Abeloff's Clinical Oncology, 4th Edition

Part I – Science of Clinical Oncology

Section D – Preventing and Treating Cancer

Chapter 33 – Gene Therapy in Oncology

James E. Talmadge,Kenneth H. Cowan


Recent Major Improvements in Gene Therapy



Vector development



Vector targeting



Regulated transgene expression



Clinical development strategies

Ideal Vector Attributes



Can be targeted either physically or via promoter expression, and is nontoxic, noninflammatory, and nonimmunogenic



Should have the potential to incorporate a large transgene and result in high levels of both transduction and transgene expression



Duration of transgene expression and/or genomic integration ought to be regulatable

Current Concerns Regarding Gene Therapy



Gene therapy has been implicated in the death of at least one patient, resulting in the temporary suspension of clinical trials (2000) with adenovirus (Adv) vectors in the United States. The first gene therapeutic to be approved (China, 2003) was an Adv vector with a p53 transgene used in combination with chemotherapy for the treatment of head and neck squamous cell carcinoma (HNSCC). A second, conditionally replicative Adv vector, H101 (ONYX-015), was approved in China in December of 2005.



Leukemic transformation by insertional mutagenesis occurred in several patients treated with retroviral vectors. However, because retroviral vectors have been successful in the treatment of patients with severe combined immune deficiency (SCID-X1) for whom no other therapy is available, their use continues to be approved on a case by case basis.

Future Directions of Gene Therapy



The choice of disease, clinical implementation, and vector are critically important to the future development of successful gene therapy.



Because of deficiencies in gene delivery and targeting, as well as expression levels, it is critical to pair protocols with specific vector attributes.


The development of nucleic acid technologies has provided insight into the molecular basis of neoplasia. The resultant detection of phenotypic and genotypic alterations in neoplastic diseases has increased optimism that molecular intervention might lead to improved clinical care for patients with cancer. One such approach is gene therapy, which is the introduction of a nucleic acid sequence into a target cell. The objectives are the delivery of a transgene to an adequate number of cells and at an effective level of expression sufficient to result in therapeutic outcomes. Both criteria require the use of a vector and potentially, a formulation such that these objectives can be met. Although this approach is both simple and attractive, thus far gene therapy has promised much and delivered little because of the technical hurdles. Several preclinical studies and clinical trials have been undertaken to improve gene transfer systems. In 2003 the first gene therapeutic was approved in China. This was an adenovirus (Adv) serotype 5 vector engineered to express TP53 (Gendicine) for treatment of patients with head and neck squamous cell carcinoma (HNSCC).[1] A second gene therapy product, H101 (ONYX-015), an Adv vector modified to replicate in and kill cancer cells with TP53 mutations, was approved in December 2005.[2] Regardless of these approvals, the primary challenges in gene therapy remain improvements in the targeting of our existing vectors and increasing gene transduction efficiency. Overcoming these obstacles will facilitate the development of targetable vectors and, given the systemic nature of most malignancies, help in the development of vectors that can be administered intravenously (IV). This chapter focuses on strategies to improve efficacy, as well as ongoing gene therapeutic strategies. We also will examine and discuss recent advances and indicate areas that require further development for clinical gene therapy to become a widely used treatment modality.


Viral Gene Transfer Vectors

Viral gene delivery has developed from a virus’ innate ability to infect cells, which offers many intrinsic advantages: [3] [4]



Specific cell-binding and cell-entry properties



Efficient targeting of the transgene to the nucleus of the cell



Ability to avoid intracellular degradation

Most viral vectors are based on the principle that an intact wild-type (wt) virus can be modified for safe and effective gene transfer. In general, the more severely attenuated the viral vector is from the wt, the safer the virus is for use in gene therapy protocols, yet the poorer the yield obtained after propagation. Typically, two (or preferably, three) noncontiguous partial or complete gene sequences are deleted to reduce the potential for homologous recombination. Specific genes critical to viral replication are then modified or deleted, resulting in a recombinant viral vector that is “replication defective.” The transgene to be delivered by the virus is then inserted into the viral genome at the site created by the removal of the viral replication genes. The transgene must be a smaller size to fit within the available space, which is a critical characteristic because the transgene cannot be packaged into an infectious particle if the new viral genome is too large. Many of the viruses that are used as vectors lack genes for replication in normal cells; therefore, the recombinant virus and its transgene must be grown in a packaging cell line that provides the complementary genes required for viral replication. The recombinant viral particles are purified as live infectious viruses and are replication incompetent in the absence of the packaging cell line. Alternatively, the packaging cell line can be used to infect (transduce) cells or tissues in vitro.


The Retroviridae is a large family of ribonucleic acid (RNA) viruses including Moloney-murine-lentivirus-related viruses (e.g., Moloney murine leukemia virus [MMLV]) and lentiviruses (e.g., human immunodeficiency viruses 1 and 2 [HIV-1 and HIV-2]).[5] Their genomes consist of two identical positive-sense, single-stranded RNA molecules (∼3.5 kb), and are encased in a capsid along with integrase and reverse transcriptase enzymes. Initially, retroviral vectors were the most widely used viral vectors, a distinction that has been replaced by Adv vectors in recent years. Retroviruses can transduce only those cells that are actively undergoing mitosis, limiting their utility with certain cell populations, especially hematopoietic stem cells. Retroviral vectors provide good gene expression and are technically easy to produce, although the titers obtained are suboptimal. In addition, the production of retroviral vectors requires careful monitoring because of the potential for helper virus contamination.


Most of the retroviral vectors that are used for gene therapy are based on MMLV. Vector replication is prevented by the deletion of the gag, pol, and env gene regions. The gag region encodes the capsid proteins, the pol region encodes reverse transcriptase and integrase, and the env region encodes proteins required for receptor recognition and envelope anchoring ( Fig. 33-1 ). The genome includes long terminal repeats at either end that play a vital role in initiating deoxynucleic acid (DNA) synthesis and regulating transcription of the viral genes. The gag, pol, and env gene products are supplied by a complementary packaging cell line. When a retroviral vector plasmid is introduced into a packaging cell line, viral RNA is produced, packaged into virions, and secreted into the medium. Each resultant viral particle is able to integrate itself into the genome of the host cell, but is unable to produce additional viral particles because it lacks the gag, pol, and env genes. The transduced DNA sequences are stably integrated into the chromosomal DNA of the target cells and in this way are transferred to cellular progeny of transduced cells. Highlights of results obtained to date with retroviral vectors include the therapeutic studies in children with severe combined immune deficiency (SCID-X1), which will be discussed later in this review, as well as the gene-marking studies of Malcolm Brenner and others. [6] [7]In the latter studies, it was shown that tumor cells within autologous stem cell transplant products could be responsible for tumor relapse, at least in leukemia patients.


Figure 33-1  Retrovirus proviral genome and gene product functions. Overview of the 9-kb genome of the HIV provirus and a brief summary of the functions for the 9 genes encoding 15 proteins.




The most recently discovered members of the retrovirus family are the human and simian immunodeficiency viruses (HIVs and SIVs, respectively), which belong to a subclass of retroviruses known as lentiviruses. [8] [9] The development of HIV gene therapy vectors has several potential advantages based on the following characteristics:



Transduce actively dividing and nondividing cells.



Long-term, stable transgene expression occurs due to genetic integration.



Inherent tropism for CD4 T cells, macrophages, and hematopoietic stem cells.

Genetic modifications, such as the introduction of vesicular stomatitis virus G protein into the lentiviral envelope, can widen the tropism of this vector. Until it has been demonstrated that HIV-based vectors are safe, however, the use of these vectors for therapies targeting diseases other than HIV could be difficult to initiate clinically.[10] The in vitro efficiency of lentiviral vectors is at an acceptable level; however, in vivo expression has not been demonstrated at an acceptable level for clinical utility. In addition, there is a need to find protocols and/or procedures that can elevate the expression levels of the HIV virus in nondividing cells.[11]

The first clinical study using a lentivirus vector was undertaken in HIV-infected patients.[12] This study investigated the safety of infusing autologous T cells modified with an HIV type 1 (HIV-1)–based lentiviral vector expressing an antisense gene against the HIV envelope. Five patients with HIV infections that were resistant to antiviral therapy and had viral loads of more than 5000 copies/mL and CD4+T cells counts between 200 and 500 cells/mm3 were treated. The primary endpoints included adverse events, viral load, CD4+ counts, and the emergence of replication-competent lentivirus derived from the vector. In this phase I study, one subject was reported to have a sustained decrease in viral load. The CD4 counts remained steady or increased in the other four subjects, and sustained gene expression was observed. These preliminary studies support the safety of lentivirus vectors.

