**5. Cells as the carriers of cancer gene therapy vectors**

generation vectors and up to 36 kbp with gutless third generation adenoviral vectors [29]. However, along with the immunogenic potential, the broad range of host cells by adenovirus limits its systemic use in human cancer gene therapy trials [30]. Targeting strategies have enabled the use of adenoviral vectors in human gene therapy trials. Adenoviral vectors cannot integrate to cellular genome and express the transgene episomally. They cannot induce random mutations. However, the transgene expression is limited to 7–10 days postinfection [31]. Therefore, repeated administrations of the vector are needed to achieve sustainable responses in cancer treatment. Adenoviruses could be engineered either as replication deficient by deleting the immediate early genes of E1 or replication-competent keeping the E1 region. Replication-competent adenoviral vectors will be further discussed in the section of

*Adeno-associated viruses (AAV)* are simple viruses with approximately single-stranded DNA of 4.7 kb in size [32]. They belong to parvovirus family and require a helper virus such as adenovirus or herpes virus for lytic replication and release from the cell [33]. They can infect a wide variety of cells independent of cell cycle. This property makes AAV as suitable vectors for cancer gene therapy. Furthermore, unlike adenoviruses, they elicit little immune response when infect the normal host cells. Another advantage of AAV over adenoviruses is their ability to integrate the transgene into a particular spot on the 19th chromosome of human cells [34]. Unlike retroviruses, AAV cannot induce mutations. However, the major drawback of AAV is its limited cargo capacity of approximately 4 kbp of therapeutic genes. AAV could transduce certain cell types. Therefore, targeting strategies such as modification of viral capsid proteins, binding monoclonal antibodies, or bispecific proteins have been developed to improve the

*Baculoviruses* are enveloped viral particles with a large dsDNA of approximately 80–180 kb. They naturally infect insect cells. There have been no diseases related to baculoviruses in humans. Along with their highly safety profile in humans, they seem very useful gene therapy vehicles with their highly large cargo capacity of approximately 40 kb with possible multiple inserts, easy manipulation, and production [37]. *Autographa californica* multiple nucleopoly‐ hedrovirus (*Ac*MNPV) is the most widely used types of baculovirus in gene therapy studies. It has a circular dsDNA genome of 135 kb [38]. They can easily transduce mammalian cells, including many types of cancer cells, and cause high transgene expression in the host cell [39]. They are already approved for the production of human vaccine components such as Cervarix (GlaxoSmithKline) in cervical cancer and Provenge (Dendreon) in prostatic cancer [40].

*Herpes simplex virus (HSV)* is a large DNA virus with approximately 152 kb of dsDNA genome. It has a natural tropism to nerve tissues and cannot integrate into the host genome [41]. The HSV vectors can be designed in three different types as amplicons, replication-defective, and replication-competent vectors [42]. In general, the replication-competent HSV vectors are used

*Poxviruses* were the first viruses to be used as gene therapy vectors. They have been used in the in vitro production of proteins and as live vaccines. The attenuated forms of poxviruses have been developed and used in the development of genetic cancer vaccine trials [44]. The immunostimulatory properties of poxviruses make them preferable agents to induce immun‐

efficiency of AAV systems in cancer gene therapy [35,36].

as oncolytic agents in cancer gene therapy studies [43].

oncolytic viruses.

6 Gene Therapy - Principles and Challenges

The systemic administration of the gene therapy vectors usually failed because of low titer achieved in the target tissue and insufficient transgene expression. The clearance of the vector by the immune system, sequestration, and nonspecific binding to nontarget tissues are the major drawbacks of viral and nonviral vectors [46,47]. In general, in vivo targeting has relied mainly upon the enhanced leakiness of the tumor vessels, allowing the extravasation and access to tumor cells. Besides, the target tropism, extravasations in tumor site, and poor penetration of the vectors into the tumor tissue are the major problems for the vectors to eradicate the metastatic tumor deposits.

Cell carriers have the potential of eliminating those problems. They are stable and most of them have tumor homing properties and can be administered locally, such as intraperitoneal or intratumoral injections or systemically. In case of the use of autologous cells, they will not be cleared by the immune system. Macrophages, bone marrow mesenchymal stem cells (MSC), T cells, NK cells, and eosinophils are the known cells infiltrating the tumor tissues. Also, the tumor cells themselves naturally have the potential of homing to the tumor deposits through‐ out the body.

Macrophages have been used to deliver therapeutic genes because of their naturally trafficking ability to sites of neoplastic diseases [48]. Further refinement of the targeting of these cells by using transcriptional promoters could avoid the transgene expression in other parts of the body where the macrophages naturally traveled [49].

T cells can be used to transfer the therapeutic genes to target tissues because of their ability to circulate through the body and arrest in tumor tissues [50]. T cells have the advantage of the release of vectors that they carry in an antigen-binding-specific manner. The T cells could also provide further antitumoral activity by their cytotoxic effects. Tumor infiltrating lymphocytes (TIL) are the first example of cell-based carriers in cancer therapy in which they were trans‐ fected with cytokine genes [51].

Mesenchymal progenitor cells from either bone marrow (MSC) or adipose tissue (PLA) have the potential to expand in culture and the differentiation along the adipogenic, osteogenic, chondrogenic, and myogenic lineages [52,53]. It has been shown that lentivirally transfected mesenchymal progenitors from the adipose tissue have sustained transgene expression, even after the differentiation into adipogenic and osteogenic lineages [54]. Further modifications of PLA cells transfected ex vivo in order to target tumor tissues of their natural potential differentiation would provide an efficient gene delivery vehicle.

Some other cells such as fibroblasts and allogeneic cells have also been used as cell carriers for gene therapy vectors [55,56]. Because of their homing properties to the tumor cell deposits, tumor cells could be good candidates to target the established metastases. An animal model of MDA-MB-231 cells, transduced ex vivo by a CD carrying Ad vector, has been shown to reduce the tumor volumes in the established metastases of the tumor [57].
