**1. Introduction**

104 Non-Viral Gene Therapy

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#### **1.1 Viral vectors for gene therapy**

Gene therapy is a way to correct mutated genes in vivo by transferring normal genes into cells of patients with genetic diseases or cancers, or to introduce new genes into cells to express therapeutic proteins. Several viruses like adenoviruses (Nayak & Herzog, 2010; Raper et al., 2003), alphaviruses (Lundstrom, 2001, 2005), retroviruses (Aiuti et al., 2009; Bordignon et al., 1989, 1995; Cavazzana-Calvo et al., 2000; Ferrari et al., 1991; Halatsch et al., 2000), lentiviruses (Dupré et al., 2004; Mortellaro et al., 2006; Nayak & Herzog,

2010), adeno-associated viruses (AAV) (Jayandharan et al., 2011; Nayak & Herzog, 2010; Terzi & Zachariou, 2008), herpes simplex viruses type 1 (HSV-I) (Epstein, 2009), have been used as vectors to deliver normal genes into cells of patients for gene therapy. However, there were limitations and hurdles in using these vectors. Some viruses like retroviruses, lentiviruses might integrate into human genomic DNA and cause cancers (Dave et al., 2004; Du et al., 2005; Hacein-Bey-Abina et al., 2003a, 2003b; Z. Li et al., 2002; Modlich et al., 2005; Seggewiss et al., 2006). Most viruses can infect both normal cells and defective/cancer cells of patients, as long as the cells have receptors of the viruses (Antar et al., 2009; K. Holmes et al., 1997; Norkin, 1995; L. Song, 2010; L. Song et al., 2009; van den Wollenberg et al., 2008; van Houdt et al., 2008), and this might lead to serious infections, inflammatory responses, and immunological reactions (Nayak & Herzog, 2010).

#### **1.2 Highly pathogenic (virulent) viruses, moderately pathogenic viruses, and lowly or mildly pathogenic viruses**

Some viruses like rabies virus, Lassa fever virus, smallpox virus, Eastern equine encephalitis virus, Ebola virus, Marburg virus, and human immunodeficiency virus are highly pathogenic and dangerous; they can cause very severe to fatal diseases in humans. For example, 399 patients had Marburg hemorrhagic fever in Angola in 2005, and 335 of them dead of the fatal disease. The human fatality rate of Ebola virus infection ranged from 50% to 89% (Balter, 2000; Peters, 2005; Rouquet et al., 2005; L. Song & Chen 1995, 1996; Virgin, 2007). Some viruses like some serotypes of seadornavirus isolated from mosquitoes in China have moderate pathogenicity, and they can cause clinical and subclinical infections.

Gene Therapy of Some Genetic Diseases by Transferring

Normal Human Genomic DNA into Somatic Cells and Stem Cells from Patients 107

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,

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 humans (Csatary et al., 1985).

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