Functional human CFTR produced by a stable minichromosome

Abstract

Artificial chromosomes have been claimed to be the ideal vector for gene therapy, but their use has been hampered by an inability to produce stable and well designed molecules. We have used a structurally defined minichromosome to clone the human cystic fybrosis transmembrane conductance regulator (CFTR) locus. To guarantee the presence of the proper regulatory elements, we used the 320 kb yeast artificial chromosome (YAC) 37AB12 with the intact CFTR gene and upstream sequences. The resulting minichromosome was analyzed for the presence of the entire CFTR gene and for its functional activity by molecular and functional methods. We have identified clones showing the presence of both the transcript and the CFTR protein. Moreover, in the same clones, a chloride secretory response to cAMP was detected. Mitotic and molecular stability after prolonged growth without selection demonstrated that the constructs were stable. This is the first example of a structurally known minichromosome made to contain an active therapeutic gene.

Reconstructing large regions of an ancestral mammalian genome in silico

“…"Humanized" mice, which have specific individual genes replaced by their human versions, have been produced by these methods. Multimegabase transgenic sequences have been introduced in mammalian artificial chromosomes, e.g., for the human CFTR locus (Auriche et al 2002). However, these methods of introducing foreign DNA are expensive even when using available genomic sequences, and new methods for synthesizing large segments of DNA de novo would be needed to apply them to ancestral genomic reconstruction, e.g., to produce what might be called "retrovolved" mice that harbor the ancestral versions of specific gene loci.…”

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects

“…[155][156][157] Problems in the use of minichromosomes derive from difficulties in manufacture, handling and delivery to target cells owing to their enormous size (a few megabase pairs), as well as to their tendency to rearrange. Not only have mammalian artificial chromosome (MAC) vectors now become available, but transfer of a MAC into human cord blood CD34 + cells with an efficiency of 2.5-4.0% has very recently been achieved.…”

Gene Therapy Progress and Prospects: Episomally maintained self-replicating systems

“…It was constructed in CHO cells containing MC1 by yeast protoplast fusion. 35 The CFTR activity was assayed in the three selected clones that showed the presence of the MC with the expected structure by means of molecular and functional assays. Northern and Western analysis showed the presence of the transcript and the protein at similar level to that of the positive controls (human cells expressing the CFTR gene).…”