can be directly phosphorylated by hexokinase to produce mannose-6-phosphate, independent of MPI, a dose dependent correction of the IB3 deficiencies was observed when cultured in variable mannose rich media. These data indicate an important role of MPI in bacterial clearance. Using an in vivo model for P. aeruginosa colonization in the upper airways, I observed a significant increase in bacterial burden in the trachea, oropharynx and, the lungs in untreated Whitsett mice compared to mice treated with an AAV5 viral vector expressing murine MPI or a hyper-mannose water diet. Analysis of lung cell suspensions from these mice revealed a significant increase in N-glycosylation in treated Whitsett mice compared to those untreated. Finally, an increased lung inflammatory response occurred in untreated Whitsett mice compared to treated mice. MPI involvement in the chronic colonization of P. aeruginosa in the CF airways is a novel concept for a hallmark disease condition in CF. Treating the N-glycosylation deficiency in the CF lung provides another therapeutic avenue for improving bacterial clearance.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ashley T Martino.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Jin, Shouguang.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021557:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021557/00001

Material Information

Title: Treating an N-Glycosylation Abnormality in Airways with a Cystic Fibrosis Transmembrane Conductance Regulator Protein Deficiency Reduces Pseudomonas aeruginosa Colonization; A Hallmark Symptom of Cystic Fibrosis
Physical Description: 1 online resource (134 p.)
Language: english
Creator: Martino, Ashley T
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aav, abnormal, adeno, aeruginosa, associated, bacterial, chronic, clearance, colonzation, cystic, deficiency, desquamation, fibrosis, gene, genetics, glycosylation, mannose, mpi, pmi, pseudomonas, therapy, virus
Genetics (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Chronic colonization of Pseudomonas aeruginosa is a hallmark symptom in cystic fibrosis (CF) patients. Abnormal glycosylation has been implicated as a contributing factor for this condition. A recent study reported that Mannose-6-Phosphate isomerase (MPI), a glycosylation enzyme involved N-glycosylation, is down regulated in a CFTR deficient airway epithelial cell line, suggesting MPI as a potential contributor to this pathogenesis. I observed a 40% decrease in N-glycosylation on the surface of CFTR deficient cells (IB3) compared to CFTR corrected cells (S9) along with a 2-fold lower attachment of P. aeruginosa laboratory strain PAO1 to IB3 cells compared to S9 cells. Blocking N-glycosylation in S9 cells prior to PAO1 binding significantly decreased the bacterial attachment, revealing a role of N-glycosylation in cell adhesion. Further analysis of the PAO1 ingestion by IB3 and S9 cells revealed a 2-fold lower uptake by IB3 cells. I also discovered an ensuing bacterial clearance deficiency in IB3 cells with a reduction in programmed cell death response as a mechanism for clearance. Transfecting IB3 cells with a MPI or CFTR over-expressing plasmid reversed these IB3 deficiencies. Additionally, as mannose can be directly phosphorylated by hexokinase to produce mannose-6-phosphate, independent of MPI, a dose dependent correction of the IB3 deficiencies was observed when cultured in variable mannose rich media. These data indicate an important role of MPI in bacterial clearance. Using an in vivo model for P. aeruginosa colonization in the upper airways, I observed a significant increase in bacterial burden in the trachea, oropharynx and, the lungs in untreated Whitsett mice compared to mice treated with an AAV5 viral vector expressing murine MPI or a hyper-mannose water diet. Analysis of lung cell suspensions from these mice revealed a significant increase in N-glycosylation in treated Whitsett mice compared to those untreated. Finally, an increased lung inflammatory response occurred in untreated Whitsett mice compared to treated mice. MPI involvement in the chronic colonization of P. aeruginosa in the CF airways is a novel concept for a hallmark disease condition in CF. Treating the N-glycosylation deficiency in the CF lung provides another therapeutic avenue for improving bacterial clearance.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ashley T Martino.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Jin, Shouguang.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021557:00001


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1 TREATING AN N-GLYCOSYLATION ABNORMALI TY IN AIRWAYS WITH A CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANC E REGULATOR PROTEIN DEFICIENCY REDUCES PSEUDOMONAS AERUGINOSA COLONIZATION; A HALLMARK SYMPTOM OF CYSTIC FIBROSIS By ASHLEY THOMAS MARTINO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Ashley Thomas Martino

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3 To Antony; my son, my stars and my moon and to Maria; my love, my life

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4 ACKNOWLEDGMENTS There are many people to thank who have helped m e in matters of emotional, educational and structural support. To all my family members, I want to say thanks for always supporting me in all my lifes decisions. You never doubted my abilities or questioned my choices and you were always supportive. To all my committee member s I want to say thanks for all the valuable guidance and education you provide d during this journey. To my educational colleagues, we worked together to achieve our goals and the experience was very rewardi ng. Finally, a thanks to all the administrative workers that constantly ma de sure I was up to date on all my requirements for graduation you helped minimize my burden. Specifically, in the area of research support I would like to thank Ch ris Mueller Ph.D., who was my senior lab mate that pr ovided me with guida nce throughout my project. I would like to thank Sofia Braag M.S., who provided me with the mice breeding support to ensure that I had mice to do my in vivo projects. I would like to thank Kevin Foust Ph.D. who was, at the time of assistance, a Ph.D. candidate who provided me with the SMN control plasmid I used in many transfection experiments. I w ould like to thank Pedro Cruz Ph.D., who provided me with valuable cloning and siRNA knowledge as well as vector backbones for me to do my subcloning. Thomas Conlon Ph.D. and the toxicology lab provided me with data analysis of viral genomes that could be found in tissue from my treate d mice. Martha Campbell-Thompson Ph.D., and the pathology core provided me with all the immunohistochemistry s upport required for my project. Neal Benson in the Flow Cytometry lab provid ed me with the initial training and machine support to collect and analyze FACS data. Finally I would like to thank Roberto Kolter Ph.D. from Harvard who provided me with a critical bacterial strain that expressed a fluorescent protein and Hudson Freeze Ph.D. at the Burhnam In stitute in California who provided me with a critical PMI (MPI) antib ody that is not commercially available.

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5 In the areas of administrative support I would like to thank ma ny folks that were so critical in my completion of this project. Amy Poirier was the lab manager and she was proactive about ensuring that I had the money and supplies necessa ry to complete this project. Joyce Conners was the departmental assistant to the graduate students and she did so much to relieve me of any concerns I might otherwise have had regarding registration, class requi rements, defense and dissertation requirements and so many other countle ss things that when put together gave me peace of mind that I was not missing a thing come graduation time. Other administrators that were helpful were those in the ID P graduate office, the administrato rs in the Pediatrics office and the administrators in the Powell Gene Therapy office. The Committee is probably the most vital s ource of support and guidance and there is no doubt that this project was made easier th rough their trust, guidance, patience and professionalism. My Committee cons isted of six well-established primary investigators all with their own productive labs. Terence Flotte M. D. and Shouguang Jin Ph.D. were the primary mentors on the Committee. In addition to the primary mentors were 4 equally important investigators: Ken Berns, M.D. / Ph.D ., Venna Antony M.D., Peggy Wallace Ph.D., and Laurence Morel Ph.D.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 LIST OF ABBREVIATIONS........................................................................................................ 15 ABSTRACT...................................................................................................................................17 CHAP TER 1 BACKGROUND.................................................................................................................... 19 Cystic Fibrosis........................................................................................................................19 CFTR Protein..........................................................................................................................19 CFTR Structure...............................................................................................................20 CFTR Function................................................................................................................20 Direct chloride effl ux by CFTR protein ...................................................................21 Regulation of ENaC and ORCC by CFTR protein .................................................. 21 Other effects of CFTR protein.................................................................................22 Mutations.........................................................................................................................22 Patho-Physiology....................................................................................................................23 Role of Airway Surface Liquid (ASL)............................................................................24 P. aeruginosa Interaction with the Epithelium ................................................................25 Increased attachment of P. aeruginosa to CF airway epithelial cells ......................25 Decreased attachment of P. aeruginosa to CF airway epithelial cells ..................... 25 Role of Immune Response in CF Airway........................................................................ 26 Secondary Pathology....................................................................................................... 26 Glycosylation Abnormalities In CF........................................................................................ 27 P. aeruginosa Binding Studies ........................................................................................27 MPI (Mannose-6-Phosphate isomerase)................................................................................. 28 MPI Contribution to P. aeruginosa P redilection in CF airway.......................................29 Proposed linkage of MPI downregulation to CFTR deficiency............................... 29 Adeno-Associated Viral (AAV) Gene Therapy..................................................................... 30 siRNA for Specific Downregula tion of mRNA Transcripts................................................... 31 siRNA Mechanism..........................................................................................................31 Developing siRNA Constructs for Expre ssion of siRNA In-vivo and In-vitro ............... 32 2 PROJECT GOALS................................................................................................................. 38 Verify N-glycosyl ation Deficiency......................................................................................... 38 N-glycosylation Abnormality Contributes to Bacterial Clearance Deficiency ............... 38 MPI and Hyper-mannose Treatments for Deficiencies................................................... 39

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7 3 IN-VITRO STUDIES............................................................................................................. 40 Materials and Methods...........................................................................................................40 Verifying N-glycosylation Abnormalities.......................................................................40 Experiments to verify abnormalities........................................................................ 40 Analysis of Bacterial Adhesion....................................................................................... 42 Experiments to test abnormal adhesion.................................................................... 42 Verify that N-glycosylation Contributes to PAO1 Binding ............................................43 Experiment to block PAO1 attachment using lectins............................................... 44 Analysis of Ingested Bacteria and Clearance..................................................................44 Experiments to measure intra-cellular bacteria be fore and after clearance period....................................................................................................................44 Analysis of Host Cellular Death to Show Clearance Deficiency.................................... 47 Experiment to analyze host cellu lar death after PAO1 ingestion .................................... 48 Ensuring that gentimicin during cl earance period is not therapeutic ....................... 49 Measuring mRNA Expression Levels in Treated and Untreated IB3 and S9 Cells ........ 49 Sybr green real-time PCR to m easure expression levels..........................................50 In-Vitro Treatments............................................................................................................ ....51 Treatment of IB3 Cells with Therapeutic Plasmids........................................................ 52 Constructing the therapeutic over-expression plasmids...........................................52 CFTR siRNA Treatment of S9 Cells to Reve rt the Healthy C ells to Diseased State...... 58 CFTR siRNA construct............................................................................................58 Therapeutic CFTR and MPI Correction in IB3 Cells...................................................... 59 Testing Therapeutic Benefit of Mannose-Rich Media.................................................... 59 Knocking down CFTR in S9 cells with siRNA construct...............................................60 Results.....................................................................................................................................61 Correction of the N-glycosylat ion Deficiency in IB3 Cells ............................................61 Testing or Clearance Deficiency and Correction in Untreated IB3 Cells ....................... 62 Binding data and gene augmentation data from MPI and CFTR correction............ 62 Mannose treatment of bacteria l attachm ent to IB3 cells.......................................... 63 Demonstrate that N-glycosylation is a candidate for PAO1 adhesion ..................... 63 Ingestion, clearance and gene augm e ntation with MPI and CFTR by CFU counting................................................................................................................64 Mannose treatment of bacterial inge stion and clearance in IB3 cells ...................... 65 Ingestion and clearance from MPI an d CFTR transfection by fluorescent detection ................................................................................................................65 Mannose rich media therapy for IB3 cells............................................................... 66 Decrease and Correction of Host Cellula r Death as a Mechanism for Clearance........... 67 Gene augmentation data from MPI and CFTR correction....................................... 67 Mannose rich media therapy for IB3 cells............................................................... 68 siRNA Treatment of S9 cells to Verify CFTR Role in Clearance Deficiencies ............ 68 Testing CFTR and MPI mRNA Levels in Treated and Untreated IB3 and S9 cells....... 69 MPI expression pattern.............................................................................................70 CFTR expression pattern..........................................................................................70 4 IN-VIVO STUDIES...............................................................................................................88

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8 Materials and Methods...........................................................................................................88 Mouse Model Methods....................................................................................................88 The CFTR deficient mouse...................................................................................... 89 Mouse MPI treatment by rAAV5-m MPI intra-tracheal delivery ............................. 89 Hyper-mannose treatment of Whitsett mice............................................................. 91 Clearing the airway prior to controlled infection..................................................... 91 Controlled infection of P. aeruginosa ......................................................................92 Data Collection................................................................................................................93 Results.....................................................................................................................................95 Bacterial Burden in the Oropharynx................................................................................ 95 Weight Loss/Gain Trend in Trea ted and Untreated W hitsett Mice................................. 96 Bacterial Burden in the lung and Trachea....................................................................... 96 AAV5-pTR2-CB Vector Genomes in the Lung Tissue.................................................. 97 Extracting DNA from the lung homogenate............................................................97 Real-Time TAQMAN PCR to analyze v ector genom es in genomic DNA from the lungs................................................................................................................ 98 Sybr Green Analysis of MPI mRNA Fr om the Lungs of Transduced Whitsett Mice......................................................................................................................99 N-glycosylation Abnormality Analysis in Lung Cell Suspensions................................. 99 Pathology Analysis........................................................................................................ 100 Hematoxylin-eosin (H&E) staining....................................................................... 101 Staining GFP in treated lungs to determ ine transduction efficiency of AAV5 viral vector ..........................................................................................................101 5 DISCUSSION.......................................................................................................................114 Defective Bacterial Clearance is a H allmark Symptom in the CF Lung.............................. 114 MPI is Involved in Global Abnormal Glycosylation.................................................... 114 Reduced Bacterial Adhesion is Linked to N-glycosylation Deficiency ........................ 115 Bacterial Ingestion Defect is Li nked to Abnorm al N-Glycosylation............................ 116 In-vitro Conclusions......................................................................................................118 In-vivo Studies...............................................................................................................118 Bacterial load in the airways.................................................................................. 119 Minor weight loss in un corrected Whitsett m ice................................................... 120 N-Glycosylation abnormality of lung ce ll suspensions from control Whitsett mice.....................................................................................................................120 Analysis of lung inflammation...............................................................................121 Viral vector transduction efficiency....................................................................... 121 In-vivo Conclusions.......................................................................................................122 LIST OF REFERENCES.............................................................................................................124 BIOGRAPHICAL SKETCH.......................................................................................................134

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9 LIST OF TABLES Table page 4-1 Percentage of lungs with in the given study groups with detectable P. aeruginosa mucoid strain for analysis of a clearance deficiency. ...................................................... 112 4-2 Estim ated viral genome copies pe r transduced mice lung cells....................................... 112 4-3 Inflamm atory rankings by H&E staining of lung sections from mice treated with AAV5-mMPI or AAV5-GFP (Control)...........................................................................112 4-4 Inflamm atory rankings by H&E staining of lung sections from mice treated with 5mg/ml of mannose or 5mg/ml of glucose (Control)...................................................... 113

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10 LIST OF FIGURES Figure page 1-1 Wild-type CFTR within a membrane. Transmembrane domains (TMDs) span the membrane groups of six. Nucleotide bindi ng domains (NBDs) and the regulatory domain (R domain) lie within the cytoplasm (5)............................................................... 33 1-2 N-glycosylation enzym atic pathway. A: MPI (PMI) converts fructose-6-phosphate into mannose-6-phosphate B: Direct phosphor ylation of external mannose for the production of mannose-6-phosphate i ndependent of MPI (PMI)(33)............................... 34 1-3 AAV genes, mRNA and proteins....................................................................................... 35 1-4 RNAi m echanisms. A) Shows duplex of microRNAs that a nneal in the absence of a hairpin B) Dicer degradation of DS RNA without a hairpi n to develop ssRNA interference on a specific translated mRNA by anti-sense annealing and mRNA cleavage by the RISC complex. C) DS hair pin precursor for the production of small anti-sense RNA by dicer cleavage for transl ation inhibition but not destruction of mRNA (105)......................................................................................................................36 1-5 siRNA expression vector for the selective destruction of mRNA from any target gene....................................................................................................................................37 3-1 Pictorial representation of th e FACS detectors, lasers a nd commons stains using a 4color flow cytom ery machine (This im age is not subject to copyright)............................ 72 3-2 Construction of GFP expressi on plasm id for expression in P. aeruginosa strain PAO1 (68)..........................................................................................................................73 3-3 Confocal image of GFP+ PAO1 ingestion in IB 3 cells (green)......................................... 74 3-4 Verifying that the gentim icin treatment during the clearance period of ingested GFP+ PAO1 in IB3 or S9 cells does not c ontribute to bacteria clearance in S9 cells or contribute to the clearan ce deficiency of IB3 cells........................................................ 74 3-5 GFP expression in IB3 cells to as an indicator of succe ssful transfection.........................75 3-6 Western blot of hMPI from S9 and IB3 cells transfected with the therapeutic plasmid pTR2-CB-hMPI or the control plasmid pTR2-CB-GFP....................................................75 3-7 siRNA oligo to subclone into universal siRNA expression construct for selective knockdown of CFTR mRNA transcripts. .......................................................................... 76 3-8 FITC conjugated Con A lectin binding to N-glycosylation profile of untreated S9 cells and untreated and treated IB3 cells............................................................................ 76

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11 3-9 FITC conjugated W GA lectin bi nding to the N-glycosylati on profile of untreated S9 cells and untreated and treated IB3 cells............................................................................ 77 3-10 FITC conjugated SBA lectin binding to the O-glycosylation profile of untreated S9 cells and untreated and treated IB3 cells............................................................................ 77 3-11 Binding of PAO1 to untreated S9 cells, unt reated IB3 cells an d IB3 cells transfected with pTR2-CB-hMPI with pRT2-CB-GFP as a control....................................................78 3-12 PAO1 binding to untreated S9 cells, untreat ed IB 3 cells and IB3 cells transfected with pTR2264hCFTR with pTR2-CB-GFP as a control............................................... 78 3-13 PAO1 binding to untreated S9 cells, untreat ed IB 3 cells and IB3 cells grown in a range of mannose rich medias............................................................................................ 79 3-14 Dose dependent inhibition of PAO1 bindi ng to untreated S9 and IB3 cells after blocking with ConA, WGA and SBA at various concentrations. ...................................... 79 3-15 Intra-cellular PAO1 prior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to dem onstrate a change in intra-cellular PAO1 after the clearance period from untreated S9 and IB 3 cells and IB3 cells transfected with pTR2-CB-hMPI with pTR2-CB-GFP as a control............................................................ 80 3-16 Intra-cellular PAO1 prior to the 4 hour clearance peri od (light) and after the 4 hour clearance period (dark) to dem onstrate a change in intra-cellular PAO1 after the clearance period from untreated S9 and IB 3 cells and IB3 cells transfected with pTR2-CB264hCFTR with pTR2-CB-GFP as a control................................................. 80 3-17 Intra-cellular PAO1 prior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to dem onstrate a change in intra-cellular PAO1 after the clearance period from untreated S9 and IB3 cells and IB3 cells grown in a range of mannose rich medias.......................................................................................................... 81 3-18 Intra-cellular GFP+ PAO1 by FACS analysis prior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to de monstrate a change in intracellular PAO1 after the clearance period fr om untreated S9 and IB3 cells and IB3 cells transfected with pTR2-CB-hMPI with pTR2-CB-SMN as a control........................81 3-19 Intra-cellular GFP+ PAO1 by FACS analysis prior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to de monstrate a change in intracellular PAO1 after the clearance period fr om untreated S9 and IB3 cells and IB3 cells transfected with pTR2-CB264hCFTR with pTR2-CB-SMN as a control............. 82 3-20 Intra-cellular GFP+ PAO1 by FACS analysis prior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to de monstrate a change in intracellular PAO1 after the clearance period fr om untreated S9 and IB3 cells and IB3 cells grown in a range of mannose rich medias................................................................. 82

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12 3-21 Analysis of 7AAD staining by FACS of unt reated IB3 and S9 cells and IB3 cells treated with pTR2-CB-hMPI with pTR2 -CB-SMN as a transfection control to determine the percent of the host cell population under going apoptosis after PAO1 infection.............................................................................................................................83 3-22 Analysis of 7AAD staining by FACS of unt reated IB3 and S9 cells and IB3 cells treated with pTR2-CB264hCFTR with pTR2-CB-SMN as a transfection control to determine the percent of the host cell population u ndergoing apoptosis after PAO1infection...................................................................................................................83 3-23 Analysis of 7AAD staining by FACS of unt reated IB3 and S9 cells and IB3 cells grown in mannose rich medias to determin e the percent of th e host cell population undergoing apoptosis afte r PAO1 infection....................................................................... 84 3-24 PAO1 binding to untreated S9 cells, untreat ed IB 3 cells and S9 cells transfected with pTR2-U6-CFTRsiRNA with pTR2-U6Scrambled siRNA as a control........................... 84 3-25 Intra-cellular PAO1 prior to the 4 hour clearance peri od (light) and after the 4 hour clearance period (dark) to dem onstrate a change in intra-cellular PAO1 after the clearance period from untreated S9 and IB3 cells and S9 cells tr ansfected with pTR2U6-CFTRsiRNA with pTR2-U6-Srambled siRNA as a control....................................... 85 3-26 Intra-cellular GFP+ PAO1 by FACS anal ysis prior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to de m onstrate a change in intracellular PAO1 after the clearance period fr om untreated S9 and IB3 cells and S9 cells transfected with pTR2-U6-CFTRsiRNA with pTR2-U6-scrambled siRNA as a control................................................................................................................................85 3-27 Analysis of 7AAD staining by FACS of unt reated IB3 and S9 cells and S9 cells treated with pTR2-U6-CFTRsiRNA with pTR2-U6-Scrambled siRNA as a transfection control to determine the percent of th e host cell population undergoing apoptosis after GFP+ PAO1 infection............................................................................... 86 3-28 In-vitro experimental design overview: Binding, Ingestion and clearance description of events and treatm ent...................................................................................................... 86 3-29 Fold change of MPI m RNA levels of experimental and control groups from in vitro cell culture studies from Sybr green r eal time data collected at cycle 20.......................... 87 3-30 Fold change of CFTR mRNA levels of experim ental and control groups from in vitro cell culture studies from Sybr green r eal time data collected at cycle 20.......................... 87 4-1 Mouse MPI Western blot anal ysis of untreated IB 3 cells and IB3 cells transfected with pTR2-CB-mMPI vector........................................................................................... 102 4-2 Glucose and m annose treatment of IB3 cells to ensure that Glucose does not correct PAO1 binding deficiency.................................................................................................103