Recombinant Adenovirus

Recombinant Adv is a nonenveloped, icosahedral, double-stranded DNA virus with a capsid containing 252 capsomeres (240 hexons and 12 pentons).[13] The large genome of Adv (36 kilobases, kb) allows large genes to be inserted into an Adv-based vector. Transgenes in Adv vectors are not incorporated into the genome of transduced cells but instead remain as an extrachromosomal entity in the nucleus. First isolated from U.S. army recruits who had acute respiratory symptoms, Adv vectors have been found to be common human pathogens. To date, 49 serotypes have been characterized and associated with a variety of symptoms, ranging from a mild cold to acute febrile pharyngitis.[14] Replication-defective recombinant Adv vectors are currently the most commonly used viral vectors in clinical trials. Ad2 and Ad5 are used primarily for gene therapy applications. Recently, however, the Ad11 and Ad35 serotypes were shown to exhibit a unique tropism that includes hematopoietic stem cells, a finding that potentially widens their utility. [15] [16] [17]

The Adv vector's genome ( Fig. 33-2 ) can be divided into two main regions: early (E) and late (L) according to the time at which their genes are expressed during virus replication. There are four regions of early genes that are termed E1, E2, E3, and E4, and one region of late genes comprising the five coding units termed L1, L2, L3, L4, and L5. The E1 region is essential for viral replication; therefore, recombinant Advs without the E1 region are considered replication defective. In a replication-defective Adv vector, the E1 region can be replaced with a transgene for expression. Moreover, removal of genetic material from the vector, such as the E3 and/or the E4 region(s), allows for larger genes to be inserted and reduces the viral immunogenicity.[18] Viruses without the E3 and E4 regions are referred to as “gutless” and have decreased antigenicity.[19]


Figure 33-2  Adenovirus genome. The Adv genome is composed of early and late genes. The E1A gene encodes the initial viral transcription unit and must be deleted to prevent the recombinant virus from replicating. In most of the original Adv vectors, E1A and E1B are deleted. The second-generation vectors (known as gutless vectors) typically also have the E3 and/or E4 genes deleted. This deletion allows larger transgenes to be inserted into the Adv vector, and the deletion of E4 significantly reduces vector immunogenicity with the potential for a more prolonged transgene expression.



The E1 region of Adv vectors is subdivided into E1A and E1B. The E1A gene product is a viral transcription unit that activates the expression of other Adv transcription units by binding to viral promoters. The E1B region codes for a 55-kd protein that interacts with the cellular p53 tumor suppressor protein and regulates the host cells’ cycle progression supporting viral replication. E1B also binds to viral E4proteins and to p53, which together act to depress host protein synthesis. The E2 region codes for viral DNA polymerase and the Adv single-stranded DNA-binding protein. The E3 region is not required for in vitro replication; however, it does offer the virus some protection against host defense mechanisms. The E4 region codes for proteins involved in



Regulation of viral and cellular protein expression



Replication of viral DNA



Switching off of host protein synthesis

The late genes (L1–L5) are expressed at the onset of viral DNA replication and encode structural polypeptides that are needed for virion assembly. This understanding of viral replication has allowed the development of extremely elaborate, conditionally replicative Adv vectors capable of replication only in cancer cells.[20]

The transduction efficiency of Adv vectors is high as compared with that of most other viral vectors. Because of the structural stability of the capsid polypeptides of Adv, viral particles can be purified and concentrated to a very high titer of ∼1 × 1012 plaque-forming units (pfu)/mL. This is in contrast to retroviral titers that achieve much lower titers (∼1 × 107 pfu/mL) because of their envelopes’ instability. Another distinguishing characteristic of Adv vectors is their lack of integration into the human genome. The Adv genome remains in the nucleus of the target cells as a nonreplicating extrachromosomal entity, thereby avoiding any potential for mutagenic effects caused by random integration into the host.

However, Adv vectors have potential shortcomings, including



Transient expression because the viral DNA does not integrate into the host



Viral protein expression by the Adv vector after administration into a host



A common pathogen and in vivo delivery could be hampered by the prior induction of host immunity.[3]

The period of Adv transgene expression is relatively short; therefore, this is a suboptimal vector if expression is desired for longer than 10 to 14 days. This short expression time is due primarily to the induction of a cytotoxic T lymphocyte (CTL) response to viral polypeptides, as well as potentially to the transgene itself, especially if it is not expressed normally. Because the Adv genome does not integrate into the target cell, only one of the daughter cells (if the target cells are dividing) will contain the transgene. Manipulation of the immune response can result in longer expression; however, Adv gene delivery is ideally suited to those situations that require only a single period of transgene expression in which transient expression is desired, for example, growth factor therapy. A second major disadvantage of Adv vectors used in vivo is the immune response (CTL and antibody [Ab]), both endogenous and induced, which can preclude infection and cause the destruction of transduced cells, resulting in local tissue damage and inflammation. This shortcoming was demonstrated in the initial studies with intrabronchial delivery of Adv for the treatment of cystic fibrosis.[21] Host cells presenting peptides from Adv-encoded transgene products target the host cell for CTL-mediated destruction. A third major disadvantage of Adv vectors is that most humans are primed against at least one serotype, because Adv is a naturally occurring virus. Using the same serotype in a gene therapy context will probably result in a rapid and vigorous immune response such that high levels of Adv-specific Abs occur in the sera within days of Adv vector administration. Another similar problem is the potential secondary immune response induced by the readministration of a vector. It must be stressed that transgene expression can occur during a boost, although a shortened duration is observed. The augmentation of a CTL response by an Adv vector suggests the utility of Adv vectors as vaccine adjuvants ( Box 33-1 ).

Box 33-1 


Adenoviral (Adv) vectors have a number of positive and negative attributes. The positive attributes include the transduction of a wide profile of cellular phenotypes such as epithelial, carcinoma, and hematopoietic cells. Further, the use of Adv vectors results in a high frequency of transduction and high levels of transgene expression. A negative attribute of Adv vectors is transient expression, although for appropriate targets such transitory infection is a positive attribute. The transient expression is due, in part, to the high level of innate vector immunogenicity, which can limit multiple cycles of transduction and chronic transgene expression. The resulting Adv profile of activity is ideal for the transduction of dendritic cells (DCs) as vaccines, the purging of tumor cells from stem cell products, and intralesional injection of carcinomas. Further, the ability to develop Adv vectors that are conditionally replicative has great potential for the treatment of neoplastic disease. It is noted that two Adv vectors have received regulatory approval in China. These controversial studies have resulted in the treatment of several thousand patients, supporting the safety of these vectors.

Recombinant Adeno-associated Virus

Adeno-associated virus (AAV) vectors offer many of the same advantages as Adv vectors, including a wide host-cell range and a relatively high transduction efficiency. [22] [23] AAV vectors stably integrate at specific sites in the host genome, resulting in a longer lasting transgene expression. In addition, these stable vectors can infect a variety of dividing and nondividing cells without inducing an immune response. AAV vectors cause little damage to target cells—unlike Adv vectors that can cause a high degree of cytopathogenicity. There is evidence, however, to suggest that AAV vectors are significantly less efficient than retroviral vectors at transducing primary cells, because most of their DNA remains extrachromosomal and does not integrate into the host genome. Furthermore, they cannot incorporate genes larger than 5 kb and must be screened closely for Adv contamination.

Recombinant Herpes Simplex Virus

Herpes simplex virus (HSV) vectors are developed primarily for protocols that target neuronal tissue.[24] Similar to Adv vectors, HSV vectors are maintained as an extrachromosomal DNA element in the nucleus of host cells, but can establish long-lived asymptomatic infections in the sensory neurons of the peripheral and central nervous tissue.[25] HSV vectors also have a wide host range and are similar to Adv vectors in that they allow large gene inserts of up to 20 kb. These vectors are infective even with multiple deletions of immediate-early (IE) genes that are essential for replication, resulting in less cytotoxic vectors, thereby reducing safety concerns.[26] HSV vectors can be produced at high titers and express transgenes for a long period of time in the central nervous system.[27] The major concern associated with HSV is the potential for wt virus to replicate lytically in the human brain, resulting in encephalitis. Other significant disadvantages with HSV vectors include



Requirement for additional engineering to increase efficiency[26]



Transient expression associated with lytic infection and viral protein expression



Relatively low transduction efficiency

Recombinant Pox Vectors

The origin of vaccinia virus (VV), the virus used for vaccination against smallpox, is not known, but it was probably derived from cowpox virus, variola virus, or a hybrid of the two. [28] [29] Percutaneous VV vaccine administration results in protective cellular and humoral immune responses in greater than 95% of primary vaccinees. Recombinant VV vectors are highly attenuated, host-restricted, and non- or poorly replicating poxvirus strains (including the modified vaccinia Ankra [MVA] and canarypox or avipox vector [Alvac]) and thus do not create productive infections. [30] [31] MVA is avirulent in normal and immunosuppressed animals, and safe in humans.[32] Recent studies using transgenic mice provided a comparison of VV immunogenicity, including MVA and Western Reserve (WR). These studies demonstrated that MVA vaccines elicited CD8+ T-cell responses that are comparable to those induced by the replication-competent WR strain. Furthermore, MVA vaccination was shown to be protective against a lethal respiratory challenge with the virulent WR strain.[33] The most frequent adverse complication of VV vaccination is inadvertent inoculation (usually autoinoculation) at other sites. Serious complications, which are more common among primary vaccinees and infants than among revaccinees and adults, include the following:



Generalized vaccinia in otherwise healthy individuals, which is generally self-limiting



Eczema vaccinatum, which consists of disseminated cutaneous lesions in highly susceptible patients with eczema or other chronic skin diseases, which can be severe or even fatal



Progressive vaccinia (vaccinia necrosum), which is a severe, potentially fatal illness seen in patients with immunodeficiency, whether congenital, acquired (e.g., via leukemia or lymphoma), iatrogenic (e.g., via chemotherapy or glucocorticoid treatment), or HIV induced



Postinfectious encephalitis, which is rare (three cases per million primary vaccinees), but can be fatal in 15% to 25% of cases and can leave 25% of patients with permanent neurologic sequelae

Similar to Adv vectors, VV vectors are used for immune manipulation and as a vector for vaccines.[34] VV vectors have been used worldwide to eradicate smallpox and as discussed earlier, provide a relatively safe live vaccine. Vaccinia vectors do not integrate into the genome of the host cell; however, they can accommodate large transgenes and are extremely immunogenic. VV vectors are used to immunize patients against tumor antigens (Ags) by cloning Ags and/or genes encoding proteins with adjuvant activity (e.g., cytokine or costimulating factor genes) into the viral genome. Most transgenes are expressed at high levels in vivo, eliciting an Ag-specific response. Vector-induced immunity, however, can limit the ability of the vaccinia transgenes to boost an immune response, which is an observation similar to that seen with Adv vectors. The current emphasis is on VV infection of dendritic cells (DCs) using a vector with an antigenic transgene. [34] [35]

