**3.2 Induced pluripotent stem cells**

110 Non-Viral Gene Therapy

Gene targeting (gene knockout) is a technique that a vector is used to deliver a fragment of mutated DNA into embryonic stem cells and to target its homologous DNA in the genome by homologous genetic recombination. Thousands of genes in mice were knocked out by using this method. Mario R. Capecchi, Martin J. Evans, and Oliver Smithies were awarded jointly the Nobel Prize in Physiology or Medicine in 2007 for their discoveries of principles for creating knockout mice (Bradley et al., 1984; Bronson & Smithies, 1994; Capecchi, 1989a,

It was assumed that plant tissue grafts did not have gene exchange, but this is not true. A recent discovery found that even in plant tissue grafts, some of their genes were exchanged

Based on the above observations and experiments, I proposed a possible approach for gene

Somatic cells and stem cells from a patient

In vitro serumfree cell culture

The corrected cells would be given back to the same patient

Fig. 1. Gene therapy of some genetic diseases by transferring normal human genomic

Briefly, normal human genomic DNA or genomic DNA fragments from a healthy donor can be transferred into somatic cells and stem cells from a patient by microinjection, gene electrotransfer (electroporation), and improved transfection. After in vitro serum-free cell culture, the defective genes could be repaired by homologous genetic recombination, since the genomic DNAs of the two persons are considerably similar, although there are deletions, insertions, rearrangements, loss-of-function variants, and copy number variations (CNV) (Fujimoto et al, 2010; Levy et al., 2007; H. Park et al., 2010; The 1000 Genomes Consortium,

Stem cells were first discovered by Ernest Armstrong McCulloch (April 27, 1926–January 20, 2011) and James Edgar Till in 1963 **(**Becker et al., 1963; McCulloch et al., 1964; Siminovitch et al., 1963; Till et al., 1964; Weissman & Shizuru, 2008). Stem cells have the ability to selfrenew and to differentiate into different cell types. Early stage embryonic stem cells can

DNA/DNA fragments into cells from patients.

Improved transfection of DNA fragments

Microinjection

Electrotransfer of DNA fragments

2010, 2011; J. Wang et al., 2008; Yim et al., 2010).

**3. Discussion** 

**3.1 Stem cells, somatic cells** 

1989b; Koller & Smithies, 1992; Kuehn et al., 1987; Robertson et al., 1986).

therapy of some genetic diseases as indicated in figure 1 (L. Song, 2009).

(Stegemann & Bock, 2009).

Genomic DNA from a normal person

In recent years, a few transcription factors (genes) were introduced into somatic cells by lentiviral or retroviral vectors, to reprogram somatic cells into pluripotent stem cells (Liao et al., 2008; I. Park et al., 2008; Takahashi et al., 2007; J. Yu et al., 2007).

Some of the genes like c-Myc that used to form induced pluripotent stem cells are oncogenes, and as above revealed, retroviral or lentiviral vectors might integrate into genome DNA randomly, these risk factors might lead to producing cancer cells. A recent study revealed that human induced stem cells were easier and faster to form tumors than human embryonic stem cells (Gutierrez-Aranda et al., 2010).

Some recently published papers disclosed that induced pluripotent stem cells could induce more immune responses and immunological rejections in the recipient mice, and had more protein-coding point mutations, more abnormal epigenomic reprogramming, and more copy number variations than normal somatic cells and normal embryonic stem cells (Gore et al., 2011; Hussein et al., 2011; Laurent et al., 2011; Lister et al., 2011; Zhao et al., 2011).
