help button home button The Oncologist
HOME HELP CONTACT US SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow eLetters: Submit a response to this article
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article link to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vorburger, S. A.
Right arrow Articles by Hunt, K. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vorburger, S. A.
Right arrow Articles by Hunt, K. K.
The Oncologist, Vol. 7, No. 1, 46-59, February 2002
© 2002 AlphaMed Press

Adenoviral Gene Therapy

Stephan A. Vorburger, Kelly K. Hunt

Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA

Correspondence: Kelly K. Hunt, M.D., Chief, Surgical Breast Section, Department of Surgical Oncology, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 444, Houston, Texas 77030, USA. Telephone: 713-792-7216; Fax: 713-792-4689; e-mail: khunt{at}mdanderson.org


   LEARNING OBJECTIVES
Top
 Learning Objectives
Abstract
 Introduction
 Adenoviral Vectors
 Clinical Applications of Gene...
 Improvement of the Adenoviral...
 Other Points of Interest
 Future Directions
 References
 
After completing this course, the reader should be able to:

  1. Understand the advantages and disadvantages of recombinant adenoviral vectors for gene delivery.
  2. Review the current biological strategies employed in clinical trials utilizing adenoviral mediated gene therapy for cancer.
  3. Understand modifications that may improve the utility of adenoviral vectors for gene therapy approaches.
Access CME test online and receive one hour category 1 credit at CME.TheOncologist.com


   ABSTRACT
Top
 Learning Objectives
 Abstract
Introduction
 Adenoviral Vectors
 Clinical Applications of Gene...
 Improvement of the Adenoviral...
 Other Points of Interest
 Future Directions
 References
 
As of May 2001, 532 gene therapy protocols had been approved for evaluation in clinical trials; however, only five of those had been evaluated in phase III clinical trials. Among the most commonly used vectors for the delivery of genetic material into human cells are the adenoviruses. Remarkable progress has been made with these vectors in the last decade, but some shortcomings continue to challenge investigators. The newly acquired knowledge of the adenoviral life cycle and the positive outcomes from phase II clinical trials have led to the application of vectors engineered to selectively target tumor tissue under controlled promoters.

Key Words. Gene therapy • Adenoviridiae • Genetic vectors • Clinical trials • Review literature


   INTRODUCTION
Top
 Learning Objectives
 Abstract
 Introduction
Adenoviral Vectors
 Clinical Applications of Gene...
 Improvement of the Adenoviral...
 Other Points of Interest
 Future Directions
 References
 
The perfect vector system for use in gene therapy would be administered by a noninvasive route, would target only the desired cells within the target tissue, and would express a therapeutic amount of transgene products with desired regulation for a defined length of time. No single vector system is likely to be optimal for all potential gene therapy applications and, thus, in the future we will see gene therapy with adapted vector systems and gene therapy in combination with standard antitumor regimens. Adenoviruses are among the most commonly used vectors for gene therapy, second only to retroviruses. This article reviews the ongoing clinical trials of adenoviral gene therapy for the treatment of cancer and discusses the current limitations and future potential of this approach.


   ADENOVIRAL VECTORS
Top
 Learning Objectives
 Abstract
 Introduction
 Adenoviral Vectors
Clinical Applications of Gene...
 Improvement of the Adenoviral...
 Other Points of Interest
 Future Directions
 References
 
The adenoviruses have several features that make them well suited for use in gene therapy. First, they are ubiquitous: adenoviruses have been isolated from a large number of different species, and more than 100 different serotypes have been reported, some 43 in humans. Most adults have been exposed to the adenovirus serotypes most commonly used in gene therapy (serotypes 2 and 5). Second, adenoviral vectors rapidly infect a broad range of human cells and tend to yield high levels of gene transfer compared to levels achieved with other currently available vectors. Third, adenoviral vectors have low pathogenicity in humans: they cause few and only mild symptoms associated with the common cold. Fourth, adenoviral vectors can accommodate relatively large segments of DNA (up to 7.5 kilobase pairs [kb]) and transduce these transgenes in nonproliferating cells. Fifth, the viral genome does not undergo rearrangement at a high rate, and inserted foreign genes are generally maintained without change through successive rounds of viral replication. Finally, adenoviral vectors are relatively easy to manipulate using recombinant DNA techniques.

