**6. Gene targeting in cancer gene therapy**

In order to maximize the therapeutic index of cancer gene therapy, the expression of thera‐ peutic genes could be restricted to the target tissues. Therefore, the targeting of gene therapy vectors is the major key for the success of those treatments. There are two main targeting strategies: physical targeting and biological targeting.

#### **6.1. Physical targeting**

The first one is physical targeting by means of some physical methods such as local injections, catheters, gene guns, and electroporation. This strategy is usually used for local delivery of gene therapy vectors and is therefore not suitable for most of the cancer patients who may have cancer spread throughout the body. Supercoiled DNA molecules and oligonucleotides are also successfully delivered to the cells of the skin following intradermal injection to the tumor deposits accessible by local injections. However, intratumoral injection might have only the transducing capacity of the cells neighboring the needle. The tumor deposits in the body cavities such as peritoneum, pleura, and meninges and in subcutaneous tissues are the potential targets for the physical targeting of the gene therapy vectors in the clinic [58,59].

#### **6.2. Biological targeting**

In a second strategy, the viral or nonviral carriers of the genes are modified in such a way that they can only bind to tumor cells but not the normal cells. Because of the low transduction efficiency of the currently used gene therapy vectors in distant tissues when administered systemically, the specific transgene expression or viral replication in target tissues could provide an opportunity to achieve sufficient antitumor activity. To achieve this goal, *tran‐ scriptionally* and *transductionally* targeted vectors have been developed. For safety reasons, mostly the replication defective vectors have been used to transfer the therapeutic genes into tumor cells. However, because of limitations of vector delivery and relatively low levels of gene transfer capacity, replication-deficient vector systems are usually inefficient for the treatment of large solid tumors. Therefore, replicating vectors could efficiently transfer genes and also increase the therapeutic efficiency by means of its oncolytic effect. Such vectors could be targeted in such a way that they can replicate within the tumor cells but not in normal cells and cause no local or systemic toxicity.

#### **6.3. Transcriptional targeting**

The clinical utility of a cancer gene therapy program will be dependent on its therapeutic index. In order to maximize the therapeutic index, the expression of therapeutic genes could be restricted to the target tissues. The selective targeting of gene therapy vectors to specific cells enables the delivery of therapeutic genes to the target cancer cells while sparing the normal tissues. This has the potential of the reducing the dose of vectors and toxicity.

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

In order to maximize the therapeutic index of cancer gene therapy, the expression of thera‐ peutic genes could be restricted to the target tissues. Therefore, the targeting of gene therapy vectors is the major key for the success of those treatments. There are two main targeting

The first one is physical targeting by means of some physical methods such as local injections, catheters, gene guns, and electroporation. This strategy is usually used for local delivery of gene therapy vectors and is therefore not suitable for most of the cancer patients who may have cancer spread throughout the body. Supercoiled DNA molecules and oligonucleotides are also successfully delivered to the cells of the skin following intradermal injection to the tumor deposits accessible by local injections. However, intratumoral injection might have only the transducing capacity of the cells neighboring the needle. The tumor deposits in the body cavities such as peritoneum, pleura, and meninges and in subcutaneous tissues are the potential targets for the physical targeting of the gene therapy vectors in the clinic [58,59].

In a second strategy, the viral or nonviral carriers of the genes are modified in such a way that they can only bind to tumor cells but not the normal cells. Because of the low transduction efficiency of the currently used gene therapy vectors in distant tissues when administered systemically, the specific transgene expression or viral replication in target tissues could provide an opportunity to achieve sufficient antitumor activity. To achieve this goal, *tran‐ scriptionally* and *transductionally* targeted vectors have been developed. For safety reasons, mostly the replication defective vectors have been used to transfer the therapeutic genes into tumor cells. However, because of limitations of vector delivery and relatively low levels of gene transfer capacity, replication-deficient vector systems are usually inefficient for the treatment of large solid tumors. Therefore, replicating vectors could efficiently transfer genes and also increase the therapeutic efficiency by means of its oncolytic effect. Such vectors could be targeted in such a way that they can replicate within the tumor cells but not in normal cells

The clinical utility of a cancer gene therapy program will be dependent on its therapeutic index. In order to maximize the therapeutic index, the expression of therapeutic genes could be

reduce the tumor volumes in the established metastases of the tumor [57].