In association with the immunogenicity of VV vectors and their ability to deliver an antigenic transgene, they have been used clinically as a melanoma vaccine. In clinical studies by Wallack and colleagues,[36] a phase III trial of a vaccinia melanoma oncolysate, delivered as an active specific immunotherapy, was found to increase the disease-free or overall survival of patients with stage III melanoma in a surgical adjuvant setting. Other studies have used VV mutants that are conditionally replicative and can lyse cancer cells after viral replication. These vectors have been used in a strategy whereby insertional inactivation of the VV thymidine kinase (tk) gene was used to limit viral replication in cells with large intracellular nucleotide pools, such as tumor cells. In a similar approach, Mastrangelo and coworkers[37] inserted the gene for granulocyte-macrophage colony-stimulating factor into the VV tk gene locus as a strategy to generate an oncolytic virus that induced antitumor immunity after infection of malignant melanoma. This vector is currently in a clinical trial of intralesional administration to patients with refractory recurrent melanoma. In the first seven patients studied, two patients had a complete response and three other patients had partial responses. Other oncolytic VV vectors have been engineered with complementary DNAs for cytokines such as interleukin 2 or with prodrug-activating enzymes such as cytosine deaminase to augment antineoplastic efficacy. [38] [39]

The role of VV vectors as vaccines has focused predominantly on carcinoembryonic Ag (CEA) as the vaccine Ag. CEA is a glycoprotein self-Ag found on breast, lung, gastric, colon, and ovarian tumors. One such vector is a recombinant VV containing the CEA gene (rV-CEA). [40] [41] In a phase I clinical trial, the safety of rV-CEA was demonstrated; however, no significant antineoplastic effects were observed.[42] Possible reasons for the lack of clinical efficacy in these trials include



Prior exposure to the VV, leading to the development of anti-vaccinia immune responses after repeated vaccinations



Advanced state of the patients’ tumors



Potentially compromised immune status of the patients

Another phase I rV-CEA vaccine study demonstrated that CEA-specific T-cell responses could be generated in humans after vaccination.[42] A second recombinant anti-CEA vaccine, Alvac-CEA, has been developed. [43] [44] Similar to rV-CEA, Alvac-CEA contains the CEA gene; however, unlike rV-CEA, it cannot replicate in mammalian cells. The safety of Alvac-CEA has been documented in phase I trials in patients with advanced carcinomas.[45] A moderate but statistically significant increase in the number of CEA-specific CTL precursors was observed in seven of nine HLA-A2+ patients treated with Alvac-CEA, although objective anticancer effects were not observed. Preclinical studies have suggested that the combination of rV-CEA and Alvac-CEA in a prime and boost protocol can induce a more vigorous T-cell response than either vaccine alone.[44] In a clinical prime-and-boost study, 18 patients with advanced tumors expressing CEA were randomized to receive either rV-CEA followed by three Alvac-CEA vaccinations, or Alvac-CEA (three times) followed by one rV-CEA vaccination. In this study, vaccination with rV-CEA followed by Alvac-CEA resulted in an increased frequency of Ag-specific interferon γ cells by enzyme-linked immunospot assay (ELISPOT) relative to the reverse order of vaccination.[46]

Another method to enhance the responses to a vaccine is to incorporate a costimulatory signal. In the absence of a costimulatory signal, presentation of an Ag to T cells can result in anergy.[47] B7.1, which binds to CD28 on T cells, is one such costimulatory signal that results in the production of interleukin 2 and interferon γ by T cells. In a vaccine study using VV vectors, 39 patients were treated with Alvac-CEA B7.1.[48] In one study using the Alvac-CEA-B7.1 vaccine, patients with metastatic CEA-expressing adenocarcinomas received vaccine intradermally every 2 weeks for a total of four injections. In this phase I trial, 27% of the patients had disease stabilization after four vaccinations. Six of 31 patients with elevated serum CEA levels had a temporary decline in CEA. In addition, HLA-A2+ patients demonstrated increased CEA-specific T-cell frequencies after three vaccinations. Based on these studies and additional phase II data, a phase III trial was initiated in 255 patients with advanced pancreatic cancer at approximately 60 medical centers.[49] The protocol was powered to detect a 2-month improvement over control chemotherapy based on a median overall survival of 6 months. Unfortunately, this study did not meet its primary endpoint of improving overall survival compared with palliative chemotherapy or best supportive care. However, this outcome is not unexpected, because patients with advanced pancreatic cancer have a rapid disease progression and are poorly responsive to intervention in general ( Box 33-2 ).

Box 33-2 


Vaccinia viral (VV) vectors have a profile of activity analogous to that of adenoviral (Adv) vectors. That is, they can easily transduce a wide range of cells, resulting in transient expression, and have as a negative attribute a brief transgene expression due to the innate antigenicity of the vector. In contrast to Adv vectors, VV vectors almost inevitably lyse the transduced cell, rendering it potentially less attractive as a vector (especially for dendritic cell [DC] transduction) due to the shorter half-life of the transduced cell. Significant experience with the administration of both VV and Adv vectors as vaccines has provided a strong safety profile for both. In theory, the concomitant use of VV and Adv vectors makes possible cycles of vaccine delivery via transduced DCs, allowing a prime and boost immunization with DCs, which have a high frequency of transduction and levels of transgene expression, while reducing the concerns associated with the innate antigenicity of the viral vectors.

Recombinant Alphavirus Vectors (Sindbis)

High-titer alphavirus vectors can provide efficient gene delivery both in vitro and in vivo. In addition, efficient central nervous system infections via intranasal and vascular injections with virulent and avirulent replication-competent Semliki Forest virus (SFV) strains have been shown in animal models. [50] [51] [52] Replication-deficient alphavirus particles have a high local and transient transgene expression in rodent brains. Furthermore, repeated SFV injections are possible in the absence of an immunogenic response against SFV, which is in contrast to Adv and VV vectors. Modifications to the envelope structure of Sindbis virus are possible with resultant changes in host range and targeting. The favorable characteristics of alphavirus vectors include



Rapid production of high-titer virus



Broad host range



High RNA replication rate in the cytoplasm



High transgene expression levels

Negative attributes include



Short-term expression



Strong cytotoxic effects on host cells

Nonetheless, both these properties are advantageous for certain indicators, particularly vaccine production.

Nonviral Gene Transfer Vectors

Nonessential genes can be removed from viral vectors to allow room for transgene(s) to reduce inflammatory responses and to increase safety. [53] [54] This process involves simplifying the virus, sometimes to an extreme. After undergoing such a process, a virus vector can be an artificial “vector shell” allowing the gene of interest to be expressed at high levels, in a highly regulated manner, and for a controlled period of time. Another approach to achieve the same result is to produce a vector that can introduce genetic material to the nucleus of cells. [53] [55] [56] This strategy has resulted in the development of several nonviral vector systems; however, the efficiency of “naked DNA” as a therapeutic is suboptimal without some form of carrier or formulation.

Direct DNA Injection and Transduction

One form of nonviral gene delivery is the use of purified DNA plasmids.[55] The transgene expression is low following intramuscular or intratumoral injection; however, high levels are observed if hydrodynamic injection is used. [57] [58] The approach of naked DNA injection is typically done as an intramuscular or intratumoral injection. Despite the simplicity of this approach, transfection efficiency is low and results in limited expression. Various formulations, including lipid or pluronic formulations, and incorporation into nanoparticles or liposomes, have been used to improve transduction efficacy and gene expression ( Box 33-3 ). [59] [60] [61]

Box 33-3 


The transduction efficiency of plasmid vectors is low, even with the use of formulations to improve transfection efficiency and increase transgene expression. Further, this approach appears to work better in vitro than in vivo. In contrast to viral vectors, plasmid vectors offer little innate antigenicity, although there have been reports of immune responses to bacterial genes. Positive attributes of plasmid vectors include the low level of innate immunogenicity and the potential for genomic integration. The use of hydrodynamic delivery in rodents has provided a powerful preclinical tool. However, clinical translation is problematic with the potential for utilization in an isolated limb. In contrast, retroviral and lentiviral vectors provide the same characteristics with higher levels of transgene expression and improved transduction efficiency relative to plasmids. However, the improved transgene expression and transduction levels of retroviral and lentiviral vectors remain significantly lower than those of adenoviral and vaccinia vectors.