Adenoviruses are DNA viruses. They contain about 36 kb of double-stranded DNA. After entry into the nucleus, genes from the early region 1 (E1a and E1b) are quickly transcribed (Fig. 1Go). During the early phase of viral replication, four noncontiguous regions of the genome are expressed (E1 to E4) (Fig. 2Go). They serve in part as master transcriptional regulators, starting the process of viral gene expression leading to genome replication. After the onset of DNA replication, the major late promoter drives much of the viral transcription. Viral-encoded functions can be separated into cis and trans elements. Whereas the cis genes, such as those responsible for the origin of replication or the packaging signal that condenses the DNA (protein IX, {psi}), must generally be carried by the virus itself, the trans genes can be complemented or replaced by inserted "foreign" DNA.



View larger version (62K):
[in this window]
[in a new window]
  		Figure 1. Life cycle of adenoviruses. Replication-defective adenoviral vectors express the proteins encoded on their DNA but do not enter the lytic phase.

 


View larger version (9K):
[in this window]
[in a new window]
  		Figure 2. Structure of adenoviral DNA. About 8 kb of foreign DNA can be introduced. The protein IX proved to be crucial for the packaging and limits the insertion at E1.

 
At least three regions of the viral genome can accept insertions or substitutions of DNA to generate a helper-independent virus: a region in E1, a region in E3, and a short region between E4 and the end of the genome. In the first-generation vectors, the E1 region was removed to make room for the therapeutic transgene and to prohibit viral replication. However, even in the absence of E1 gene products, there was low-level transcription of the remaining viral genes, resulting in early innate host cytokine transcription followed by antigen-dependent immune responses. This resulted in a reduction of the period of gene expression because of cell-mediated destruction of the transduced cells [1–3]. This strong immune response was a major drawback of early adenoviral gene therapy and might well have contributed to the decrease of gene expression in a number of adenovirus gene-transfer studies in patients. Early trials studied the effect of suppression of the immune response through concomitant administration of low-dose etoposide, but this strategy had limited success [4]. Today's second- and third-generation adenoviral vectors have deletions of various E1, E2, and E4 genes, because viral proteins encoded by these DNA sequences were shown to induce most of the host immune response. These new vector constructs have decreased toxicity and result in prolonged gene expression in vivo [5]. However, an important limitation in the use of recombinant adenoviruses has been the difficulty in obtaining efficient gene transfer upon a second administration of virus due to formation of neutralizing antibodies. This T-helper and B cell-dependent process that develops as a result of major histocompatibility complex (MHC) class II presentation of input viral proteins cannot be prevented directly by redesigning the vectors. In contrast to retroviral vectors, however, an inhibition by circulating complement was not noted in most recent adenoviral vector constructs [6].

Adenoviral vectors allow for transmission of their genes to the host nucleus but do not insert them into the host chromosome. Therefore, there is a low probability of disturbance of vital cellular genes or processes. On the other hand, the adenoviral-vector approach limits gene therapy to treatment strategies in which only temporary protein expression is needed. Because the viral DNA eventually disappears, treatments for chronic conditions, such as cystic fibrosis, would have to be repeated at specific intervals. However, if only short-term activity of a gene is needed—for example, to arouse the immune system against cancer cells or induce apoptotic stimuli—such nonintegrating delivery vehicles are desirable.

In preclinical settings, it has been shown that adenoviral vector DNA is expressed in liver, skeletal muscle, heart, brain, lung, pancreas, and tumor tissue [7]. When adenoviral vectors are given intravenously, most of the virus accumulates in the liver. Treatment close to reproductive organs, such as treatment for prostate and cervical cancer, has been shown to be safe. Even with replication-competent adenoviral vectors, which persist longer in the target tissue and liver, no offspring have shown germline transmission [8]. Table 1Go displays advantages and disadvantages of other vectors commonly used today.


View this table:
[in this window]
[in a new window]
  		Table 1. Pros and cons of other common vectors in current clinical trials
 

   CLINICAL APPLICATIONS OF GENE THERAPY WITH ADENOVIRAL VECTORS
Top
 Learning Objectives
 Abstract
 Introduction
 Adenoviral Vectors
 Clinical Applications of Gene...
Improvement of the Adenoviral...
 Other Points of Interest
 Future Directions
 References
 
Today, adenoviral vectors are used in suicide gene therapy, in gene-based immunotherapy, in gene replacement strategies, and in approaches that combine gene therapy with chemotherapy. In addition, treatment with replication-competent adenovirus vectors are showing promising results in clinical trials.

Suicide Gene Therapy
The fascinating concept of suicide gene therapy (also known as prodrug therapy) refers to the delivery into tumor cells of enzymes that metabolize systemically administered nontoxic prodrugs to locally active chemotherapeutic agents. Treatment efficacy is enhanced by the death of neighboring, nontransduced cells (bystander effect). In studies in different animals, adenoviral delivery of the herpes simplex virus thymidine kinase (HSV-tk) gene, which activates the prodrug ganciclovir, has been one of the most effective approaches in treating experimental brain tumors [9–14]. Recent phase I clinical trials, however, showed substantial toxicity at high levels of vector particles due to a multifactorial cellular and humoral immune response to the first-generation vectors [15].

