**2. A possible approach for gene therapy of some genetic diseases**

It is well known that a bacterium can obtain foreign DNA from another bacterium through a process of bacterial conjugation (Lederberg & Tatum, 1946). In this process, DNA is directly transferred from one cell into another cell via direct cell-to-cell contact or via a bridge-like structure between two cells. By this way, a bacteium's genomic DNA could be changed by homologous recombination with another bacterium's genomic DNA.

As above pointed out, multisegmented RNA viruses like influenza virus can reassort their genomic segments in vivo, if an animal is infected by two or more different strains of a virus in the same time period. This can create new strains of the virus, and the new strains carry the gene segments of their parental strains. There are some triple reassorted influenza virus strains in nature (V. Gregory et al., 2001; Ma et al., 2010; Octaviani et al., 2011; Rambaut et al., 2008; Smith et at., 2004; Vincent et al., 2006; Webby et al., 2000; X. Xu et al., 2002, 2004; Zhou et al., 1999).

Complementation test was used to study genetic subtypes (complementation groups) of a genetic disease like Fanconi anemia. If cells from two patients with Fanconi anemia were from two different complementation groups, the defective genes could be repaired after fusion of the two genetic complementation cells; and the hybrid cells were able to resist the attack of DNA cross-linking agents such as mitomycin C and diepoxybutane. Whereas, if the cells of two patients were from the same complementation group, the hybrid cells would still be sensitive to the attack of DNA cross-linking reagents (Buchwald, 1995; Duckworth-Rysiecki et al., 1985; Fanconi Anaemia/Breast Cancer Consortium, 1996; Giampietro et al., 1997; Joenje et al., 1995; Joenje & Patel, 2001; Levitus et al., 2004; Tischkowitz & Hodgson, 2003; Whitney et al., 1995). 14 different complementation groups of Fanconi anemia have been discovered, and 14 distinct Fanconi anemia genes have been identified (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FNACI, FANCJ, FANCL, FANCM, FANCN, FANCP) (Alderton, 2011; Kim et al., 2011; Levitus et al., 2006; Reid et al., 2007; Smogorzewska et al., 2007; L. Song, 2009; Stoepker et al., 2011; Taniguchi & D'Andrea, 2006; Tischkowitz et al., 2008).

 It is very interesting that a number of patients with some genetic diseases were cured or improved naturally later in their lives. Somatic mosaicism was involved in this form of miracle "natural gene therapy" process. Somatic mosaicism means that there are genetically different somatic cells exist in a given organism resulting from in vivo reversion of a

1994; Tai at el., 2005), and there are increasing evidences indicating that reovirus can infect normal human cells in vivo and cause some mild to serious diseases like upper respiratory illnesses, meningitis in humans (Johansson et al., 1996; Schiff et al., 2007; Tyler et al., 2004); the old concept that reoviruses were "orphan" viruses, and they were not associated with

Wild-type reovirus should not be considered as an oncolytic virus, and it is unlikely that

Non-viral vectors such as peptide, polymer mediated gene therapy can only produce transient expression of genes, and the transfection efficiency is much lower compared to viral vectors (Al-Dosari & Gao, 2009; Cartier & Reszka, 2002; X. Gao et al., 2007; Niidome &

It is well known that a bacterium can obtain foreign DNA from another bacterium through a process of bacterial conjugation (Lederberg & Tatum, 1946). In this process, DNA is directly transferred from one cell into another cell via direct cell-to-cell contact or via a bridge-like structure between two cells. By this way, a bacteium's genomic DNA could be changed by

As above pointed out, multisegmented RNA viruses like influenza virus can reassort their genomic segments in vivo, if an animal is infected by two or more different strains of a virus in the same time period. This can create new strains of the virus, and the new strains carry the gene segments of their parental strains. There are some triple reassorted influenza virus strains in nature (V. Gregory et al., 2001; Ma et al., 2010; Octaviani et al., 2011; Rambaut et al., 2008; Smith et at., 2004; Vincent et al., 2006; Webby et al., 2000; X. Xu et al., 2002, 2004;