Nonviral liposomal delivery systems can be IV injected with limited vector-associated toxicity, but with transgene expression, especially in the lungs.[62] Tumor targeting using tumor-specific promoters, ligandation of receptors to the liposome surface, and pegylation of liposomes have all been studied. [63] [64] [65] [66] [67] [68] [69] Although some degree of tumor targeting has been observed using these delivery systems, the level of transgene expression is generally low. Studies have revealed that liposome-DNA complexes can also elicit an inflammatory response when injected systemically, resulting in suppression of transgene expression. [70] [71] [72] Furthermore, failure to achieve increased or sustained gene expression after repeated injections has been a major obstacle in the development of liposomes.[70] [73] Recently, it was shown that cationic liposome (DOTAP : cholesterol or DOTAP : Chol)–DNA complexes can achieve effective levels of transgene expression in tumor-bearing lungs and when injected IV can achieve levels sufficient to cure immunocompetent mice with disseminated experimental metastases.[74] Furthermore, repeated daily injections can result in a dose-dependent increase in transgene expression in tumor-bearing lungs.[75]

Hydrodynamic Gene Delivery

Hydrodynamic tail-vein plasmid delivery results in high levels of transgene expression in the livers of rodents.[76] Lower levels of transgene expression (100- to 1000-fold) are found in the spleen, heart, kidneys, and lungs. This simple nonviral gene transfer procedure entails the rapid delivery of naked plasmid DNA in a relatively large volume of physiologic saline.[57] In a typical mouse, weighing 20 g, the plasmid is delivered in a total volume of 2.0 mL over a period of 5 to 7 sec. Although there are toxicity issues, clinical studies are under discussion for the delivery of dystrophin into the arms of Duchenne muscular dystrophy patients.[77]

Liposomes and Virosomes

In their most basic form, liposomes consist of two lipid species: a cationic amphiphile and a neutral phospholipid. [75] [78] Liposomes spontaneously bind to and condense DNA to form complexes that have a high affinity for the plasma membranes of cells, resulting in the uptake of liposomes to the cytoplasm by endocytosis. Many variations of this approach are used, resulting in varying levels of gene expression. Unfortunately, liposome-facilitated gene delivery is relatively ineffectual in vivo. More recently, some of the advantages of viral delivery vectors have been combined with the safety and “simplicity” of the liposome to produce fusigenic virosomes.[78] Virosomes are engineered by forming complexes of the membrane fusion proteins with liposomes that have already-encapsulated plasmid DNA. The inherent ability of the viral proteins in virosomes to fuse with cell membranes results in the efficient introduction of DNA to the target cell, providing improved gene expression. Viral vectors have limitations based on the size of transgene that can be incorporated; in contrast, no such limit exists for virosome or liposome technology (at least in theory).

Ballistic Delivery (Gene Gun)

This physical method of gene delivery involves microcarriers (usually gold particles) coated with DNA and “fired” at high velocity using an explosive or gas-powered ballistic device called a “gene gun.”[79] [80] [81] Once the particles are inside the target cell, the DNA is slowly released from the microcarriers, resulting in gene transcription and translation. This application has been used extensively in vivo, but its clinical use is restricted to exposable surfaces or ex vivo transduction because the fired particles do not penetrate tissues deeply.[82]


Novel polymeric delivery systems (e.g., nanospheres) that can be administered in novel ways are being developed. [83] [84] [85] These particles are potentially useful because the smaller the condensed DNA particles are, the better will be their in vivo diffusion toward target cells and the trafficking within the cell. Individual plasmid molecules can be collapsed into a nanoparticle using detergents. For example, nanoparticle-based gene delivery was targeted to the neovasculature of mice using an integrin-targeting ligand, resulting in tumor regression.[86] Nonetheless, the size of the transgene that can be delivered by nanoparticles is limiting, and the primary focus has been on small interfering RNA (siRNA) delivery, which will be discussed in the following section.

Nucleic Acid-Based Therapeutics

DNA Transduction

High-molecular-weight, double-stranded DNA constructs containing transgenes, which encode specific proteins, are classically used in gene therapy to introduce transgenes into cells that inherently lack the ability to produce a protein of interest. In addition to being used to treat congenital diseases, DNA vectors can be used as vaccines for genetic immunization.[87] Suicide gene therapy is another rapidly emerging strategy for the induction of transgenes. [88] [89] In this approach, chemosensitization genes are delivered to tumor cells, which upon gene expression convert a separately administered, nontoxic prodrug into a chemotoxic drug. Because only the transfected tumor cells can convert the prodrug, the susceptibility to the chemotoxic entity is limited to the tumor cells—hence the term suicide gene therapy.

RNA Transduction

Thus far the primary transgene source for DC transduction is DNA, although other Ag sources are also used with DCs, including peptides, recombinant or purified proteins, cellular extracts from tumorcells, apoptotic bodies, and RNA or DNA plasmid vectors. Nevertheless, the carrier of choice for loading DCs with tumor Ags is DNA or RNA.[90] Nucleic acid transfection leads to the display of multiple antigenic epitopes by both class I and II major histocompatibility complex via the Ag-processing machinery of the patients’ DCs, resulting in the display of the “most appropriate” peptides. This is in contrast to vaccine strategies based on synthetic peptides, which require the knowledge of the patient's unique peptide epitopes. Thus, nucleic acid transfection of DCs offers several advantages for both immunologic and practical considerations.

The bias for the use of DNA vectors includes an increased stability as compared with RNA, the ability to produce plasmids in large quantities, and the ease with which the sequence can be modified to regulate expression.[91] In several respects, however, RNA vectors are also advantageous when compared with DNA transfection. RNA vector advantages include the ability to use total messenger RNA (mRNA) isolated from tumors to transfect DCs with no intervening cloning steps and the ability to express several or potentially all tumor-derived genes within DCs. Transfected RNA need only reach the cytoplasm of DCs, whereas DNA requires entry into the nucleus and subsequent transcription. Furthermore, it has been suggested that the low level of antigenic epitope expression that occurs with RNA-transfected DCs could be advantageous, provided that expression levels are sufficient to generate a T-cell response. [90] [91] When low levels of antigenic peptides are presented by DCs, only those T cells with high-affinity recognition are activated, thus skewing the response toward T cells that can better recognize the tumor cells. Conversely, when DCs present high levels of antigenic peptides, T cells of low affinity may be activated, thus masking or even preventing the activation of high-affinity T cells. This could result in T cells that kill cells with high Ag expression but cannot kill tumor cells, which typically express low Ag levels. Thus, RNA-transfected DCs might have greater efficacy for the activation of high-affinity T cells.[91]


Oligonucleotides are short single-stranded segments of DNA or RNA that upon cellular internalization can selectively inhibit the expression of a single protein.[92] Multiple forms of oligonucleotides are used in gene therapy including antisense, siRNA, and ribozymes. Most of these constructs form a duplex with the mRNA or the pre-mRNA and inhibit their translation or processing, consequently inhibiting protein biosynthesis. This occurs by multiple mechanisms, as discussed in the following section.

Small Interfering RNA

RNA interference is a recently discovered mechanism for silencing the transcription of mRNA. siRNA is generated by dicer, an endonuclease that cleaves long double-stranded RNA molecules into fragments of 21 to 23 base pairs (bp) and is highly specific for the nucleotide sequence of its target mRNA siRNAs. [93] [94] [95] [96] These siRNAs associate with helicase and nuclease molecules and form a large complex, which is termed RNA-induced silencing complex that unwinds siRNA and directs precise, sequence-specific degradation of mRNA. Although RNA interference was discovered only recently, the field has exploded.[97] It is now apparent that RNA interference is a highly conserved molecular mechanism that is used by eukaryotic organisms to control gene expression during development and to defend their genomes against invaders, such as transposons and RNA viruses. Recently, it was shown that siRNA is active in vivo with resultant therapeutic activity.[98] In one study, siRNA knockdown of the mutant K-ras oncogene had pronounced antitumor activity.[99] In this study, siRNA was delivered as a nonreplicative retroviral transgene and was shown to inhibit the relevant mutant K-ras and prevent anchor-independent growth and tumorigenicity.

Antitumor activities can also be induced in vivo through siRNA knockdown of other critical components of tumor growth, metastasis, angiogenesis, and chemoresistance.[100] Stable transfection and expression of siRNA is obtained with nonreplicating viruses [101] [102]; however, oncolytic virus vectors provide a potential method to extend bioactivity. The tumor-selective infectivity has the potential to restrict transgene expression to the cancer microenvironment, potentially reducing toxicity and extending transgene expression via viral replication and multiple cycles of the infection of permissive cancer cells. [103] [104] Furthermore, viral oncolysis has the potential to augment antitumor outcomes by siRNA-mediated therapeutic activity. In a recent study,[105] the replication-competent, oncolytic adenovirus, ONYX-411, was used to deliver a mutant K-ras siRNA transgene. In this study, additive tumor growth-inhibitory responses via siRNA-mediated K-ras knockdown and ONYX-411–mediated oncolysis were observed. Therapy with ONYX alone or ONXY-411 with green fluorescent protein siRNA as controls had significantly lower therapeutic activity.


The principles of antisense technology are conceptually simple. Oligonucleotides are designed to hybridize to a defined target mRNA and to inhibit its translation into protein. [106] [107] This approach was first employed in 1978 by Stephenson and Zamecnik[108] to inhibit the Rous sarcoma virus expression in chicken fibroblasts. Several antisense oligonucleotides are in clinical trials, and one has received Food and Drug Administration (FDA) approval for the treatment of cytomegalovirus retinitis. Currently, an antisense to Bcl2 has been submitted for licensing by the Food and Drug Administration for the treatment of leukemia.[109] Although it is relatively easy to synthesize phosphodiester oligonucleotides, they cannot be used as drugs because of their sensitivity to nuclease degradation. To improve their resistance to nuclease digestion, different chemical modifications are used, including phosphorothioates, methylphosphonates, and phosphoramidates.[110] These modifications increase the stability of oligonucleotides, but they also alter the capacity to hybridize with RNA and reduce cellular internalization.


Ribozymes are RNA molecules capable of sequence-specific cleaving of mRNA molecules.[111] They selectively bind to target mRNAs and form a duplex that is easily hydrolyzed, suppressing specific genes.[54] Two types of ribozymes, the hammerhead and hairpin, have been extensively studied.[112] However, the RNA backbone in ribozymes is an easy target for RNases, so they are biologically unstable in vivo.[54] Ribozymes have been used primarily for gene suppression, the induction of apoptosis, and antiproliferative effects. Phase I clinical trials using ribozyme gene therapy to treat AIDS patients are ongoing.[113]


Targeted gene therapy of cancer can be achieved through



Targeted gene expression



Vector targeting [13] [114] [115]

Although it is less important during ex vivo or intratumoral gene delivery, targeted gene therapy becomes crucial with systemic gene transfer. Impediments to gene therapy include the poor selectivity of existing vectors and the low efficiency of gene transfer. Overcoming these hurdles is critical to achieving vectors that can be targeted and injected IV—an important goal given the systemic nature of cancer.