Gene-Based Immunotherapy
Because cancer patients frequently develop both humoral and cellular immune responses to tumor-associated antigens, enhancement of these reactions by gene therapy has been proposed as another potential gene therapy strategy. Dendritic cells have emerged as the key cell type responsible for initiating and controlling cellular immune responses. They are the best equipped and most powerful antigen-presenting cells (APC) and the only cell type able to stimulate naïve T cells [16]. Various methods have been developed to "prime" dendritic cells against tumor antigens. In tumor-lysate pulsing, the dendritic cells are collected from peripheral blood and fed with whole tumor antigen proteins from lysates. The dendritic cells take up and process the proteins, then present their antigenic epitopes through the exogenous MHC class II pathway and in MHC class I molecules. Another method that proved to protect mice from tumor spread was fusion of dendritic cells with tumor cells. This enabled the tumor cell to present antigens in the manner of an APC [17].

Yet another approach to the production of patient-specific vaccines involves transducing autologous tumor cells so that they secrete GM-CSF. The clinical potential of this approach has been demonstrated in metastatic melanoma, and in May 2001, a phase II study of vaccination with adenoviral vector in patients with non-small cell lung cancer (NSCLC) was under way (Table 2Go).


View this table:
[in this window]
[in a new window]
  		Table 2. Ongoing phase II clinical trials of gene therapy with adenoviral vectors for treatment of cancer
 
Several issues surrounding immunostimulating gene therapies remain to be resolved. One is the need for production of autologous dendritic cells under good manufacturing conditions that are contamination free. Currently, production of autologous dendritic cells requires expensive in vitro techniques. The optimal source of dendritic cells, the optimal method of antigen loading, and the optimal source and preparation of tumor antigen remain to be determined. The optimal immunization strategy also needs to be clarified, because dendritic cells can also induce immune tolerance, a highly unwanted effect in antitumor therapy [18].

Gene Replacement and Combinations of Gene Therapy with Standard Antitumor Strategies
Roth et al. showed that the delivery of wild-type p53 to cells with p53 mutations increased their radiation sensitivity [19]. Tumor cells expressing wild-type p53 are more sensitive to chemotherapeutic agents and radiation than are cells that lack functional p53. The heightened sensitivity of cells with wild-type p53 is thought to be attributable to their propensity to undergo p53-mediated apoptosis (programmed cell death) after insult [20–23]. Abnormality and blocking of the apoptosis pathway are often associated with resistance of cancer cells to current antitumor treatment strategies. Gene therapy has the potential to overcome this block by re-establishing apoptosis-inducing regulators (E2F-1, wild-type p53, interleukin-6 [IL-6], interferon-{alpha}, Bax) or by transferring genes that induce cell suicide directly (e.g., FAS-ligand, Caspase-8, tumor necrosis factor-{alpha}) [24–26]. In animal models, treatment with a combination of cisplatin and adenovirus-p53 led to enhanced apoptosis and suppression of tumor growth [27]. We have found increased apoptotic cell death in breast cancer cell lines treated with a combination of adenoviral vectors with the E2F-1 transcription factor gene plus paclitaxel and doxorubicin, indicating a strong synergistic effect [28]. Chemotherapy, in return, enhances expression of transduced genes from adenoviral vectors with a wide range of promoters, and radiation improves immediate transduction efficiency and the duration of transgene expression [29].

Treatment with Replication-Competent Adenoviral Vectors
Treatment with replication-competent adenoviral vectors specific to the target tissue has been considered as a means of enhancing the transduction and expression rate of adenoviral vectors. The major advantage of these cytolytic vectors is their potential to reach widespread metastases. Delivery of replication-competent vectors in combination with incorporated suicide genes or prodrugs could reduce systemic cytotoxic effects and inflammatory responses [30]. However, the mouse systems in which replication-incompetent adenoviral vectors have been studied so far might not be adequate models for replication-competent human adenoviruses. It is not known whether treatment with these replicating vectors results in DNA integration in the host cell genome more often than with their nonreplicative counterparts [31]. The prolonged persistence and higher titers of replication-competent vectors and their induction of the S phase of the cell cycle (where adenovirus-associated virus and retrovirus integrate) might be indicative of a higher DNA integration rate [32,33]. A very recent publication reports a model for replication-competent adenovirus vectors based on mouse epidermal cells [34].