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13 4-3 Total am ount of the mucoid strain CFU collected from the oropharnyx swabs for the assessment of a bacterial clearance trend from the control group (GFP) or experimental group (MPI) of mice transduced by rAAV5-GFP or rAAV5mMPI viral vector by IT delivery. 6 total mice used; p value < 0.05 using one-way anova with repeat sampling................................................................................................................ 103 4-4 Total am ount of the mucoid strain CFU collected from the oropharnyx swabs for the assessment of a bacterial clearance trend fr om the control group (High Glucose Diet) or experimental group (High Mannose Diet ) of mice transduced by rAAV5-GFP or rAAV5mMPI viral vector by IT delivery. 4 total mice used; p value < 0.05 using one-way anova with repeat sampling...............................................................................104 4-5 Weight change data used to determ ine any weight loss trends from the control (GFP) or experimental (MPI) group during and after the controlle d infection period with the mucoid strain. 6 total mice used; p value < 0.05 using one-way anova with repeat sampling....................................................................................................................... ....104 4-6 Weight change data used to determ ine any weight loss trends from the control (High Glucose Diet) or experimental (High Mannose Diet) group during and after the controlled infection period w ith the mucoid strain. 4 total mice used; p value < 0.05 using one-way anova with repeat sampling.....................................................................105 4-7 Total am ount of mucoid CFU in the lung to determine bacterial load from mucoid infection 6 weeks after the last day of in fection in efforts to assess any clearance deficiency in treated (MPI) or untreated (GFP) CFTR deficient mice using rAAV5mMPI or rAAV5-GFP respectively.................................................................................105 4-8 Total am ount of mucoid CFU in the trachea to determine bacterial load from mucoid infection 6 weeks after the last day of in fection in efforts to assess any clearance deficiency in treated (MPI) or untreated (GFP) CFTR deficient mice using rAAV5mMPI or rAAV5-GFP respectively.................................................................................106 4-9 Total am ount of mucoid CFU in the lung to determine bacterial load from mucoid infection 6 weeks after the last day of in fection in efforts to assess any clearance deficiency in treated (Ma nnose diet) or untreated (Glu cose diet) CFTR deficient mice..................................................................................................................................106 4-10 Total am ount of mucoid CFU in the trachea to determine bacterial load from mucoid infection 6 weeks after the last day of in fection in efforts to assess any clearance deficiency in treated (Ma nnose diet) or untreated (Glu cose diet) CFTR deficient mice..................................................................................................................................107 4-11 Vector genomes from 500ng of genom ic DNA is olated from mouse lung tissue transduced with either AAV5-GFP or AAV5 -mMPI or an untransduced lung as a negative control............................................................................................................... .107 4-12 Sybr green real-tim e PCR analysis of MP I mRNA extracted from lungs collected from the mice transduced with AAV5-MPI and AAV5-GFP......................................... 108

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14 4-13 FITC conjugated Lectin anal ysis by FACS to determ ine N-glycosylation profile in control (AAV5-GFP) or therapeutic (rAAV5 -MPI) lung cell suspensions using Con A and WGA..................................................................................................................... 108 4-14 FITC conjugated Lectin anal ysis by FACS to determ ine N-glycosylation profile in control (Glucose diet) or therapeutic (Mannose diet) l ung cell suspensions using Con A and WGA..................................................................................................................... 109 4-15 H&E staining to judge infl ammation in the lungs of W hitsett mice treated with AAV5-GFP (control) or AAV5-mMPI............................................................................ 110 4-16 H&E staining to judge infl ammation in the lungs of W hitsett mice treated with 5mg/ml of mannose or 5mg/ml of glucose (control).......................................................111

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15 LIST OF ABBREVIATIONS AAV Adeno-Associated Virus; a co mmon virus used in gene therapy. ASL Airway Surface Liquid; the thin liqui d layer that lines the extra-cellular surface of the epithelium that particip ates in the muco-ciliary clearance and normal function of lung anti-microbial agents. CB Chicken Beta Actin Promoter; a st rong constitutive promoter that is utilized in the viral and plasmid vect ors for increasing expression levels of the transgene. CF Cystic Fibrosis. CFTR Cystic Fibrosis Transmembran e Conductance Regulator, which is the protein involved in Cystic Fibrosis. CFU Colony Forming Unit; used to define a single bacterium that is cable of forming colonies on bact eria culture plates. Con A Concanavalin A; a common lectin is olated from Jack Bean Seeds, which binds to -mannose glycosylation subunits. ENaC Epithelial Sodium Channel; involved in Na+ absorption of epithelial cells. FACS Fluorescent Activated Cell Sorting; an experimental procedure to analyze differences in cells by fluorescent markers. GFP Green Fluorescent protein; a crit ical control prot ein with native fluorescence in the 530 nm range when excited by a 488 nm wavelength and is a valuable non-therapeutic protei n that can be utilized to verify efficient transduction and transfection as well as ensure the transduction or transfection procedure does not cont ribute to therapeutic benefit when using a therapeutic protein. IB3 CFTR deficient human ai rway epithelial cell line. IT Intra-Tracheal injections; injection procedures that use the trachea as the route of administration to the lower airways (lung). ITR Inverted Terminal Repeats; re gions of the AAV genome, which are necessary for the lifecycle of the virus and critical for the construction of recombinant viral vectors. LPS Lipopolysaccharide; a common struct ure on the surface of gram-negative bacteria that can mediate attachment to host cells.

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16 MOI Multiplicity of Infection; the term used to describe the ratio of the infecting particle to the host cells. MPI Mannose-6-Phosphate Isomerase protein. ORCC Outwardly Rectifying Chloride Ch annel; transmembrane channels that allow for the selective efflux Clanions. PAO1 A common Pseudomonas aeruginosa laboratory strain. S9 IB3 cell line corrected with functional human CFTR gene. SBA Soy bean agglutinin; a common lectin isolated from soy that binds to Nacetyl galactoseamine (GalNAc) glycosylation residues. WGA Wheat Germ Agglutinin; a common l ectin isolated from wheat that binds to N-acetyl glucoseamine (Glc NAc) and N-acetyl neuraminic acid(NeuAc) glycosylation residues.

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17 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TREATING AN N-GLYCOSYLATION ABNORMALI TY IN AIRWAYS WITH A CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANC E REGULATOR PROTEIN DEFICIENCY REDUCES PSEUDOMONAS AERUGINOSA COLONIZATION; A HALLMARK SYMPTOM OF CYSTIC FIBROSIS By Ashley Thomas Martino December 2007 Chair: Shouguang Jin Major: Medical Sciences Genetics Chronic colonization of Pseudomonas aeruginosa is a hallmark symptom in cystic fibrosis (CF) patients. Abnormal glycosylation ha s been implicated as a contributing factor for this condition. A recent study reported that Mannose-6-Phosphate isomerase (MPI), a glycosylation enzyme involved N-glycosylation, is down regulated in a CFTR deficient airway epithelial cell line, suggesting MPI as a potential c ontributor to this pathogenesis. I observed a 40% decrease in N-glycosylation on the surface of CFTR deficient cells (IB3) compared to CFTR corrected cells (S9) along with a 2-fold lower attachment of P. aeruginosa laboratory strain PAO1 to IB3 cells compared to S9 cells Blocking N-glycosylation in S9 cells prior to PAO1 binding significantly decreased the bact erial attachment, revealing a role of Nglycosylation in cell adhesion. Further analysis of the PAO1 ingestion by IB3 and S9 cells revealed a 2-fold lower uptake by IB3 cells. I also discovered an ensuing bacter ial clearance deficiency in IB3 cells with a reduction in programmed cell death response as a mechanism for clearance. Transfecting IB3 cells with a MPI or CFTR over-e xpressing plasmid reversed these IB3 deficiencies. Additionally,

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18 as mannose can be directly phosphorylated by hexokinase to produce mannose-6-phosphate, independent of MPI, a dose dependent correctio n of the IB3 deficiencies was observed when cultured in variable mannose rich media. These data indicate an important role of MPI in bacterial clearance. Using an in vivo model for P. aeruginosa colonization in the uppe r airways, I observed a significant increase in bacterial burden in the trachea, oropharynx and, the lungs in untreated Whitsett mice compared to mice treated with an AAV5 viral vector expressing murine MPI or a hyper-mannose water diet. Analysis of lung cell suspensions from these mice revealed a significant increase in N-glycosylation in treated Whitsett mice compared to those untreated. Finally, an increased lung inflammatory response occurred in untreated Whitsett mice compared to treated mice. MPI involvement in the chronic colonization of P. aeruginosa in the CF airways is a novel concept for a hallmark disease condition in CF. Tr eating the N-glycosylation deficiency in the CF lung provides another therapeutic avenue for improving bacterial clearance.

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19 CHAPTER 1 BACKGROUND Cystic Fibrosis Cystic fibrosis (CF) is an autosom al homozygous genetic disease that leads to premature death occurring at a frequency of roughly 1/3,300 births in North America. The range of premature death extends from early in childhood to adult middle age with the average life expectance hovering around the mid 30 s. Early detection is critical to increase life expectancy by initiating life-extending treatment as soon as possible to slow the pace of lung tissue destruction, which is the primar y cause of mortality. There are a host of other factors that influence life expectancy which can include the type of mutations, fre quent access to modern therapies, discipline of the patient to follow phys icians instructions, diet and exercise. Although CF affects multiple organs, which includes pancreas liver, digestive tract and reproductive tract, the primary pathology occurs in the lung th at accounts for 90% of the mortality (2). In 1989 CF was linked to mutation in the Cy stic Fibrosis Transmembrane Conductance Regulator (CFTR) gene (1). Appr oximately 1 in every 20 people of European descent is a carrier of a mutant CFTR allele, amounting to an es timated 12 million American carriers and roughly 40 million European carriers (2). The CFTR gene is 230 kb in length and is located on the short arm of human chromosome 7 (19q13). Transcription and splicing produces a 6.1 kb mRNA with a coding region of 4.4 kb that encodes a 1480 am ino acid membrane bound glycoprotein with a molecular mass of 170,000 Daltons (3-5). CFTR Protein The cystic fibrosis transm embrane conductan ce regulator (CFTR) is in the ATP binding cassette (ABC) transporter family. It is primarily located in the apical membrane of epithelia cells and regulates the transepithelial salt a nd liquid movement. A 90% to complete CFTR

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20 protein function loss causes the genetic disease cy stic fibrosis with less dysfunction causing mild to no disease state. CFTR Structure The CFTR protein is composed of fi ve domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), a nd a regulatory (R) domain that together forms a novel phosphorylation dependent Clefflux channel protein (figure 1-1) (5). The (R) domain in the CFTR protein is unique to the ABC transporter family. The two membranespanning domains (MSD1 and MSD2 ) are each composed of 6 -helices. Together these domains function primarily to stabilize the prot ein in the cell membrane and provide pore assembly for Clspecific transport across the cell me mbrane. The two nucleotide-binding domains (NBD1 and NBD2) provide the ATP nucleotide binding sites and utilize the ATP energy to open and close the ion gate. Finally, the domain that makes the CFTR protein unique amongst the ABC transporter family is the regulat ory domain ( R ). This R-domain is a highly charged region of the CFTR protein that ha s multiple consensus phosphorylation sites for regulating the activity of the CFTR protein (5,6) CFTR Function Clefflux is the primary function of the CFTR pr otein (5-10). The CFTR protein is critical in maintaining salt balance and fluid flow in the cell and the surrounding extra-cellular environment. Although Clefflux has been determined to be the main process of this protein, it is not just a transmembrane channel protein. There are well-character ized, secondary functions that include regulation of the Epith elial Sodium Channel (ENaCs) (6-10) and Outward Rectifying Chloride Channel (ORCCs) (6,9,10). There is a range of loss of function mutations in the

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21 CFTR gene leading causing different sources of protein dysfunction from abnormal trafficking to improper regulation within the membrane (5). Direct chloride efflux by CFTR protein The m aintenance of salt levels in the intra-cel lular and extra-cellular environment is critical for normal physiology. There is a large range of membrane bound proteins responsible for the movement of these salt ions for the purposes of maintaining this salt balance. CFTR is a multidomain pore transmembrane protein that uses the energy from ATP hydrolysis to regulate Clanion movement between the intra-cellular and ex tra-cellular environment. The 2 TMDs of the CFTR protein stabilize the protein in the transmembrane and comprise the pore for passive movement of Cldown the electrochemical gradient. Additionally, there are charge pockets in these domains that provide the selective permeability toward Clanions of the CFTR protein. Although ion movement is not restricted to Cl-, the large difference in permeability of Cl compared to other ions shows the preference towards Clanions (5,9). Movement of the Clanions is regulated by the active and quiescent state of the channel gate. ATP binds to the NBDs of the CFTR prot ein. Hydrolysis of the ATPs is regulated by phosphorylation of the preferenti al serines with in the R-dom ain by cAMP dependent protein kinase A or C (PKA or PKC). When ATP hydrolysis occurs th e CFTR protein is activated and allows the passive flow of Clanions. Dephosphorylation of the R-domain at preferential sites restores the CFTR to quiescent state and stops the passive flow of Clanions (5,9,10). Regulation of ENaC an d ORCC by CFTR protein The epithelial sodium channel (ENaC) protein is a multi-subunit membrane transport protein for the absorption of Na+ across the apical surface of ep ithelial cells (11). Abnormal Na+ absorption into the airway epithelium occurs in pa tients with cystic fibros is (7,12). Under normal

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22 conditions, CFTR has an inverse effect on Na+ absorption in cells c ontaining functional ENaC and CFTR proteins (7). Na+ absorption was analyzed in a can ine kidney epithelial clone (MDCK cell line) that expresse s ENaC but not CFTR. Na+ absorption was reduced when functional CFTR was expressed in this cell line (7). Furt her secondary regulation f unction of CFTR extends to another type of chloride channel; the Outward Rectifying Chloride Channels (ORCC) (6,9,10). ORCC Clefflux activity is defective in the presence of mutant but not f unctional CFTR showing a positive regulation of CFTR on the ORCCs (13,14). Other effects of CFTR protein Additional studies have shown th at CFTR is involved in othe r cellular mechanism s outside of the Na+ / Clregulation. CFTR has been shown to regu late vesicle trafficking (15,16), regulate intracellular compartment acidification and prot ein processing (17,18) and modulate the renal outer medullary potassium channel (R OMK) sensitivity to sulfonylurea (19,20). Mutations Over 1300 different m utations have been discovered in the CFTR gene. The F508 mutation is the most common mutation. Roughl y 70% of patients have at least one F508 allele. F508 is a three-base in-frame deletion resulting in the loss of a single ph enylalanine residue at amino acid position 508. The resultant protein in a partially functional, misfolded protein that undergoes degradation early in the ERAD pathway and is not properly tr afficked to the cell membrane (21,22). Due to the large amount of known mutations and the 5 domain structure of the protein these mutations have be divided into 5 classes: 1) splice mutations, leading to reduced amounts of intact CFTR in the various tissues; 2) mutations producing Clchannels with reduced single-

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23 channel conductance; 3) mutations producing Clchannels that cannot be activated normally; 4) mutations resulting in the prot ein being caught and degraded in the cells quality control machinery (the class for the F508 allele) and 5) mutations lead ing to protein truncations (23). Parallel to the wide array of mu tations is a range of pathology with some mutations leading to mild form of cystic fibrosis (9). Patho-Physiology Although the CF pathology is very com plex, affecting many organs with ranges of severity, hallmark lung pathologies that account for 90% of mortality from CF have been revealed. The Clefflux deficiency and a secondary increase in Na+ absorption caused by the deficient CFTR protein in airway epithelial cells leads to altera tions in the airway surface liquid (ASL). Increased liquid flow from ASL into the epithelium results in a dehydrated ASL containing mucus with elevated viscosity leading to deficient muco-ciliary clearance of airway pathogens leaving the airway vulnerable to opportunistic pathogens. (23-25). For reasons that are not entirely clear, the CF lung environment has a strong predilection for the opportunistic pathogen Pseudomonas aeruginosa The vast majority of CF patients have a chronic infection with P. aeruginosa (25,26) This chronic state of pers istent lung infection leads to a constant influx of cellular infiltrates from the immune system that ultimately leads to destruction of lung tissue and may progress to respiratory failure. CFTR deficiency affects other organs, including the pancreas, small and large in testine, liver, sweat gland ducts, and male reproductive tract, but the primar y cause of morbidity and mortal ity is the lung condition (27). Although most of focus has been on CFTRs role in regulating fluid and electrolyte fluxes within the airways, CFTR has also been shown to be involved directly with the binding, ingestion and finally clearance by desquamation of P. aeruginosa (26,28,29). This brings to light a deficiency,

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24 not only in general muco-ciliary clearance but in a specific clearance by cellular means as well. This may account for the predilection of CF patie nts not seen in ciliary dyskinesia; a mucociliary lung condition due to immotile cilia cause d by a dynein motor protein function loss (30). Role of Airway Surface Liquid (ASL) The airways epithelium is covered with a thin la yer of liquid that provid es a barrier gel that allows for normal ciliary movement. It has been established that normal ASL is critical in maintaining host defense against invading pathog ens (80,81). It is widely accepted that the CF lung has severe abnormalities in th e ASL that contributes to the increase in pathogen load with in the airways. Unfortunately, there are differing theo ries on the type and cause of the abnormality. One theory suggests that the ASL is hypertonic in the CF airway that leads to an impairment of anti-microbial agents in the lung. This theory suggests that the CFTR dysfunction leads to a decreased salt absorption by the ep ithelium (82). This hypertonic ASL environment prevents the optimal defense from lysozymes, lactoferrin and -defensins generating a predisposition to pulmonary infection (83-85). An opposing, and more widely accepted, theory s uggests that the ASL is isotonic in the CF lung leading to a dehydrated muco-ciliary envi ronment and ciliary dysfunction. This theory suggests that fluid absorption into the epithelium is increased due to increased salt absorption into the surrounding cells do to lo ss of regulation of th e ENaC protein. As previously mentioned the CFTR protein regulates sodium absorption by controlling ENaC f unction. When CFTR is defective, the ENaC sodium channels are not regulated and there is an increase of Na+ absorption with consequent water absorption. These abnor malities result in a th ick mucus layer with increased viscosity limiti ng ciliary and cough clearan ce of pathogens (7,86,87).

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25 P. aeruginosa Interac tion with the Epithelium There are opposing but not mutually exclus ive theories on the role of the airway epithelium and P. aeruginosa. The basic points behind these two theories are findings that have revealed an increased attachment of P. aeruginosa and an opposing (88,89) decreased attachment of P. aeruginosa to CFTR deficient airway epithelial cells (29,90,91). Increased attachment of P. aerugin osa to CF airway epithelial cells Asiaylated glycoproteins (aGM1) have asialoGM1 glycolipids and are airway epithelial cell surface receptors for P. aeruginosa It has been shown th at these are increased in the CF airway (88,89). Studies to verify asialoGM1 is a P. aeruginosa receptor were performed by blocking asialo-GM1 adhesion molecules on P. aeruginosa with free asialo-GM1 or by blocking the asialo-GM1 on glycoproteins usi ng anti-asialo-GM1 to inhibit P. aeruginosa attachment (88, 92, 93). The increased binding of P. aeruginosa to the CF airway could also be corrected in vitro by correcting the CFTR deficient airway epith elial cells with functional CFTR (94). Decreased attachment of P. aeruginosa to CF airw ay epithelial cells Contrary to the previous theo ry, opposing evidence has revealed that there is a decrease in host-cellular clearance by cellu lar death in the absence of CFTR protein. The proposed mechanism involves the CFTR protein as a receptor for P. aeruginosa via the lypopolysaccharide (LPS). When CFTR is deficient a decreased uptake of P. aeruginosa and a reduction in host cellular death as a mechanis m for clearance will oc cur (29,90,91). Therefore, this mechanism involves more than just attachme nt of bacterial but also a host cell clearance response. To add evidence to this theory, it has been shown that a bacterial internalization increase occurs in a CFTR dose-dependent manner in a CFTR mouse model (95).

