Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

Amplification of the DFR1 gene in Saccaromyces cerevisiae

A new system was employed to study amplification of t,he DHF'R gene DFB,1 ) in Sa<,:;charoillYCB§. .Q~~Yi...S!i<;1~. . This system consists of a series of yeast strains containing a casset,te which encodes t he yeast, D..ERl gene ttghtly linked tjO a f usion of the yeast 1EU2. regulat,ory region wi tJ1 the LAQZ str ctural gene from E. cO.1-1 (,) . M. Clement , unpubl i,::;hed) . Th's casset;t e was shown t.o be integrat,ed int o a unj que chromosomal l ocati on in each strain . Yeast cells were se l ected for MTX-resistance and overproduction of ~ galac t osi d se ( B-gal ). Since the inserted DF'Rl and ~ACZ genes are independently regulated, it was thought that cel l s with this phenotype probably contain e d ampl if ications of the cassette. A lar ge variat ion in the f requn y o f MTX-resistance was found between the di ff e r ent str ains. These freqlen c ~ es r anged from about 2 x 10 - 7 fo r a population of cells containing the cassette integrated at, the BI J2.l gene in t,he middle of the long arm of chromosome V, to about 5 x 10-4 for a strain with the cassette i nserted in the r DNA cluster Abo It 85% of the MTX- res i stcmt iso l ates examined showed enhanced B·-gal act i v ity rel a t ive t o the parental strain . For the ma jorit y of strains, the mean B- gal activity in drug-r sistant clones was about 3 times that o f the parent following a single se l ect i on step . I n con t r ast, primary MTX-resistant derivat~ves of cells with the cassette inserted 3 at the rDNA cluster showed inc r eases in B- gal activity ranging from 9 - 14 f old r elative to the parent. Analysis of the latte r s train by Southe rn hybr idization indicated that the cassette was inde e d amplified several fold in MTX-re sistant derivatives. A sing l e strain, in which the cassette was inserted at the !lEA;], loc u.s , was used to examine in more detai 1 , the parameters affecting DFRl gene amplificat~ion in yeast . The mean B- gal activity in drug-resistant derivatives of this strain could be increased from 3 to 6 or 7 fold relative to the parent, by stepwise sel ection using increasing MTX concentrations. B-gal overproduction was found to be un stable in all primary and highly -resistant isolates examined. There was no indication, h owever, of a decrease i n growth r a t e in MTX-res i s tant cells which overproduced B - gal.

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.

Welland Canals Society fonds, 1979-1991

The Welland Canals Society was a coalition of business, tourism, heritage, and recreational groups that joined with the Regional Government in 1986 to promote the redevelopment of the Welland Canals Corridor. The mandate of the Society was to provide leadership and assistance to the public and private sectors in achieving heritage-sensitive and tourism/recreation-related economic development in the Welland Canals Corridor. The Society folded in 1991 due to government funding cuts.

Niagara River (Ontario) Remedial Action Plan fonds, 1984-1997, n.d.

The Niagara River Remedial Action Plan was part of an initiative to restore the integrity of the Great Lakes Basin ecosystem. In 1972, the Great Lakes Water Quality Agreement was signed by both Canada and the United States to demonstrate their commitment to protecting this valuable resource. An amendment in 1987 stipulated that Remedial Action Plans (RAPs) be implemented in 43 ecologically compromised areas known as Areas of Concern. The Niagara River was designated as one of these areas by federal and provincial governments and the International Joint Commission, an independent and binational organization that deals with issues concerning the use and quality of boundary waters between Canada and the United States. Although the affected area included parts of both the Canadian and American side of the river, Remedial Action Plans were developed separately in both Canada and the United States. The Niagara River (Ontario) RAP is a three-stage process requiring collaboration between numerous government agencies and the public. Environment Canada, the Ontario Ministry of the Environment, and the Niagara Peninsula Conservation Authority are the agencies guiding the development and implementation of the Niagara River (Ontario) RAP. The first stage is to determine the severity and causes of the environmental degradation that resulted in the location being designated an Area of Concern; the second stage is to identify and implement actions that will restore and protect the health of the ecosystem; and the third stage is to monitor the area to ensure that the ecosystem’s health has been restored. Stage one of the RAP commenced in January 1989 when a Public Advisory Committee (PAC) was established. This committee was comprised of concerned citizens and representatives from various community groups, associations, industries and municipalities. After several years of consultation, the Niagara River (Ontario) Remedial Action Plan Stage 2 Report was released in 1995. It contained 16 goals and 37 recommendations. Among them was the need for Canadians and Americans to work more collaboratively in order to successfully restore the water quality in the Niagara River. Stage three of the Niagara River (Ontario) RAP is currently ongoing, but it is estimated that it will be completed by 2015. At that point, the Niagara River Area of Concern will be delisted, although monitoring of the area will continue to ensure it remains healthy.

Letters to Reverend William Proudfoot, 1823, 1842-1843, 1846

A 2 ½ page letter addressed to The Editor of the Presbyterian Magazine, care of [illegible], London, C.W. The writer describes the Village of Chippawa and its location in Ontario. He writes that there are many people there of Scotch [Scottish] descent. He says that a congregation was formed and 39 names were on the roll. The letter is from J.P. [John Porteous] with an added note from Wm. Porteous. The letter is from St. Catharines. There is one postmark – St. Catharines, April 6, 1823 A 1 ½ page letter addressed to Rev. W. Proudfoot, Ed. Of Presbyterian Mag., London, C.W. This letter is from Walter Mitchell in St. Catharines. He sends a list of peoples’ names and the amounts that they have paid toward the Presbyterian Magazine. Mr. Mitchell is acting as an agent for the magazine. This letter has 1 postmark – St. Catharines, Sept. 13, 1842 A 2 page letter addressed to Rev. W. Proudfoot, London, C.W. This letter is from John Jennings of St. Catharines. The writer claims that he is ill but he makes plans to meet Reverend Proudfoot in Toronto in order to go to a meeting in Rochester. The writer expects that Reverend Proudfoot will preach in Rochester. The letter has 1 postmark – St. Catharines, Aug. 14, 1843. A 2 page letter addressed to The Rev. Professor Proudfoot, London, C.W. from John Porteaus of St. Catharines. The writer says that he will not preach in Detroit. He says that the people of Detroit are expecting Mr. Dalrymple [who was sent as a missionary to Canada from Scotland in 1846] and also, he doesn’t want to leave his congregation for 2 Sabbaths. The letter has 2 postmarks – St. Catharines, August 1846 [this postmark is very faint] and Hamilton, August 2, 1846.