Clinical Trials of Gene Therapy with Adenoviral Vectors
An excellent source for data on clinical trials of gene therapy is the Journal of Gene Medicine website at http://www.wiley.co.uk/wileychi/genmed. The site allows users to scan investigator names and results for ongoing and recent clinical trials and has hyperlinks to frequently updated sites listing adverse effects of gene therapy. Most of the data presented in this review are derived from this database.

Figure 3Go shows the recent increase in the number of clinical trials based on gene therapy strategies. Over the last 3 years, the number of gene therapy trials targeting cancer has increased by about 50% each year. At the moment, there are 96 ongoing clinical trials of adenoviral gene therapy for cancer.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. Number of gene therapy trials worldwide, 1999-2001. Increasing number of gene therapy trials in the last 3 years.

 
Overview of Trials by Phase
Table 3Go provides details of the six completed gene therapy studies using adenoviral vectors. All these studies were phase I clinical trials designed to evaluate the toxicity of treatment. Grade III toxic effects were very rare, and there were no instances of grade IV toxic effects. Even though the number of patients entered in these trials was low, it can be concluded that the intratumoral application of up to 1 x 1012 adenoviral particles is safe.


View this table:
[in this window]
[in a new window]
  		Table 3. Closed phase I trials of gene therapy with adenoviral vectors for treatment of cancer
 
The ongoing phase II clinical trials of adenoviral gene therapy (Table 2Go) illustrate the growing variety of transgenes and methods of action that can be used in gene therapy. This variety underscores the exceptional flexibility of adenoviral vectors. Today, most vectors are administered intratumorally. Histologic assessment of treated tumors has shown that, with the intratumoral approach, maximal transgene expression occurs along the needle tract. Expression of the therapeutic gene decreases with increasing distance from the injection site, reflecting the need for more advanced vector targeting or tissue-specific promoters that allow for systemic application of the vectors.

Only two phase III clinical trials of adenoviral gene therapy are listed on the Journal of Gene Medicine website. Both trials are for women with advanced ovarian cancer and use intraperitoneal application of adenoviral vectors carrying wild-type p53 to attempt to restore p53 function, thereby improving the efficacy of chemotherapeutic treatment. The multicenter phase II study by Swisher (Table 2Go), which utilizes adenoviral vector to carry wild-type p53 into the tumor cells of NSCLC, uses a Bayesian study design allowing patients to be evaluated for the phase III study at the same time.

Adverse Events Reported to Date
Adenoviral gene therapies have been associated with very good tolerance and minimal toxicity in most phase I clinical trials. The most common side effects with vector treatment up to 1 x 1011 plaque-forming units (pfu) (correlating with a viral particle load about 20 times higher) administered intratumorally or subcutaneously are reported to be shivering, light fever, chills, local pain, and diarrhea. Even in the case of adenoviral load detected in the liver, only one event of hepatotoxity and no symptoms of hepatitis have been reported. However, in phase I clinical trials in which a combination of adenoviral HSV-tk and chemotherapeutic agents was used to treat advanced malignant intracranial tumors, more serious adverse effects have been reported, such as severe headache, relapsing seizures, and transient or persistent change of mental status.

In September 1999, an 18-year-old patient with ornithine-cytosine transferase deficiency died as a direct consequence of gene therapy at the University of Pennsylvania. A first generation replication-defective adenovirus administered through the hepatic artery was used to transduce the DNA sequence of the missing enzyme. However, the viral titer was very high (1 x 1011 pfu/kg, equivalent to 1 x 1014 viral particles per kilogram). This high viral load is believed to have initiated the systemic activation of the innate immune response mediated by APCs and macrophages leading to the release of cytokines. Within 2 hours, the patient developed a fever, which was quickly followed by coagulopathy and signs of liver dysfunction. Because of the underlying metabolic deficiency, ammonia accumulated in the blood followed by coma and ultimately multi-organ failure with adult respiratory distress syndrome. In a very recent study, increased liver enzyme levels, surge of cytokines, particularly IL-6, and development of coagulopathy were reported in monkeys that received systemically high doses of adenovirus vector [35]. There is some evidence that the protein coat of the vector triggers this immune response. Interestingly, in these rhesus monkeys, APC in the liver and spleen were full of vectors. The incident at the University of Pennsylvania added considerably to the negative press concerning gene therapy and led to further considerations of treatment strategies: A) the purification of the viable viral particles and stripping of the vectors from transcribed viral genes should be further improved (E1-, E3-, E4-deleted vectors, gutless vectors) to minimize toxic vector proteins; B) vector concentration should be measured not only in pfu but also in viral particle load, which is about 10 to 100 times higher, and C) host factors, like liver function and other underlying diseases, must be taken into account in patients selected for systemically administered adenoviral vector treatment [36,37].