Conditional Gene Targeting

Vector targeting is a goal for both viral and nonviral vectors [13] [114]; however, the current emphasis is on tissue- or target-specific promoters. Transcriptional regulatory sequences are used because they are responsible for protein production in carcinoma cells such as oncogene products. One example is the use of tissue-specific promoters to facilitate tumor-specific killing via expression of a suicide gene (such as the HSV-tk) followed by exposure to ganciclovir, or the expression of the cytosine deaminase gene and exposure to 5-fluorocytosine. In addition, transcriptional targeting is used to achieve conditionally targeted transgene expression.

Tissue-Specific Promoters

The production of proteins within a cell requires that the appropriate gene be transcribed into mRNA and then translated to protein.[116] This process is under multiple levels of control, with the regulation of transcription mediated by interactions between the enhancer/promoter region of the appropriate piece of DNA and the specific proteins or transcription factors that bind to this region. Activation or repression of promoters is achieved through interactions with specific transcription factors. Thus, some tissues might express specific proteins because the promoter for that gene is activated in that tissue alone. The success of transcriptional targeting is dependent on achieving a differential gene expression in cancer cells as compared with normal cells. Transcriptional control of gene therapy is an important goal for two reasons:



Current gene transfer vectors can be inefficient in gaining entry into the types of cells needing treatment.



Many therapeutic genes can be toxic if delivered to an unintended cellular target.

Criteria for selecting a promoter for use in a gene therapy protocol include consideration of the promoter's strength, tissue specificity, and size. Promoter candidates include regulatory elements that are already expressed by the malignant cell, tissue-specific promoters, or externally inducible sequences. Unfortunately, any of these candidate promoters can lack sufficient activity, specificity, or both. To address promoter potency, promoters and enhancers that retain cell-specific function are often linked to transactivators. Additional strategies to enhance promoter activity in malignant tissues include the use of cell-cycle elements, normal or abnormal tissue differentiation factors, hormones, cytokines, chemicals, or physical stimuli.

A convenient classification of candidate promoters for cancer gene therapy ( Table 33-1 ) includes tumor-associated, tissue-specific, and inducible promoters. These are further discussed in the ensuing sections, as is the role of transcriptional regulation of replication-competent viruses. Specific examples are provided that are the most mature developmentally but should be viewed as representative. The most focused reviews and articles are referenced.

Table 33-1   -- Transcriptional Regulation for Cancer Gene Therapy

Transcriptional Mechanism


Target Tumor


PSA, Kallikrein







Hepatocellular carcinomas (HCC): breast, lung, and pancreas cancers


α-fetal protein (AFP)



c-erb B2







Lung cancer



Small cell lung carcinoma



Small cell lung cancer


Immunogloblin heavy chain

B lymphomas



Breast cancer



Breast cancer









β-islet cells


Whey acidic protein

Breast cancer


Cirulatory leukoprotease inhibitor (CLPI)

Lung, colon, breast, bladder, oropharyngeal, ovarian, and endometrial carcinomas


Glial fibrillary acidic protein

Brain astrocytes, glioma cells





T-cell receptor

T lymphocytes


Her 2/neu

Breast, pancreatic, and gastric carcinomas


Myc-Max responsive element

Lung cancer






Urinary bladder and HCC



Melanoma, fibrosarcoma and breast tumor vessels



Tumor vasculature



Lung cancer


Hexokinase II

Lung cancer


c-erb B2

Breast and pancreas tumors



Small cell lung cancer



Ovarian carcinoma



Lung and ovary tumors






Prostate, breast, and melanomas









Tumor-Associated Promoters

Selective delivery of a transgene to a tumor is not currently an achievable clinical goal. Consequently, tissue and tumor promoters are used to regulate transgene expression in a given tumor tissue with the goal of reducing nonspecific transgene expression. However, this approach retains challenges, including the infection of only a small fraction of tumor cells within the target tissue. As such, tumor-specific and associated promoters are also extensively utilized to target transgene expression, especially ones associated with the tumor vasculature. [117] [118] [119]


Telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is expressed in high levels in malignant tumors, stem cells, and germ cells, but not in normal tissues. It is thought to be essential for the maintenance of the proliferative capacity of tumor cells and, for this reason it represents an attractive target for gene therapy. The human telomerase reverse transcriptase is regulated primarily at the transcriptional level, and its promoter has the potential for targeted cancer gene therapy. [120] [121]


Another target for gene therapy is provided by the tumor's vasculature. The tumor vasculature has excellent accessibility to systemic delivery across all solid tumor types.[122] Indeed, high levels of vascular endothelial growth factor (VEGF), a growth stimulus for endothelial cells, have been correlated with a poor prognosis for specific tumor histotypes. VEGF activity is mediated by two high-affinity receptors: the tyrosine kinases VEGFR-1/fms-like tyrosine kinase (Flt-) 1 and VEGFR-2/flk-1. These ligand-stimulated tyrosine kinases are induced in a tumor stage-dependent manner during cancer progression and are expressed exclusively in tumor vascular endothelial cells.[123] This suggests that VEGF receptors are promising targets for tumor endothelial cell-specific therapy. [122] [124] Thus, the 939-bp Flk-1 promoter fragment and an enhancer element located in a 2.3-kb fragment upstream have been used to induce tumor endothelium-specific reporter gene expression in transgenic mice.[125]Targeting of the VEGF receptor/ligand system has been shown to be a useful approach with which to inhibit tumor growth and prolong survival in colon cancer.[124] The human preproendothelin-1 promoter has also been shown to have specificity for breast microvascular endothelial cells using a recombinant retroviral vector.[126]

Tumor-Specific Promoters


Prostate-specific Ag (PSA) is expressed at a high level in the luminal epithelial cells of the prostate and is absent or expressed at low levels in other tissues. The PSA promoter is usually regulated by androgens, but it might retain its activity in an androgen-free environment. The minimal PSA promoter, however, is weak in both PSA+ and PSA- cells and does not respond to androgenic stimuli. Nonetheless, the PSA promoter has been used to target the delivery of therapeutic genes to prostate tumors. [127] [128]


Specificity for malignant melanoma may be conferred by the human tyrosinase promoter. [129] [130] Driven by this promoter, in vitro and in vivo melanoma transduction by constructs results in selective transgene with the potential to induce tumor regression. Similarly, a construct consisting of the human tyrosinase promoter linked to two enhancer elements causes high-level, melanoma-specific expression of a reporter gene in transient transfection assays. The murine tyrosinase promoter-enhancer expression cassette expressed by an Adv vector maintains transcriptional specificity for pigment cell lineages, especially human melanoma cell lines.

Conditional Replication and Inducible Promoters

During evolution, various stress response genes developed, and their promoters are now considered as gene therapy transcriptional regulators. Heat, hypoxia, glucose deprivation, irradiation, and chemotherapeutic agents upregulate stress response genes. Because of the relative weakness of tissue- and tumor-specific promoters, these inducible promoters are attractive as mediators of transient transgene activation. Promoters of these genes are also attractive for cancer gene therapy because they depend to a large extent on the biology of the tumor or are already induced by various therapeutic modalities. [131] [132]


Genes that are upregulated during stress include ABCB1, human heat-shock protein (HSP), VEGF, irradiation-inducible EGR1 (early growth response gene), and the tissue plasminogen activator (tpa) promoters. Irradiation-responsive promoter sequences have been identified for the tpa and EGR1 genes.[133] The first irradiation-inducible promoter system used in combination with gene therapy involved the EGR1 promoter driving either the radiosensitizing cytokine tumor necrosis factor α (TNF-α) or tk. The HSP family is induced by a variety of environmental conditions, including heat, irradiation, photobeam irradiation, hypoxia, acidosis, hypoglycemia, and osmotic changes. These conditions can exist in poorly vascularized tumors and can trigger anticancer gene expression linked to the HSP70promoter.[134] It is significant that HSP70 expression is upregulated in p53-deficient tumor cells, thereby providing transcriptional targeting.


ABCB1 encodes a membrane effluxing glycoprotein, whose expression is induced by vincristine, actinomycin D, and doxorubicin. Its promoter is indirectly transactivated by these compounds and induces transcription and expression of therapeutic genes, such as TNF-α in tumors exposed to chemotherapy.[135] Chemotherapy can also induce another mechanism of drug resistance, namely, activation of the glutathione detoxification system and apoptosis-controlling gene alterations (especially p53 and bcl-2). Because the ABCB1 promoter contains heat-responsive elements, it is also activated by HSP. In addition to its promoter activity, ABCB1 can be transduced into hematopoietic stem cells to reduce the myelosuppressive effects of chemotherapy and radiotherapy.[136]


Several drug-related gene expression systems are available to control target gene transcription through the use of small-molecule-inducing compounds. [137] [138] Although the utility of such systems has been demonstrated in vitro and in transgenic mice, they are also targeting use in a therapeutic context. [139] [140] Dexamethasone, a synthetic glucocorticoid, can selectively activate the p21 promoter in rat hepatoma cells via a glucocorticoid-responsive region between nucleotides 21481 and 21184.[141] This region does not contain a canonical glucocorticoid response element, but it confers specific dexamethasone responsiveness to heterologous prostate promoters.