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26 Role of Immune Response in CF Airway The chronic infection that plagues the CF ai rway prom otes a chronic hyper-inflammatory response that is dominated by neutrophils and polymorphonuclear leukocyt es. These neutrophils and polymorphonuclear leukocytes c ontain granules that release proteases, anti-microbial peptides and reactive oxygen species (ROS) (9698). Coordinating this chronic influx is the excessive increase in proinflammatory cytokines, most importantly IL-8. The elevated level of IL-8 is directed by the NF-KB transcription factor activation du e to the chronic persistence of bacteria in the lung (99, 100). This chronic inflammatory response pr oves to be destructive to the lung tissue and in most cases, leads to a lethal pulmonary challenge (101). Secondary Pathology The presence of secondary pathologies has al ways been known and the severity of these pathologies increase as the CF patients life extends into ad ulthood. Additionally, there is an occurrence of other agerelated pathologies that was not co mmonly observed when patients had a lower life expectancy. Pancreatic insu fficiency has been well characteri zed in CF and now many older CF patients are developing CF related diabetes. Liver and di gestion deficiencies have been well documented and also progress in severity as CF patients increase in age. In fact liver disease is the second most common cause of death in CF patients. CF patients also commonly suffer from malnutrition, vitamin E deficiency, maldigesti on and inadequate weight gain, related to the secondary symptoms in the digestive tracts and pa ncreas. Finally, males are sterile due to the bilateral absence of th e vas deferens (31).

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27 Glycosylation Abnormalities in CF The com plexities of glycosylation abnormalities are well established in the CF lung. The presence of abnormal gylcosylation in the CF lung is widely accepted bu t what contribution the abnormal glycosylation has on the CF lung pathol ogy and what causes this abnormality is still under debate. There are two contrary theories that have been we ll pursued. The majority of the research in the field of abnormal gylcosylati on has focused on how the abnormality contributes to the chronic colonization of P. aeruginosa. Reduction of sialic acid terminal glycosylation residues by the increase in the asialo-GM1 glycolipid is widely accepted as a common abnormality in glycosylation (39,40). If and how this abnormality affects P. aeruginosa attachment in the airway is still widely deba ted. Additionally there is a global reduction in Nglycosylation of glyco-proteins in CF patients (41). To account for this N-glycosylation deficiency an in vitro micro-array study of CF TR deficient IB3 cells revealed a reduction in MPI mRNA expression levels (involved in N-linked glycosylation produc tion) compared to the CFTR corrected S9 cells (42). P. aeruginosa Binding Studies Unfortunately, it is not clear why P. aeruginosa targets the CF airways so effectively. Som e authors have reported that P. aeruginosa adheres better to CFTR deficiency airway epithelial cells than wild type cells vi a an interaction mediated by as ialo-GM1 binding, though others do not report similar results (43,44). Some investigators have demonstrated that CFTR is a receptor for P aeruginosa lipopolysaccharide (LPS) and is deficient in the CF airway (45). Furthermore, i n vitro studies have reported that CFTR plays a role in bacterial ingestion into airway epithelial cells for clearance by desquamation and is reduced by defective CFTR in (46). This demonstrates a cellular clearance deficiency in the CF airway by a re duction in sloughing and

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28 clearing of bacteria-laden epithe lial cells. Further studi es demonstrate that mucins have strong adhesion to FliD, the flagellar cap protein associated with P. aeruginosa by an unknown mechanism (47-49). Additionally, in vitro studies report that this P. aeruginosa attraction to mucins enhances biofilm formation (49,50). MPI (Mannose-6-Phosphate isomerase) Mannose-6-phosphate isom erase (MPI), also known as phosphomannose isomerase (PMI), catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate and plays a critical role in mainta ining the supply of D-mannose derivatives, which are required for most glycosylation reactions (32) Mannose-6-phosphate is a crucial substrate that is used for the production of N-glycans, which are necessary for N-linked glycosylation of glyco-proteins (Figure 1-2). A very rare autosomal homozygous genetic disorder; C ongenital Disorder of Glycosylation 1b is linked to MPI. Patients present with severe loss of n-glycosylation units on serum glyco-proteins analyzed by the levels of Carbohydrate Deficient Transferin (CDT) in the blood. The symptoms are variable but some consis tencies do exist. Patie nts commonly exhibit cyclic vomiting, profound hypoglycemia, failure to thrive, liver fibrosis and protein-losing enteropathy and is occasionally associated with coagulation disturban ces. No neurological involvement has been characterized which is unique amongst the glycosylation disorders (3335). Fortunately, there is a wellconserved pathway for the produc tion of N-glycans independent of MPI by direct phosphorylati on of cellular ingested mannose by hexokinase yielding mannose6-phosphate. Therefore, oral supplement with a hyper-mannose diet is used to treat the MPI genetic disorder in patients with great success (33,34). MPI or PMI is well conserved through the different prokaryot ic and eukaryotic organisms (33). In yeast there is roughly 40% conservation of MPI compared to humans and loss of MPI in

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29 yeast in lethal useless growth is supplemented with mannose (36). In most bacteria, the MPI gene is roughly 30% conserved compared to hum ans and expression levels are severely reduced by the high salt stresses (37). Finally MPI nu ll mice are embryonic lethal at E11.5 (38). MPI Contribution to P. aeruginosa Predilec tion in CF airway MPI deficiencies contribute to reduction in N-acetyl glucosamine; which has adhesion properties for P. aeruginosa It has been demonstrated that MPI down regulation occurs in IB3 cells; A CFTR deficient bronchial epithelial cell line (42). Furthermore, decreased binding to IB3 cells compared to S9 cells that express f unctional CFTR has been demonstrated (46). Additionally, CFTR patients presen t with an increase in carbohydrat e deficient transferin (CDT) in serum (41). Increase of CDT is used as a marker for N-glycosylation deficiency (51). Proposed linkage of MPI down regulation to CFTR deficiency Stress conditions have traditionally played a role in abnorm al expression on a global level. Many intracellular stress conditions exist in CFTR deficient cells such as abnormal intracellular pH levels and increased salt content (52). These stress conditions can play a role in the misregulation in CFTR deficient IB 3 cells (53). As mentioned prev iously, MPI is well conserved through eukaryonic and prokaryotic organisms (54). A focused oligonucleotide microarray study in Lactocccus lactis to profile the expression of 375 ge nes was performed. Among the focused study was PMI (MPI) expression levels. Lactocccus lactis showed a 5-fold decrease in MPI expression under a high salt stress condition (54). Although this maybe tenuous in linking to CFTR deficiency to MPI mis-regu lation it does provide precedence.

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30 Adeno-Associated Viral (AAV) Gene Therapy Adeno-associated virus (AAV) is a parvoviru s with a sm all single strained genome (4.7kb) that consists of two inverted terminal repeats (ITRs) flanking the two genes, rep and cap The rep gene encodes the Rep78, Rep68, Rep52 and Rep40 proteins that provide functions necessary for viral DNA replication. The cap gene encodes the VP1, VP2 and VP3 proteins, which are the structural components of the viral capsid. AAV is naturally defective for replication, and cells generally require co-infection with a helper virus in order to support productive replication of AAV. In the absence of a helper virus co-i nfection, AAV enters a la tent phase, which may include episomal or integrated forms. Wild type AAV2 integration in tiss ue culture is unique in that it is often site-specific on human chromosome 19; the AAVS1 site (55-57). In recent years, naturally occurring variations in the cap gene account for the discovery of many different genomic variants and serotypes of AAV. These variants provide flexibility of AAV as a viral vector by providi ng multiple serotypes of the vira l capsid. These serotypes have different tropisms allowing a wide array of tissu e types to be infected depending on the variant used. AAV2 was the first serotype to be investig ated (58-60). The vast majority of humans are seropositive for AAV2 exposure ye t it has not been c onsistently associated with any human disease (61-64). Typica l rAAV vectors contain only the ITR sequences, providing roughly 4.7 Kb of packaging capacity for the transgene of inte rest and any promoter or expression enhancer. rAAV does not retain the site-speci ficity of integration seen with wt-AAV2 in tissue culture, but rather persists primarily in episomal form (55,56). On a whole, AAV as a viral vector has many a dvantages. With the wide array of serotypes capable of infecting dividing and non-dividing cells there is a library of viral vectors to work with that gives us a foreseeable promise that a ll tissue types can be poten tial targets for viral

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31 transduction with this delivery system. The ab sence of any observed pathology from infection provides solace to safety concerns using viral vectors for gene therapy. Although the packaging limits of the viral genome are constrained, with only 4.7 kb available for the packaging size of the expression cassette, most cDNAs are sm all enough to accommodate the addition of expression enhancing elements w ithout having to modify the cDNA size. Finally, if subsequent infections are necessary different serotypes can be used to evade the immune system. siRNA for Specific Down regulation of mRNA Transcripts The siRNA technique to selectively reduce th e m RNA expression levels of a specific gene has been well established. This t echnique has become so invaluab le that many scientific supply companies have developed siRNA libraries for co mplete coverage of the common genomes used in medical research such as Homo sapiens and Mus musculus (common house mouse). siRNA technique can be used in vitro or in vivo and will knockdown your mRNA of choice to develop a direct relationship to your gene and a diseased or normal physiological ev ent. This technique is also ideal for knocking down genes that are norma lly embryonic lethal when attempts to develop knockout animals have been pursued. siRNA Mechanism Sm all interfering RNAs (siRNA) utilize the natural occurring double stranded RNA interference (RNAi) mechanis ms first discovered in Caenorhabditis elegans (C. elegans) for sequence specific destruction of mRNAs (102). It was found that untranslated micro ds-mRNAs could cause a significant decrease in translated mRNA expression levels compared to a micro ssmRNA. miRNAs (microRNAs) occur naturally in a large range of organisms. Over 3000 miRNAs have been discovered in species rangi ng from plants to humans and are largely involved in embryonic development. Temporal miRNAs expression is critical in embryonic

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32 development to silence genes that, if unregulated, would have the potential to cause deformities during critical points of developm ent(103). It is estimated that 600 miRNAs exist in mammalian cells and that 30% of human gene expr ession is regulated by miRNAs (104,105). miRNAs are small double-stranded (DS) mRNAs us ually 21 nt in length either with a stem loop between the sense and anti-sen se strands to develop the DS structure or with separately expressed sense and anti-sense micro RNAs for DS annealment in the absence of a stemloop. DS miRNA structures are cleaved with dicer and beco me associated with the RISC protein complex. Additionally, the cleaved DS mR NA can associate with the siR NA protein complex. The RISC complex with the DS siRNA has the potential to introduce these small fragments of DS miRNA to target mRNAs for antisense attachment to a small portion of the mRNA and the RISC complex cleaves the DS portion of the mRNA causing degradation. The other mechanism of limiting protein levels involves the siRNA protein complex with the DS microRNA stem loop complex. This complex becomes associated w ith the mRNA for translation and inhibits translation without degrading the mRNA. (Figure 1-4 (105)) Developing siRNA Constructs for Expre ssion of siRNA In-vivo and In-vitro We can utilize the naturally occu rring RNAi mechanism to selectively degrade mRNA levels of a target gene by engineering specific siRNAs for our gene of interest and subcloning this siRNA into an expression vector with a spec ial U6 promoter; a naturally occurring promoter involved in the expression of tRNAs. The U6 prom oter is efficiently used for the expression of small untranslated mRNAs. The siRNA will have a sense strand a stem loop and an anti-sense strand that will target our mRNA of interest (figure 1-5).

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33 Figure 1-1. Wild-type CFTR within a membrane. Transmembrane domains (TMDs) span the membrane groups of six. Nucleotide bindi ng domains (NBDs) and the regulatory domain (R domain) lie within the cytoplasm (5).

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34 Figure 1-2. N-glycosylation enzymatic pathway. A: MPI (PMI) converts fructose-6-phosphate into mannose-6-phosphate B: Direct phosphor ylation of external mannose for the production of mannose-6-phosphate i ndependent of MPI (PMI)(33). A B

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35 Figure 1-3. AAV genes, mRNA and proteins. Rep78 VP1 VP2 Rep52 Rep40 Rep68 mRNA

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36 Figure 1-4. RNAi mechanisms. A) Shows duplex of microRNAs that anneal in the absence of a hairpin B) Dicer degradation of DS RNA without a hairpi n to develop ssRNA interference on a specific translated mRNA by anti-sense annealing and mRNA cleavage by the RISC complex. C) DS hair pin precursor for the production of small anti-sense RNA by dicer cleavage for transl ation inhibition but not destruction of mRNA (105).

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37 Figure 1-5. siRNA expression vect or for the selective destructi on of mRNA from any target gene.

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38 CHAPTER 2 PROJECT GOALS Verify N-glycosylation Deficiency My dissertation began with a claim that the MPI deficiency in CF TR deficient airway epithelial cells generates an overall deficiency in the N-glycosylation profile on the membrane surface and contributes to abnormal bacteria adhesi on. The first step in this study was to verify that N-glycosylation deficiency plagues the epithe lial profile in CFTR deficient airway epithelial cells. N-glycosylation Abnormalit y Contributes to B acterial Clearance Deficiency Abnormal adherence of P. aeruginosa is a widely accepted consequence of CFTR deficiency. Abnormal glycosylation is one of the agreed upon contributors to the abnormal binding. Unfortunately, the exact glycosylation abnormality has yet to be agreed upon, although reduced sialic acid residues, increased fucosylatio n and an increase in asialoGM1 have been well documented in CF there is still some dispute if these are the sources of abnormal binding. With the discovery of N-glycosylation deficiencies in CFTR defective airway epithelial cells in vitro I had a candidate that may contribute to abnormal bacterial adhesion. A decrease in bacteria adhesion, ingestion and subsequent clear ance by desquamation of ai rway epithelial cells has been previously revealed. I suggest that the N-glycosylation abnormality contributes to these deficiency in bacterial adhesion. Th e next step of this project is to verify that the decrease in bacterial adhesion by CFTR deficiency, di scovered previously, can be replicated in vitro Additionally, in will develop an in vivo model to demonstrate that this deficiency can be observed in CFTR deficient mice.

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39 MPI and Hyper-mannose Treatments for Deficiencies The final step of this project will be to corre ct th is bacterial clearance deficiency in cell culture and in the developed mous e model to show a bacterial burde n increase in the CF airways. I am going to show that reversing the MPI defi ciency in a CFTR deficient cell line and the proposed deficiency in CFTR null mice will provide partial or, in the most optimistic outcome, complete correction of the bacterial clearance de ficiency. Additionally, I am going to show that a non-evasive hyper-mannose liquid diet or treatment can provide co rrection of this clearance deficiency independent of MPI correction.

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40 CHAPTER 3 IN-VITRO STUDIES Materials and Methods This m aterial and methods section provides a complete description of all the experiments and the necessary materials to complete the in vitro related project goals. This section will be followed by a description of the expect ed results and the actual results. Verifying N-glycosylation Abnormalities I proposed that the MPI deficiency discovered in CFTR de ficient ai rway epithelial cells contributes to a global N-glycos ylation deficiency in the epithelial membrane profile. These experiments were used to test this theory. Experiments to verify abnormalities Fluorescen ce activated cell sorting (FACS) analysis of fluorescein (FITC) conjugated lectins was used to quantify the N-glycosylation profile of IB3 (CFTR deficient / diseased) and S9 (CFTR corrected / healthy) cell lines. The lectins used to specifically attach to Nglycosylation portion of the membrane bound glyco-proteins were Wheat Germ Agglutinin (WGA) and Concanavalin A (Con A), which are common lectins to analyze N-linked glycosylation (65-67) IB3 and S9 cells were grown in T-75 tissue cu lture flasks with 15mls of epithelial cell culture media until 75% 90% confluency was ac hieved. Normal growth conditions were used for all cell culture experiments: 37 C with 5% carbon dioxide (CO2) in a VWR signature water jacketed incubator. The growth media utilized for all the in vitro studies (unless otherwise noted) was LHC-8 media from Invitrogen with 5% fetal bovine serum (FBS) and 1% penicillin and streptomycin antibiotic mixt ure both obtained from CellGrow incubated at 37C to warm

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41 media prior to adding. The cells were detached by removing growth media, adding 1ml of Trypsin-EDTA solution from CellGrow and incubating at 37 C for 10 minutes. The detached cells were collected in 5mL of growth media in a 15ml conical tube The cell suspension was centrifuged at 4 C for 8 minutes at 650 gs. The supernat ant was poured off and the cell pellet was washed with 5mls of room temperature Phosphate Suffered Saline (PBS) and centrifuged using the same condition. Again, the supernatant was poured off and the cell pellet was resuspended in 5ml of growth media. 5.0e5 cells were added each well of a 6-well cell culture plate and growth media was added to a final vol ume of 2ml. The cells were incubated for 48 hours to reach optimal confluency. After the cell growth reached optimal conflu ency, n-glycosylation profiling was performed using a common FITC conjugated lectin staining procedure. The staining measurements were collected by fluorescent detecti on. As a control for background fluorescence by FACS analysis some IB3 and S9 cells were blocked with unconjugated lectins prior to adding the FITC conjugated lectins. To block for background cont rol 1ml of 100 ug/ml of either unconjugated WGA or Con A in Pharmingen Stain Bu ffer (BSA) from BD Biosciences was added to each well and incubated at 37 C for 1hr. After the blocking period blocked and unblocked epithelial cells were stained with FITC-conjugated lectin s by adding 1ml of 50ug/ml of either FITCconjugated WGA or Con A to either blocked or unblocked cells and incubating at 37 C for 1hr. Following these incubations, the staining solution was removed and 1ml of just stain buffer was added to the cells. The cells were mechanically det ached from the surface of the culture plates by using a cell scrapper and collected in the 1ml of stain buffer. Finally, quantification of FITC stained cells was performed by using FACS analysis to obtain a percentage of stained cells using the FL 1 fluorescent detector channel. FITC stain uses

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42 Fluorsecein molecules that ar e excited at 494 nm and emit fluorescence at 512 nm. FACS analysis of the stained cells was performe d on a 4-color Becton Dickinson FACS caliber machine which utilizes 2 lasers at 488 and 635 nm to excite fluorescence and 4 fluorescent detectors at 530 15 nm (FL1), 585 21 nm (FL2), 670 nm (FL3), 661 8 nm (FL4) (see Figure 3-1). To determine that the abnormal glycosylation pr ofile of the CFTR deficient epithelial cells was confined to the N-linked glycosylation units staining using an O-glycosylation lectin was performed. The previously described staini ng procedure was performed using Soy Bean Agglutinin (SBA); a common lectin used to anal yze O-linked glycosylation (65). FACS analysis proceeded the IB3 and S9 staining with FITC SBA to analyze any O-linked glycosylation changes. Analysis of Bacterial Adhesion Bacteria adh esion analysis is the first step in bacterial ingestion and subsequent clearance by the host cell. I proposed that th is first stage mechanism of b acterial adhesion is reduced in CFTR deficient airway epithelial ce ll followed by reduced clearance. Experiments to test abnormal adhesion IB3 and S9 cells were utilized to test the adhesion of P. aeruginosa strain PAO1. IB3 and S9 cell were grown first in T-75 and 6-well plates as previously described. Additional wells were used for counting the amount of IB3 and S9 cells present in each well of the 6-well plate after 48 hours of growth to determ ine the correct amount of PAO1 colony forming units (CFU) used for incubation with the IB3 and S9 cells to analyze adhesion. After the 48 hours of growth of 5.0e5 IB3 and S9 cells in a 6-well tissue culture plate, 20X multiplicity of infection (MOI) of PAO1 in 1m l of warm Opti-Mem media was added to each

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43 well and incubated at 37 C for 1 hour. After the 1hr infect ion period the infection media was removed and the wells were rinsed twice of any free floating PAOI bacterium that did not attach using 2ml of warm Hanks media. Collection of th e attached and ingested bacteria was performed by adding 500ul of lysis buffer with 1.0% IPE GAL (NP40 substitute) in 100 mM NaCl, 100 mM Tris-Cl pH 8.0 and 25 mM EDTA pH 8.0. This ly sis buffer selectively de stroyed the host cells, while the bacteria remained viable. The isolated bacteria was diluted and plated on ampicillin selection plates for counting as follows. First, 100ul of the 500ul lysis buffer samp le with the collected bacteria was added to 900ul of tryptic soy broth and 1:10 serial dilutions were made. Diluted bacteria were plated on selective tryptic soy bacterial growth plates with 100 ug/ml of ampicillin for selection of P. aeruginosa, which is naturally resistant to ampicillin. The dilutions used were 1:1000, 1:3300, and 1:10,000. The bacteria were grown to form colonies overnight at 37 C and the colonies were counted the next day. Multiplying by the dilution f actors gave the total nu mber of bacterial CFU that was collected from the IB3 and S9 cells. A pe rcentage of attached bacteria was obtained by dividing this number by the to tal amount of bacteria CFU incubated during infection. Verify that N-glycosylation Contributes to PAO1 Binding A link would have to established between the proposed N-glycosylation deficiency in the CF cell line and the proposed reduction in PAO1 binding. This link would dem onstrate that the n-glycosylation deficiency has an over all consequence of host cellular death in vitro as a mechanism for clearance that would mimic desqua mation in the lung epithelium (26). To prove the concept that N-glycosylation contributes to PAO1 adhesion, the n-glycosylation profile was blocked with WGA or Con A lectin prior to infecting with PAO1.