Therapeutic Outcomes Reported to Date
Ironically, the first clinical success of gene therapy against cancer occurred with a vector that did not contain a therapeutic gene. The promising results came from a phase I and a phase II clinical trial started in 1996 by Khuri that used a replication-conditional adenovirus, dl1520, later called ONYX-015 [30]. This adenovirus is modified in the early regulatory protein E1b region (normally allowing the virus to bind and inactivate the host p53 gene to promote its own replication [38]). This mutated adenovirus replicates in and lyses human cells with a defective p53 pathway, including cells with loss of p14ARF (a protein that can induce apoptosis by activation of p53) function. However, the modified virus cannot replicate in cells carrying wild-type p53 and an intact p53 pathway [33,39,40]. As p53 is mutated in 34%-70% of adenocarcinoma cells, ONYX-015 was developed as a tumor cell-specific therapeutic agent. The standard first-line treatments of squamous cell carcinoma of the head and neck induce a response in only 30%-40% of patients, and the recurrence rates are high. The combination of intratumoral application of ONYX-015 with cisplatin and 5-fluorouracil in 30 patients caused an objective response (at least 50% reduction in tumor size) in 19 patients and a complete response in eight patients. Tumors as large as 10 cm regressed completely. None of the tumors that responded had progressed after a mean follow-up of 5 months.

In a phase I/II study, the same vector was applied in combination with fluorouracil for the treatment of unresectable primary and secondary liver tumors. Intratumoral, intravenous, and intraarterial administration of up to 3 x 1011 pfu showed two distinct cell death patterns of tumor cells: necrosis and apoptosis. This trial demonstrated only limited clinical response [41]. In another study from New Zealand, researchers investigated the induction of cell death by ONYX-015. They, too, found that the killing of the cells demonstrated two distinct patterns: A) a rapid killing in cells transfected with adenovirus containing the E1b region, through formation of a p53-E1b complex, and B) a delayed killing (after 10 days) in p53 mutant cells or cells treated with E1b-deficient adenovirus vectors such as ONYX-015, independent of the p53 status. These results support earlier reports that questioned the dependency of induced cell killing by ONYX-015 on the p53 status of the tumor cells [42,43].

One reason that might explain the lack of expected treatment success in some clinical trials with ONYX-015 is the fact that the absence of the primary cellular receptor for adenovirus, coxsackie-and-adenovirus receptor (CAR), on the tumor cells restricts the oncolytic potency of a replicating adenovirus vector. The potential therapeutic advantages afforded by viral replication could be negated by poor intratumoral spread of the viral progeny due to the failure to infect non-CAR-expressing tumor cells [44].


   IMPROVEMENT OF THE ADENOVIRAL VECTOR
Top
 Learning Objectives
 Abstract
 Introduction
 Adenoviral Vectors
 Clinical Applications of Gene...
 Improvement of the Adenoviral...
Other Points of Interest
 Future Directions
 References
 
The combination of adenoviral vectors with other viral vectors and the targeting of adenoviral vectors to the tumor cells with controlled expression of the therapeutic transgene have reached the preclinical stage. These improvements finally will allow us to exploit the whole potential of gene therapy.

"Gutless" or "Helper-Dependent"Adenoviral Vectors
Recently, "gutless" adenoviral vectors—vectors that are devoid of all viral-protein-coding DNA sequences—have been developed [5,45–47]. In this helper-dependent vector system, one vector (the helper) contains all the viral genes required for replication but has a conditional gene defect in the packaging domain making it less likely that its DNA is packaged into a virion. The second vector contains only the ends of the viral genome, therapeutic gene sequences, and the normal packaging recognition signal, which allows this genome to be selectively packaged and released from cells (Fig. 4Go). In addition to offering further reduced toxicity and prolonged gene expression in animals, this helper-dependent system allows the introduction of up to 32 kb of foreign DNA [48–51]. Processing of gutless adenoviral vectors is currently labor intensive, but it is anticipated that the adaptation of antibody-based purification systems that use affinity chromatography can further enhance the purity of vectors and their large-scale fabrication [52–55].



View larger version (37K):
[in this window]
[in a new window]
  		Figure 4. Construction of a gutless, helper-dependent adenoviral vector by cotransfection of a cell line (CIN1004) that excises the packaging gene of an E1-deleted adenovirus, which is otherwise capable of producing all necessary viral proteins for viral replication but cannot pack its genome in the virion.

