**1.3 Reovirus is not an oncolytic virus**

There are Some scientists in the world have been trying to use a few so-called oncolytic viruses to cure cancers (Pennisi, 1998). One of the major problems of these therapies is that it is hard to find an ideal wild-type oncolytic virus, which only target cancer cells but not normal cells.

Normal rhesus monkey kidney LLC-MK2 cell line was established in 1955 (Evans et al., 1959; Hull et al., 1956, 1962). Normal mouse L929 cell line was first described by Stanford et al. in 1948. The L929 cell line was a cloned strain of its parental mouse cell strains L. L cell strain was made from the normal subcutaneous areolar and adipose tissue of a male mouse (Earle, 1943; Stanford et al., 1948). These two cell lines were widely used to isolate, grow, and multiplicate many types of viruses including reoviruses.

Seadornavirus can cause mild encephalitis and fever. Multisegmented RNA viruses like influenza virus (Garten et al., 2009; E. Holmes, 2005; Karasin et al., 2000; Sun et al., 2011), rotavirus (Matthijnssens et al., 2010; Maunula & Von Bonsdorff, 2002), bluetongue virus (Batten et al., 2008), kemerovo virus (Nuttall & Moss,1989), Thogoto virus (C. Davies et al., 1987; Jones et al., 1987) are able to reassort their genomic segments in vivo, if a cell is infected by two or more different strains of a virus. This is the major reason why these viruses have multiple serotypes and subserotypes. As seadornavirus genome consists of 12 distinct segments of double-stranded RNA, it is easy to create new genotypes of seadornavirus through the reassorment event among different strains of the virus in nature. There are at least 6 different genotypes of seadornavirus in China, and there are various serotypes and subserotypes within the Chinese isolates (Q. Li et al., 1992; L. Song et al., 1995; L. Song & Chen, 1995, 1996; Tao et al., 1999; L. Xu et al., 2003; P. Xu et al., 1990; You et al., 1990). Similar seadornaviruses were isolated from mosquitoes collected in Indonesia (Brown et al., 1993) and Vietnam (Nabeshima et al., 2008). The virus was classified as a probable member of the genus Coltivirus previously, and later it was renamed as a member of a novel genus-Seadornavirus within the family of Reoviridae (Mohd Jaafar et al., 2005). Some viruses like M14-a nonpathogenic twelve-segmented double-stranded RNA virus isolated from mosquitoes in China (C. Huang et al., 1985, Liang et al., 1985) are lowly or mildly virulent viruses. The majority of viruses like hepatitis A, B, C, D, and E virus, polio virus, measles virus, mumps virus, West Nile virus, influenza virus, Coxsackie A virus, enterovirus 71, rhinoviruses, coronaviruses, norovirus, rubella virus, and the newly isolated member of bunyavirus which caused severe fever and thrombocytopenia syndrome in China (X-J. Yu et al., 2011), have moderate pathogenicity. There are very few human viruses are truly nonpathogenic viruses in nature, except some animal viruses that mainly infect animals but not humans. These mildly virulent viruses cannot cause obvious infections in

We should be aware that even some mild viruses which do not cause serious infections in normal people still can be dangerous to those with weakened immune systems, like late stage cancer patients, very elderly or critically ill patients, and patients with immunodeficiency disorders. Most viruses were modified and attenuated before being used as vectors for gene therapy, but in very rare situations, even those modified viral vectors can cause problems. An 18-year-old young man with partial ornithine transcarbamylase deficiency died after a clinical trial of gene therapy, even though the vector used in that trial

There are Some scientists in the world have been trying to use a few so-called oncolytic viruses to cure cancers (Pennisi, 1998). One of the major problems of these therapies is that it is hard to find an ideal wild-type oncolytic virus, which only target cancer cells but not

Normal rhesus monkey kidney LLC-MK2 cell line was established in 1955 (Evans et al., 1959; Hull et al., 1956, 1962). Normal mouse L929 cell line was first described by Stanford et al. in 1948. The L929 cell line was a cloned strain of its parental mouse cell strains L. L cell strain was made from the normal subcutaneous areolar and adipose tissue of a male mouse (Earle, 1943; Stanford et al., 1948). These two cell lines were widely used to isolate, grow,

was a modified human adenovirus type 5 virus (Raper et al., 2003).

and multiplicate many types of viruses including reoviruses.

humans (Csatary et al., 1985).

**1.3 Reovirus is not an oncolytic virus** 

normal cells.

