Input DNA Ratio Determines Copy Number of The 33 kb Factor IX Gene on De Novo Human Artificial Chromosomes

Abstract

Human artificial chromosomes (ACs) are non-integrating vectors that may be useful for gene therapy. They assemble in cultured cells following transfection of human centromeric alpha -satellite DNA and segregate efficiently alongside the host genome. In the present study, a 33 kilobase (kb) Factor IX (FIX) gene was incorporated into mitotically stable ACs in human HT1080 lung derived cells using co-transfection of a bacterial artificial chromosome (BAC) harboring synthetic alpha -satellite DNA and a P1 artificial chromosome(PAC) that spans the FIX locus. ACs were detected in >or=90% of chromosome spreads in 8 of 19 lines expanded from drug resistant colonies. FIX transgene copy number on ACs was determined by input DNA transfection ratios. Furthermore, a low level of FIX transcription was detected from ACs with multiple transgenes but not from those incorporating a single transgene, suggesting that reducing transgene number may limit misexpression. Their potential to segregate cross species was measured by transferring ACs into mouse and hamster cell lines using microcell-mediated chromosome transfer. Lines were obtained where ACs segregated efficiently. The stable segregation of ACs in rodent cells suggests that it should be possible to develop animal models to test the capacity of ACs to rescue FIX deficiency.

Organization of Synthetic Alphoid DNA Array in Human Artificial Chromosome (HAC) with a Conditional Centromere

“…The spectrum of the fragments rescued by TAR cloning corresponded to the spectrum of multiple bands seen on Southern blots after genomic digestion by an endonuclease that cuts the input DNA only once. This phenomenon seems general, as similar irregular-size Southern profiles suggesting rearrangements in the input DNA have been reported for several de novo formed HACs 17, 27, 29, 32, 33 . However, the detailed structural basis of the multiple bands in those HACs had not been investigated.…”

Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells

“…S8). Although additional studies are needed to determine whether genes inserted into the alphoid tetO -HAC may eventually be subject to silencing, our results and those obtained with other HACs (21,41) support the view that proximity to a functional kinetochore does not negatively affect gene expression. It is possible that, in the human genome, centromeric chromatin may be a privileged region for Pol II-transcribed genes, as previously shown for rice centromeres (43) and suggested by the resemblance of the centromeric chromatin profile to the downstream region of transcribed genes in human cells (39,44).…”

A new generation of human artificial chromosomes for functional genomics and gene therapy

“…Analysis revealed a specific pattern of the input DNA molecules assembled into a megabase-size array that provides a good control of the HAC integrity during HAC manipulations. Similar patterns had been previously reported for other de novo formed HACs [43, 54, 72, 73]. For therapeutic purposes, it is very important to know whether the HAC structure changes over many months of culture and after multiple transfers from one cell line to another.…”

“…A comprehensive list of genes loaded on HAC vectors is available in the recent review by Kazuki and Oshimura [22]. Among them, the following genes that are represented as genomic copies that include all regulatory elements: human beta-globin [46], CFTR [52, 53], Factor IX [54], STAT3 [55], TP53 [56], DYS [57], VHL , and NBS1 [58]. However, in almost all cases, the copy number of the gene in the HAC was not precisely controlled either because of the presence of multiple gene accepter sites in the HAC or because the gene was inserted into the HAC during its de novo formation, i.e.…”