**3.11 Graft-versus-host disease (GVHD)**

It is often hard to find a human leukocyte antigen (HLA)-identical sibling or a well-matched HLA unrelated donor when a patient needs hematopoietic stem cell transplant (HSCT). Sometimes, a patient had to receive a mismatched or partially matched bone marrow transplant and cord-blood transplant, when there was no HLA-matched unrelated donor available, and when a transplant was needed urgently. Acute and chronic graft-versus-host disease is the most severe and common long-term side effect of allogeneic hematopoietic stem cell transplantation (HCT). Acute GVHD was more likely to occur after mismatched marrow transplantation. Chronic GVHD was the major cause of late death of HSCT patients (Eapen et al., 2010; Laughlin et al., 2001, 2004; Mastaglio et al., 2010; Rocha et al., 2004).

Cells seem to be able to tolerate foreign DNA without immunological reactions; this is proved by the animal cloning experiments, transgenic animal models, and human and animal replication phenomena. Therefore, the possible approach I described above might have great benefits and advantages. Hopefully some genetic diseases listed below could be cured or improved by using this gene therapy method.

#### **3.12 Fanconi anemia**

116 Non-Viral Gene Therapy

two β chains, and four heme groups (Feng et al., 2001; Groudine et al., 1983; Hardison, 1996; Yin et al., 2007). The human α-globin gene cluster lies on chromosome 16 (16p13.3), and is about 30 kb, it has 7 genes: zeta, pseudozeta, mu, pseudoalpha-1, alpha-2, alpha-1, theta (Barbour et al., 2000; Entrez Gene, 2011; Feng et al., 2001; Higgs et al., 1989). The human β-globin gene cluster is about 100 kb; it locates on chromosome 11 (11p15.5), and has 5 genes in the order of epsilon, gamma-G, gamma-A, delta, and beta. Both of α-globin gene and

Typically, in a viral or plasmid vector mediated gene therapy, normal mRNAs are reverse transcribed into cDNAs; and specific cDNAs are amplified by PCR method; the PCR products are purified and digested by restriction enzymes; the digested PCR products are inserted into the viral or plasmid vectors; the viral or plasmid vectors containing the normal genes are transfected/transformed into cells, in order to express normal proteins, or to

This procedure has a problem. As the above described, the mutated genes might be separated by several introns and located in several places of the genomic DNA, the cDNA clones of the normal genes are too short to match and find the mutated genes, therefore, it is hard to correct the mutated genes in vivo, although the cloned genes might express normal proteins transiently. By transferring normal human genomic DNA into cells from patients, it

We are living in an age that many important organisms have been sequenced (S. Ahn et al., 2009; Fujimoto et al., 2010; Holmes et al., 2005; International Human Genome Sequencing Consortium, 2001, 2004; International Silkworm Genome Consortium, 2008; Levy et al., 2007; O'Brien et al., 1999; Venter et al., 2001; J. Wang et al., 2008; Xia et al., 2004). We gained some valuable information from the genome sequence data of these organisms, but we are far away from knowing the secret of lives. A silkworm has a short but magical life cycle, and it proceeds in the following processes: it starts from a tiny egg; in a suitable environment, the egg turns into a small worm (larva); the small worm eats mulberry tree leaves greedily and thoroughly days and nights, and after 4 times of shedding its skin, it grows bigger and bigger; one day it starts to weave a silk house-a cocoon for itself, in about 2 days, a beautiful and perfect white colored cocoon is made by itself; the silkworm pees before weaving a cocoon, this makes its body smaller, so as to let itself be able to fit in the cocoon; inside the cocoon, the worm changes to a pupa, and before this happens, the worm poops, this makes its body further smaller; after about two weeks, the pupa becomes a moth, and it is time to get out of the cocoon; the moth is very smart, it pees inside the cocoon, the chemicals of the urine are so powerful-one of the chemicals is a special enzyme which can break down the cocoon wall, and it makes one end of the cocoon softer, so the moth can get out without trouble; the female moth comes out of the damaged cocoon, and releases sex pheromones to attract males, and mates with a few males, lays eggs after mating; and a new life cycle is started again if the environment is appropriate; or it will go through a

When you think about this miracle life cycle of a silkworm, you have to believe that these abilities, talents, and skills of a silkworm are not learned from others or from the environment, because actually no one teaches it to do this step by step, especially for the first silkworm who started doing these things earlier than all the others in a group of

β-globin gene have three exons and two introns (Higgs et al., 1989; Yin et al., 2007).

**3.10 Non-coding sequences of genome sequences, and the miracle silkworm** 

correct the mutated genes in vivo.

can overcome this difficulty.

period of hibernating.