Complementation test was used to study genetic subtypes (complementation groups) of a genetic disease like Fanconi anemia. If cells from two patients with Fanconi anemia were from two different complementation groups, the defective genes could be repaired after fusion of the two genetic complementation cells; and the hybrid cells were able to resist the attack of DNA cross-linking agents such as mitomycin C and diepoxybutane. Whereas, if the cells of two patients were from the same complementation group, the hybrid cells would still be sensitive to the attack of DNA cross-linking reagents (Buchwald, 1995; Duckworth-Rysiecki et al., 1985; Fanconi Anaemia/Breast Cancer Consortium, 1996; Giampietro et al., 1997; Joenje et al., 1995; Joenje & Patel, 2001; Levitus et al., 2004; Tischkowitz & Hodgson, 2003; Whitney et al., 1995). 14 different complementation groups of Fanconi anemia have been discovered, and 14 distinct Fanconi anemia genes have been identified (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FNACI, FANCJ, FANCL, FANCM, FANCN, FANCP) (Alderton, 2011; Kim et al., 2011; Levitus et al., 2006; Reid et al., 2007; Smogorzewska et al., 2007; L. Song, 2009; Stoepker et al., 2011; Taniguchi & D'Andrea,

 It is very interesting that a number of patients with some genetic diseases were cured or improved naturally later in their lives. Somatic mosaicism was involved in this form of miracle "natural gene therapy" process. Somatic mosaicism means that there are genetically different somatic cells exist in a given organism resulting from in vivo reversion of a

any human diseases, is not true anymore, and it should be revised.

**1.4 Non-viral vectors for gene therapy** 

Huang, 2002).

Zhou et al., 1999).

2006; Tischkowitz et al., 2008).

reovirus could be an effective and practical anticancer agent (L. Song, 2010).

**2. A possible approach for gene therapy of some genetic diseases** 

homologous recombination with another bacterium's genomic DNA.

mutated allele to wild type. Somatic mosaicism has been found in several genetic disorders, such as hemophilia B, tyrosinemia type I, Bloom syndrome, adenosine deaminase deficiency, epidermolysis bullosa, Wiskott-Aldrich syndrome, androgen insensitivity syndrome, T-cell immunodeficiency, leukocyte adhesion deficiency type 1, Duchenne muscular dystrophy, atypical X-linked severe combined immunodeficiency, and Fanconi anemia. The mechanism of somatic mosaicism is complicated; it might be due to epigenetic alterations of DNA, copy number variations, back mutation, gene conversion, framerestoring mutation, DNA polymerase slippage, and compensatory mutation in cis. Homologous genetic recombination between paternally and maternally derived chromosomes also plays a vital role in somatic mosaicism (Darling et al., 1999; J. Jr. Gregory et al., 2001; M. Gross et al., 2002; Hirschhorn, 2003; Lo Ten Foe et al., 1997; Mankad et al., 2006; Müller & Williams, 2009; Notini et al., 2008; Piotrowski et al., 2008; L. Song, 2009; Wada et al., 2001, 2003; Waisfisz et al., 1999; Youssoufian & Pyeritz, 2002).

Homologous genetic recombination was first discovered in bacterium *Escherichia coli* strain K-12 in 1946 by Joshua Lederberg (May 23, 1925 – February 2, 2008), winner of the 1958 Nobel Prize in Physiology or Medicine (Lederberg & Tatum, 1946; Lederberg, 1947, 1987a, 1987b; Tatum & Lederberg, 1947).

Homologous recombination happens when two homologous DNA molecules meet in vivo. They pair up and exchange some sequences. Homologous genetic recombination occurs in the processes of mitosis and meiosis. Gene recombination takes place between two nonsister chromatids of the two homologous chromosomes by crossover during meiosis. Homologous recombination happens much more often in distal regions of chromosomes and on shorter arms of chromosomes. Crossover occurs at least one time per chromosome in each of the process of meiosis. Gene sequences are exchanged during the process of meiosis by crossover (Creighton & McClintock, 1931, 1935; Holliday, 1974; International Human Genome Sequencing Consortium, 2001; Weil, 2002; Whitby, 2005).

Crossover during the process of meiosis is really a smart and fair way to let a female's eggs or a male's sperms to have genetic information from both of her/his parents; and when an egg and a sperm form a new life in the womb, the new baby carries the genetic information from both of his/her grandparents on his/her father's side and grandparents on his/her mother's side; so a baby's genetic traits are inherited from his/her four biological grandparents, this could make the baby more diversity, flexible, and fit.