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44 Experiment to block PAO1 attach ment using lectins This experiment was performed the same as th e previously described protocol to analyze bacterial adhesion with the exceptio n of a dose dependent pre-treatm ent with lectins to block the attachment of PAO1 CFU. Prior to adding 20X MO I of PAO1 I incubated IB3 and S9 cells for 1 hr at 37 C with 1 ml of Opti-Mem containing either no lectins (as a positive control) or 500ng/ml, 5ug/ml and 50ug/ml of either Con A, WGA or SBA lectin. Con A and WGA will block N-glycosylation and SB A will block O-glycosylation. Analysis of Ingested Bacteria and Clearance I had to further develop the story of n-glycos ylation deficiency contributing to a reduction in bacterial clearance in the CF TR deficient epithelial cells. The next experim ental procedures would be to analyze ingestion of bacteria by the host epithelial cells. This would be followed by measuring the clearance of the in ternalized bacteria after a 4hr period of intra-cellular activity on the ingested bacteria. My proposal was that, initially, there is d ecreased number of bacteria CFU ingested by the CFTR deficient IB3 cells compared to the CFTR corrected S9 cells. Additionally, I theorized that after 4 hours of intra-cellular activity on the ingest ed bacteria, there would be an increased number of bacterium in the IB3 cells compared to the S9 cells. Experiments to measure intra-cellular bact eria before an d after clearance period Fortunately, two experimental-f riendly protocols could be es tablished in the lab for the purpose of analyzing intra-cellular bacteria before and after the clearance period. Therefore, two experimental procedures could be performed that focused on the proposed IB3 cell deficiency in bacterial ingestion and subsequent clearance. The first experiment al procedure involved counting the amount of intra-cellular bacteria by plating th e CFUs collected from the host cell on selective

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45 plates, similar to the previously mentioned protocol for analyzi ng bacteria adherence. The second experimental procedure utilized the same type of experimental design. The difference would be in the use of fluorescent detection as means to measure the intra-cellular bacteria. This procedure utilized a PAOI strain expressing the green fluorescent protein (GFP) (68). GFP fluorescence from the ingested bacteria could be analyzed by FA CS analysis of the host cells after ingestion of the GFP expressing bacteria. The first protocol (called the CFU counting protocol) was sim ilar to the bacteria adhesion protocol. IB3 and S9 cells were grown in 6-well culture plates starting at 5.0e5 cells and grown for 48 hours. After the 48 hours of growth, the cells were infected with 40X MOI of PAO1 strain for 1hr in 1ml of Opti-Mem. The infection medi a was removed and the cells were rinsed twice with Hanks media. Following the rinsing procedure 300ug/ml of gentimicn was added to the growth media of the infected IB3 and S9 cells for 1 hours at 37 C to kill the extra-cellular bacteria that was not ingested af ter the 1 hour infection. After the a ntibiotic treatment 500ul of the previously described lysis bu ffer was added to the IB3 and S9 cells to collect only ingested bacteria from the host cells. The collected PAO1 CFU was cultured and analyzed as previously described in the bacteria adhesi on protocol. This portion of the experiment gave me the total ingested CFU prior to the clearance period To get the data for intra-cellular CFU following the clearance period I added a 4 hour clearance period after the antibiotic treatment period following the infection/ingestion period. Briefly, side-by side e xperiments were performed with IB3 and S9 cells grown for analysis of intra-cellular CFU prior to and after the clearance period. The CFU ingested after the clearance period was analyzed by allowing intra-cellular activity to continue fo r four hours following the antibiotic treatment period with gentimicin. The IB3 and S9 cells with intra-cellu lar bacteria were incubated for 4 hours in growth media with 300

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46 ug/ml of gentimicin. The gentimicin during the clearance period was to kill any viable bacteria that were released from dead or dying host ce lls to minimize re-infection the remaining viable host cells. This clearance period followed infection and antibiotic death of extra-cellular bacteria. After the clearance period intra-cellular bacterial CFU was collected as previously described with 500ul of lysis buffer. The collected bacteria were grown and analyzed using the same methods to analyze bacteria adherence. The green fluorescent protein (G FP) is excited by the same FACS laser that excites Fluorescein (FITC) and emits fluorescence that is absorbed by the FL1 fluorescent detector in 4color FACS caliber flow cytometr y machine from Becton Dickinson A quantitative fluorescence measurement could be collected using the P. aeruginosa strain that expresses GFP. Host IB3 or S9 cells that have ingested GFP expressing bacteria could be sent through the flow cytometer and the GFP intensity of these host ce lls could be measured to provide comparative study between disease (IB3) and healthy (S9) ce lls. A PAO1 strain with a GFP expressing plasmid was obtained from Roberto Kolter at Ha rvard Medical School (Fi gure 3-2) (68). This strain produces a high intensity GFP P. aeruginosa pathogen (see Figure 3-3). The bulk of the procedure to analyze GFP fl uorescence from host cell ingestion of PAO1 expressing GFP is the same as the procedure to analyze the amount of PAO1 CFU in the host cells before and after the clearance period To refresh, IB3 and S9 cells were grown in 6-well plates and infected the host cells with G FP expressing PAO1 at 40X MOI for 1hr at 37 C. After the infection period the host cells were treated with gentimicin to kill the remaining extra-cellular bacteria that did not get ingest ed. This was followed by the 4hr clearance period during which the host cells containing intracellular GFP expressing PAO1 ar e incubated with gentimicin

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47 growth media to kill any viable b acteria that get released from dead or dying host cells so that these bacteria cannot be re-digested by surviving host cells (Figure 3-28). Collecting the host cells for GFP fluorescen ce by FACS analysis differs from the procedure to count CFU on bacteria l plates. Cells were collected at two stages of the procedure. Cell group 1, which is the initial analysis of ingested GFP expressing PAO1 prior to the clearance period was collected after the antibiotic period and before the clearance period Cell group 2, which analyzes the change in ingested GFP expressing PAO1 and is collected after the 4 hour clearance period With the data from the two groups we can analyze the initial amount of GFP expressing CFU ingested into the host cell and the change in the amount of intra-cellular GFP expressing CFU after the clearance period. To collect the host cells, 100ul of TrypsinEDTA was added to the cells after the growth me dia was removed to detach the cells from the surface of the plate. The detach ed cells were collected in 1 mL of Pharmingen Stain Buffer (BSA) from BD Biosciences This GFP fluorescent host cell suspension was analyzed by FACS analysis and the GFP fluorescent was meas ure by the FL1 fluorescent channel on the flow cytometry machine. A background control is required to measure auto-fluorescence from bacteria and host cell interact ion. The background control used was normal PAO1 strain, which was added to the group of host cells used to establish background fluorescence instead of the GFP expressing PAO1 strain. Analysis of Host Cellular Deat h to Show Clearance Deficiency Previously, experiments have been designed to show that there is a N-glycosylation deficiency in the CFTR defective IB3 cells with a Mannose-6-phosphate (MPI) deficiency compared to S9 cells and that this deficiency l eads to a decrease in ba cterial binding, ingestion and a deficiency in bacterial clearance from th e host cells. To finalize this story I needed to

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48 analyze host cellular death as a mechanism for ba cterial clearance. My theory was that the IB3 CFTR deficient airway epithelia l cell with abnormal MPI regulat ion would show a reduction in host cellular death afte r bacterial ingestion. Experiment to analyze host cellu la r death after PAO1 ingestion This procedure was the same as the proce dure to determine ingested GFP expressing PAO1 into the host cells by anal ysis of GFP fluorescence from IB 3 and S9 cells with ingested bacteria. The GFP expressing PAO1 strain was used to infect those cells for 1 hr. The infection period was followed by the gentimicin antibiotic period to kill undigested b acteria and then one group of host cells was collected for initial analysis of cell death prior to the clearance period Finally, the host cells with intra-cellular bacteria were collected after the clearance period for analysis of the change in host cellular death after the clearance period The collection of the host cells was the same as previously described fo r analysis of intra-cel lular bacteria by FACS analysis. Briefly, the host-cells we re detached from the surface of the culture plate and collected in 1ml of Pharmingen Stain Buffer (BSA) from BD Biosciences After collection the cells were stained by the large molecule nucleic acid stai n 7AAD. 10ul of the 7AAD stain solution from BD Biosciences was added to the host cells suspended in 1ml of from Pharmingen Stain Buffer (BSA) from BD Biosciences and incubated at room temperat ure for 10 minutes in the dark. The cells were centrifuged for 8 minutes at 650 gs to collect the cell pellet. The cell pellet was resuspended in 1ml of Pharmingen Stain Buffer (BSA) from BD Biosciences and this cell suspension was used for FACS analysis of both GFP and 7AAD fluorescence. A background control was established by staini ng non-infected IB3 and S9 cells. 7AAD is used as marker for early and late cellular death. 7AAD is a large molecule with native fluorescence. Much like Propidium iodide, 7AAD can only penetrate th e cell membrane and stain the DNA when the

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49 membrane is compromised by pores that occur during programmed cellular death. 7AAD fluorescence is detected at 647 nm by the FL3 fl uorescent detector on the 4-color FACS caliber machine from Becton Dickinson Ensuring that gentimicin during clearance period is not therapeutic The 4hr clea rance period previously described was done in the presence of gentimicin in hopes of preventing re-infection of viable bacteria liberated from dying or dead host cells. Unfortunately, it was discovered that there was a po ssibility that this treatment could contribute to any differences observed in bacterial load w ithin the host cells when comparing the diseased (IB3) cells to the healthy (S9) cel ls. S9 cells could be more sensitiv e to gentimicin than IB3 cells and decrease the amount of intra-cellular bacter ia in S9 cells by an antibiotic mechanism not a host cellular death mechanism. To rule out this possibility, the same ingestion and clearance experiments previously desc ribed were performed to an alyze ingested bacteria without the gentimicin during the 4hr clearance period Just as a reminder, the 1hr antibiotic period prior to the clearance period was still performed to ensure that there was not free floating or undigested bacteria prior to the beginning of the clearance period Data was collected as previously described by analyzing GFP fluoresce nce with FACS analysis from GFP positive intra-cellular bacteria in the hos t cells. There was no difference when measuring the intra-cellular bacteria when the cells where incubated without gentimicin during the clearance period compared to the cells treated with gentimicin during the same clearance period (Figure 3-4). Measuring mRNA Expression Levels in T reated and Unt reated IB3 and S9 Cells The foundation of this dissertation was built fr om the discovery that the MPI expression level is significantly decreased in the IB3 cells compared to the S9 cells by Isabel Virella-Lowell M.D, HenryBaker Ph.D, Terence Flotte M.D a nd others (42). Although, th ere is no question to

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50 credibility of this discovery, I wanted to verify this deficiency. Additionally, I wanted to confirm that the IB3 cells transfected w ith MPI, CFTR and the S9 cells treated with the siRNA CFTR indeed show an increase in the CFTR and MPI expression levels (in the transfected IB3 cells) and a decrease in the CFTR expr ession in the S9 siRNA cells. Sybr green real-time PCR to measure expression levels Real-Tim e PCR is used to measure the amount of amplified product af ter each cycle using fluorescent detection. Syber green is a dsDNA dye that binds to all DS products. A comparative analysis of mRNA levels from treated samples can be compared to untreated samples when normalized to the expression of a housekeeping gene that remains unchanged when comparing the treated and untreated samples. This analysis will return a fold change between untreated and treated samples. The first step was to isolate the total mRNA from the samples to analyze. This was performed using the mRNA mini extraction kit from QIAGEN Following the extraction of the procedure the total mRNA profile was converted to a total cDNA profile using the Quantitect reverse transcription kit from QIAGEN The final step was to specifically amplify the cDNAs of interest using cDNA specific pr imers. Along with this amplifi cation step is the addition of Syber green detection dye for meas uring the levels of the amplif ied cDNA at each cycle. This final step was achieved by using the Sybr Green Quantitect PCR kit from QIAGEN and predesigned primers from QIAGEN that when engineered for use with the Sybr Green PCR Kit. The PCR portion of this expe riment was performed using the 7700 ABI sequence detection system using the standard conditions that were provided with the PCR kit. Now the focus shifts to analyzing the data colle cted from this procedure. To understand the methods to analyze the data there has to be a di scussion about how the raw data is presented. The

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51 common amplification curve begins with the exp onential phase usually from cycle 1 to cycle 15 then the linear phase usually from cycle 16 to 35 and the plateau phase at the end. The fluorescent intensity in the linear phase of the amp lification is region of the real time PCR curve that is used to collect the data that will be anal yzed. In this phase there is a linear increase from one cycle to next and any differe nces in mRNA levels when comp aring experimental and control samples can be extracted by a simple mathema tical calculation. The known data points required for this mathematical calculation to determine the fold differences between experimental and control group are the ct values from the targeted mRNA and the selected housekeeping mRNA. The ct value is the cycle in which the amplifica tion product reaches a selected fluorescent intensity with in the linear phase of the curv e. The first calculation is to determine the ct between either the control or experimental targeted ct value and the control or experimental housekeeping ct value ( ct= ct of the MPI mRNA from IB 3 untreated ct from housekeeping mRNA in the IB3 untreated). The second calculation is the ct calculation. The ct value is calculated by subtracting the ct control value from the ct experimental value. The fold change is determined by this formula; Fold = 2ct. In-Vitro Treatments Several treatm ents were used to verify that CFTR and down regulation of MPI plays a role in abnormal glycosylation and a resultant clea rance deficiency by IB3 cells. I proposed that reversing either the CFTR or MPI deficiency in IB3 cells would provide therapeutic benefit for the cellular based clearance deficiency by IB3 ce lls. Additionally, I theo rized that IB3 cells grown in mannose rich media would show part ial correction of the clearance deficiency providing a non-evasive therapeutic alternative to gene therapy in CFTR deficient mice (the

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52 presumption was that I would see a similar defici ency in bacterial clearance by an increase of bacterial burden in vivo following a period of b acterial infection). CFTR and MPI correction of IB3 cells was pe rformed by transfecting IB3 cells with an over-expressing plasmid that was later utilized for the production of a rAAV viral vector. For therapeutic delivery, the over-expressing plasmid encoded either full length MPI or a function CFTR mini-gene. For transfecti on controls, the over-e xpressing plasmid encoded either GFP or spinal motor neuron (SMN), which were presumed to have no therapeuti c benefit for bacterial clearance in IB3 cells. A final experiment was performed to demonstrate that decreasing CFTR expression in S9 cells would revert the S9 ce ll to the IB3 state. To accomplish this, a siRNA study was performed using a siRNA expressi ng plasmid to target the CFTR mRNAs. Treatment of IB3 Cells wi th Thera peutic Plasmids The construction of a therapeutic plas mid is necessary for any standard in vitro experiment involving augmentation of a specifi c transgene. For this study an over-expressing plasmid was constructed expressing either a function human CFTR cDNA or a functional human MPI cDNA. A specialized plasmid was used that could transfect cells in vitro and also be used to package the AAV viral vector for gene therapy studies that would proceed the in vitro studies. Constructing the therapeutic over-expression plasmids A backbone for the over-expression of any c DNA for purposes of transfecting cells in vitro and for the production of a rAAV viral vector was pr eviously engineered (69) This very efficient over-expressing plasm id was developed to increase the efficiency of gene therapy transduction using the rAAV viral vectors but could also be utilized for gene augmentation for in vitro studies by direct transfection of the plasmid DNA. The ov er-expressing cassette w ithin this vector was engineered around the AAV2 viral genome. As previously described, the AAV genome is

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53 converted to a viral vector expression cassette for any candidate cDNA that can fit within the viral ITRs by removing all of the viral genes. The ITRs are necessary to stabilize and possibly integrate the vector cassette in the host cells. This vector has a well developed over-expressing cassette that begins and ends with the AAV viral ITRs. This v ector utilizes a multiply cloning site (MCS) for subcloning any desired cDNA pa ired to the CBA prom oter with intron-exon sequences (324 bp) prior to the AT G translation initiation site added to increase expression and finally the SV40 poly adenalation segment. To summarize, the over-expression cassette features are arranged in this fashion; 5-ITR-CBA promoter-intron-exon sequences-MCS-cDNA-Polya tail-ITR-3, with the plasmi d backbone containing the amp resistant gene for ampicillin selection. A viral vector cassett e for expression of a functional CF TR mini gene was previously constructed (70). The key elements in this plas mid are the: chicken beta-actin promoter, intronexon sequences, the 264CFTR mini gene subcloned into the MCS, the ampicillin resistant gene and the origin of replication. This plasmid backbone was utilized for s ubcloning the human MPI cDNA, the GFP and SMN control cDNAs. The pTR2-CBGFP plasmid was previously subcloned and donated to me by Pedro Cruz Ph.D at the University of Florida. This plasmid has been widely used as a control vector for countless transfec tion experiments. The pTR2-CB-SMN control plasmid was constructed by Kevin Foust Ph.D at the Ohio Stat e University. This plasmid was constructed as a potential therapeutic plasmid for the treatment of Spinal Muscular Atrophy (SMA). The function of SMN in motor neurons suggests that SMN expression airway epithelial cells will not affect the targeted clearance mechanisms. For the construction of the MPI over expressi ng plasmid the human MPI was amplied from the carrier vector and subcloned into the pTR2-CB vector I received the human MPI cDNA in

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54 the pCMV-Sport6 plamsid from Invitrogen The first step was to prepare a PCR cDNA fragment of the human MPI transgene to subc lone into the pCR2.1-TO PO TA cloning vector from Invitrogen The PCR2.1 TA cloning vector is supp lied as a linearized plasmid with a single T 5 overhang at both e nds. Attached at the ends of the linearized plasmid is a topoisomerase to ligate the appropriate cDNA in to the plasmid producing a circular plasmid. The critical features of this plasmid are the lacZ ope ron, the F1 and pUC origin of replication sites, the ampicillin resistant gene, the kanamycin re sistant gene and the MCS. To prepare a cDNA fragment for cloning into this ve ctor the 3 ends of the frag ment must contain a single A overhang. To produce such a fragment a PCR re action using human MPI specific primers was performed to amplify the entire coding re gion of the cDNA using the pCMV-Sport6-hMPI plasmid as a template. The polymerase mixture used in the reaction was a triple mix from Eppendorf This triple mix uses a mixture of hi gh fidelity polymerases to reduce point mutations during replication and a taq polyermase to attach a single A to the 3 ends of the fragment. The cDNA of human MPI is roughly 1.3 kb so the addition of a high fidelity polymerase to the taq polymerase was necessary to prevent point mutations. This fragment was gel purified for use in subcloni ng into the pCR2.1 cloning vector. This fragment was cloned into the pCR2.1 plasmid using the standard protocol for TA cloning by Invitrogen Following the cloning reaction 2-5ul of the cl oning reaction was incubated with 50ul of chemically competent TOP10 cells from Invitrogen This mixture was incubated on ice for 30 minutes. Following the 30 minute incubation pe riod, the mixture was heat shocked at 42C for 30 seconds and then immediately transferred it to ice for 5 minutes. SOC media was added to the mixture and incubated at 37 C while shaking at 225 RPMS fo r 1 hr. The solution containing potential ecoli bacteria transformed with the pCR2.1 plasmid was plated onto standard LB

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55 selection plates with 100 ug/ml of am picillin and incubated overnight at 37 C. Any colonies that grew were picked and incubated in 5ml of standard ecoli LB broth with 100ug/ml of ampicillin. The next day 800ul of the culture was used for glycerol storage at -80 C and 2mls were used to purify the plasmid by using the Eppendorf Mini-Prep Kit To verify that the plasmid contained the human MPI cDNA an EcorV digestion enzyme was used to cut in the multiple cloning site a nd the human MPI cDNA. After verifying that the MPI cDNA was present the MPI cDNA was restricted out from the pCR2.1 vector using EcorI and NotI and subcloned it into the pTR2-CB vector with compatible sticky ends. This plasmid was transformed into the SURE2 cells from Stategene that are necessary to prevent recombination by the viral ITRs. The presen ce of the human MPI cDNA was verified by digestion and sequencing. Next, it was necessary to verify that MPI over-expression occurred when a transfection was performed with the pTR-CB-hMPI plasmid. A dditionally, it was necessary to verify that there was a decrease in MPI protei n levels in IB3 compared to S9 cells prior to transfection. IB3 and S9 cells were grown in 6-we ll plates as previously described. After 48 hours of growth the cells were ready for transfection and showed about 75% confluency. Both IB3 and S9 cells were transfected with the pTR2-CB-hMPI plasmid as a potential therapeutic vector or the pTR2-CB-GFP as a tran sfection control to ensure that the transfection was successful and that the transfection pr ocedure does not influence the production of endogenous MPI production. For optimal transfection the cells were grown to 75% confluency. After the cells reached there targ et confluency (typically 48 h ours after plating 5.0e5 cells) we added the transfection media mixed with the pl asmid DNA to move the plasmid DNA into the intra-cellular environment for transcription and translation of the transgene. We used the

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56 standard procedure from Invitrogen to transfect the cells. The procedure requires 10ul of the transfection media Lipfectamine2000 and 4ug of the plasmid DNA. Lipofectamine2000 is a cationic liposome that has a positively charge d head that can bind the negatively charged DNA and a lipid bi-layer to fuse with the cell membrane to move the DNA into the cell. This procedure is the standard procedure used for a ll transfection used through out this study. 10ul of Lipofectamine2000 was added to 250ul of Opti-Mem pe r sample and 4ug of plasmid DNA was added to 250 ul of Opti-Mem. These two mixt ures were combined and incubated at room temperature for 20 minutes. After the incubation period the growth media was removed from the cells in the 6-well plates and the transfection me dia was added to the cells with an additionally 1mL of Opti-Mem and incubated for 4hrs in norma l growth conditions. Af ter the 4hr incubation period the transfection media was removed and 2ml of growth media was added. The transfection media has cytotoxic properties so a longer incubation period with the transfection media may kill the cells. Forty-ei ght hours after transfection the G FP control cells were viewed under a Fluorescence microscope for GFP expressi on (Figure 3-5). The othe r cells are collected for experimental purposes. For western blot analysis of the MPI pr otein in the IB3 and S9 transfected and untransfected treated and untreated IB3 and S9 cells were collected using 100ul of TrypsinEDTA solution to detach the cells from the surface of the culture plate an d collected in 1mL of PBS and centrifuged at 650 gs for 8 minutes at 4 C. The supernatant was poured off and the pellet was resuspended in 200ul of PBS and stored at -80 C until western blot procedure was performed. MPI is an intra-cellular protein so a lysis procedure is necessary for the release of the MPI protein. The cells were thawed at room te mperature and frozen with liquid nitrogen 3 times to lyse the cells. 15ul of the lysis sample was used for western blotting. The Criterion Western

