Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts

Human artificial chromosomes (HACs) segregating freely from host chromosomes are potentially useful to ensure both safety and duration of gene expression in therapeutic gene delivery. However, low transfer efficiency of intact HACs to the cells has hampered the studies using normal human primary cells, the major targets for ex vivo gene therapy. To elucidate the potential of HACs to be vectors for gene therapy, we studied the introduction of the HAC vector, which is reduced in size and devoid of most expressed genes, into normal primary human fibroblasts (hPFs) with microcell-mediated chromosome transfer (MMCT). We demonstrated the generation of cytogenetically normal hPFs harboring the structurally defined and extra HAC vector. This introduced HAC vector was retained stably in hPFs without translocation of the HAC on host chromosomes. We also achieved the long-term production of human erythropoietin for at least 12 weeks in them. These results revealed the ability of HACs as novel options to circumvent issues of conventional vectors for gene therapy.

[1]  J. E. Mejía,et al.  Advances in human artificial chromosome technology. , 2002, Trends in genetics : TIG.

[2]  F. Ruddle,et al.  Chromosome-mediated gene transfer results in two classes of unstable transformants. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Killary,et al.  [9] Microcell fusion , 1995 .

[4]  J. V. Moran,et al.  Initial sequencing and analysis of the human genome. , 2001, Nature.

[5]  Mark A. Kay,et al.  Progress and problems with the use of viral vectors for gene therapy , 2003, Nature Reviews Genetics.

[6]  M. Oshimura,et al.  Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice , 1997, Nature Genetics.

[7]  B. Osborne,et al.  Cloned transchromosomic calves producing human immunoglobulin , 2002, Nature Biotechnology.

[8]  H. Willard,et al.  Formation of de novo centromeres and construction of first-generation human artificial microchromosomes , 1997, Nature Genetics.

[9]  Martin Fussenegger,et al.  Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells , 1998, Nature Biotechnology.

[10]  Charles Lee,et al.  Generation of an ∼2.4 Mb Human X Centromere-Based Minichromosome by Targeted Telomere-Associated Chromosome Fragmentation in DT40 , 1999 .

[11]  A. Telenius,et al.  Transfer and Stable Transgene Expression of a Mammalian Artificial Chromosome into Bone Marrow‐Derived Human Mesenchymal Stem Cells , 2004, Stem cells.

[12]  H. Masumoto,et al.  Distribution of CENP-B boxes reflected in CREST centromere antigenic sites on long-range alpha-satellite DNA arrays of human chromosome 21. , 1994, Human molecular genetics.

[13]  N. Mahmud,et al.  Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo. , 2001, Human gene therapy.

[14]  G. Andriole,et al.  Erythropoietin therapy. , 1997, The New England journal of medicine.

[15]  M. Goldberg,et al.  Regulation of the erythropoietin gene , 1993, Blood.

[16]  M. Oshimura,et al.  Construction of a novel human artificial chromosome vector for gene delivery. , 2004, Biochemical and biophysical research communications.

[17]  M. Oshimura,et al.  Construction of Mouse A9 Clones Containing a Single Human Chromosome (X/Autosome Translocation) via Micro‐cell Fusion , 1989, Japanese journal of cancer research : Gann.

[18]  H. Kondoh,et al.  Improved mammalian vectors for high expression of G418 resistance. , 1987, Cell structure and function.

[19]  H. Masumoto,et al.  Construction of YAC–based mammalian artificial chromosomes , 1998, Nature Biotechnology.

[20]  A. Telenius,et al.  Efficient in-vitro transfer of a 60-Mb mammalian artificial chromosome into murine and hamster cells using cationic lipids and dendrimers , 2004, Chromosome Research.

[21]  M. Oshimura,et al.  Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Oshimura,et al.  Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts , 2000, Nature Biotechnology.

[23]  E. Loboa,et al.  Human mesenchymal stem cells express palladin , 2005 .

[24]  M. Oshimura,et al.  Stability of transferred human chromosome fragments in cultured cells and in mice , 2004, Chromosome Research.

[25]  W. Earnshaw,et al.  Functional complementation of a genetic deficiency with human artificial chromosomes. , 2001, American journal of human genetics.

[26]  K. Choo,et al.  Construction of neocentromere-based human minichromosomes by telomere-associated chromosomal truncation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[27]  A. Killary,et al.  Microcell fusion. , 1995, Methods in Enzymology.

[28]  B. Ebert,et al.  Regulation of the erythropoietin gene. , 1999, Blood.