Radioactive materials like radon gas in some basement rooms, ultraviolet (UV) light, toxic chemicals, reactive-oxygen compounds, polluted air, and smoking, some viral infections all can damage human genomic DNA, cause mutations, and lead to cancers. Human cells are able to cope with outside and inside challenges, and to repair the damaged DNA molecules by several ways. One of the DNA repair mechanisms is homologous recombination to repair DNA gaps, DNA double-stranded breaks, and DNA interstrand crosslinks. A damaged chromatid can be repaired by its undamaged sister chromatid or its homologous nonsister chromatid through homologous recombination during mitosis. Sister chromatids are the preferred templates over homologous or heterologous chromosomes for recombination repair in yeast and mammalian cells. Instead of crossover, gene conversion is the major result of homologous recombination. Cells seem reluctant to crossover their chromatids unnecessarily to maintain their genome's integrity and stability (Hope et al., 2007; Jackson & Bartek, 2009; Johnson & Jasin, 2001; Kadyk & Hartwell, 1992; X. Li & Heyer, 2008; Lorenz & Whitby, 2006; Willers et al., 2004).

Gene Therapy of Some Genetic Diseases by Transferring

et al., 1998; Weissman & Shizuru, 2008).

**3.2 Induced pluripotent stem cells** 

al., 2011).

& Wente, 2006).

al., 1999).

Normal Human Genomic DNA into Somatic Cells and Stem Cells from Patients 111

form all types of cells. Adult stem cells only can differentiate and generate specialized cells, like bone cells, liver cells, blood cells, skin cells. The stem cells that produce all the blood cell types are called hematopoietic stem cells (Bordignon, 2006; Spangrude et al., 1988; Thomson

Stem cells and their differentiated cells (somatic cells) maintain a balance-homeostasis. When under stress, stem cells are activated, and start to produce more differentiated cells

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

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

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

Both a eukaryotic cell and a prokaryotic cell have a flexible lipid bilayer plasma membrane that controls movement of molecules in and out of the cell. A eukaryotic cell has a nucleus, while a prokaryotic cell does not have a nucleus; this is the characteristic difference between

The eukaryotic cell nucleus is surrounded by a nuclear envelope with nuclear pores. The nuclear envelope has two layers: the out nuclear membrane which faces the cytoplasm, and the inner nucleic membrane which faces the nucleoplasm. The nuclear pores are formed by nuclear pore complexes (NPCs) that span the double lipid bilayer of the nuclear envelope. The NPCs are formed by about 30 proteins. NPCs are gatekeepers of the nucleus (Alber et al., 2007; D'Angelo et al., 2006; D'Angelo & Hetzer, 2008; Devos et al., 2006; Fernandez-Martinez & Rout, 2009; Lam & Dean, 2010; Terry et al., 2007; Theerthagiri et al., 2010; E. Tran

Ions and small molecules and DNA smaller than 200 bp can diffuse through the nuclear pore freely; while the transport of DNA molecules between 310 bp and 1500 bp from the cytosol to the nucleus is through an active transport process. DNA greater than 2 kb can rarely be seen in the nucleus (Cartier & Reszka, 2002; Hagstrom et al., 1997; Ludtke et

A foreign DNA molecule has to go through the human cell membrane, cytoplasm, and the nuclear envelope to reach the genomic DNA in the nucleus. This process can be performed

and prompted by microinjection, electroporation, and transfection.

(Jiang et al., 2009; Martinez-Agosto et al., 2007; Till et al., 1964; Wilson et al., 2004). Somatic cells are the end products of stem cells, they are unable to self-renew.

al., 2008; I. Park et al., 2008; Takahashi et al., 2007; J. Yu et al., 2007).

human embryonic stem cells (Gutierrez-Aranda et al., 2010).

**3.3 Cell membrane and nuclear envelope** 

a eukaryotic cell and a prokaryotic cell.

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

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 (Stegemann & Bock, 2009).

Based on the above observations and experiments, I proposed a possible approach for gene therapy of some genetic diseases as indicated in figure 1 (L. Song, 2009).

Fig. 1. Gene therapy of some genetic diseases by transferring normal human genomic DNA/DNA fragments into cells from patients.

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