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57 Blot system from Biorad was used for all protein analysis studies. Briefly the sample was prepared by adding 15ul of Laemmle buffer with 0.2% b-mercaptoethanol to 15ul of the protein sample. This sample was incubated at 95 C for 5 minutes. This sample was added to a 12% TrisHCl Criterion gel in 1X Tris\Gly cine\SDS running buffer from Biorad and ran at 150 Volts for 1hr. A protein ladder was added to the gel to determine the size of the detected protein as well as to verify that protein transfer to the me mbrane was successful. The gel was removed and transferred to the blotting membrane using the wet transfer system. This requires a Blotting Sandwich with the membrane facing the anode pl ate and the gel facing th e cathode plate. Prechilled transfer buffer is added to the transfer ap paratus. The transfer buffer was 1X Tris\glycine from Biorad with 20% methanol. The transfer wa s performed at 100 Volts for 60 minutes while mixing the transfer buffer with a frozen bl ock of blue ice to pr event overheating of the transfer procedure and increased electrical resistance. The membrane with transferred proteins was blocked with 5% dried milk solution in 0.1 % PBS Tween solution and shook for 1hr at room temperature. This was followed by primary detection with the MP I antibody (or CFTR antibody). The MPI antibody is not commercially available and was obtained from Hudson Freeze PhD at the Burnham Institute (71). The primary antibody was diluted at 1:500 in 0.1% PBS Tween for 1hr at room temperature and atta ched only if the MPI protein band was present. A horse radish peroxidase (HRP) conjugated seco ndary antibody was used to detect rabbit-IgG from the primary antibody and was diluted at 1:5000 in 0.1% PBS tween and incubated for 1hr at room temperature. Next, chemiluminescence de tection was performed by developing the HRP conjugated 2 antibody using the ECL Detection System from Amersham The membrane with the developed HRP 2 antibody was exposed to Hyper Film ECL from Amersham to detect chemiluminescence. A clear band at the anticipa ted protein mass will appear if the targeted

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58 protein is present. A more intense band indicated a higher level of targeted protein. This western blot procedure showed an increase in intensity from IB3 and S9 cells transfected with pTR2-CBhMPI compared to the cell lines transfected with pTR2-CB-GFP as a control (see Figure 3-6). CFTR siRNA Treatment of S9 Cells to Revert the Health y Cells to Diseased State In the background there is a thorough descripti on of how DS siRNAs can utilize the RNAi mRNA degradation pathway to selectively reduce th e transcripts of a target ed gene to performed knockdown experiments. CFTR in the correcte d S9 cells is a good candidate for a knockdown study. We have proposed that the CFTR de ficiency is involved in the downregulation of MPI mRNA and has an adverse aff ect of the bacterial clearance mechanism by host cellular death and contributes to the chronic lung colonization phenotype in cystic fibrosis. Targeting the CFTR transcripts in S9 cells will revert these CFTR corrected cells back to the their initially diseased state and will mimic the results obta ined from the CFTR deficient IB3 cells. CFTR siRNA construct A universal siRNA construct was previously engineered by Pedro Cruz, P hD., at the University of Florida (107). The construct utiliz es the same AAV expression cassette with 5 and 3 endcaps with some modifications. The CBA promoter was removed and replaced with the strong U6 promoter for efficient expression of small untranslated mRNAs. The MCS site gave me the flexibility to develop a suitable siRNA c DNA with compatible sticky ends for subcloning easily into the universal constr uct. A previously developed hC FTR siRNA sequence was used to target the hCFTR mRNAs. The siRNA targets a 19 bp region 76 bps downstream of the ATG start site using NCBI accession number NM_000492 as a template for blasting (108). The targeted 5 sense sequen ce was GGATACAGACAGCGCCTGG. A sense-stemloop-antisense

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59 oligo was constructed using the AMBION website with the desired sticky ends for subcloning. To make a negative control siRNA the 5 sense sequence for targeting the desired region of the CFTR transcript was scrabbled a nd than blasted against the human ESTs in the NCBI database to verify that no complete sequence alignment occurred. The BamH1 restriction sticky end was used at the sense 5 end and the HindIII restrict ion sticky end at the sense 3 end. The entire siRNA oligo for subcloning included a BamhI Sticky End-Sense Sequence-Stem loop- Anti-sense Sequence-HindIII Sticky End (F igure 3-7). The negative control utilized the same sticky ends and stem loop sequence but the sense sequence was scrambled and a complementary anti-sense sequence was developed from the scrambled sense sequence. Therapeutic CFTR and MPI Correction in IB3 Cells All the exp erimental procedures utilized for the in vitro portion of this study were previously described. The experiments described to this point were to reveal an abnormality in cellular based bacterial clearance of PAO1 P. aeruginosa strain in IB3 cells with a deficiency in both MPI and CFTR protein levels and a global defect in the epithelial profile of N linked glycosylation. Additionally, I proposed that gene augmentation of either a function CFTR minigene or human MPI cDNA in the IB3 cells would ha ve a therapeutic benefit. To demonstrate this claim the therapeutic plasmids would have to be transfected into the IB3 cells for correction of the deficiencies in n-glycosylation, bacterial adhesion, bacterial i ngestion and bacterial clearance by host cellular death. Testing Therapeutic Benefit of Mannose-Rich Media Extra-cellular m annose can be utilized for the production of mannose-6-phosphate independent of MPI. Mannose-6-phosphate is th e substrate for the production of N-glycans. Additionally, it has b een shown that mannose supplement in pmi (mpi) null yeast rescues the

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60 yeast from lethality (54). Moreover, the conge nital disorder of glycosylation 1b (CDG 1b) caused by homozygous mutation of MPI in human is corrected by a hyper-mannose diet (72,73). Having an established precedent of a hyper mann ose treatment supplementing a MPI deficiency an effort to test a non-invasive treatment of ch ronic bacterial colonizatio n was pursued. IB3 cells where grown in variable mannose media as a trea tment for the purposed IB3 deficiencies in a dose dependent fashion. We tested a range of mannose concentrations in the growth media to determine the optimal concentration for therapeutic benefit. The IB3 cel ls were grown in the following concentrations to test correction: 50 nM, 500 nM, 5 uM, 500 uM, 1mM and 50 mM. Mannose rich growth media for the IB3 cells was tested in the N-glycosylation deficiency, reduced bacterial adhesion, ingestion and defect in bacteria l clearance by host cellular death. Knocking down CFTR in S9 cells with siRNA construct S9 cells were transfected as previously described with the pTR2-U6-CFTRsiRNA and negative control constructs. I ve rified that the CFTR protein was reduced in the S9 cells transfected with the pTR2-U6-CFTRsiRNA cons truct compared to the scrambled negative control construct by using the western blotting technique prev iously described. The primary antibody was the commercially available hum an CFTR antibody from Santa Cruz Biotech ; sc20074 and the secondary antibody was an anti-mous e IgG HRP labeled antibody. After verifying that the siRNA successfully knocked down the CFTR protein level the S9 cells were treated with this construct and the N-glycosylation profile was measured. Next, S9 siRNA treatment was tested for deficiencies in the bacterial attachment, ingestion and host cell deat h phenotypes to determine if the data mimicked that obtained from the CFTR deficient IB3 cells. The procedures for these experiments were previously described.

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61 Results Correction of the N-glycosylation Deficiency in IB3 Cells I predicted that there was a deficien cy in Nglycosylation and not Oglycosylation in the IB3 cell line. Additionally, I suggested that CF TR and MPI gene augmentation in IB3 cells by transfection of either pTR2-CB-MPI or pTR2-CB264CFTR with pTR2-CB-SMN as a transfection control will correct the deficiency in N-glycosylation without affecting the O-glycan profile. I also claimed that IB3 cells grown in mannose rich media would provide some correction of this deficiency. The first set of data to test these theories was the N-glycosylation values collected from using FITC conjugated lectins to bind to th e membrane profile in untreated and treated IB3 and S9 cells as previously de scribed using: WGA, C on A and SBA. The data was presented as a percentage of the detected FITC fluorescence from attached lectins. The data from untreated IB3 cells were nor malized to 100% and the data from S9, MPI treated IB3 cells, CFTR treate d IB3 cells, 500uM Mannose treated IB3 cells, siRNA treated S9 cell and SMN treated IB3 and S9 as controls were presented as a percent increase or decrease over untreated IB3 cells (See Figur e 3-8, 3-9 & 3-10). The untreated S9 and S9 cells transfected with the scrambled CFTR siRNA control showed a 1.5 fold increase over the untreated IB3 cells using the FITC conjugated WGA and Con A lect ins while the FITC conjugated SBA showed no significant difference. The IB3 cel ls transfected with MPI or CF TR showed a 1.3 fold increase over the untreated IB3 cells us ing the WGA and Con A lectins while the FITC conjugated SBA lectin showed no significant difference. To con tinue, there was a 1.3 fold increase from IB3 cells cultured in 500mM mannose in rela tionship to the IB3 cells that we re not treated using the FITC conjugated WGA and Con A while the control, FITC conjugated SBA lectin, showed no difference. Finally, S9 cells transfected with the CFTR siRNA showed no significant difference

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62 when measured against the untreated IB3 cells using the WGA and Con A lectins as well as the SBA lectins. Testing or Clearance Deficiency a nd Correction in Untreated IB3 Cells The theory is that the IB 3 cells will show a reduction in bacterial attachment, bacterial ingestion and subsequent clearance by host ce llular death. I developed previously described experiments to test these proposed deficiencies. Included in these designed experiments were the proposed therapeutic conditions to correct thes e suggested IB3 defects. I predicted that transfecting IB3 cells with pTR2-CB-hMPI or pTR2-CB264hCFTR with pTR2-CB-GFP as a transfection control or gr owing IB3 cells in a range of mannose rich growth media would protect the IB3 cells from these disease conditions. Additi onally, I suggested that treating the healthy S9 cells with the siRNA against CFTR will reverted this corrected cell line to the disease state. Binding data and gene augmentation data from MPI and CFTR correction CFU counting data was collected from the bacterial adhesion protocol previously described. T his data includes PAO1 CFU bound to untreated IB3 and S9 cells as well as gene correction data from IB3 cells transfected with pTR2-CB-hMPI or pTR2-CB264 with pTR2CB-GFP as a transfection control. The data from untreated IB3 cells were normalized to 100% and the data from S9, MPI treate d IB3 cells, CFTR treated IB3 cel ls and GFP treated IB3 control were presented as a percent in crease or decrease over untreated IB3 cells. This data shows a 3.0 fold increase in attached PAOI to untreated S9 cells compared to untreated IB3 cells. Additionally, the IB3 cells transfected with CFTR and MPI s howed a 2.0 fold increase compared to untreated IB3 cells (See Figure 3-11 and 3-12).

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63 Mannose treatment of bacterial attachment to IB3 cells CFU counting data was collected from the bacterial adhesion protocol previously described. T his data includes PAO1 CFU bound to untreated IB3 and S9 cells as well as correction data from IB3 cells grown in a range of mannose-rich growth media. The data from untreated IB3 cells were normalized to 100% a nd the data from S9 and IB3 cells grown in mannose rich medias were presented as a percent increase or decrease over untreated IB3 cells. The data showed a bell curve type of data trend with the highest amount of attached bacteria occurring in the mean of the va rying concentrations and the lo west amount of bacteria binding occurring at the lowest and hi ghest concentrations of mannose of bound PAOI to the IB3 cells grown in a range of mannose rich media when co mpared to untreated IB3 cells (See Figure 313). Demonstrate that N-glycosylation is a candidate for PAO1 adhesion De monstrating a proof of concept that N-glycosylation plays a role in PAO1 adhesion is critical for this study. Data was collected from the previously described bacteria adhesion protocol by counting total PAO1 colonies grown on the tryptic soy growth plates and multiplying by the dilution factors. To show that N-glycosyl ation and not O-glycosyla tion plays a role PAO1 attachment the N-glycosylation and O-glycosyl ation profile was blocke d in a dose dependent fashion by treating untreated IB3 and S9 cells with unconjugated WGA, Con A and SBA at varying concentrations prior to infection with 20X MOI of PAO1 to analyze bacteria adhesion. This data showed that S9 cells displayed a decrease in attached bacteria in a dose dependent manner from the WGA and Con A blocking compared to untreated S9 cells while there was no significant change blocking w ith SBA (See Figure 3-14).

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64 Ingestion, clearance and gene augmentation w ith MPI and CFTR by CFU counting These data where collected to show any defi ciencies in the IB3 cell lines when testing bacterial ingestion and clearance compared to th e S9 cell line. Additionally, I wanted to check that there was a therapeutic affect when the IB 3 cells where transfected with the MPI or CFTR over-expressing plasmid. The amount of intra-cellu lar bacterial CFU was counted after the 1hr infection/ingestion period and 1hr antibiotic period to establish the initial amount of ingested bacteria. Additionally, the intra-cellular bacterial CFU was measured after the clearance period that follows the antibiotic period and collected as the clearance da ta and compared to the initial ingested CFU. This data was used to compare th e initial ingested bacteria to the intra-cellular bacteria after the clearance period to verify a clearance deficiency in the IB3 cells compared to S9 cells. Therapeutic treatment was included in this data by transfecting IB3 cells with pTR2CB-hMPI and pTR2-CB264hCFTR with pTR2-CB-GFP as a transfection control. The data from untreated IB3 cells were nor malized to 100% and the data from S9, MPI treated IB3 cells, CFTR treated IB3 cells and GFP treated IB3 control were presented as a percent increase or decrease ove r untreated IB3 cells. Untreate d S9 cells showed a 1.7 fold increase in initial ingested b acteria compared to untreated IB 3 cells. The therapeutic treatment showed a 1.5 fold increase of the initial ingested bacteria in IB3 cells transfected with MPI as well as the CFTR mini-gene when measured ag ainst untreated IB3 cells. When analyzing the clearance data the amount of intra-cellular bacteria in S9 cells reduced to half after the clearance period while the intra-cellular bacteria almost doubled in the IB3 cells af ter the same clearance period. The IB3 cells transfected with the CFTR mini-gene showed a 30% reduction in the intracellular bacteria and the IB3 cel ls transfected with the MPI pl asmid showed a 20% reduction in intra-cellular bacteria af ter the clearance period (See Figure 3-15 and 3-16).

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65 Mannose treatment of bacterial inge stion and clearance in IB3 cells CFU counting data was collected from the b acte rial ingestion and clearance protocol previously described. This data includes PAO1 CFU ingested by untreated IB3 and S9 cells as well as correction data from IB3 ce lls grown in a range of mannoserich growth media. The data from untreated IB3 cells were nor malized to 100% and the data from S9 and IB3 cells grown in a range of mannose rich medias were presented as a percent increase or decrease over untreated IB3 cells. The data for the initial intracellular bacteria in the IB3 cel ls grown in a range of mannose rich media showed the same bell curve trend seen in the data for analyzing attachment previously mentioned of the compared to the un treated IB3 cells. When analyzing the data after the clearance period there was no change in the in tra-cellular bacteria in the IB3 cells grown in all the mannose concentrations te sted while the bacteria almost doubled in the untreated IB3 cells (See Figure 3-17). Ingestion and clearance from MPI and CFTR transfection by fluorescent detection This data was used to confirm the ingestion and clearance data co llected from the CFU counting protocol. Utilizing the data from GFP expressing PAO1 ingestio n protocol by FACS analysis I could confirm the data collected from the CFU counting protocol in regards to the initial bacteria ingestion and subsequent clearance. This da ta was collected by fluorescent detection from the flow cytometer by using host ce lls with ingested GFP expressing bacteria at the same periods as the CFU counting protocol. To refresh, untreated IB3 and S9 cells with ingested GFP expressing PAO1 where measured for initial ingested bacteria as well as the intracellular GFP expressing bacteria after the cleara nce period by FACS anal ysis. Additionally, IB3 cells where transfected eith er pTR2-CB-hMPI and pTR2-CB264hCFTR with pTR2-CB-SMN

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66 as a transfection control. The data from untreat ed IB3 cells were normalized to 100% and the data from S9, MPI treated IB3 cells, CFTR treat ed IB3 cells and GFP treated IB3 control were presented as a percent increase or decrease over untreated IB3 cells. Untreated S9 cells showed a 1.9 fold increase in initial ingested b acteria compared to untreated IB3 cells. The therapeutic treatment show ed a 1.7 fold increase of the initial ingested GFP expressing bacteria in IB3 cel ls transfected with MPI and a 1.5 fold increase in the initial ingested GFP expressing bacteria in IB3 cells transfected with the CFTR mini-gene when measured against untreated IB3 cells. When anal yzing the clearance data the amount of intracellular bacteria in S9 cells reduced to half after the clearance period while the intra-cellular bacteria almost doubled in the IB3 cells after th e clearance period. The IB3 cells transfected with the CFTR mini-gene showed a 30% reduction in th e intra-cellular bacteria and the IB3 cells transfected with the MPI plasmid showed a 40% reduction in intr a-cellular bacteria after the clearance period (See Figure 3-18 and 3-19). Mannose rich media therapy for IB3 cells FACS data was collected from GFP fluorescen ce by ingested GFP+ PAO1 CFU in the host cells following the bacterial inges tion and clearance protocol that was previously described. This data analyzed the fluorescent intensity dependi ng on the amount of GFP+ PAO1 ingested by untreated IB3 and S9 cells as well as GFP fl uorescence from intra-cellular GFP+ PAO1 in IB3 cells grown in a range of ma nnose-rich medias. The data from untreated IB3 cells were normalized to 100% and the data from S9 and th e IB3 cells treated with mannose rich medias were presented as a percent increase or decr ease over untreated IB3 cells. The data from the initial intra-cellular bacteria in the IB3 cells grown in a range of mannose rich media showed an increase in the median range of the varying ma nnose concentrations compared to the untreated

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67 IB3 cells. When analyzing the da ta after the clearance period ther e was a range of almost double the amount of intra-cellular bacteria to no change in the intra-cellular bact eria in the IB3 cells grown in variable mannose rich media while the bacteria almost doubled in the untreated IB3 cells (See Figure 3-20). Decrease and Correction of Host Cellular Death as a Mechanism for Clearance This data was collected to analyze ho st cellular death as a mechanism for bacterial clearance that mimics in vivo epithelial desquamation. Data was collected from the fluorescence of a large molecule nucleic acid stain; 7AAD by the previously describe d protocol designed to determine the percentage of host cells in an apoptotic state after the infection/ingestion and clearance period of GFP + PAO1. I predicted that there is a deficiency in host cellular death after the infection/ingestion and clearance period by untreated IB3 cells compared to S9 cells. FACS analysis data of fluorescence from the 7A AD staining protocol was collected to confirm this deficiency. Additionally, I predicted that transfecting IB3 cells with pTR2-CB-hMPI and pTR2-CB264hCFTR with pTR2-CB-GFP as a transfection c ontrol and growing IB3 cells in a range of mannose rich growth medias would protect the IB3 cells from this deficiency. Gene augmentation data from MPI and CFTR correction FACS data was collected from 7AAD fluor escence of host cells after ingestion and clearance of GFP+ PAO1 CFU in the host cells using the 7AAD staining protocol previously described. This data analyzed the fluorescent intensity of 7AAD depending on the apoptotic state of the population of host cells after the infection/ingestion and clearance period of GFP+ PAO1 ingestion by untreated IB3 and S9 cells as well as 7AAD fluorescence from the population IB3

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68 cells transfected with pTR2-CB-hMPI or pTR2-CB264 with pTR2-CB-SMN as a transfection control. The data from untreated IB3 cells were nor malized to 100% and the data from S9, MPI treated IB3 cells, CFTR treated IB3 cells and GFP treated IB3 control were presented as a percent increase or decr ease over untreated IB3 cells. The S9 untreated cells showed a 1.7 fold increase in cellular death compared to the unt reated IB3 cells. Additionally, the MPI corrected IB3 cells showed a 1.5 fold increas e in cellular death compared to the untreated IB3 cells while the CFTR corrected IB3 cells had a 1.6 fold incr ease in cell death (See Figure 3-21 and Figure 322). Mannose rich media therapy for IB3 cells FACS data was collected from 7AAD fluor escence of host cells after ingestion and clearance of GFP+ PAO1 CFU in the host cells using the 7AAD staining protocol previously described. This data analyzed the fluorescent intensity of 7AAD depending on the apoptotic state of the population of host cells after the infection/ingestion and clearance period of GFP+ PAO1 ingestion by untreated IB3 and S9 cells as well as 7AAD fluorescence from the population IB3 cells grown in a range of ma nnose rich medias. The data from untreated IB3 cells were normalized to 100% and the data from S9 a nd IB3 cells grown mannose rich media were presented as a percent increase or decrease over untreated IB3 cells. There was a dose dependent increase of cellular death in th e IB3 cells treated with a range of mannose media compared to the untreated IB3 cells (See Figure 3-23). siRNA Treatment of S9 cells to Verify CFTR Role in Clearance Deficiencies The experim ental designs to test for a bacteria clearance deficiency in the IB3 cells have been previously described. Additionally, potential therapeutic benefit was characterized using

