Development of human papillomavirus plasmids capable of episomal replication in human cell lines

Abstract

Gene delivery into human cells has been facilitated by the plasmids containing the viral E1 and E2 genes (or the E1 development of viral vector systems. These vectors have gene alone) and an origin of replication were shown to repshown great potential for the efficient delivery of theralicate to significant levels in the transfected human cervical peutic genes into human cells. A problem with many of the carcinoma C-33A cell line. Since approaches towards the existing systems, however, is the integration of these vecpossible gene therapy of cystic fibrosis (CF) are currently tors into the chromosome which affects the length of gene under intensive investigation, we have also tested shortexpression and may promote oncogenic transformation. HPVs are small circular double-stranded DNA viruses of approximately 8-kb that infect squamous epithelial cells and produce benign warts and papillomas, as well as cancer of the cervix. 6-8 The viral genome exists in an extrachromosomal form as a chromatin and is maintained in the basal cells of the squamous epithelium in its latent state in a stable copy number estimated to be approximately 50. 9 In the upper differentiating cells of the epithelium, vegetative replication of HPV occurs resulting in a high copy number. 9 HPV types such as 1a and 2 infect cutaneous epithelium whereas types such as 6, 11, 16 and 18 infect the mucosal epithelium. 6 HPVs require the viral E1 and E2 proteins for their replication, and a cis-acting sequence termed the long control region (LCR) contains the promoter and enhancer elements as well as the origin of replication (ori). 10 The ori includes one E1 and multiple E2 binding sites. Previous studies using three-plasmid systems, one expressing the HPV E1 protein, another the E2 protein and a plasmid containing the HPV ori have demonstrated HPV DNA replication in transfected fibroblast and epithelial cell lines. [10][11][12][13][14] In addition, in the case of HPV type 1a, the E1 protein alone was shown to be sufficient for replication of ori plasmids. 15 The E6 and E7 proteins of high-risk HPVs such as types 16 and 18 are involved in cellular transformation and target the cellular p53 and RB proteins, respectively. 16 However, it is important to note that the transforming E6 and E7 genes of HPVs are not required for HPV DNA replication, at least in short-term assays. [10][11][12][13][14]

Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia

“…The pZF HpV  + F N genotype may be sub-cloned using similar PCR-primers to the ones proposed above for consortia plasmids, with tailored modifications to HPV plasmids or PsV. Sverdrup et al [43] have previously generated such human papillomavirus (HPV) plasmids containing the viral E1 and E2 genes (or the E1 gene alone) and an origin of replication, which they simultaneously demonstrated to replicate to significant levels in the transfected human cervical carcinoma C-33A cell line. Alternatively, HPV PsV encapsidation of the zinc finger consortium’s plasmids carrying the pZF HpV  + F N (see results of modeling above) may suffice.…”

Chapter 4 Replication and Partitioning of Papillomavirus Genomes

“…For example, newer BPV-1 based vectors contain only the LCR and the E1 and E2 genes (Mannik et al, 2002;Ohe et al, 1995) and are maintained in transgenic mice for several generations (Mannik et al, 2003). Human papillomavirus-based vectors have also been explored and shown to be able to replicate, at least transiently, in human cell lines and in the lungs of mice (Gadi et al, 1999;Sverdrup et al, 1999). However, at this point these studies have shown mainly "proof of principle" of papillomavirus based vectors and they have not yet been widely or practically used to express foreign genes.…”

Μελέτη Του Ενζύμου Της RNA Πολυμεράσης ΙΙΙ Σε Δείγματα Καρκίνου Του Τραχήλου Της Μήτρας Που Έχουν Μολυνθεί Από Τον Ιό Του Θηλώματος Του Ανθρώπου (HPV)

“…Η πρωτεΐνη αυτή είναι απαραίτητη για την ιική DNA αντιγραφή, καθώς προσδένεται στο σηµείο έναρξης της αντιγραφής του ιικού γονιδιώµατος στην LCR περιοχή[Stunkel & Bernard 1999]. Έχει δειχτεί ακόµη, πως προσδένει ATP καθώς και την πλήρους µεγέθους E2, η οποία ονοµάζεται και E2 διεγέρτης της µεταγραφής (E2 transcription transactivator, E2TA), ώστε να εντείνει την ιική µεταγραφή[Sverdrup et al 1999]. Ωστόσο, κάποια προϊόντα εναλλακτικής ωρίµανσης αυτού του ORF, λειτουργούν σαν καταστολείς της µεταγραφής[Chiang et al 1992].…”