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Figure 17.3 Plasmid map of pEPI:

17.7 Human artificial chromosome

Artificial chromosomes are replica of normal chromosomes that are capable of replication and retention at low, defined copy number and within host cells. They have three main parts: a centromere, telomeres at both termini, and ori of replication. These chromosomes have exceptionally high cloning capacity and thus provide an excellent approach to be used as vectors in gene therapy. The first prototype HAC was generated in1997.

Human Artificial Chromosomes (HAC) can be synthesized de novo by assembly of the major components or by truncation of mammalian chromosome down to mitotically stable minichromosomes of 1-10 Mb, consisting of alpha satellite DNA arrays. Also neocentromeres based chromosomes provide another alternative of synthesizing HAC. Neocentromeres are functional variant of human centromeres and naturally present in non-centromeric region that are devoid of alpha satellite DNA. Neocentromere based human minichromosomes are 0.7-2 Mb in size that bind to centromere protein and are mitotically stable. HAC have been used in several gene therapy studies, for example in long term expression of 40 kb HPRT gene in Lesch-Nyhan syndrome, 250 kb CFTR gene and regulatory sequence in treating cystic fibriosis.

Because of its large size, microcell mediated chromosome transfer and microinjection are used to transfect HAC into different cell types in ex vivo approaches. Conventional non viral approaches make use of liposomes along with polyethylenimine or polylysine to deliver artificial DNA but are of lesser efficiency in vivo . The use of lipids with ultrasound has recently been demonstrated to induce better membrane pore formation allowing entry of DNA. Addition of NLS sequence either to the delivery agent or HAC might further improve nuclear entry or help stable transfection.