Engineering an improved adenovirus packaging cell line

Recombinant human adenovirus (Ad) vectors are being extensively explored for their use in gene therapy and recombinant vaccines. Ad vectors are attractive for many reasons, including the fact that (1) they are relatively safe, based on their use as live oral vaccines, (2) they can accept large transgene inserts, (3) they can infect dividing and postmitotic cells, and (4) they can be produced to high titers. However, there are also a number of major problems associated with Ad vectors, including transient foreign gene expression due to host cellular immune responses, problems with humoral immunity, and the creation of replication competent adenoviruses (RCA). Most Ad vectors contain deletions in the E1 region that allow for insertion of a transgene. However, the E1 gene products are required for replication and thus must be supplied in trans by a helper ceillille that will allow for the growth and packaging of the defective virus. For this purpose the 293 cell line (Graham et al., 1977) is used most often; however, homologous recombination between the vector and the cell line often results in the generation of RCA. The presence of RCA in batches of adenoviral vectors for clinical use is a safety risk because tlley . may result in the mobilization and spread of the replication-defective vector viruses, and in significant tissue damage and pathogenicity. The present research focused on the alteration of the 293 cell line such that RCA formation can be eliminated. The strategy to modify the 293 cells involved the removal of the first 380 bp of the adenovirus genome through the process of homologous recombination. The first step towards this goal involved identifying and cloning the left-end cellular-viral jUl1ction from 293 cells to assemble sequences required for homologous recombination. Polymerase chain reaction (PCR) was performed to clone the junction, and the clone was verified through sequencing. The plasn1id PAM2 was then constructed, which served as the targeting cassette used to modify the 293 cells. The cassette consisted of (1) the cellular-viral junction as the left-end region of homology, (2) the neo gene to use for positive selection upon tranfection into 293 cells, (3) the adenoviral genome from bp 380 to bp 3438 as the right-end region of homology, and (4) the HSV-tk gene to use for negative selection. The plasmid PAM2 was linearized to produce a double strand break outside the region of homology, and transfected into 293 cells using the calcium-phosphate technique. Cells were first selected for their resistance to the drug G418, and subsequently for their resistance to the drug Gancyclovir (GANC). From 17 transfections, 100 pools of G418f and GANCf cells were picked using cloning lings and expanded for screening. Genomic DNA was isolated from the pools and screened for the presence of the 380 bps using PCR. Ten of the most promising pools were diluted to single cells and expanded in order to isolate homogeneous cell lines. From these, an additional 100 G41Sf and GANef foci were screened. These preliminary screening results appear promising for the detection of the desired cell line. Future work would include further cloning and purification of the promising cell lines that have potentially undergone homologous recombination, in order to isolate a homogeneous cell line of interest.

Second Welland Canal - Book 1, Survey Map 1 - Entrance, Lock 1 and Port Dalhousie

Survey map of the Second Welland Canal created by the Welland Canal Company showing the areas in and around Port Dalhousie and Grantham Township. Identified structures associated with the Canal include Lock 1, Lighthouse, Lighthouse Keeper's House, East and West Piers, Harbour, Waste Weir, Store House, Collector's Office, Collector Assistant Office, Lock Tender's House and the new towing path. Features of the First Welland Canal are noted in red ink and includes the old Harbour, old Lock 1, old towing path and the original bed of the Twelve Mile Creek. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks and businesses are also identified and include streets and roads (ex. Lock Street and Colonel Clark's Cattle Road), Alex Muir's Dry Dock, RandJ Laurie Flouring Mill, R. Laurie and Company Grist Mill, A. Morrison Saw Mill, Johnson's Tavern, a store and a church. Properties and property owners of note are: Concession 1 Lots 21 and 22, John Christie, John Clark, N. Pawling, William Pawling, W. Carter, G.A. Clark, J. Maven, Mrs. Wood, James Drabble and J. Woodall.

Second Welland Canal - Book 1, Survey Map 2 - Lock 1 and Port Dalhousie

Survey map of the Second Welland Canal created by the Welland Canal Company showing the areas in and around Port Dalhousie and Grantham Township. Identified structures associated with the Canal include Lock 1, East and West Piers, Collector's Office, Lock Tender's House and the new towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks and businesses are also identified and include streets and roads (ex. Road to St. Catharines, Side Line, Old Road to Port Dalhousie, Road to Niagara), the Welland Railway and its structures (ex. freight sheds, wood shed, raised platform, elevator, cranes, water tank, turn table, and passenger station), G. A. Clark's Wood Yard, Clark's Wood Office, Alex Muir's Dry Dock, Donald, Andrews and Ross' Dry Dock, RandJ Laurie Flouring Mill, R. Laurie and Company Grist Mill and A. Morrison Saw Mill. A New Road to St. Catharines is featured in red ink. Properties and property owners of note are: Concession 1 Lots 19, 20 and 21, John Christie, and John Clark.

Second Welland Canal - Book 1, Survey Map 9 - Lock 2 in Grantham Township

Survey map of the Second Welland Canal created by the Welland Canal Company showing a portion of the Grantham Township sometimes referred to as the Welland Vale. Identified structures associated with the Canal include Lock 2, several weirs, and the Lock Tender's House. The surveyors' measurements and notes can be seen in red and black ink and pencil. Features of the First Welland Canal are noted in red ink and include the old towing path and the Old Canal itself. Local area landmarks and businesses are also identified and include streets and roads (ex. Side Line and Old Road to Port Dalhousie), J. C. Clark's Ice House, J. L. Ranney Store House, a burnt mill, barrel shed, a building leased to Michael Kerrins, and a number of unidentified structures (possibly houses or cabins) belonging to D. Cain, R. Cain, W. Weaver and W. Huddy. A New Road to St. Catharines is featured in red ink. Properties and property owners of note are: Concession 5 Lots 20, 21 and 22, Concession 6 Lots 20 and 21, Thomas Adams, John Gould, George Rykert, Theophilus Mack, William H. Merritt, J. L. Ranney, and the Board of Works.