 
Hybrid Vector Systems
Hybrid vector systems that combine the highly efficient infection capacity of adenoviruses with the long-term genome-integrating potential of retroviruses and adeno-associated viruses are currently being tested. Such hybrid systems showed efficacy in murine cancer models [56]. Proof of the great potential of vector engineering is the construction of a two-vector adenoviral system that causes the recipient cell, after its coinfection with both viruses, to become a retrovirus-producing cell and spread a high-titer retroviral vector to neighboring cells [57,58].

Tissue Targeting
Adenoviruses are taken up by epithelium-derived cell types, which makes adenoviral vectors suitable for solid tumors but less so for hematologic malignancies. In addition, this natural tropism makes it impossible to control the systemic delivery of adenovirus in vivo. Until recently, the mechanism of binding and internalization of adenoviruses was not known. Studies on cell receptors revealed that the CAR plays a crucial role in the infection of human cells, many of which express this 46-kDa membrane glycoprotein [59,60]. After anchoring at the CAR by virtue of the knob domain (the CAR ligand domain at the end of the capsid protein of the virus), the adenoviruses achieve internalization through interaction of the capsid penton protein with integrins {alpha}vß3 and {alpha}vß5 present on the target cells [61]. Studies using cells not expressing {alpha}v integrins indicated that the presence of these integrins is obligatory for viral integration [62]. The identification of the mechanism of adhesion and uptake of adenovirus provided important leads for retargeting adenoviral vectors to different cell receptors.

Several authors reported experimental work on retargeting adenoviral vectors to non-CAR-expressing cells by removing critical CAR/binding residues in the fiber knob and inserting new sequences that facilitate binding to alternative receptors [63–67]. Michael et al. first demonstrated that new cell-type specificities could be achieved with a retargeted adenovirus fiber [68]. Very promising is the replacement of CAR binding fiber with bispecific antibodies. In this method, one antibody is directed against a penton base target sequence on the virus, and the other antibody is directed against the novel antigen on the cell [69,70].

The potential of retargeted adenoviral gene therapy lies not only in its systemic application but also in the potential for new clinical applications in gene transfer to hematopoietic cells. Retargeted adenoviral gene therapy enabled successful transduction of foreign DNA into hematopoietic stem cells that lack CAR [71]. This technique will allow production of immunovaccines for residual leukemias, the induction of apoptosis in selected leukemic cell populations, or the expression of apoptosis-blocking proteins in hematopoetic stem cells preventing cell death during high-dose chemotherapy.

For retargeting approaches to succeed, not only must the vector bind to a specific target cell type, but also receptor-mediated endocytosis must take place. In many cases, manipulating endocytosis could be far more complicated than altering the tropism of the vector. The currently used in vitro models may not accurately predict biology relevant to the gene delivery in situ. For example, most immortalized tumor cell lines express CAR and are, therefore, readily transduced by adenoviral vectors, but these models do not necessarily reflect what happens at the tumor site in the clinical setting. A retrospective analysis of primary epithelial neoplasms revealed profound alteration or deficiency of CAR in about 50% of cases [72–74]. These findings might, in part, explain the inadequate efficiency of adenoviral gene therapy in clinical trials. Some investigators, therefore, concentrate on CAR-independent gene delivery by adenovectors. Other ways to achieve internalization without CAR binding is by physical complexing of the adenoviral particles [75–77], or by using recombinant fusion protein-retargeting complexes [70,78,79]. Because of the diversity of adenoviruses, of which there are more than 100 serotypes, changing only the capsid of the adenoviral vector can alter the tropism of the vector to the desired cells [49,80].

Regulation of Promoter Activity
To set off transcription of the induced transgene, this DNA sequence has to have a promoter gene in front (upstream) on the DNA strand. Very promising preclinical studies have concentrated on regulation of the promoter. One of the common promoters for adenovirus transmitted genes is the promoter for cytomegalovirus, which can start the reading of the transgene at a high rate. The use of tumor- or organ-specific promoters, such as the carcinoembryonic antigen, prostate-specific antigen, and myelin basic protein promoters, has already proven to be effective in animal models, and will soon be investigated in phase I clinical studies [81,82]. The limitation of these promoters is their need for a specific antigen expressed by the targeted tumor or organ tissue.

The idea of using an external switch to start and stop transgene transcription is very tempting. Of particular interest is promoter control by irradiation, a technique pioneered by Weichselbaum as early as 1992. This technique allows the systemic application of vectors, which can then be activated in a specified location by external irradiation [83,84]. Moreover, combining radiotherapy with transfer of genes that induce apoptosis, has beneficial synergistic, not just additional effects. So far, radiation-sensitive promoters have shown relatively low expression rates and are not yet applicable in clinical trials. Another promoter displaying heat-inducible qualities might overcome this shortcoming. In a preclinical mouse model, hyperthermia-mediated intratumoral expression of IL-12 without systemic toxicity has been achieved at tissue temperatures between 39°C and 42°C [85].