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69 CFTR and MPI correction or rich mannose treatment independent of MPI. Now, efforts to bring another dimension to this well developed experi mental story will be tested. The CFTR siRNA construct engineered to target human mRNA transcripts was previ ously described. This construct was developed to show that a CFTR correct ed cell line under the previously described experimental parameters: N-glycosylation, bact erial adhesion, bacteria l ingestion, bacterial clearance and host cell death could be reverted to a disease state by knocking down CFTR expression. S9 cells were transfected with the pTR2 -U6-CFTRsiRNA or the pTR2-U6-scrambled CFTR-siRNA control. These tran sfected S9 cells were sent th rough the series of previously described experiments to test all the parameters of cellular based bacterial clearance. These transfected cells were compared against S9 unt reated (normal) and IB 3 untreated (diseased) cells. The data from untreated IB3 cells were normalized to 100% and the data from S9 untreated, S9 CFTRsiRNA and S9 siRNA negative control were presented as a percent increase or decrease compared to untreated IB3 cells. In all aspects the S9 cells treated with the CFTR siRNA plamsid showed a reversion to the untr eated IB3 state (See Figure 3-24 3-27). Testing CFTR and MPI mRNA Levels in T reated and Untreated IB3 and S9 cells Theoretically, the MPI and CFTR expression pa tterns are going to be variable depending on whether or not the IB 3 and S9 cells are treated or untreated. It is critical to verify this proposed variability to ensure that differences se en in the treated and untreated IB3 or S9 cells when testing the parameters of bacterial clea rance is influenced by MPI and CFTR expression levels are paralleled wi th expression changes. I anticipate an increase in the MPI expression from the IB3 cells transfected with pTR2CB-hMPI and untreated S9 cells when compared to untreated IB3 cells Additionally, I would

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70 expect to see a modest fold increase in MPI e xpression from IB3 cells transfected with pTR2CB264hCFTR and only a small fold increase fro m S9 cells transfected with pTR2-U6CFTRsiRNA. The CFTR expression pattern predic tion is an significant increase from IB3 cells transfected with pTR2-CB264hCFTR and a significant decrease fr om S9 cells transfected with pTR2-U6-CFTRsiRNA when compar ed to IB3 untreated cells. MPI expression pattern Syber green m easurements were taken fr om MPI mRNA levels in untreated S9 and untreated IB3 cells as well as IB3 cells transfec ted with MPI or CFTR and IB3 cells grown in 500uM mannose. Additionally, MPI mRNA levels were measured from S9 cells transfected with CFTR siRNA. The methods for this proce dure where previously described using GAPDH expression as the house keeping re ference gene. The data was pres ented with untr eated IB3 cells normalized to a fold value of 1 (no change) whil e the other values collected from the untreated and treated S9 cells and treated IB3 cells where shown as a fold increase or decrease compared to the untreated IB3 cells. The untreated S9 cells showed a 7 fold increase of MPI expression levels compared to untreated IB3 cells at cycl e 20 while the S9 siRNA treatment showed only a 2.5 fold increase over the untreated IB3 cells. Add itionally, the CFTR treated IB3 cells showed a 3.5 fold increase of MPI expression level and the MPI treated IB3 cells had a 7.2 fold increase compared to untreated IB3 cells. All other trea tments; controls and mannose showed no change (Figure 3-29). CFTR expression pattern Syber green m easurements were taken from CFTR mRNA levels in untreated S9 and untreated IB3 cells as well as IB3 cells transfec ted with MPI or CFTR and IB3 cells grown in

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71 500uM mannose. Additionally, CFTR mRNA levels were measured from S9 cells transfected with CFTR siRNA. The methods for this pro cedure where previously described using GAPDH expression as the house keeping reference gene. The methods for this procedure where previously described using GAP DH expression as the house keeping reference gene. The data was presented with untreated IB 3 cells normalized to a fold va lue of 1 (no change) while the other values collected from the untreated and tr eated S9 cells and treated IB3 cells where shown as a fold increase and decrease compared to the untreated IB3 cells. Th e IB3 cells transfected with the CFTR mini-gene showed a 8.6 fold incr ease of CFTR expression levels compared to untreated IB3 cells at cycle 20. Additionally, th e S9 siRNA treatment showed an 80% reduction of CFTR mRNA compared to the untreated IB3 cells. A ll other treatment type s and the untreated S( cells showed no change in the fold change (Figure 3-30).

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72 Figure 3-1. Pictorial representati on of the FACS detectors, lase rs and commons stains using a 4-color flow cytomery machine (This image is not subject to copyright).

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73 Figure 3-2. Construction of GFP ex pression plasmid for expression in P. aeruginosa strain PAO1 (68).

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74 Figure 3-3. Confocal image of GFP+ P AO1 ingestion in IB3 cells (green). 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 S9 w/GenS9 w/o GenIB3 w/GenS9 w/o gen% Change of ingested GFP + PAO1 Initial Change Figure 3-4. Verifying that the gentimicin treatment during the clearance period of ingested GFP+ PAO1 in IB3 or S9 cells does not cont ribute to bacteria clearance in S9 cells or contribute to the clearan ce deficiency of IB3 cells.

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75 Figure 3-5. GFP expression in IB3 cells as an indicator of successful transfection. Figure 3-6. Western blot of hMPI from S9 a nd IB3 cells transfected with the therapeutic plasmid pTR2-CB-hMPI or the control plasmid pTR2-CB-GFP.

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76 Figure 3-7. siRNA oligo to subc lone into universal siRNA expr ession construct for selective knockdown of CFTR mRNA transcripts. 90.0 100.0 110.0 120.0 130.0 140.0 150.0 160.0 170.0 180.0 190.0S9 Untrt S9 Contr IB3 untrt IB 3 S M N S 9 siRNA IB3 2 7/264hCFTR IB3 hMPI I B 3 500 u M mann os e% Bound ConA Figure 3-8. FITC conjugated Con A lectin binding to N-glycosyl ation profile of untreated S9 cells and untreated and treated IB3 cells.

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77 80.0 90.0 100.0 110.0 120.0 130.0 140.0 150.0 160.0 170.0 180.0S 9 Untrt S 9 Contr IB 3 untrt IB3 S MN S 9 siRNA I B 3 27/26 4hCFT R IB 3 hMPI IB3 500 uM m an nos e% change of WGA binding Figure 3-9. FITC conjugated WGA l ectin binding to the N-glycosyl ation profile of untreated S9 cells and untreated and treated IB3 cells. 50.0 60.0 70.0 80.0 90.0 100.0 110.0 120.0 130.0 140.0S 9 U n t rt S 9 C o n t IB3 u ntr t IB3 SM N S 9 s iRN A IB3 2 7/2 6 4h C F T R IB3 h MP I IB3 5 00 u M ma n n o s e% change of SOY binding Figure 3-10. FITC conjugated SBA lectin binding to the O-glycosylation profile of untreated S9 cells and untreated and treated IB3 cells.

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78 100 120 140 160 180 200 220 240 260 280 300 320 S9 IB3 IB3 MPI IB3 GFP% binding of PAO1 to Host Cell Figure 3-11. Binding of PAO1 to untreated S9 cell s, untreated IB3 cells and IB3 cells transfected with pTR2-CB-hMPI with pRT2-CB-GFP as a control. 100 120 140 160 180 200 220 240 260 280 300 320 S9 Untr IB3 untr IB3 CFTR IB3 GFP% Binding PAO1 to Host Cells Figure 3-12. PAO1 binding to untreated S9 cells, untreated IB3 cells and IB3 cells transfected with pTR2264hCFTR with pTR2-CB-GFP as a control.

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79 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0 S9 Untr IB3 Untr 50 nM500 nM 5 uM50 uM300 uM 1 mM50 mM% change of bound CFU Figure 3-13. PAO1 binding to untreated S9 cells, untreated IB3 cells and IB3 cells grown in a range of mannose rich medias. 0 5 10 15 20 25 30 35 40 S9 IB3% of Bound CF U Unblocked 500 ng ConA 5 ug ConA 50 ug ConA Unblocked 500 ng WGA 5 ug WGA 50 ug WGA Unblocked 500 ng Soy 5 ug Soy 50S Figure 3-14. Dose dependent inhi bition of PAO1 binding to untr eated S9 and IB3 cells after blocking with ConA, WGA and SB A at various concentrations.

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80 40 60 80 100 120 140 160 180 200 220 S9 UntrIB3 UntrIB3 MPIIB3 GFP% change of GFP + cells Ingestion Clearance Figure 3-15. Intra-cellular PAO1 pr ior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to demonstrate a change in intra-cellular PAO1 after the clearance period from untreated S9 and IB 3 cells and IB3 cells transfected with pTR2-CB-hMPI with pTR2-CB-GFP as a control. 50 70 90 110 130 150 170 IB3S9 CFTRGFPPercent Change of Ingested CFU Prior Clearance Figure 3-16. Intra-cellular PAO1 pr ior to the 4 hour clearance pe riod (light) and after the 4 hour clearance period (dark) to demonstrate a change in intra-cellular PAO1 after the clearance period from untreated S9 and IB 3 cells and IB3 cells transfected with pTR2-CB264hCFTR with pTR2-CB-GFP as a control.

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81 50 100 150 200 250 300 350 S9 UntrIB3 UntrIB3 50nM IB3 500nM IB3 5uMIB3 50uM IB3 500uM IB3 2.5mM% Change of ingested PAO1 CFU Prior Clearance Figure 3-17. Intra-cellular PAO1 pr ior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to demonstrate a change in intra-cellular PAO1 after the clearance period from untreated S9 and IB3 cells and IB3 cells grown in a range of mannose rich medias. 50 70 90 110 130 150 170 190 210 230 S9 Untr IB3 Untr IB3 MPI IB3 SMN% Change of GFP + Ingested PAO1 Initial Change Figure 3-18. Intra-cellula r GFP+ PAO1 by FACS analysis prio r to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to de monstrate a change in intracellular PAO1 after the clearance period fr om untreated S9 and IB3 cells and IB3 cells transfected with pTR2-CB-hMPI with pTR2-CB-SMN as a control.

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82 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0 220.0 S9 Untr IB3 Untr IB3 SMNIB3 CFTR% Change of ingested Bacteria Initial Change Figure 3-19. Intra-cellula r GFP+ PAO1 by FACS analysis prio r to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to de monstrate a change in intracellular PAO1 after the clearance period fr om untreated S9 and IB3 cells and IB3 cells transfected with pTR2-CB264hCFTR with pTR2-CB-SMN as a control. 60 80 100 120 140 160 180 200 220 240 S9 UntrIB3 UntrIB3 50nM IB3 500nM IB3 5uMIB3 100uM IB3 500uM IB3 2.5mM% change of GFP + cells Initial Change Figure 3-20. Intra-cellula r GFP+ PAO1 by FACS analysis prio r to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to demonstrate a change in intracellular PAO1 after the clearance period fr om untreated S9 and IB3 cells and IB3 cells grown in a range of mannose rich medias.

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83 90 100 110 120 130 140 150 160 S9 untrIB3 untrIB3 hMPIIB3 SMN% of cellular death Figure 3-21. Analysis of 7AAD staining by FACS of untreated IB3 and S9 cells and IB3 cells treated with pTR2-CB-hMPI with pTR2 -CB-SMN as a transfection control to determine the percent of the host cell population under going apoptosis after PAO1 infection. 100 120 140 160 180 200 220 240 S9 Untr IB3 untr IB3 SMN IB3 CFTR% difference of cell death by 7AAD Figure 3-22. Analysis of 7AAD staining by FACS of untreated IB3 and S9 cells and IB3 cells treated with pTR2-CB264hCFTR with pTR2-CB-SMN as a transfection control to determine the percent of the host ce ll population undergoing apoptosis after PAO1infection.

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84 95 105 115 125 135 145 155 165 175 185 195 S9 untrIB3 untrIB3 50nMIB3 500nM IB3 5 uMIB3 100uM IB3 500uM IB3 2.5mM% of cellular death Figure 3-23. Analysis of 7AAD staining by FACS of untreated IB3 and S9 cells and IB3 cells grown in mannose rich medias to determin e the percent of th e host cell population undergoing apoptosis after PAO1 infection. 100 120 140 160 180 200 220 240 260 280 300 S9 Untr S9 Contr IB3 untr S9 siRNA% change in bound CFU Figure 3-24. PAO1 binding to untreated S9 cells, untreated IB3 cells and S9 cells transfected with pTR2-U6-CFTRsiRNA with pTR2-U 6-Scrambled siRNA as a control.

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85 50 70 90 110 130 150 170 190 210 S9 S9 Contr IB3 S9 siRNAPercent Change of Ingested CFU Prior Clearance Figure 3-25. Intra-cellular PAO1 pr ior to the 4 hour clearance pe riod (light) and after the 4 hour clearance period (dark) to demonstrate a change in intra-cellular PAO1 after the clearance period from untreated S9 and IB 3 cells and S9 cells transfected with pTR2-U6-CFTRsiRNA with pTR2-U6Srambled siRNA as a control. 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 S9 untr S9 ContrIB3 untrS9 siRNA% Change of ingested GFP + PAO1 Initial Change Figure 3-26. Intra-cellular GFP+ PAO1 by FACS analysis prior to the 4 hour clearance period (light) and after the 4 hour clearance period (dark) to de monstrate a change in intracellular PAO1 after the clearance period fr om untreated S9 and IB3 cells and S9 cells transfected with pTR2-U6-CFTRsiRNA with pTR2-U6-scrambled siRNA as a control.

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86 100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 S9 S9 Contr IB3 untr S9 siRNA% difference of cell death by 7AAD Figure 3-27. Analysis of 7AAD staining by FACS of untreated IB3 and S9 cells and S9 cells treated with pTR2-U6-CFTRsiRNA with pTR2-U6-Scrambled siRNA as a transfection control to determine the percent of th e host cell population undergoing apoptosis after GFP+ PAO1 infection. Figure 3-28. In-vitro experimental design overvie w: Binding, Ingestion and clearance description of events and treatment.

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87 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 IB3 untrS9 untrIB3 ContS9 ContrS9 siRNAIB3 CFTRIB3 MPIIB3 MannMPI Expression Fold Change Figure 3-29. Fold change of MPI mRNA levels of experimental and control groups from in vitro cell culture studies from Sybr green real time data collected at cycle 20. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 IB3 untrS9 untrIB3 ContS9 ContrS9 siRNAIB3 CFTRIB3 MPIIB3 MannCFTR Expression Fold Change Figure 3-30. Fold change of CFTR mRNA levels of experime ntal and control groups from in vitro cell culture studies from Sybr green real time data collected at cycle 20.

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88 CHAPTER 4 IN-VIVO STUDIES Materials and Methods This chapter will discuss the CF mouse model used in this study and the therapies that I proposed would provide some correction. A de pendable CF mouse model that mimics the bacterial clearance deficiency in human patients has yet to be established. A reasonable model was developed at Harvard in Gera ld Piers lab that has been te rmed the Drinking Water Model because the bacterial infecti on to colonize the mice is given through the drinking water; unfortunately, it has been difficu lt to replicate (74). I based my airway colonization model of P. aeruginosa around this model with some modificatio ns that I hoped would establish some consistency using this promising model. Treatm ents included AAV5-MPI gene therapy directed at the airways and a non-invasive diet of hype r-mannose through the drinking water of the CFTR deficiency mice. Mouse Model Methods The purpose of this m odel was to establish a colonization of P. aeruginosa in the airways of CFTR deficient mice. There were four distinct periods in this model. The first period began prior to the infection period (Day 0). This peri od involved the therapeuti c treatment period prior to infection with either the hi gh-mannose diet or the constructed viral vector expressing mouse MPI targeting the ai rways and begins at wk or wk respectively. The second period was a preliminary antibiotic period that began two weeks prior to the infection period (wk ). This antibiotic treatment period was es tablished to clear the airways of endogenous infection so there would be no competition for the selected P. aeruginosa strain to help colonization of the airways. The infection period of the selected P. aeruginosa strain began at wk 0 and continued for two weeks. Following the 2 week infection pe riod there was a mild antibiotic regimen with

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89 the hyper-mannose treatment if using mannose as a treatment for the length of the study. The mice were sacrificed at wk +8 and the endpoi nts for the model were performed. To note, I changed the antibiotics and bacterial tainted dr inking water (during the infection period) daily. The CFTR deficient mouse I used the W hitsett strain of mice for this study; CFTRm1Unc /(FABP-hCFTR). This mouse was engineered by John Whitsett by cross br eeding a homozygous CFTR deficient C57/BL6 mouse strain which has a premature stop codon in the CFTR mRNA developed at University of North Carolina (UNC) (109) and a transgenic FVB mouse expressing no rmal hCFTR using the Fatty Acid Binding Promoter (FAB P) for directed expression in the digestive system only leaving the airways still vulnerabl e to CF pathologies. This CFTR deficient mouse does not have the failure to thrive characteristic of the CFTRm1Unc / mouse, can be breed as homozygotes and can be given solid food without developing a fatal gut infection but still shows susceptibility to terminal lung conditions (75). Mouse MPI treatment by rAAV5-mMPI intra-tracheal delivery First I developed a rAAV5mMPI viral vector to selectively target the airway for highlevel expression of mouse MPI. AAV5 serotype has been previously shown to have strong tropism for the apical surface of the airway epit helial cells. I was targeting the airway epithelium by gene therapy transduction to correct any cl earance deficiency so the use of rAAV5 was optimal for this gene therapy study. The University of Florida Vector co re developed the rAAV5 viral vector expressing mMPI fr om the vector genome. The Vect or Core was provided with a plasmid for packaging the expression cassette with the mouse MPI cDNA. This plasmid was generated this by the same met hods previously described to develop the pTR2-CB-hMPI for transfecting the IB3 ce lls for in-vitro therapy. The m ouse MPI cDNA was received from Invitrogen in the pCMV-Sport6 carrier vector. Mous e MPI specific primers were utilized to

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90 PCR amplify the cDNA using the tr iple master mix from Eppendorf for high fidelity amplification and for adding a sing le A overhang at the 3 ends for subcloning into the pCR2.1 TA cloning vector from Invitrogen as previously described. After the mMPI cDNA was subcloned in the pCR2.1 backbone the Multiple Cloning Site (MCS) was used to excise out the mMPI cDNA with Ecor1 and Hi ndIII providing compatible stic ky ends for cloning into the pTR2-CB vector used for AAV5 packaging. Afte r subcloning the mMPI cDNA into the pTR2CB backbone, the IB3 cells were tran sfected with this vector to en sure increased levels of mMPI using the western blot procedure previously to verify mouse human MPI production from the pTR2-CB-hMPI (Figure 4-1). The antibody used to detect the human MPI cross-reacts with the mouse MPI protein with more efficiency. This developed pTR2-CB-mMPI vector cassette was packaged as the viral genome in the rAAV5 viral vector for gene therapy purposes. After obtaining a sample of the rAAV5-mMPI vi ral vector from the vector the next step was to treat the Whitsett Mice. A 50ul sample containing 2.312 vector genomes of the rAAV5mMPI mixed with phosphate buffer saline (PBS) was injected into the trachea of 6 Whitsett mice with a small 25 gauge butterfly needle to allow the sample to spray into the lungs. For the control group, 6 Whitsett mice were injected with 2.312 vector genomes of a rAAV5-GFP control viral vector obtained from the Vector Core. Trans duced cells will produce a non-therapeutic green fluorescent protein (GFP) and can be used to m onitor the transduction efficiency using intratracheal delivery of the AAV5 v ector in the mice airways and ru le out the possibility of an artificial therapy due to the pr ocedure not the treatment. The in jection was done 3 weeks prior to the infection period to allow for sufficient production of recombinant MPI or GFP.

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91 Hyper-mannose treatment of Whitsett mice The m annose treatment is a non-invasive tr eatment involving a hyper mannose liquid diet through the drinking water. The mice were given drinking water with 5ug /ml of mannose for 4 weeks prior to the infection period. The dosage was based on the previous in vivo studies developed to test treatment of the mpi -/disorder CGD1b with a hype r-mannose diet (110). It is widely accepted that CFTR deficient airway cells turnover every 4 w eeks in CF patients. Therefore, a 4 week treatment peri od prior to infection is optimal to ensure that all the epithelial cells present in the airway would have gone through a replication cycle during the hypermannose liquid diet. The hyper mannose diet was temporarily suspended during the 2 week infection period and restarted for the remainde r of the study along with a mild antibiotic treatment. The control group used was mice treat ed with a hyper-glucose diet instead of mannose. In vitro PAO1 binding study showed no therapeuti c effect of glucose when IB3 cells were grown in rich glucose media (Figure 42). An additional note is that the mannose and glucose treated drinking water was changed daily. Clearing the airway prior to controlled infection Enterobacter is a common pathogen that infects the airw ay of m ice. It is vital to clear the majority or all of this pathogen as well as other pathogens from the airways prior to the controlled infection of P. aeruginosa An aggressive antibiotic treatment was started at WK and continued for two weeks. This aggressive antibiotic treatment was stopped at the beginning the bacterial infection period. Th is antibiotic mixture consisted of 400ug/ml of nitrofuratoin, 200 ug/ml ampicillin and 100 ug/ml of gentimicin and was delivered through the drinking water of the mice, which was changed daily.