The Tet-controlled transcription system is made up of Tet-off and Tet-on transcriptional regulation, derived from the Escherichia coli Tet-resistance operon.[142] The Tet-R system can be used to suppress or induce cytotoxic and reporter gene expression. [143] [144] The latter selects gene expression to p53-deficient tumor cells. Similar to Tet-R, mifepristone is an orally bioavailable antiprogestin that can switch on gene expression in allosteric systems, whereby a chimeric transactivator activates a target gene.[145] This system can circumvent constitutive expression of transgenes in normal tissues by drug-specific and temporal regulation of the target gene. In addition, the replacement of the activation domain of the chimeric transactivator with a transcriptional repressor domain results in inducible repression of the transgene.[146]

Conditionally Replicative Viruses

Toxic or tumor suppressor gene expression from nonreplicative vectors, as a single therapeutic, is inadequate to control solid-tumor growth in humans. [147] [148] Thus, replication-competent viruses have been developed and tested as therapeutic agents in cancer. Adv vectors are the most commonly used agents in this context, although retrovirus, reovirus, HSV, and vesicular stomatitis virus are all used for the treatment of malignancies.

The criteria governing the utility of replication-competent viruses include infection efficacy, replication selectivity, viral dispersion from the injection site, and evasion of the host immune response. Augmented gene transfer efficiency has been reported for Adv vectors based on the coxsackievirus Adv receptor (CAR)-independent cellular entry pathways.[149] Propagation of these vectors within tumor tissue remains a challenge, however. Recent improvements in our understanding of cancer biology have made possible the development of viral vectors with improved tumor-selective replication and the restriction of lytic effects to cancer cells. Dysregulation of the normal control over cell cycle and circumvention of physiologic apoptotic signals might allow tumor-selective replication of an engineered virus and, subsequently, direct oncolysis by viral cell killing.[150]

Conditionally Replicative Adenoviruses

Conditionally replicative Ads (CRAds) are designed by the deletion of Adv natural genes encoding cell-cycle regulatory proteins and/or by placing a tissue-specific promoter to control a viral gene essential for viral replication. An example of a vector with a deleted Adv gene is the deletion of CRAd E1A ( Table 33-2 ). This results in a loss of its conserved region 2, which precludes binding to the retinoblastoma gene (Rb) and eliminates the inhibitory effect of Rb on E2F. Consequently, the engineered Adv replicates selectively within cells in which the G1-S phase checkpoint is impaired (i.e., tumor cells). [151] [152] Deletion of the Adv E1B 55-kd protein was initially suggested to be selective for replication in p53-mutant cells, but this hypothesis has since been questioned. [153] [154] Despite the mechanistic uncertainty, the E1B-deleted ONYX-015 virus selectively infects head and neck tumor cells and could show a clinical benefit in patients with recurrent carcinomas.[155] Although ONYX-015 does not have a therapeutic transgene and relies on its lytic effect, this is the first clinical utility of a CRAd for cancer therapy. It should be remarked that development of ONYX-015 by the American biotechnology company, ONYX Pharmaceuticals, was halted primarily because of financial concerns. Shanghai Sunway Biotech Co. licensed world rights to ONYX-015 and obtained regulatory approval in China in December 2005.[2] This gene therapy product, H101 (ONYX-015), is a recombinant Adv modified to selectively replicate in and kill tumor cells with TP53 mutations. This involves a loss-of-function mutation at the E1B locus. The E1B locus product is a 55-kd protein that binds to and inactivates the p53 tumor suppressor protein. Thus, the vector is crippled in its ability to grow in cells with wt TP53, and replicates and causes the death of cells with mutant TP53.[156] Phase I, II, and III clinical trials undertaken in China have been reported to show the safety of H101 with efficacy demonstrated in patients with HNSCC. Approval was based on a study that combined H101 and chemotherapy, which was reported to be effective in 78.8% of patients with this disease.[157]

Table 33-2   -- Transcriptional Regulation of Adv Replication

Genetic Modification

Biologic Result

Deletion of E1A (AA 121–127)

Transformation deficiency

Deletion of E1B 55 K protein

Susceptibility to apoptosis

E1A control by the αFP promoter

E1A transcription limited to αFP+ cells

E1A control of the PSA promoter

E1A transcription limited to PSA+ cells

E1A control by the DF3/MUC1 promoter

E1A transcription limited to DF3/MUC1+ cells

E1A control by the pS2 promoter

E1A transcription limited to estrogen receptor+ cells

E1A control by the Sp-β promoter

E1A transcription limited to surfactant producing cells

E1 deletion

Selective DNA replication of Adv vectors in trans-complementing tumor cells



Recently, mutants of human Adv 5 (Ad5) with enhanced oncolytic activity have been isolated using a procedure termed bioselection. In this process, Ad5 is mutagenized and repeatedly passaged in a human colorectal cancer cell line. From such a cell line, mutants can be found that replicate more rapidly than wt Ad5 and that lyse cells up to a thousand-fold more efficiently.[158] Another strategy for designing CRAds uses tissue-specific promoters to drive expression of E1A, thereby restricting viral replication to specific tissues or tumors.[159] The application of heterologous promoters in Adv vectors is difficult because their activity and specificity are often affected by viral enhancers and promoters. The E1A gene expressed from the alpha fetoprotein (AFP) gene promoter induces relatively selective replication in hepatocellular carcinoma cells.[159] Control of Adv E1A expression under the minimal PSA enhancer/promoter has also been shown to confer prostate-specific oncolytic viral replication.[160] Recently, a reengineered Adv vector with enhanced oncolytic efficacy was developed. This vector contained a novel regulatory circuit in which p53-dependent expression of an antagonist of the E2F transcription factor inhibits viral replication in normal cells. In tumor cells, however, the combination of the p53 pathway defects and deregulated E2F allows replication at near-wt levels. This Adv vector also has significantly enhanced efficacy for the treatment of human xenograft tumor models compared with the extensively studied E1B-deleted Adv vectors.[20]

CRAds for breast tumors have been created using the DF3/MUC1 promoter (which is abnormally activated in breast tumors) and are used to drive the expression of E1A. This CRAd selectively replicates in MUC1+ cells and can inhibit the growth of human breast cancer xenografts.[161] Another approach is to target CRAd replication within estrogen receptor (ER)-positive tumors based on replacing the E1Aand E4 promoters with a portion of the pS2 promoter containing two estrogen-responsive elements.[162] This promoter induces transcriptional activation of the E1A and E4 in response to estrogen in cells that express an ER. This CRAd is able to lyse ER+ human breast cancer cell lines as efficiently as Adv, with decreased capacity to affect ER- cells.

Another strategy that has been reported recently makes use of the generation of a functional promoter/gene constellation only on Adv DNA replication, thereby providing selective transcriptional activation.[163] These strategies to discriminate between tumor and normal tissue are based on selective DNA replication of Adv vectors with the entire E1 gene in tumor cells deleted. An E1 deletion is considered to abolish Adv replication; however, human tumor cell lines apparently can support DNA replication of Ad with an E1 deletion. Inverted repeats insert into the E1 region of AdE1 vectors can mediate genomic rearrangements, and bring a transgene into control of a promoter. Thus, formation of a functional expression cassette depends on viral DNA replication, which is expected to occur specifically in tumor cells.

Vector Targeting

Targeted in vivo gene transfer is becoming a reality as a result of an improved understanding of influences that govern gene delivery. [13] [14] [114] Viral-based vectors are designed to avoid gene transfer through their native receptors and are redirected to tissue- and tumor-specific receptors. In most therapeutic applications, the vector is introduced into a mixed population of cells with the goal of delivering the therapeutic transgene to specific cells. Transduced stem cells can also be targeted to treat certain genetic diseases, improve tolerance to chemotherapy, or assist in tissue repair and remodeling. DCs can also be targeted for the development of improved vaccines. Finally, a systemically administered, targeted vector can potentially reach systemic disease. Nevertheless, these vectors require additional development, including clinical testing, reduced liabilities (including innate and acquired immune augmentation), and an improved understanding of the mechanisms that govern biodistribution and pharmacokinetics.

Ligand-directed targeting of gene vectors allows control of the site at which genes are expressed by imparting the capacity to distinguish between target and nontarget tissue(s). These ligand-directed targeting vectors achieve this capability through the addition of ligands to the vector that recognize receptors specific for a tissue or disease. This approach has met two goals:



Improved efficiency for the current gene transfer vectors in transducing the targeted cells that need treatment



Reduction in the toxicity due to delivery of therapeutic genes to unintended target cells

Thus, ligand-directed targeting can potentially improve both the safety and the efficacy of gene transfer and make possible therapies that could not be envisioned with standard gene transfer vehicles.

Although targeting gene transfer to specific cells and tissues holds promise, it is a challenge for vector design. Regardless of the vector, three variables are critical to ligand-directed targeting. These include cellular specificity, physical barriers, and the host innate or acquired response, which could eliminate the vector from the circulation. Cellular specificity can be achieved by the use of ligands that recognize cell-specific receptors. For viral-based vectors, specificity requires a targeting element plus modification of the vector so that it no longer binds to its native cellular receptors. In the case of nonviral vectors, targeting requires modification of the vector to avoid nonspecific uptake. Apart from cellular specificity, physical barriers (e.g., the cellular matrix) can limit access of the vector to the target cell. Finally, avoiding elimination and neutralization of vectors by innate and acquired immunity is critical to gene transfer. This is critical, because Abs and serum proteins can directly inactivate the vector or direct it to the liver for rapid clearance, if the vector is given systemically.

Adenoviral Vectors

Our improved understanding of the attachment and entry processes of Adv vectors has facilitated the development of Adv-targeting vectors. [13] [34] [114] The nonenveloped subgroup C Adv vectors use at least two coat proteins to gain entry into cells. The knob portion of the fiber coat protein binds to the cellular receptor, CAR, and mediates virus attachment. [164] [165] At the base of the fiber protein, the penton base coat protein contains an Arg-Gly-Asp (RGD) motif that binds to integrins and facilitates vector uptake into the cell.[166] Compared with a vector with native receptor binding interactions intact, gene expression in the liver and other organs is substantially reduced after systemic administration of a vector containing mutations that ablate CAR and integrin binding.[167] This observation suggests that these receptor interactions are important for in vivo gene transfer. The loss of CAR and integrin binding also reduces gene transfer after direct injection. Furthermore, it allows Adv vectors to be retargeted genetically. [17] [168] Ablation of CAR binding alone does not significantly reduce liver gene transfer, which suggests that the standard two-step model of attachment via the CAR and entry by means of integrins does not apply to in vivo gene transfer to the liver, which instead probably involves Kupffer cells.