**6. Gene targeting in cancer gene therapy**

strategies: physical targeting and biological targeting.

**6.1. Physical targeting**

8 Gene Therapy - Principles and Challenges

**6.2. Biological targeting**

and cause no local or systemic toxicity.

**6.3. Transcriptional targeting**

Transcriptional targeting, which utilizes DNA regulatory (promoter/enhancer) elements that enable the expression of transgenes within specific cells, would probably decrease the toxicity of the treatment while increasing the specificity. The promoters used to drive the transgenes in viral or nonviral vectors targeted in cancer therapy could be tumor-selective, inducible, or cell cycle regulated. Certain genes have been expressed specifically in tumors such as L-plastin, survivin, telomerase, and midkine [60]. The vector constructs carrying tumor-specific pro‐ moters such as L-plastin, survivin, and midkine have been shown to efficiently eradicate tumor cells while sparing normal cells [61–63].

Likewise, the tumor-type-specific group of selective promoters shows a pattern of tumor tissue specificity. The promoters of oncofetal antigens such as carcinoembryonic antigen (CEA) and alpha-feto protein (AFP), mucin 1 (muc1), and oncogenes such as c-erbB2 and MYC have been used widely in the transcriptional targeting of gene therapy vectors to achieve specific transgene expression in tumor tissue [64–68].

The phenotypically heterogeneous expression of certain genes in certain tissues constitutes the basis of tissue-specific promoters in cancer gene therapy. The tissue specificity of those genes is largely regulated at the transcriptional level. Therefore, the promoters of those genes have been used to target cancer gene therapy vectors to specific tumor types in a specific manner of their origin of tissues. The tissue-specific promoters such as PSA in prostatic cancer [69], tyrosinase in melanoma [70], albumin in hepatocellular carcinoma [71], thyroglobulin (TG) in thyroid cancers [72], glial fibrillary acidic protein (GFAP) in glioblastoma [73], and osteocalcin (OC) in osteosarcoma [74] have been used to specifically target those tumors.

The inherent problems of tissue or tumor-specific promoters such as relative weakness and lack of true restriction of gene expression to the tumor tissues have led to the use of new promoters whose activity can be controlled exogenously. These systems also provide temporal control of gene expression. Various stress genes of the body are usually silent under normal conditions, but they are activated during the stress to protect the tissues. The stress genes upregulated during stress such as heat, hypoxia, glucose deprivation, irradiation, and chemo‐ therapy have opened a new avenue to the development of the tumor-specific targeting of gene therapy. The use of human heat shock protein (HSP)-driven HSV-TK or CD suicide gene therapy vectors has been a significant activity when combined with hyperthermia [75]. The promoter region of the hypoxia-inducible factor (HIF-1), a key regulator of the transcriptional response to oxygen, has been successfully used to target tumor cells [76]. MDR-1 gene encodes a 170-kDa P-glycoprotein and belongs to the ATP-binding cassette (ABC) family of transport‐ ers, which mediates the transport of some chemo drugs out of the cells thereby decreasing the efficacy of the treatment [77]. Therefore, the use of vector constructs carrying therapeutic genes under the control of MDR-1 promoter could efficiently target chemo-resistant tumor cells [78].

Uncontrolled cell proliferation is the prominent feature of cancer cells. The retinoblastoma family of proteins and their upstream regulators such as cyclin D, CDK4, and p16/INK4 regulate the G1 checkpoint in the cell cycle. Tumor suppression by Rb has been linked to its ability to repress E2F-responsive promoters such as E2F-1 promoter. It has been shown that Ad vectors that contain transgenes driven by E2F-1 promoter can mediate tumor-selective gene expression in vivo in glioma cells [79]. The promoters of cell cycle genes such as cyclin D, cyclin A, cdc25c, cyclin-dependent kinase inhibitors, p16/INK4, p27, and p14 could be expected to exert cell cycle arrest, thereby increasing the apoptosis when used in vector targeting strategies in proliferating tumor or endothelial cells [80–83]. Additionally, drug-inducible systems such as tat-on/tat-off regulated by tetracycline or rapamycin could provide a wide-dose response range in the treatment [84].