   OTHER POINTS OF INTEREST
Top
 Learning Objectives
 Abstract
 Introduction
 Adenoviral Vectors
 Clinical Applications of Gene...
 Improvement of the Adenoviral...
 Other Points of Interest
Future Directions
 References
 
At present, adenoviruses are the most effective vectors with regard to percentage of transduced tumor cells and gene expression levels. In many apoptotic-transgene delivery systems, more cells undergo apoptosis than are genetically altered because of the killing of neighboring cells through, thus far, not fully understood mechanisms (the so-called bystander effect) and, therefore, transduction of all cancer cells is not needed. Nevertheless, to effectively shrink large tumors, transduction and expression rates must be increased. The insufficient gene expression rate might explain the lack of efficacy seen in some clinical trials of gene therapy. Transcriptionally active drugs like retinoic acid and histone deacetylase inhibitor trichostatin A, a potent inhibitor of histone deacetylases, enhance adenoviral transgene expression up to sevenfold [86]. This can be explained by recent advances indicating that chromatin remodeling is important for the active transcription of expressed genes [87,88]. Condensed chromatin, in fact, negatively influences DNA accessibility to transcription factors, and histone acetylation is required for DNA opening and active transcription.


   FUTURE DIRECTIONS
Top
 Learning Objectives
 Abstract
 Introduction
 Adenoviral Vectors
 Clinical Applications of Gene...
 Improvement of the Adenoviral...
 Other Points of Interest
 Future Directions
References
 
Most patients treated in clinical trials of gene therapy to date have been treated in phase I studies in which the primary objectives are the delineation of safety, toxicity, and feasibility. The trials of adenoviral gene therapy have shown this approach to be generally safe and promising. However, clinical efficacy has been demonstrated only with replication-competent, tumor-cell-specific adenovirus, indicating the need for further improvement of this treatment modality. The advances in adenoviral gene therapy to date have set the stage for clinical trials with new generation adenoviral vectors that exhibit less stimulation of the host immune system and that can be selectively targeted to the desired tissue. If careful and objective trials are conducted, we will witness the expansion of the armamentarium against cancer by a versatile instrument in the very near future.


   ACKNOWLEDGMENT
Top
 Learning Objectives
 Abstract
 Introduction
 Adenoviral Vectors
 Clinical Applications of Gene...
 Improvement of the Adenoviral...
 Other Points of Interest
 Future Directions
 References
 
The authors would like to thank Stephanie Deming for her editorial assistance with the manuscript.


   REFERENCES
Top
 Learning Objectives
 Abstract
 Introduction
 Adenoviral Vectors
 Clinical Applications of Gene...
 Improvement of the Adenoviral...
 Other Points of Interest
 Future Directions
 References
 

