Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells

Abstract

The expression of genes from genomic loci can be relatively complex, utilizing exonic, intronic and flanking sequences to regulate tissue and developmental specificity. Infectious bacterial artificial chromosomes (iBACs) have been shown to deliver and express large genomic loci (up to 135 kb) into primary cells for functional analyses. The delivery of large genomic DNA inserts allows the expression of complex loci and of multiple splice variants. Herein, we demonstrate for the first time that an iBAC will deliver and correctly express in human glioma cells the entire CDKN2A/CDKN2B genomic region, which encodes for at least three important cell-cycle regulatory proteins (p16 INK4a , p14 ARF and p15 INK4b ). Two of these proteins are expressed from overlapping genes, utilizing alternative splicing and promoter usage. The delivered locus expresses each gene at physiological levels and cellular responses (apoptosis versus growth arrest) occur dependent on cellular p53 status, as expected. The work further demonstrates the potential of the iBAC system for the delivery of genomic loci whose expression is mediated by complex splicing and promoter usage both for gene therapy applications and functional genomics studies.

Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis

“…S/MARs are associated with most of the characterized mammalian origins of replication [172,173]. The 30 nm fiber of DNA, histones and chromatin proteins appears to be organized into loops by interaction of S/MARs with the nuclear matrix [174].…”

“…To achieve physiological conditions of expression, correct alternative splicing and promoter usage, an efficient vector should be based on sequences derived from the human genome, which include the native promoter and all regulatory sequences of a gene [172,173].…”

HSV-1 amplicon viral vector-mediated gene transfer to human bone marrow-derived mesenchymal stem cells

“…These viral elements are needed for the amplicon plasmid vectors to be packaged into replication defective infectious virions in the presence of HSV helper functions. 27,28 HSV-1 amplicon viral vectors excel in their capacity to package large transgene insert of up to 150 kb, 13,[29][30][31] which is in contrast to the 8 kb allowable insert size of lentiviral vector. The large transgene capacity of HSV-1 amplicon viral vectors enabled for the insertion of therapeutic genes such as dystrophin (full-length cDNA of 17.3 kb) and frataxin (encoded by the 135 kb gene FRDA) for the treatment of Duchenne's muscular dystrophy 32 and Friedreich's ataxia, 33 respectively, which is not possible with other vector system.…”

Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

“…2,[9][10][11] The focus of the present study was on stable transduction and expression of the BGAL gene in cells, ideally through integration at the AAVS1 locus.…”

“…[5][6][7][8] Amplicon vectors can incorporate foreign DNA of up to about 150 kb. [9][10][11] These vectors are generated with the aid of replication-conditional helper-virus or in a helper virusfree packaging system with titers between 10 7 and 10 11 transducing units/milliliter (tu/ml). 8,[12][13][14] DNA delivered by HSV amplicon vectors persists only temporarily in the nucleus of the infected cell.…”