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92 Controlled infection of P. aeruginosa The selection of a strain of P. aeruginosa that will be op timal for establishing an infection in the lung of Whitsett mice is vital. Establishi ng colonization in the l ung of CFTR deficient mice rarely occurs naturally. Therefore, a pragma tic approach to selectin g the correct bacterial strain is necessary. The flagella of P. aeruginosa has a cap protein, FliD th at strongly attaches to mucins in the airways. Mucin production in the mouse lung is minimal in comparison to mucin production in humans, which constrains the ability of Pseudomonas to colonize th e lung of mice. Therefore, it was necessary to use a P. aeruginosa strain that was more virulent. This lead to the suggestion of using of a mucoid strain of Pseudomonas because of the low motility characteristic of mucoid strains as well as the increase d production of polysaccharides, which enhances bacterial attachment and microcolony formation in the airway of mice. The mucoid strain used was isolated from a CF patient in Europe that wa s sent to use from Niels Hoiby at the University of Denmark. Unfortunately, this strain has not been thoroughly charac terized but increased bacterial burden was observed in the lung of Whitsett mice infected by nasal administration compared to C57/Bl6 mice (76). The mice were infected for two weeks with 5.07 mucoid CFU in 250 mL of drinking water with 5mM MgCl to prevent the bacteria from bursting in a hypotonic so lution and an antibiotic mixture of 400ug/ml of nitrofur atoin / 200 ug/ml of ampicillin. Pseudomonas is naturally resistant to many antibiotics including nitrofuran toin and ampicillin. Following the two weeks of infection, the infection was stopped and the mild antibiotic treatment of 400ug/ml of nitrofuratoin and 200 ug/ml of ampicillin wa s continued for the remainder of the study.

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93 Data Collection Data was continually collected during the course of the st udy to m easure the bacterial burden in the upper airways and to monitor the health of the mice. The idea was that the mucoid strain would develop microcolonie s of bacterial aggregates that quickly develop into a biofilm and enhance colonization. Attached bacteria have limited motility th rough the use of the Type IV pili and the swarming motility of the flagella when attached to a solid or semi-solid surface. These properties of the P. aeruginosa encourages the spread of mi crocolonies which can lead to bacterial colonizing in the orophary nx; the most distal part of th e airway in relation to the lung. The oropharynx is accessible for rep eat culturing of the bacteria by throat swabs to measure the bacterial burden in the upper airw ay. To monitor the health of the mice were weighed weekly. Sick mice will present with weight loss while healthy mice will maintain their optimal weight and possibly gain weight. The oropharynx swabs were taken weekly starti ng at Day 0 (the first day of infection) using 1mm calcium alginate swabs while the mi ce where under 3% isoflurane anesthesia. The cultured alginate swab was placed in a 14ml cultu re tube with 1ml of st erile tryptic soy broth. The bacterial content from the alginate swab wa s determined by counting the amount of bacterial CFU after plating a 1:10 and 1:30 d ilution of the bacterial suspensi on from the alginate swab in the tryptic soy. Non-antibiotic tryptic soy plates and plates with 100 ug/ml of ampicillin were used to for plating the bacteria. The non-antibiot ic plates were used to determine whether other bacterial pathogens were in the airway by differences in colony morphology as well as comparing the bacterial content on the non-antibiotic plates to the ampicillin plates. If there was ampicillin sensitive bacteria in th e airway there would have been an increase in bacterial content on the non-antibiotic plates.

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94 Unfortunately, the most anticipated endogenous bacterial contaminant, Enterobacter, was resistant to ampicillin. It was necessary to determine whether Enterobacter was present in the airway. Fortunately, the Enterobacter colony morphology is notic eable different than P. aeruginosa colony morphology after overnight growth at 37 C. Pseudomonas colonies are very tiny and white, while the enterobacter colonies are very large with a yellowish tint. Additionally, P. aeruginosa colonies will become blue by cytochrome C oxidation of -naphtol in 95% ethanol in the presence of N ,N -dimethyl-p-phenylenediamine as performed by the Gaby-Hadely oxidase procedure from Sigma providing a great screeni ng method to measure only P. aeruginosa. The first step of this screening procedur e was to transfer the colonies onto a 9 cm, low flow, Whatman filter paper from Fisher Scientific The Whatman filter paper with the transferred colonies was immersed in a sma ll volume Gaby-Hadely Oxidase Solution A and B mixture from Sigma The final volume of the solution wa s 1ml with a 1:1 mixture of solution A and B. This 1ml solution was added to the top portion of a 9 cm bacterial culture plate and the side of the Whatman paper that does not have the colonies was placed on top of the solution in the dish. This procedure allows the oxidase solution mixture to fl ow slowly to the side of the Whatman filter paper with the colonies and turn the Pseudomonas colonies blue. Finally, the P. aeruginosa colonies can be counted with great confidence to accurately determine the Pseudomonas burden in the oropharynx. On the sacrifice date, 8 weeks after the first day of controlled inf ection with the mucoid strain, the lungs and trachea were collected for an array of end point experiments. One lung of the mouse has three equal lobes and the other lung is one large lobe. The larger lobe was fixed in 10% formalin, transferred to 70% ethanol and then embedded in paraffin for future histochemistry analysis. One small lobe of the other lung was used for homogenization to

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95 determine the bacterial burden and to isol ate mouse genomic DNA for Real-Time PCR to determine the total number of AAV5 vector ge nomes present in the genomic DNA from the lung. Another small lobe was used to get a lung cell suspension to determ ine the N-glycosylation profile by FACS analysis with FITC conjugated l ectins. The third small lobe was preserved in RNAlater solution from Ambion and stored at -80 C for the possibility of future analysis of mouse MPI mRNA content using Sybr Green Te chnique. The trachea was divided into two sections. The section most distal from the lung was used for homogenization to determine bacterial burden in the upper airway. The lower half was fixed in 10% formalin, transferred to 70% ethanol and embedded in paraffin for future histochemistry analysis. Results Bacterial Burden in the Oropharynx Oropharynx cultures were taken weekly to dete rm ine bacterial burden in the upper airway. The bacterial burden directly from the trachea an d lung cannot be analyzed in live mice without the use of severally invasive procedures that wi ll constrain the ability for repeat sampling. In order to see a true clearance tr end the bacterial burden was m onitor over the course of many weeks. The data collected from the oropharynx sw abs was used to develop an 8 week trend to determine the clearance capacity of untreated Whitsett mice (AAV5-GFP) compared to treated Whitsett mice (AAV5-mMPI). Six mice where used in the control and tr eatment group utilizing the rAAV5-mMPI therapy and four mice where used in the control and treatment groups utilizing the Hyper-Mannose diet therapy. Thes e groups of mice were enough to generate statistically significant data fr om the oropharynx swabs. Using a one-way anova statistical test there was a significant increase in bacterial lo ad from the untreated Whitsett mice (AAV5-GFP

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96 or Glucose) compared to the treated Whitsett mice (AAV5-MPI or mannose) (Figure 4-3 and 44). Weight Loss/Gain Trend in Treated and Untreated Whitsett Mice Weight loss data over the course of an in vivo stu dy is critical to determine the health of the mice. The mice were infected over a 2 week period with a vi rulent mucoid strain of P. aeruginosa that has previously caused premature deat h in CFTR deficient mice (76). Monitoring the health of these inf ected mice by weight loss is a valid data point that mimics the failure to thrive phenotype in CF patients. Weekly weight data was collected over the course of both rAAV5 and mannose studies. Using a one-way anova statistical test there was a significant weight loss from the untreated Whitsett mice ( AAV5-GFP or Glucose) compared to the treated Whitsett mice (AAV5-MPI or mannose) (Figure 4-5 and 4-6). Bacterial Burden in the lung and Trachea On sacrifice day a portion of the m ouse lung and trachea was hom ogenized, diluted and then plated for colony formation overnight as pr eviously described in the oropharynx swab data analysis. This provided an accurate measurement of the bacterial burden in the lung and trachea after a 6 week clearance period that followed the last day of controlled infection with the P. aeruginosa mucoid strain. There was doubl e the amount of bacteria in both the lung and trachea isolated from the control groups (AAV5-GFP and gl ucose) compared to the experimental groups (AAV5-MPI and mannose) but this difference was only statistically signifi cant in the tracheas (Figure 4-7, 4-8, 4-9 and 4-10). A dditionally, a table was prepared to show what percentage of the lungs with in a given study group had bacter ial burden from the cont rolled infection. In the AAV5-GFP / AAV5-MPI study group the 83% of th e GFP mice had bacter ia in the lungs

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97 compared to 50% of the MPI mice. In the gl ucose / mannose study, 75% of the glucose mice had bacteria in the lungs compared to 50% of the mannose mice (See table 4-1). AAV5-pTR2-CB Vector Genomes in the Lung Tissue Previously, a sm all piece of lung tissue was homogenized to determine the bacterial burden of the lung. The volume of homogenate was 1mL and only 100ul was used to determine the bacterial burden. The remaining 900ul was used for genomic DNA extraction to determine the total amount of vector genome present in 500ng of mouse gDNA and to make an estimation of the amount of vectors genomes present in each cell based on the estimation that there is roughly 6 picograms of DNA in each cell (the total amount of genomes will be from roughly 83,000 cells). Extracting DNA from the lung homogenate The Qiagen DNeasy e xtraction kit was used to isol ate the total gDNA from the mice lung homogenates. First, 200ul of ATL tissue homogenization buffer and 20ul of proteinase K solution was added to the remaining 900ul of the lung homogenate and in cubated overnight at 55C overnight shaking at 1200 RPM in a thermomixer from Eppendorf After complete homogenization, 200ul of AL cell lysis buffer and 200ul of 100% ethanol was added and vortexed immediately. This mixture was added to the DNA isolation column that uses a low pH membrane to collect the DNA. The elution buffer is a high pH that allows fo r the isolation of the DNA collected on the membrane. A 200ul volume of DNA was obtained at roughly 25ng/ul concentration. These samples were used to analyze the amount of vector genomes in 500ng of DNA.

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98 Real-Time TAQMAN PCR to analyze vector genomes in genomic D NA from the lungs The toxicology core directed by Thomas Conlon PhD at the University of Florida performed the TAQMAN PCR to determine the total amount of vector genomes in 500ug of gDNA isolated from the mice lungs treated w ith AAV5-CB viral vector and a lung from the mannose treated mice as a negative control. TaqM an real-time PCR is one of the two types of quantitative PCR methods. TaqMan uses a fl uorogenic probe that is a single stranded oligonucleotide of 20-26 nucleotides and is desi gned to bind only to the DNA sequence between the two PCR primers. Therefore, only specific PCR product can generate fluorescent signal in TaqMan PCR. To do TaqMan PCR, besides reagents required for regular PCR, additional things required are a real-time PCR machine, two PCR primers with a preferred product size of 50-150 bp, a probe with a fluorescent reporter or fluor ophore such as 6-carboxyf luorescein (FAM) and tetrachlorofluorescin (TET) and quencher such as tetramethylrhodamine (TAMRA) covalently attached to its 5' and 3' ends, respectively. The CBA (chicken beta actin) promoter is th e target for the sequen ce specific probe and primers. The CBA promoter is used for expressi on of the recombinant mouse MPI or GFP within the viral genome of the AAV5 viral vector. The CBA is not an endogenous mouse promoter so the detection signals from TAQMAN real-time PCR analysis will be from the vector genome within genomes DNA isolate from the mice lungs. The Primer sequences were: Forward primer: 5-CATCTACGTATTAGTCATCGCTATTACCA-3 and the Reverse primer: 5CCCATCGCTGCACAAAATAATTA-3 and the probe sequence was 5'-(FAM)-TCA GAGCTGCAGTGACCCCGGGAAG-(TAMRA)-3'. The toxicology core did TAQMAN realtime PCR analysis using the ABI PRISM Sequence Detection Systems 7900. This uses realtime laser scanning coupled with a fluorogenic

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99 probe that enables quantification of large numbe r of amplified products rapidly and accurately. Quantification of the number of vector genomes is based on serial dilutions of a cloned equivalent of the target sequence. In this case the pTR2-CB-mMPI plasmid was used to develop the standard curve. This collected data was used to determine the total amount of vector genomes in 500ng genomic DNA. More importantly, with this data, an estimation of the total amount of vector genomes per cell could be determined. This data no detectable levels of vector genomes in the control lung tissue sample while the lung tissue transduced with AAV5-GFP or MPI had levels ranging from 1.05 to 3.55 (Figure 4-11 and Table 4-2). Sybr Green Analysis of MPI mRNA From the Lungs of Transduced Whitsett Mice The m ethods used to isolate total mRNA, convert to a total cDNA profile and specifically amplify MPI for real-time PCR analysis using Sybr green was previously described in Chapter 3. This same method was used to in this mouse study. To ensure that mRNA stability from the collected lung tissue one of the small lobes from the lung wa s suspended in 2 ml of RNAlater solution from AMBION and stored at C until mRNA extraction was performed. The Sybr green analysis would provide data used to calculate any fold changes in MPI mRNA expression when comparing lung tissue from the AAV5-MPI and AAV5-GFP transduced Whitsett mice (Figure 4-12). N-glycosylation Abnormality Anal ysis in Lung Cell Suspensions Lung cell suspensions w ere collected from one of the small lung lobes that were removed from the mice during sacrifice. The lung cell suspen sions collected could be used to determine if the same N-glycosylation tr end and correction in the in vitro studies occurred in the in vivo study. FACS analysis of the lung cell suspension s after incubation with the FITC conjugated Con A and WGA lectins was done to determine th e N-glycosylation profile of the lung cell

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100 suspensions from the control and experimental groups in the MPI and Mannose treatment studies. The FITC WGA and C on A binding percentages were increased from the AAV5-mMPI and Mannose treated mice compared to the AAV5-GFP and glucose control treatment. The WGA showed a 1.6 fold increase from the AAV5-mMPI and a 1.8 fold increase from the mannose treated mice. Although ther e was a trend of increased binding from the Con A lectin, this increase was not statistically significant (Figures 4-13 and 4-14). Pathology Analysis All procedures besides the coll ection of the lung and fixing in for malin were performed by Martha Campbell-Thompson PhD. and Amy Wri ght MS from the University of Florida pathology core. We collected one full lung from each mouse for the purposes of immunohistochemistry. This lung wa s placed in a tissue cassette a nd fixed in 10% formalin for 24 hours than incubated in a series of ethanol ba ths. After 24 hours the fixed tissue was placed in 50% ethanol for 45 mins, transferred into 70% ethanol for 45 mins, tran sferred to 95% ethanol for 45 mins and transferred into 100% ethanol fo r 45 mins. This series of ethanol incubations was done to dehydrate the fixed lung tissue. The dehydrated tissue was transferred to Xylene for 45 min. This step was repeated twice by transferring the tissue to fresh Xylene to re moved ethanol. Next the tissue was added to melted paraffin at 60 C for 6 hours. The molten paraffin wa s changed and incubated for 12 hrs then the tissue infiltrated paraffin was finally embedded into a block of paraffin. 5 uM-thick paraffin sections were attached to glass slides and were progressively and sequentially dipped through a series of reagents to prepare for staini ng. The sections were deparaffinized with xylene for 60 seconds. The xylene was remo ved by six dips in 100% ethanol.

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101 Hematoxylin-eosin (H&E) staining H&E staining is a common m ethod for staini ng cells based. Typically the cytoplasm is stained red with the eosin and the nucleus is st ained dark blue with hemotoxylin. Additonally, red blood cells are stained bright red so the presense of red blood cells in the tissue sample is easiliy decteable. For our purposes we are interested in how H&E staining can show inflammation in the lung. Eosinophils and baso phils are acid loving white blood cells. The infiltration of these cells into tissue indicates inflammation. Fortuna tely, because of the high acid levels within the cytoplasm these cells stain dark blue and are easily detectable in H&E staining. It was previously mentioned that we had two different therapeutic groups with there respective controls to determine treatment of th e bacterial clearance de ficiency in the CFTR deficient Whitsett mice. The first therapeutic treatment was intra-tracheal delivery of AAV5mMPI with AAV5-GFP as a negativ e control. There were six Whitsett mice in each group. The other treatment group was 5mg/ml of mannose de livered in the drinking water with 5mg/ml glucose as a neagtive control. Four Whitsett mi ce were used in each group for this therapeutic study. H&E staining was used to determine the levels of inflammation in the lungs due to the increase of mucoid bacteria in the lung (Fi gure 4-15 4-17). Additionally, these H&E stained lung sections were scored for inflammati on by a trained pathologist; Martha CampbellThompson PhD and the percent and severity of focal inflammation as well as the percent of bronchiestasis were judge. (Table 4-3 and Table 4-4). Staining GFP in treated lungs to determine tr ansduction efficiency of AAV5 viral vector We previously described the procedure to m ount embedded lung sections onto the slides for staining the GFP levels in the lung. We ha d 12 lungs that were transduced with the AAV5 viral vector; six of which were transduced with AAV5-GFP. The firs t step to antibody staining of

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102 GFP in the lung is antigen retrival (GFP) from th e lungs section mounted to the slides. There are several methods to retrieve the trageted antigen for detection. The pathology core utilized a TRIS-EDTA buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0) to break protein cross-links from formalin fixing to expos e the antigens for selective detection using a specific antibody for GFP. Unfortunately, the MP I antibody is not commercially available and we were not able to acquire more from our previous source for MPI immunohistochemistry staining in the lung sections for mouse MPI. After the antigen retrieval, al l twelve lungs sections were stained first with a rabbit antiGFP antibody than with the secondary antibody anti-rabbit IgG conjuagte with horse-radish peroxidase (HRP). Developed HRP is stained br own so GFP labeling in lung sections will be visuable. We anticipated that the AAV5-GFP tr eated mice lung sections will stain while the AAV5-mMPI treated mice lung sections will not be stainined (Figure 4-16). Figure 4-1. Mouse MPI Western blot analysis of untreated IB3 cells and IB3 cells transfected with pTR2-CB-mMPI vector.

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103 80 90 100 110 120 130 140 150 160 170 180 190 200IB3 Untr S9 UntrIB3 500uM MannIB3 500uM Glucpercentage variation of bound CFU Figure 4-2. Glucose and mannose treatment of IB3 cells to ensure that Glucose does not correct PAO1 binding deficiency. -50 0 50 100 150 200 250 300 350 DAY 0 DAY 7DAY 14DAY 21DAY 28DAY 35DAY 42DAY 49Day 56total cultured CFU GFP MPI Figure 4-3. Total amount of the mucoid strain CFU collected from the oropharnyx swabs for the assessment of a bacter ial clearance trend from the control group (GFP) or experimental group (MPI) of mice transduced by rAAV5-GFP or rAAV5mMPI viral vector by IT delivery. 6 total mice used; p value < 0.05 using one-way anova with repeat sampling.

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104 -50 0 50 100 150 200 250 300 DAY 0 DAY 7DAY 14 DAY 21 DAY 28 DAY 35 DAY 42 DAY 49 Day 56total CFU cultured Mannose Glucose Figure 4-4. Total amount of the mucoid strain CFU collected from the oropharnyx swabs for the assessment of a bacteria l clearance trend from the control group (High Glucose Diet) or experimental group (High Mannos e Diet) of mice transduced by rAAV5GFP or rAAV5mMPI viral vect or by IT delivery. 4 to tal mice used; p value < 0.05 using one-way anova with repeat sampling. -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 DAY 0Day 7Day 14Day 21Day 28Day 35Day 42Day 49Day 56% of weight change GFP MPI Figure 4-5. Weight change data used to determine any weight loss trends from the control (GFP) or experimental (MPI) group during a nd after the controlled infection period with the mucoid strain. 6 total mice used ; p value < 0.05 using one-way anova with repeat sampling.

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105 -12 -10 -8 -6 -4 -2 0 2 4 6 8 DAY 0Day 7Day 14Day 21Day 28Day 35Day 42Day 49Day 56 Mannose Glucose Figure 4-6. Weight change data used to determine any weight loss trends from the control (High Glucose Diet) or e xperimental (High Mannose Diet) group during and after the controlled infection period with the mu coid strain. 4 total mice used; p value < 0.05 using one-way anova with repeat sampling. -20 0 20 40 60 80 100 120 140 160 180 GFP MPItotal CFU from Lung Figure 4-7. Total amount of mucoid CFU in the lung to determine bacterial load from mucoid infection 6 weeks after the last day of in fection in efforts to assess any clearance deficiency in treated (MPI) or untreated (GFP) CFTR deficient mice using rAAV5mMPI or rAAV5-GFP respectively.

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106 0 50 100 150 200 250 300 350 400 GFP MPItotal CFU from Trachea Figure 4-8. Total amount of mucoid CFU in the trachea to determine bacterial load from mucoid infection 6 weeks after the last da y of infection in efforts to assess any clearance deficiency in tr eated (MPI) or untreated (GFP) CFTR deficient mice using rAAV5-mMPI or rAAV5-GFP respectively. -20 0 20 40 60 80 100 120 Glucose MannoseTotal CFU from Lung Figure 4-9. Total amount of mucoid CFU in the lung to determine bacterial load from mucoid infection 6 weeks after the last day of in fection in efforts to assess any clearance deficiency in treated (Ma nnose diet) or untreated (Glu cose diet) CFTR deficient mice.