Second Welland Canal - Book 1, Survey Map 11 - St. Catharines and Locks 3 and 4

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Town of St. Catharines. Identified structures associated with the Canal include Lock 3 and its Lock House, Lock 4 and its Lock House, Hydraulic Race, and a floating tow path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include streets and roads (ex. Geneva Street, Ontario Street, St. Paul Street, and Merritville Road), Lincoln Mills and its Store House, T. M. Merritt's Store House, Cooper Shop, L. Shickluna's Dry Dock, Peter McGill's Grist Mill, J. Flint's Saw Factory, T. Hosteter's Gristing Mill, J. Dougan Builder's shop or office, Norris and Nelson Mill, G. N. Oil Foundry and its Machine Shop and Boiler, a barrel shed, woolen factory, Estate of P. Nihen (or T. Nihan), Norris and Nelson's Wharf, the W. C. Office, and structures (possibly houses) or small properties belonging to T. Adams, and A. K. Boomer. Properties and property owners of note are: Concession 6 Lots 16, 17, and 18, Concession 7 Lots 16, 17, and 18, Alva Dittrick, James R. Benson, W. B. Robinson, and C. Phelps.

Second Welland Canal - Book 1, Survey Map 12 - St. Catharines and Lock 4

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Town of St. Catharines. Identified structures associated with the Canal include Lock 4, Hydraulic Race, floating tow path, and the Canal waterway itself. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include streets and roads (ex. Geneva Street, St. Paul Street, Queenston Street, Niagara Street and Mill Street), C. Phelp's Grist Mill and Store House, Stinson's Distillery, and several unnamed bridges. Properties and property owners of note are: Concession 6 Lot 16, J. R. Benson, Calvin Phelps, J. Hudson, David Gray, A. Roberts, Mrs. McDonell, J. S. McDonell, T. B. Wragg, J. Donaldson, W. Barr Jr., C. L. Hall, G. Ward, Ridout Bros and Co., and the St. Catharines Grass Co.

Second Welland Canal - Book 1, Survey Map 13 - St. Catharines and Locks 4 and 5

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Town of St. Catharines. Identified structures associated with the Canal include Lock 4, Lock House, Lock 5, Small Lock House, the towing path, and Gasometer for Canal. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include streets and roads (ex. Geneva Street, Queenston Street, and Academy Street), C. Phelps Mill and Store House, St. Catharines and Welland Canal Gas Works, William Mahony's Tannery, Cooper Shop, a barrel shed, barn, and gas tanks. Properties and property owners of note are: Concession 6 Lots 14, 14, and 16, Concession 7 Lots 14, 15, and 16, C. Phelps, R. M. Clement, Orson Phelps, R. Collier, D. P. Haynes, W. Chace, and John Soper.

Second Welland Canal - Book 1, Survey Map 14 - St. Catharines and Lock 5

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Canal along the eastern edge of the Town of St. Catharines. Identified structures associated with the Canal include Lock 5 and the towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include bridges, streets, and roads (ex. Queenston Street, St. Catharines Macdamized Road and Suspension Bridge), a Pond, a number of unnamed bridges, Stinson's Distillery, and R. Collier's Saw Mill. Properties and property owners of note are: Concession 6 Lots 14 and 15, R. Collier, W. Gillespie, Orson Phelps, W. Chase, M. Bryant, John Soper, Winsor Chace, John Berryman, John Boyle, J. Madigan, B. F. Reynolds, W. Smaill, F. Stinson, G. Ward and Mrs. Soper.

Second Welland Canal - Book 1, Survey Map 15 - Hydraulic Race and Aqueduct in Grantham

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Canal along the eastern edge of the Town of St. Catharines. Identified structures associated with the Canal include Lock 7, Lock House Lot, and the towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include bridges, streets, and roads (ex. Queenston Road, St. Catharines Macdamized Road and Suspension Bridge), a hydraulic race, and the Hydraulic Aqueduct. Properties and property owners of note are: Concession 7 Lots 12, 13, and 14, M. Bryant, Mrs. Soper, J. Capner, O. Phelps, P. Marren, Mrs. Parnell, J. Carty, Mrs. Ward, and J. Goodenew.

Second Welland Canal - Book 2, Survey Map 1 - Locks 5, 6 and 7 in Grantham

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Grantham Township just southeast of the Town of St. Catharines. Identified structures associated with the Canal include Locks 5, 6, and 7, two Lock House Lots, Small Lock House, waste weir, and the towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include roads (ex. Road to Thorold), Dicks Creek, R. Collier's Saw Mill, a gate yard, paper mill, grist mill, store house, and a barn. Properties and property owners of note are: Concession 7 Lots 13, 14, and 15, R. Collier, Orson Phelps, and a Mill Lot leased to Richard Collier.

Second Welland Canal - Book 2, Survey Map 2 - Locks 9 and 10 in Grantham

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Grantham Township between the Town of St. Catharines and Merritton. Identified structures associated with the Canal include Locks 9 and 10, waste weirs, the towing path, a 2nd towing path, and the Canal waterway itself. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include roads (ex. Road to Centreville), hydraulic race, and the Centreville Mill. Properties and property owners of note are: Concession 8 Lots 12 and 12, Lewis Traver, Richard Ash, John Bradley, Owen Clifford, Orson Phelps, C. Bradley, the W. C. Loan Company, and T. Towers Mill Lot.