Adv vectors have been retargeted by both genetic and nongenetic means. Peptides (including the fiber, penton base, and hexon) have been functionally incorporated into coat proteins, although few functional peptide ligands have been identified thus far. [166] [169] [170] [171] [172] [173] In addition to genetic modifications for retargeting viral vectors, ligation approaches have also been used with Adv vectors. Such approaches involve a bifunctional adaptor or bridging molecule that binds to the vector and to a target receptor. Such systems have demonstrated the feasibility of targeting conventional Adv vectors to more than 20 different receptors, including αv integrins, endoglin, E-selectin, EpCAM, and folate receptors.[114] Specific targeting to the lung vasculature has also been demonstrated through a combination of receptor-based targeting via lung endothelial-specific receptor, angiotensin-converting enzyme, and promoter-based targeting through the endothelial-specific promoter, Flt-1.

Structural Modification of the Fiber Protein

One approach to Adv retargeting involves engineering of the knob domain of the fiber protein. In this domain, the introduction of heterologous cell targeting peptides requires consideration of the structural limitations of the fiber three-dimensional configuration. The fiber is synthesized as a monomer, which undergoes trimerization before its attachment to the penton base. Thus, any modification of the knob domain of the fiber must not impair trimer formation. In addition, the final quaternary configuration of the new fiber must make the incorporated ligand accessible to target cell receptor recognition and binding.

Recombinant Adv vectors have been constructed with a heparin/heparan sulfate-binding domain, consisting of polylysine residues added to the C terminus of the fiber. Gene transfer to different mammalian cells has been obtained with a level of efficiency 10- to 300-fold higher as compared with unmodified vector.[174] The main drawback with this approach is the lack of specificity, because most mammalian cells express heparin-containing cellular receptors. Genetic modification of the Adv fiber C terminus is limited, because the addition of more than 25 to 30 amino acid residues renders the fiber trimer unstable and limits function.[175] The modification of Adv vectors by placing an RGD peptide in the HI loop rather than in the C terminus of the fiber knob domain was reported recently.[176] Thismodification resulted in an increase in gene transfer to ovarian cancer cell lines (30- to 600-fold) and ovarian cancer cells (twofold to threefold).

Modification of the Penton Base

Retargeting of Adv vectors has also focused on the modification of the penton base, which mediates the second step of Adv infection (i.e., internalization). Recombinant Adv vectors have been generated in which the RGD motif in the penton base has been replaced by the FLAG peptide. A complex of this vector with a bispecific Ab—consisting of a monoclonal ab to the FLAG epitope and a monoclonal ab to integrins—was shown to target cells lacking the Adv fiber receptor, such as endothelial cells or human intestinal smooth muscle cells. Thus, the first two steps of Adv infection binding and internalization are both mediated by α integrins.[177] In addition, recombinant Adv vectors can be constructed of chimeric penton base proteins that recognize tissue-specific integrin receptors.[178]

Retroviral Vectors

Retroviral vectors were the first viral vectors to be targeted and the first to demonstrate the promise of vector targeting. [34] [114] Since that time, the challenge has been to incorporate targeting ligands without compromising vector entry into target cells. There are now several approaches used to address this problem. One approach exploits pseudotyping, classically with the γ-glycoprotein from the vesicular stomatitis virus, in which entry events are mediated through common membrane phospholipids.[179] Pseudotyped lentiviral and retroviral vectors have also been generated with glycoproteins from a variety of enveloped viruses, including Ebola virus, Marburg virus, rabies virus, lymphocytic choriomeningitis virus (LCMV), Mokola virus, human foamy virus, gibbon ape leukemia virus, murine leukemia virus, influenza virus, avian leukosis-sarcoma virus, and respiratory syncytial virus. [180] [181] [182] [183] [184] Although these pseudotyped vectors vary in terms of degree of envelope shedding, efficiency of packaging, titer, and stability, they can be concentrated to high titers for in vivo comparisons of cellular tropisms.

Another vector modification involves the ligation of polypeptides at the N terminal of env to extend the host range of ecotropic murine leukemia virus (MLV). Examples include erythropoietin, heregulin, and CD4. [185] [186] [187] It should be stressed that coexpression of the wt env protein is necessary for infection to occur, possibly because incorporation of the engineered env protein in the virons can be facilitated by the oligomerization of both wt and chimeric env proteins. Another approach for engineering of the ecotropic MLV env protein involves the display of different polypeptide binding domains to the N terminus of the ecotropic MMLV surface protein. Examples include the N-terminal moiety of the amphotropic MLV env, single-chain Abs recognizing different cell surface receptors, heregulin, and epidermal growth factor. [188] [189] [190] [191] In some of these studies, infection specificity was redefined, though with lower efficiencies than those obtained with viruses expressing wt amphotropic envelopes. [192] [193]

Bifunctional bridging agents that recognize both the retrovirus and the targeted cell surface molecule provide evidence that retroviruses can enter cells via cell surface molecules that are not viral receptors. Such bridging agents have been used to infect human cells that are naturally resistant to ecotropic MLV-based vectors. The agents used usually consisted of an αβ to the MLV envelope protein connected to either another αβ or a growth factor that would bind to the appropriate receptor such as the epidermal growth factor receptor, the insulin receptor, or major histocompatibility complex class I and class II molecules. Unfortunately, infection efficiencies for these agents are extremely low, emphasizing that binding of a retrovirus to a target other than the natural receptor cannot guarantee success. [194] [195]

Nonviral Vectors

Nonviral vectors have no native receptor binding, yet do have a high incidence of nonspecific gene transfer from the high positive charge on many nonviral vectors. [54] [55] [56] Many nonviral vectors transduce lung vasculature, potentially as a result of vector-mediated red blood cell aggregation and arrest in the lung subsequent to IV administration. The solution to the problem of nonspecific delivery is to shield the vectors, either with hydrophilic polymers such as polyethylene glycol (PEG) or with a ligand to reduce the surface charge. This type of shielding is applied successfully to lipid-based systems and to cationic polymer-based systems (polyplex).

Ligand-directed liposomes have shown some success in targeting tumors. Coupling of a synthetic αvβ3-integrin ligand to cationic liposomes permits the selective delivery of a mutant Raf gene that causes apoptosis in angiogenic blood vessels within tumors. Systemic injection of the αvβ3-targeted liposome results in apoptosis of tumor-associated endothelium and the regression of primary and metastatic tumor(s). This accomplishment highlights the extension of this approach from in vitro to in vivo efficacy.[86]

Another promising advance is to coat polyethylenimine (PEI)-DNA polyplexes with a ligand, such as transferrin or transferrin plus PEG. Shielding by PEG or transferrin prevents nonspecific interactions with plasma proteins and erythrocytes but does not interfere with target cell interactions. When systemically administered in a subcutaneous tumor mouse model, the shielded complexes were shown to selectively transduce a well-vascularized, rapidly growing tumor. Although the specificity of this approach is high, the overall level of transduction is low.[196]

In all the studies to date that use nonviral approaches, the need for relatively high dosing levels (∼100 mg/mouse) suggest that innate clearance mechanisms might have to be saturated before substantial gene transduction can occur. Other potential issues that remain include determining whether the high doses used in the animal studies can be manufactured and delivered successfully for human studies. Attaining further improvements in the efficiency of gene transfer and better defining the toxicity profiles associated with these vectors are also critical steps.


The development of gene therapy over the last decade has been on a roller-coaster ride that has yet to fulfill the promise of this exciting new research and therapeutic tool. Retroviral gene therapy has inarguably been shown to reverse congenic diseases. This was the first success for gene therapy, whereby a retroviral-based treatment was undertaken for infants suffering from X chromosome-linked SCID-X1. These studies provided the first demonstration of the potential for long-term treatment of hereditary diseases.[197] The success of this approach is due not only to gene therapy but also to improvements in our understanding of hematology and the availability of clinical-grade cytokines to support the transduction of adequate numbers of stem cells. These studies have resulted, however, in the concept of retroviral insertional carcinogenesis moving from a theoretic to a real concern in recent months. Two of the initial 11 children who received retroviral gene therapy for the treatment of SCID developed a leukemia-like condition.[198] Both of these cases, as well as a third in which leukemia has not yet developed, seem to be due to the insertion of the corrective gene near another gene called Lmo2, which helps to control cell growth and can contribute to cancer if turned on at the wrong time.[198] Nonetheless, the unique nature of this therapeutic strategy for patients who have no other viable therapeutic modality, and the responsiveness of the resultant leukemias to chemotherapy, suggest that it remains a justifiable therapeutic strategy for those patients who have no matching allotransplant donor.

Therapeutic activity has also been demonstrated[199] for retroviral vector transduced stem cells in infants with a defective gene for adenosine deaminase (ADA-SCID). In contrast to children with SCID-X1, enzyme replacement therapy has been available for children with ADA-SCID using pegylated ADA (PEG-ADA). Initially, ethical concerns required that gene therapy studies in ADA-SCID patients be undertaken concomitant with PEG-ADA therapy. A generalized schema for these gene therapy protocols is shown in Figure 33-3 . However, the SCID-X1 studies by Cavazzana-Calvo revealed that a strong selective pressure was needed to expand the transduced cells following infusion.[197] This revelation provided the impetus for studies in ADA-SCID whereby PEG-ADA was discontinued, allowing the selection of transfected stem cells and more importantly differentiated T cells. In addition, nonmyeloablative conditioning with busulfan was used to provide space in the marrow for the infused stem cells. Although insertional mutagenesis remains a concern, no clonal expansion has been reported to date. A recent follow-up at a median of 3 years was presented at the 2006 American Society of Hematology (ASH) annual conference and reported that seven of eight children responded to gene therapy, with approximately 95% of T cells expressing ADA. The one patient who did not show improvement was treated at an older age, suggesting a higher level of disease damage.