Fanconi anemia (FA) is a rare chromosomal recessive genetic disease. As above cited, there are at least 14 subtypes of Fanconi anemia, and 14 genes whose mutation can cause FA are cloned. FANCB gene is on the X chromosome, and it is the only one on sex chromosomes, the other 13 FA genes are on autosomes. FA was first described by the Swiss pediatrician Guido Fanconi (1892-1979) in 1927 (Joenje & Patel, 2001; Lobitz & Velleuer, 2006; L. Song, 2009; Tischkowitz & Hodgson, 2003).

There are mouse models of Fanconi anemia available currently; FancA, FancC, FancG, FancD1, and FancD2 genes have been deleted or mutated in the mice (Parmar et al., 2009).

Gene Therapy of Some Genetic Diseases by Transferring

al., 2004; Hacein-Bey-Abina et al., 2002, 2010).

**3.19 Wiskott-Aldrich syndrome (WAS)** 

**3.20 Other diseases** 

**4. Conclusion** 

**5. References** 

**3.18 Adenosine deaminase deficiency (ADA)-SCID** 

**3.17 X-linked severe combined immunodeficiency (SCID-X1)** 

al., 2004; Qasim et al., 2009; Ramesh et al., 1997; Zhu et al., 1997).

insulin-dependent) (Freeman et al., 1999) diabetes.

Normal Human Genomic DNA into Somatic Cells and Stem Cells from Patients 119

X-linked severe combined immunodeficiency (SCID-X1) is caused by the mutations of interleukin-2 receptor subunit gamma (IL2RG) gene. Patients with the disease lack of T cells and natural killer cells, their B cells are functionally impaired; therefore, they are extremely vulnerable to infections (Aiuti & Roncarolo, 2009; Cavazzana-Calvo et al., 2000; Gaspar et

ADA- SCID is a rare genetic disease caused by a mutation of a gene on chromosome 20; this gene encodes an enzyme called adenosine deaminase (ADA). The mutation can lead to lack of ADA enzyme, and the lack of ADA enzyme causes disorder of adenosine metabolism and severe combined immunodeficiency (Aiuti et al., 2002, 2009; Aiuti & Roncarolo, 2009; Bordignon et al., 1989, 1995; Ferrari et al., 1991; Gaspar et al., 2009; Mortellaro et al., 2006).

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive primary immunodeficiency disease caused by mutations of the WAS protein (WASP) gene. The WASP gene is located on chromosome Xp11.22–Xp11.23. It has 12 exons, and encodes 502 amino acids. Patients with Wiskott-Aldrich syndrome have smaller platelets and lymphocytes, and their platelet counts are decreased; they have bleeding problems, recurrent bacterial and viral infections, and higher risk of autoimmune diseases and cancers. This disease affects about 1-10 in 1 million of live births (Aiuti & Roncarolo, 2009; Bouma et al., 2009; Dupré et al., 2004; Jin et

This possible gene therapy method also might be used to cure other diseases such as Alzheimer's disease (Rogaev et al., 1995; Sherrington et al., 1995), Parkinson's disease (Terzi & Zachariou, 2008; Veeriah et al., 2010), X-chronic granulomatous disease (CGD) (Aiuti & Roncarolo, 2009; Kang et al., 2010), type I (insulin-dependent) (Efrat, 1998) and type II (non-

It is possible that normal human genomic DNA to be used as materials for homologous genetic recombination to repair defective genes *in vivo*. Normal human genomic DNA or normal genomic DNA fragments can be transferred into somatic cells/stem cells from a patient by microinjection, transfection, and electroporation. The corrected cells can be transplanted back to the same patient. Cells seem to be able to tolerate foreign DNA without immunological rejections; thus, the method described above may be an effective, relatively simple gene therapy method, and it may have no or less immunological reactions and rejections. Certainly, this possible approach of gene therapy should be performed only after

Ahn, A. & Kunkel, L. (1993). The Structural and Functional Diversity of Dystrophin. *Nature* 

strict and well-designed cellular and animal experiments and human clinical trials.

*Genetics*, Vol.3, No.4, (April 1993), pp. 283-291, ISSN 1061-4036

These 5 mouse models of Fanconi anemia will be used to prompt the research of gene therapy of these 5 subtypes of Fanconi anemia, because we can use these mouse models to do animal experiments. Normal genomic DNA or normal DNA fragments can be microinjected/electrotransfered, and transfected into stem cells and somatic cells from a mutated mouse; the corrected stem cells and somatic cells can be transplanted back to the same mouse, to see if the Fanconi anemia mouse model's physiological function is improved by this kind of gene therapy.