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107 0 50 100 150 200 250 Glucose Mannosetotal CFU from Trachea Figure 4-10. Total amount of mucoid CFU in the trachea to determine bacterial load from mucoid infection 6 weeks after the last da y of infection in efforts to assess any clearance deficiency in treat ed (Mannose diet) or untre ated (Glucose diet) CFTR deficient mice. 0 50000 100000 150000 200000 250000 300000 350000 400000Negative MPI n=6 GFP n=6Vector Genomes Figure 4-11. Vector genomes from 500ng of genomic DNA isolated from mouse lung tissue transduced with either AAV5-GFP or AAV5 -mMPI or an untransduced lung as a negative control.

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108 1 3 5 7 9 11 13 15 AAV5-GFP AAV5-MPIComparative Fold Change Figure 4-12. Sybr green real-time PCR analysis of MPI mRNA extracted from lungs collected from the mice transduced with AAV5-MPI and AAV5-GFP 90.0 100.0 110.0 120.0 130.0 140.0 150.0 160.0 170.0 180.0 190.0 200.0 210.0 220.0 230.0 ConA WGA% of Bound Lectin MPI GFP Figure 4-13. FITC conjugated Lec tin analysis by FACS to determ ine N-glycosylation profile in control (AAV5-GFP) or therapeutic (rAAV5 -MPI) lung cell suspensions using Con A and WGA.

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109 90.0 100.0 110.0 120.0 130.0 140.0 150.0 160.0 170.0 180.0 190.0 200.0 210.0 220.0 230.0 ConA WGA% of Bound Lectin Mann Gluc Figure 4-14. FITC conjugated Lec tin analysis by FACS to determ ine N-glycosylation profile in control (Glucose diet) or therapeutic (Mannose diet) lung cell suspensions using Con A and WGA.

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110 Severe Mild Mild Severe Mild Severe Mild NONE Mild Whitsett Mice AAV5-GFP Mild NONE Mild Mild NONE NONE Whitsett Mice AAV5-mMPI Bronchiectasis Figure 4-15. H&E staining to judge inflammation in the lungs of Whitsett mice treated with AAV5-GFP (control) or AAV5-mMPI.

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111 Mil d NON E NON E Seve r e Mil d Seve r e Mil d Seve r e Mil d W hit se t t G l ucose Seve r e Mil d NON E Mil d W hit se t t -M a nn ose Bronchiectasis Bronch iectas Figure 4-16. H&E staining to j udge inflammation in the lungs of Whitsett mice treated with 5mg/ml of mannose or 5mg/ ml of glucose (control).

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112 Table 4-1. The percentage of lungs with in the given study groups with detectable P. aeruginosa mucoid strain for analysis of a clearance deficiency. Treatment Group % of lungs with detectable levels of bacteria GFP (Control) 83 MPI (therapeutic) 50 Glucose (control) 75 Mannose (therapeutic) 50 Table 4-2. Estimated viral genome c opies per transduced mice lung cells. AAV5-MPI Treated Mice N=6 AAV5-GFP Treated Mice N=6 Treatment Estimated Vectors Genomes per Cell 3 copies 1 copies 2 copies 4 copies 5 copies 4 copies 2 copies 2 copies 4 copies 1 copies 4 copies 3 copies

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113 Mannose Treated Mice N=4 Glucose Treated Mice N=4 Treatment Inflammatory Rankings B y H&E stainin g % NONE: 50 % Bronchiectasis 0 % Mild Focal Inflammation only: 25 % Severe Focal Inflammation: 25 % NONE: 25 % Bronchiectasis 50 % Mild Focal Inflammation only: 0 % Severe Focal Inflammation: 75 AAV5-MPI Treated Mice N=6 AAV5-GFP Treated Mice N=6 Treatment Inflammatory Rankings By H&E staining % NONE: 50 % Bronchiectasis 33 % Mild Focal Inflammation only: 50 % Severe Focal Inflammation: 0 % NONE: 17 % Bronchiectasis 33 % Mild Focal Inflammation only: 50 % Severe Focal Inflammation: 33 Table 4-3. Inflammatory rankings by H&E staining of lung sections from mice treated with AAV5-mMPI or AAV5-GFP (Control). Table 4-4. Inflammatory rankings by H&E staining of lung sections from mice treated with 5mg/ml of mannose or 5mg/ml of glucose (Control).

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114 CHAPTER 5 DISCUSSION Defective Bacterial Clearance is a Hallmar k Symptom in the CF Lung This study focused on the bacterial clearance de ficiency that results from a CFTR defect. The work from this study was directed to the possibility of a novel f actor involved in this bacterial clearance defect that has not been previously pursued. Abnormal glycosylation related to terminal residues changes has been the focus of attention for this bacterial clearance defect. This abnormal global N-glycosylation profile from the membrane bound glyco-proteins is a potential target for treatment of the defect in bacterial clearance seen in almost all cystic fibrosis patients. MPI is Involved in Global Abnormal Glycosylation Mannose-6-phosphate m RNA is down regulated in the IB3 CFTR defective cell line compared to the CFTR corrected S9 cell line. This defect was confirmed by western blot of MPI in uncorrected IB3 cells and S9 cells (Figure 3-6). This figure shows a faint MPI band from the S9 cell line compared to no detectable band usin g the protein dilutions previously described. This indicates that there is an in crease in the amount of MPI prot ein in the S9 cell lines compared to the IB3 cells lines. A severe global N-glycosylation reduction occurs in the mpi deficient homozygous disease; Congenital Disorder of Glyoc sylation 1b. It was dem onstrated that a down regulation of MPI in IB3 cells can cause a signif icant decrease in the N-gl ycosylation profile and that this decrease can be corrected by tr ansfection with pTR2-CB-hMPI or pTR2-CB264CFTR or with a mannose rich media (See Figure 3-8 and 3-9). Additionally, transfecting the S9 cell line with pTR-U6-CFTRsiRNA plasmid resulted in a significant decrease in the N-glycosylation

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115 profile. These data indicate that the N-glycosylation deficiency in the IB3 cell line is dependent on CFTR expression and correction of this deficiency can occur independent of CFTR correction by correcting the MPI deficiency by MPI transfection or by s upplementing the MPI deficiency using a hyper-mannose treatment. Mannose is a sugar that can be converted di rectly to mannose-6-phophate independent of MPI by direct phosphorylation by hexokinase. A Hyper-mannose diet treats the Congenital Disorder of Glycosylation 1b cau sed by MPI defect. This provides promise that a non-invasive treatment by mannose diet may pr ovide correction of the bacter ial clearance defect in CF. Reduced Bacterial Adhesion is Link ed to N-glycosylation Deficiency Several glycosylation residues ar e common adhesion molecules for P. aeruginosa. These in vitro studies show the common P. aeruginosa laboratory PAO1 stra in binds with less efficiency to the IB3 cells compared to the S9 cells and this defect can be corrected by transfection with pTR2-CB-hMPI or pTR2-CB264CFTR and treatment with mannose rich media (see Figure 3-11, 3-12 and 3-13). Additionall y, the PAO1 bacterial adhesion to S9 cells was blocked in a dose dependent fashion by a pr e-incubation of the WGA and Con A lectins, which preferentially bind to N-glycosylation subunits (see Figure 3-14). Bacterial adhesion is the critical first step in host cellular clearan ce by programmed cell death to clear intra-cellular bacteria. This demonstrates that the N-glycosyla tion profile on the epithe lial cell surface plays a role in adhesion of P. aeruginosa and when there is a N-glycos ylation deficiency there is a deficiency in bacterial binding. Finally, correct ion of the IB3 deficiency was successful by reversing the MPI deficiency, the CFTR defici ency or providing a hyper-mannose treatment to supplement the MPI deficiency independent of MPI. The S9 CFTR corrected cell line was produced by adenoviral vector with a normal human CFTR transgene of the IB3 cell line

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116 followed by clonal expansion to esta blish a stable cell line. To ensu re that the lack of bacterial binding deficiency in the S9 cell compared to the IB3 cell was not due to the clonal expansion procedure S9 cells were treated with an hC FTR siRNA to knockdown CFTR expression. This treatment was successful in reverting the stable corrected S9 cell line to the IB3 state (Figure 324). These data indicate the first step in the bacterial clearance mechanism by host cellular death is deficient in the CFTR defective IB3 cell line and this defect is coordinated by an MPI deficiency with in the IB3 ce ll line. Additionally, the binding deficiency can be corrected by gene augmentation of either CFTR of MPI but more importantly it also indicates that this deficiency can be corrected by mannose supplement independent of gene augmentation. Therefore, correcting a glycosylation abnormality in the IB3 cell line will co rrect a deficiency in the first stage of bacter ial clearance by host cellular death. Wi th this revelation, there is hope the further stages of bacterial clearance are deficient in the IB3 cell a nd can be corrected by directly treating the N-glycosyl ation deficiency. Bacterial Ingestion Defect is Link ed to Abnormal N-Glycosylation Bacterial clearance by in tracellular ingestion and program med host cellular death has previously been demonstrated in epithelial cells (77,78). This m echanism of bacterial clearance was the focus of our study. The theory that airw ay epithelial cells play a role in bacterial clearance by bacterial binding, i ngestion and subsequent cellul ar death by desquamation was thoroughly researched in this dissertation. Reliable experiments were developed to research bacterial ingestion and clearance by host cellu lar death. It was discovered that the CFTR defective and MPI deficient IB3 cell line is hindered in this mechanism to clear bacteria compared to the CFTR corrected S9 cell line. Th e CFTR corrected S9 cells show an average 2

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117 fold increase of ingested PAO1 compared to IB 3 cells (Figure 3-15 to 3-20). The role of MPI in this mechanism by correcting the IB3 cells with the pTR2-CB-hMPI tr ansfection plasmid was confirmed (3-15, 3-18). Additionally, it was fu rther observed that a dose dependent IB3 correction by growing the IB3 cells in a range of mannose concentrations (Figure 3-17, 3-20). Finally, correcting the IB3 cells by tran sfecting the cells with the pTR2-CB264CFTR vector for corrected the CFTR deficiency in IB3 cells (3-16, 3-19). The most important step of this in vitro project was analysis of the bacterial load in the airway epithelial ce lls after a clearance period. Focusing on th is step revealed a concerning detail in the CFTR deficient IB3 cells; the amount of bacterial CFU in the IB3 cells doubled after the 4 hour clearance period while the bacterial load decreased in the S9 ce ll line after the same clearance period. This physiology deficiency has been previously observed in CFTR deficient macrophages. After bacterial ingestion by the CF TR deficient macrophage occurs the bacteria continues to survive and replicat e with in the cytoplasm of the macrophage (79). To account for this clearance deficiency in IB 3 cells the clearance by host cell ular death was targeted as a mechanism. This pursuit demonstrated that the IB 3 cells are deficient in host cellular death after bacterial infection using the 7AAD large mol ecule nucleic acid stain as a marker for programmed cell death (Figure 3-21, 3-22, 3-23 a nd 3-27). Further, the correction was achieved by a MPI or CFTR transfection treatment or a hyper-mannose treatment by restoring the bacterial clearance capabilities of the IB 3 cells by increasing host cellular death and by reducing the intracellular PAO1 after the 4 hr clearance period (Figure 3-15 to 3-23). The final step was the treatment of CFTR siRNA in S9 cells to ensure that S9 cells were a suitable wild-type control to establish a nor mal phenotype. Bacterial adhesion, ingestion and subsequent clearance by host cellu lar death were the targets for th is project. Knocking down the

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118 CFTR transcripts in the S9 cells was pursued to establish a proposed link between CFTR and bacterial adhesion, ingestion and subsequent clearance by host cellular death and that CFTR deficiency indeed leads to a defect in bacteria l clearance. siRNA treatment of hCFTR in S9 cells showed a deficiency in bacterial inges tion, bacterial clearance after the 4 hour clearance period and host-cellular death (Figure 3-25 to 3-27). In-vitro Conclusions These data confirm the theory that the N-glycos ylation deficiency in the IB3 cell line has a direct bearing on the bacterial clearance capability of these cells by host cellular death indicating the possibility of a predisposition to bacteria l colonization do to the CFTR deficiency. Additionally, it can be confirmed that directly targ eting the CFTR deficiency in the IB3 cells as an area of treatment is viable, but more important ly targeting only the N-glycosylation deficiency independent of MPI or CFTR gene augmentatio n is also viable as a therapeutic avenue. To confirm the proof of concep t, the MPI expression levels where increased in the CFTR corrected S9 cells compared to the IB3 cells. A dditionally, transfec ting the IB3 cells with either pTR2-CB-hMPI or pTR-CB264CFTR increased the mRNA levels of MPI and CFTR respectively. Moreover, CFTR gene augmentation resulted in a modest increase of MPI expression. Finally, siRNA treatment of CFTR in the S9 ce ll line indeed resulted in a significant decrease of CFTR transcripts al ong with a significant decrease in the MPI tr anscripts compared to untreated S9 cells (F igure 3-29 and 3-30). In-vivo Studies Establishing bacterial coloni zation in the airway of CF TR deficient mice has been problematic but essential to test therapies related to correcting the bacterial clearance deficiency in CF patients. Therefore, it was critical to develop a suitable mous e model to test the N-

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119 glycosylation involvement in bacterial clearance in vivo. A low-grade colonization in the upper airways of Whitsett CFTR deficient mice was ac hieved when the Whitsett mice were treated with a non-therapeutic AAV5-GFP viral vector by intra-tracheal delivery or by giving a nontherapeutic high-glucose diet. A virulent mucoid strain of P. aeruginosa isolate was used as the infecting bacteria after clearing the airways of any host infect ions to allow adequate attachment and growth of this mucoid stra in. A partial correction of this mild colonization using the two different treatment groups previously described. As a reminder there was an intra-tracheal injection of the AAV5-mMPI viral vector and a non-invasive hyper mannose diet as therapies for the mild bacterial colonization of the P. aeruginosa mucoid stain. Bacterial load in the airways To establish a bacterial clear ance trend in non-therapeuti c W hitsett mice compared to therapeutic treatments, the bacteria load wa s analyzed in weekly oropharynx upper airway swabs, lung homogenates, and trachea homogena tes. AAV5-mMPI intra-tracheal delivery and 5mg/ml mannose in the drinking wa ter were the two independent therapeutic treatments with the respective AAV5-GFP or 5mg/ml glucose controls. Th ere was an increase in bacterial load in the lung homogenates, trachea homogenates and week ly oropharynx swabs in the non-therapeutic control Whitsett mice compared to the AAV5-mMPI or mannose treatments. This increase was statistically significant in the trachea homogenate s using a two-tailed, pair ed T-Test (Figure 4-8 and 4-10) and in the weekly oropharynx swabs by a one-way anova with repeat sampling (figure 4-3 and 4-4). Unfortunately, the difference was not significant in the l ung homogenates (Figure 4-7 and 4-9) but the data did indicate a tre nd towards increased lung bacteria in the nontherapeutic Whitsett mice. This trend was verified by charting the percentage of lungs with in a given group had bacteria in the lungs. From this end point 50% of lungs from the AAV5-mMPI

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120 treated mice had bacteria compared to 83% of lungs from the AAV5-GFP treated mice while 50% of the lungs from the mannose treated mice ha d bacterial compared to 75% of the glucose treated mice (Table 4-1). These data clearly in dicate the bacterial load in the airways in decreased by treating the assumed N-glycosyl ation deficiency in Whitsett mice directly. Additionally, an non-invasive tr eatment using hyper-mannose has the potential to be viable option in treating the bacterial cl earance deficiency that plagues CF patients. Later reported data was used to verify that there was indeed an N-glycosylation deficiency in non-therapeutic Whitsett mice. Minor weight loss in uncorrected Whitsett mice The weight of these m ice were measured week ly to monitor he health of the mice during the experiments. There was chroni c weight loss in the control mi ce and with in the therapeutic mice there was an acute weight loss that restored to normalcy during the 6 week clearance period following the infection period. This data indicates that there is an acute sickness that parallels the infection period but is ameliorated followi ng the infection period in the therapeutic mice while this mild illness remains chronic in the control mice (Figure 4-5 and 4-6). N-Glycosylation abnormality of lung cell su sp ensions from control Whitsett mice The in vitro study revealed a clear N-glyocsylation de ficiency in untreated CFTR deficient IB3 cells. The carry over of this deficiency to the in vivo studies using the Whitsett CFTR deficient mice was very encouraging. This op timism was confirmed by analyzing the lung cell suspension from therapeutic and control Whitse tt mice by the analysis of FITC conjugated Con A and WGA. The AAV5-MPI and mannose treat ment showed a trend of increased Nglyocsylation by an increase of bound FITC c onjugated Con A and WGA compared to AAV5-

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121 GFP and glucose treatment respectively. This di fference was statistically significant using a twotailed, paired T-Test when analyzing WGA but not by C on A (Figure 4-13 and 4-14). Analysis of lung inflammation Typically, an increase in bacterial burden in the lung is paralleled by acute inflamm ation. The persistent lung infection in cystic fibrosis patients leads to chroni c lung inflammation. With an established mouse model that can develop a mild bacterial colonization in the lung of untreated Whitsett mice it was possible to determine if this deficiency in bacterial clearance is associated with an increase of inflammation in the lung of non-therapeutic Whitsett mice. An increase in severe focal infl ammation and bronchiectasis in th e control Whitsett mice compared to the AAV5-mMPI or mannose treatment was observed: 40% of AAV5-GFP and glucose controls combined showed bronchiectasis compared to 10% or AAV5-mMPI and mannose treatment combined. Moreover, 50% of AAV5-G FP and glucose controls combined showed severe focal inflammation compared to 10% or AAV5-mMPI and mannose treatment combined. Finally, 50% of the mannose and AAV5-mMPI mice combined showed no focal inflammation in the lungs compared to 21% in the AAV5-GFP and glucose controls combined (Figure 4-15 and Figure 4-16; Table 4-2 and Table 4-3). Viral vector transduction efficiency Transductio n efficiency is critical when perf orming gene therapy studies. It was necessary for to parallel the th erapeutic correction compared to c ontrol treatment using the AAV5 viral vector with transduction of the de livered viral vectors. The viral vector leaves its mark in the lung cells were transduction has occurred. The AAV5 viral vector, after inf ection of the host cell, uncoats and the viral genome is released into the cytoplasm and persists largely as an episome. This viral genome mark can be tracked by specific real-time TAQMAN PCR amplification by

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122 targeting a specific sequence with in the recombinant viral vect or genome unique to the mouse genome. In this study, the CBA promoter with in the genome was targeted by TAQMAN PCR. There was a range of 1 5 vector genomes pe r cell from genomic DNA isolated from roughly 83,000 cells (500ng). Additionally, 500ng of DNA isol ated from a control (untransduced lung) showed only a background level of 200 vector genomes compared to an average of 200,000 vector genomes from 500ng of DNA from tran sduced lungs (Table 4-2 and Figure 4-11). Additionally, the mRNA levels we re significantly increased in the AAV5-mMPI treated Whitsett mice when compared to the AAV5-GFP mice (F igure 4-12). These data indicate that transduction of AAV5 viral vector with pack age MPI or GFP was successful and that the transduction with mMPI increa sed the levels of mRNA from genomic DNA from lungs. In-vivo Conclusions The f irst important discovery was the ability to develop a mild bacterial colonization model in CFTR deficient mice under control cond itions. Using this mouse model a significant increase of bacterial burden in the upper airway s of control CFTR deficient mice compared to therapeutic mice was observed. 78% of the non-therapeutic CFTR deficient mice had detectable levels of mucoid CFU in the lung compared to 41 % in the therapeutic mice This deficiency in bacterial clearance was coordinate d by a N-glycosylation deficien cy from analyzing the lung cell suspension. Correcting this N-glyc osylation deficiency by MPI gene augmentation or by the noninvasive mannose treatment, decreased the amount of bacteria recovered from the airways of the Whitsett mice. Finally, this correction of the b acterial clearance deficiency was paralleled by a decrease in lung inflammation. All, these data indicate that treating the N-glycosylation deficiency in CFTR deficient mice prevents a persistent infection in the lung and minimizes inflammation. Additionally, a non-invasive mannose diet independent of MPI gene augmentation

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123 has therapeutic benefit. This novel study lays the foundation for future studies involving a noninvasive treatment for cystic fibrosis.

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134 BIOGRAPHICAL SKETCH My nam e is Ashley Martino and I am a 34 y ear old male Ph.D candidate studying medical genetics. Specifically, I am inves tigating novel therapies for the tr eatment of Cystic Fibrosis. I am America citizen born abroad in Amsterdam, Holland. My childhood was spent in Los Angeles, California. I made my first move out of California to San Antonio in 1996 when I joined the Air Force. From there I was statione d in Valdosta, Georgia. I moved back to Los Angeles after I finished my tenur e in the Air Force. Finally, I m oved to Gainesville Florida to study at the University of Florida. My high school education was spent at El Camino High School in Los Angeles Unified School District. While I was in the military I at tended Georgia Military College and the Valdosta State University. When I moved back to California after my brief military career, I completed my BA in Biology at California State University Northridge. Focusing on more personal issu es, I was married and I have an 11 year old son from the marriage named Antony. At this time, I am e ngaged to a wonderful woman from Colombia named Maria Fernanda Rojas and we are planning a spring wedding. I have many hobbies that cente r around music and sports. I have become quite an accomplished piano player and basketball player. Although, I currently have not been able to do either because my right hand is broken which has made writing this dissertation difficult. After I finish my dissertation work I plan to stay in Gainesville and continue to pursue medical research. I am very excited about what my future will bring. I have been studying for so long, it will nice to finally be able to begin my professional career.