Reovirus (Respiratory Enteric Orphan Virus) is a member of the family Reoviradae. It got the name originally because it was often isolated from human respiratory and enteric systems but no obvious human disease was associated with it. Reovirus can cause cytopathic effect (CPE) in many normal cell lines like rhesus monkey kidney LLC-MK2 and MA-104E, African green embryonic monkey kidney Vero, baby hamster kidney BHK-21, Buffalo green monkey kidney BGM, African green monkey kidney BS-C-1, Madin-Darby bovine kidney (MDBK), Madin-Darby canine kidney (MDCK), human embryonic intestinal (intestinal 407), human embryonic lung (HEL), and mouse L929 cells. After a few days of cell culture, like most other viruses, reovirus will destroy and lyse the cells it infected eventually in vitro (McClain et al., 1967; Nibert et al., 1991; Ridinger et al., 1982; Rozee & Easterbrook, 1970; Schiff et al, 2007; L. Song et al., 1995, 1999b, 2000, 2009). Reovirus can infect and kill both normal cells and human tumor cells in vitro, as long as the cells have reovirus receptor-junctional adhesion molecule (Antar et al., 2009; L. Song et al., 1999b, 2000, 2009; van den Wollenberg et al., 2008; van Houdt et al., 2008). If a small number of reoviruses are injected into tumor tissues directly, the virus will infect and kill some tumor cells locally. In the meantime, the human immune system will fight with the virus, a lot of immune cells, such as T cells, B cells, natural killer cells, neutrophils, and macrophages will be recruited to the infection site, and the immune cells will produce antibodies, chemokines, and cytokines like interferons, interleukins; and after a few days, before the virus spreading to other parts of the body, the virus will be killed, and be cleared from the human body. If a large number of reoviruses are injected into the tumor body, the viruses will infect tumor cells and nearby normal cells, and spread to other organs of the body and cause systemic infection. This could be fatal for some cancer patients, as we know that many cancer patients have unbalanced, weakened, and dysfunctional immune systems. A great number of cancer patients are treated with radiation and immunosuppressive anticancer drugs, these anticancer therapies can damage immune cells further. Cancer patients with weakened immune systems have more chances to have opportunistic infections (Baggiolini et al., 1997; Bodey, 1986; Dunn et al., 2002, 2004; Locati & Murphy, 1999; Lodish et al., 2008; Luster, 1998; Murdoch & Finn, 2000; Nibert et al., 1991; Pitisuttithum et al., 2001; Schiff et al, 2007; Sutlu & Alici, 2009; Swann & Smyth, 2007; Virgin, 2007). This is the same problem we are facing when a patient has chemotherapies nowadays; there are rare drugs that only specifically and selectively target cancer cells but not normal cells. Over doses of anticancer drugs will kill both normal cells and tumor cells of patients, and lead to serious side effects and deaths; normal doses or small doses of anticancer drugs will not kill all the cancer cells, and the remained cancer cells will overexpress a membrane protein-P-glycoprotein, and be able to resistant to the cell kill effects of multi-anticancer drugs (Arkin et al., 1989; Croop et al., 1988; De Rosa et al., 2008; Debenham et al., 1982; Deuchars et al., 1987; Endicott & Ling, 1989; Goldstein et al., 1989; Juliano & Ling, 1976; Kobayashi et al., 1994, 1998; Moscow & Cowan, 1988; Pastan & Gottesman, 1987; Riordan et al., 1985; L. Song et al., 1999a). Most people were infected by reovirus without significant symptoms, but L-H. Song et al.

isolated a reovirus from the throat swabs of a patient of severe acute respiratory syndrome (SARS) in Beijing in 2003, and the virus can cause clinical symptoms similar to SARS in guinea pigs and macaques (L-H. Song et al., 2008). Antarasena et al. isolated some avian reoviruses from chickens with sudden death in Thailand (Antarasena et al., 2002). Chua et al. reported that a reovirus of bat origin could cause acute respiratory disease in humans (Chua et al., 2007). Given the fact that more than 50% of people were infected by reovirus in their lifetimes, and many of the infections occurred in the early childhood (Selb & Weber,

Gene Therapy of Some Genetic Diseases by Transferring

1987b; Tatum & Lederberg, 1947).

Normal Human Genomic DNA into Somatic Cells and Stem Cells from Patients 109

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;

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,

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

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

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,

Wada et al., 2001, 2003; Waisfisz et al., 1999; Youssoufian & Pyeritz, 2002).

Genome Sequencing Consortium, 2001; Weil, 2002; Whitby, 2005).

grandparents, this could make the baby more diversity, flexible, and fit.

2008; Lorenz & Whitby, 2006; Willers et al., 2004).

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

Wild-type reovirus should not be considered as an oncolytic virus, and it is unlikely that reovirus could be an effective and practical anticancer agent (L. Song, 2010).