#### **6.4. Transductional targeting**

The second strategy of biologic targeting is to engineer the either viral or nonviral vectors in such a way that they can be captured only in tumor tissues, and therapeutic genes are produced only in the environment of the tumor tissue. There have been numerous attempts to modify the vectors with tumor cell-specific ligands that would increase the specific binding to tumor cells and reduce the toxicity. Therefore, targeting DNA complexes to the tumor cell-specific receptors is an attractive strategy. One of the well-known strategies is coating the surface of the complexes with transferrin, an iron-binding plasma protein that is mainly an up-regulated expression on rapidly proliferating cells as tumors [85]. Likewise, coating with EGF has also been reported to cause a 50-fold increase in the transgene expression in hepatocellular carcinoma cells [86]. The suicide gene HSV-TK/PEI complex mixed with a single chain antibody (scFv) against EGFR with a negatively charged oligopeptide tail has exhibited EGFRspecific gene transfer in vitro and in vivo [87].

The nonviral systems usually fail in promoting the delivery of DNA to the nucleus. Almost 99% of the internalized DNA from a nonviral vector is degraded in the cytoplasm [88]. Trafficking of exogenous DNA from cytosol to the nucleus may be improved by using the nuclear localization signal (NLS) found in some nuclear proteins [89]. Dermaseptins, a family of antimicrobial peptides that destabilize the membrane, have been successfully linked to NLS of SV40-T antigen and HIV-1 Rev protein [90]. Likewise, mellitin, which is a membrane-active protein, and viral protein r (vpr) of HIV-1, which binds directly to nucleoporins of the nuclear pore complex, have been successfully bound to PEI/DNA complexes to improve nuclear transport [91].

The selective targeting of viral vectors to specific cells permits the cell-specific expression of transgenes and enables the systemic administration of the vectors. Avoiding the targeting of the native receptor found on immune and inflammatory cell surfaces also reduces the immunity and inflammation to those vectors. Replication-competent retroviral vectors (RCR) based on murine leukemia virus (MLV) represent an attractive system for gene delivery through their ability to replicate and provide long term transgene expression in rapidly proliferating cells [92]. However, the uncontrolled spread of the RCR might cause the infection of nontarget cells. In order to develop tumor-selective RCR vectors, several modifications have been made such as a modification of the envelope protein by insert‐ ing single chain antibodies (scFv) [93] and peptide ligands [94]. Also, the specifically targeted entry of replication-deficient retroviral vectors has been accomplished by combin‐ ing cell-specific monoclonal antibodies [95,96].

The capability of an Ad vector to infect a cell is mainly based on CAR and integrin expression. Following the attachment of an adenoviral vector to the target cell via C-

terminal part of the fiber protein (knob) and CAR (Coxsackie's B adenovirus receptor), the alphaV beta3 and alphaV beta5 integrins mediate the internalization of the vector [97]. The CAR deficiency of the primary tumor cells limits the success of the gene therapy proto‐ cols using Ad vectors [98]. Redirecting the Ad vectors to bind other cellular receptors would allow CAR independent virus entry into the tumor cells. There are mainly two strategies to redirect the viral vectors to the cells: conjugate-based and genetically modified viral membranes. Adenoviral vectors have been targeted to different cells by genetic modifica‐ tion of the capsid or by using adapter molecules. In the conjugate-based strategy, it is aimed to complex the vector with the targeting molecule that redirects the vector to the cellspecific receptors. Bispecific molecules containing a first specificity for the fiber knob to block binding to CAR and the second specificity for a cell-specific receptor, such as bispecific fusion proteins (antibodies), bispecific peptides, polymer mediated ligand coupling, and chemical modifications (biotin–avidin bridges), have been utilized to target adenoviral vectors [99–101].

Adeno-associated vectors (AAV) possess a highly favorable safety profile and have the unique potential in certain cancer models. However, they have a restricted range of cells to transduce transgenes to the target tissues. In order to augment the transduction efficiency of AAV in various tissues retargeting strategies such as engineering of viral capsid, monoclonal antibod‐ ies and specific peptides have been used to successfully retarget the AAV vectors [102,103].