Figure 33-3  Characteristic protocol for retroviral treatment of congenital diseases. An improved understanding of stem cell culture has facilitated therapeutic efficacy for the treatment of children with common γ-chain, severe combined deficiency; SCID-X1; and ADA-SCID. Protocols for other congenital diseases are similar. In general, children have a bone marrow harvest either in utero or more commonly in the first few years of life. The harvested cells are briefly cultured, typically with a mixture of cytokines active on primitive hematopoietic stem cells; transfected with a retroviral vector; and then cultured again to allow transgene insertion into the chromosome. The patient may also undergo nonmyeloablative conditioning, typically with busulfan, before receiving the transfected cells. Several studies have shown that supportive therapy must be withdrawn to allow selection of the transfected cells. Typically within a few weeks post-transplant, a high percentage of the circulating T cells are transduced with retroviral vector, resulting in normalization of T-cell numbers and a significant decrease in infections.



The second vector type that has shown significant clinical potential is Adv vectors. In contrast to retroviral vectors, Adv vectors induce a transient gene expression and demonstrate both high transduction efficiency and high transgene expression; in addition, these vectors can be grown to high titer for virus stocks. Furthermore, the activity profile of these vectors (particularly transient gene expression) provides an attractive characteristic for many current clinical development strategies. Most notably, these protocols involve the induction of tumor apoptosis via systemic or intralesional injection of vectors with transgenes that induce apoptosis or result in the activation of cytotoxic drug precursors, such as TK or cytosine deaminase. In addition, Adv vectors are being used to deliver Ags to Ag-presenting cells (e.g., DCs), resulting in the induction of an Ag-specific immune response and (theoretically at least) therapeutic activity.

In a recent phase I/II trial of extensive stage, small cell lung cancer,[200] 29 patients received standard first-line chemotherapy and three vaccinations with Adv-transfected DCs as a vaccine. Patients with stable disease to first-line chemotherapy had DCs prepared from enriched monocytes, which were then transfected with Adv-p53 and the resultant p53-DC injected three times. Following disease progression, patients received second-line chemotherapy. The objective response rate to vaccine alone was low, although it was reported that following p53-DC vaccination one patient had lymph node metastasis regress with the remainder of patients rapidly progressing. In contrast, 60% of patients with progressive disease following vaccination had objective clinical responses to second-line chemotherapy, resulting in an overall survival of greater than 11 months after immunotherapy. Indeed, the major response rate to second-line chemotherapy (complete and partial responses) was 90% in those patients who developed an immune response to p53 as compared with 40% for those patients who did not develop an immune response. In this disease, aggressive combination chemotherapy regimens can extend the median survival time to 9 to 10 months from diagnosis, as compared with 2 to 3 months if untreated.

Similar to retroviral vectors, Adv vectors have experienced “growing pains.” Although the majority of the vectors used in current practice have been replication incompetent, the routes of administration and therapeutic targets have been variable and—in part because of this—various toxicity issues have developed. Studies using Adv-p53 vectors have shown clearly that these can be injected at doses up to approximately 7.5 × 1013 for intraperitoneal administration, and 2.5 × 1013 viral particles has been identified as the maximum total dosage.[201] This same particle number, administered via the hepatic artery for the treatment of hepatic metastasis, has also been identified as the maximum total dosage.[202] Initially, Adv vectors were delivered based on plaque-forming units, a strategy found to be less rigorous quantitatively when compared with a particle number strategy.[203] Yet, despite the known biodistribution and toxicity profile of Adv vectors—including Adv vectors delivered by vascular injection—one child with a non-life-threatening disease, ornithine transcarbamylase deficiency, was dosed with a high number of viral particles, resulting in his untimely demise.[204] The incident resulted in a regulatory hold on Adv for a period of time, which limited the use of Adv vectors. This toxicity problem has largely been overcome with increased clinical conservatism. Similarly, the immunologic reaction to the Adv vectors can be reduced as shown with some second- or third-generation vectors (gutless), providing a significant impact on safety, transgene expression, and duration of expression.[205]Indeed, Adv vectors have been used to deliver receptors for retroviral vectors to improve their transduction frequency.[206] Thus, in addition to “naked DNA” vectors, retroviral and Adv vectors have been used predominantly in the clinic thus far. Clearly, other agents are used, including AAV, a viruses, and herpes vectors, but to a lesser extent.

Several factors directly related to vectors have hampered the clinical progression of gene therapy. These include



Inefficient gene delivery, which is associated predominantly with nonviral and retroviral vectors that have reasonable gene delivery efficacy in vitro but disappointing efficacy in vivo



Poor ability to target transgene expression to either cells or tissues of interest to avoid expression of toxic gene products in healthy or unintended target tissue



Short duration of expression due to poor replication and/or stability of episomal vectors and to inefficient or inappropriate integration of vectors into the host genome



Poor production of vectors at high titer, which is developmentally limiting in the cases of retroviral and gutless Adv vectors



Safety, which is a prerequisite for clinical gene therapy trials. Safety includes not only issues of direct toxicity but also the potential for homologous recombination, which has to be maintained at theoretically acceptable levels. Furthermore, targeted genomic integration has also recently been shown to be potentially critical.

Because of the challenges associated with targeting and transfection efficiency, the therapeutic strategies currently in use take advantage of the positive aspects of the vectors and limit their deficiencies. There are four overall approaches that reduce the challenges associated with the targeting and delivery of the transgene:



Hepatic arterial delivery of Adv-p53 for the treatment of hepatic metastasis [202] [207] [208]



Intratumoral administration of Adv vectors for head and neck tumors [155] [209] [210]



Intralesional injection for the treatment of bladder cancer with Adv vectors[211]



Intralesional injection for the treatment of lung cancer

The use of Adv vectors to purge hematopoietic stem cell products is also an exciting strategy that initially targeted breast cancer. [212] [213] [214] [215] [216] It has become a historical approach, however, with the reduction in transplantation for the treatment of metastatic breast cancer. These are the types of approaches that are needed for the successful development of gene therapeutics. The future of gutless vectors or vectors with improved targeting is bright, but at present such vectors introduce additional deficiencies such as low manufacturing titers.

In 2004, Adv-p53 vectors were approved in China for the treatment of patients with HNSCC.[1] This approval was based on a single clinical study using an Adv serotype 5 vector engineered to express p53 (Gendicine). In this multicenter, randomized clinical trial, 135 patients were randomized and entered to receive Gendicine in combination with radiotherapy (GTRT) or radiotherapy alone (RT). It was reported that the response rate in the GTRT group was 93%, with 64% showing complete regressions and 29% partial regressions. This contrasts with a phase I study in the United States by Introgen that entered 106 patients, which were reported to have only a 10% tumor response rate, defined as a 30% reduction in tumor size, in patients who received gene therapy alone.[217] This response increased to 26.5% for the clinical biomarker defined population, resulting in a progression-free interval of greater than 12 months from initial treatment of patients who had prior chemotherapy. In the overall treatment population, tumor response was associated with a significant increase in survival. The median survival of responders was 16.9 months, a significant increase as compared with 5.4 months for nonresponders. Thus, there is some controversy regarding the Chinese studies; however, the resulting population of patients that have received Gendicine (≥3,000) has provided valuable information. One such observation is that Gendicine has a better response if injected directly into the tumor. This potentially minimizes any immune reaction against the Adv vector and improves the efficiency of p53 delivery.

The majority of gene therapy trials are focused on cancer, and to date, approximately 66% of the over 1000 gene therapy trials in the United States have been initiated for this indication. This represents approximately 63% of all clinical trials. The predominance of clinical vectors used is retroviral vectors, although Adv vectors are also being used extensively. The majority of therapeutic strategies are focused on immunotherapy, with the predominance of transgenes used being either cytokine or Ag. When one considers the timeline for most drug development, gene therapy is on target. Although there was considerable initial optimism, the reality is that a period of time and appropriate attention to toxicities, adverse events, and pharmacologic issues (including biodistribution and cell targeting) are required before success can be achieved. Great strides have been made as vector biology begins to catch up with improvements in vectors. It is our expectation that future successes (such as those found with SCID and retroviral vectors) can be expected, although future frustrations are also to be expected and appropriate conservatism must be maintained. One area that has a high potential for success is the utility of vectors such as VV and Adv to deliver Ags to DCs as a vaccine for the treatment of infectious diseases or tumors. Clearly, Adv and VV vectors have innate vector antigenicity, which is limiting these approaches and providing opportunities for “naked DNA” and formulated “naked DNA” for either vaccine priming or boosts. A successful clinical protocol will be achieved only if these liabilities are considered carefully, with appropriate attention to well-designed protocols that take advantage of the positive attributes of vectors and minimize their negative attributes. Within this review, we have attempted to stress the great strides have been made recently with targeting and to illustrate that the future is clearly bright.


The authors wish to thank Ms. Kirsten Stites for her assistance with the preparation of the manuscript. This research was supported in part by the Nebraska Research Initiative Programs in Molecular Therapeutics (J.E.T.) and in Gene Therapy (J.E.T.), as well as the Avon-NCI Progress for Patients (PFP) Award Program (P30 CA036727-AV-93P-A1)(K.H.C. and J.E.T.).


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