  1. Kay MA, Holterman AX, Meuse L et al. Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. Nat Genet 1995;11:191–197.[CrossRef][Medline]
  2. Yang Y, Li Q, Ertl HC et al. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 1995;69:2004–2015.[Abstract]
  3. Yang Y, Wilson JM. Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo. J Immunol 1995;155:2564–2570.[Abstract]
  4. Roth JA, Cristiano RJ. Gene therapy for cancer: what have we done and where are we going? J Natl Cancer Inst 1997;89:21–39.[Abstract/Free Full Text]
  5. Schiedner G, Morral N, Parks RJ et al. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat Genet 1998;18:180–183.[CrossRef][Medline]
  6. Zoltick PW, Chirmule N, Schnell MA et al. Biology of E1-deleted adenovirus vectors in nonhuman primate muscle. J Virol 2001;75:5222–5229.[Abstract/Free Full Text]
  7. Bramson JL, Graham FL, Gauldie J. The use of adenoviral vectors for gene therapy and gene transfer in vivo. Curr Opin Biotechnol 1995;6:590–595.[CrossRef][Medline]
  8. Paielli DL, Wing MS, Rogulski KR et al. Evaluation of the biodistribution, persistence, toxicity, and potential of germ-line transmission of a replication-competent human adenovirus following intraprostatic administration in the mouse. Mol Ther 2000;1:263–274.[CrossRef][Medline]
  9. Mizuno M, Yoshida J, Colosi P et al. Adeno-associated virus vector containing the herpes simplex virus thymidine kinase gene causes complete regression of intracerebrally implanted human gliomas in mice, in conjunction with ganciclovir administration. Jpn J Cancer Res 1998;89:76–80.[CrossRef][Medline]
  10. Lanuti M, Gao GP, Force SD et al. Evaluation of an E1E4-deleted adenovirus expressing the herpes simplex thymidine kinase suicide gene in cancer gene therapy. Hum Gene Ther 1999;10:463–475.[CrossRef][Medline]
  11. Colak A, Goodman JC, Chen SH et al. Adenovirus-mediated gene therapy in an experimental model of breast cancer metastatic to the brain. Hum Gene Ther 1995;6:1317–1322.[Medline]
  12. Colak A, Goodman JC, Chen SH et al. Adenovirus-mediated gene therapy for experimental spinal cord tumors: tumoricidal efficacy and functional outcome. Brain Res 1995;691:76–82.[CrossRef][Medline]
  13. Perez-Cruet MJ, Trask TW, Chen SH et al. Adenovirus-mediated gene therapy of experimental gliomas. J Neurosci Res 1994;39:506–511.[CrossRef][Medline]
  14. Chen SH, Shine HD, Goodman JC et al. Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. Proc Natl Acad Sci USA 1994;91:3054–3057.[Abstract/Free Full Text]
  15. Trask TW, Trask RP, Aguilar-Cordova E et al. Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors. Mol Ther 2000;1:195–203.[CrossRef][Medline]
  16. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991;9:271–296.[CrossRef][Medline]
  17. Gong J, Chen D, Kashiwaba M et al. Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells. Proc Natl Acad Sci USA 1998;95:6279–6283.[Abstract/Free Full Text]
  18. Rissoan MC, Soumelis V, Kadowaki N et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science 1999;283:1183–1186.[Abstract/Free Full Text]
  19. Roth JA, Swisher SG, Meyn RE. p53 tumor suppressor gene therapy for cancer. Oncology (Huntingt) 1999;13:148–154.[Medline]
  20. Lowe SW, Ruley HE, Jacks T et al. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993;74:957–967.[CrossRef][Medline]
  21. Lowe SW, Schmitt EM, Smith SW et al. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 1993;362:847–849.[CrossRef][Medline]
  22. Lowe SW, Bodis S, McClatchey A et al. p53 status and the efficacy of cancer therapy in vivo. Science 1994;266:807–810.[Abstract/Free Full Text]
  23. Xie Y, Gilbert JD, Kim JH et al. Efficacy of adenovirus-mediated CD/5-FC and HSV-1 thymidine kinase/ganciclovir suicide gene therapies concomitant with p53 gene therapy. Clin Cancer Res 1999;5:4224–4232.[Abstract/Free Full Text]
  24. Hunt KK, Deng J, Liu TJ et al. Adenovirus-mediated overexpression of the transcription factor E2F-1 induces apoptosis in human breast and ovarian carcinoma cell lines and does not require p53. Cancer Res 1997;57:4722–4726.[Abstract/Free Full Text]
  25. Shinoura N, Koike H, Furitu T et al. Adenovirus-mediated transfer of caspase-8 augments cell death in gliomas: implication for gene therapy. Hum Gene Ther 2000;11:1123–1137.[CrossRef][Medline]
  26. Boldin MP, Varfolomeev EE, Pancer Z et al. A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J Biol Chem 1995;270:7795–7798.[Abstract/Free Full Text]
  27. Nguyen DM, Spitz FR, Yen N et al. Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirusmediated p53 gene transfer. J Thorac Cardiovasc Surg 1996;112:1372–1376.[Abstract/Free Full Text]
  28. Yoshida K, Nishizaki M, Hunt KK. Combination therapy of adenovirus-mediated overexpression of E2F-1 transcription factor and chemotherapy enhances growth suppression in breast cancer cells. Proc Am Assoc Cancer Res 2001;42:855.
  29. Stevens CW, Zeng M, Cerniglia GJ. Ionizing radiation greatly improves gene transfer efficiency in mammalian cells. Hum Gene Ther 1996;7:1727–1734.[Medline]
  30. Khuri FR, Nemunaitis J, Ganly I et al. A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 2000;6:879–885.[CrossRef][Medline]
  31. Kim JH, Freytag SO, Menon M et al. Suicide gene therapy: a phase I study of E1B-attenuated replication-competent adenovirus vector-mediated intratumoral administration of the E. coli cytosin deaminase/HSV-1 thymidine kinase fusion gene in conjunction with two prodrugs, 5-fluorocytosin and ganciclovir, for patients with local recurrence of prostate cancer after radiation therapy. [BB-IND 8436, RAC Protocol 9906-321], 1999.
  32. Lane DP. Killing tumor cells with viruses—a question of specificity. Nat Med 1998;4:1012–1013.[CrossRef][Medline]
  33. Vollmer CM, Ribas A, Butterfield LH et al. p53 selective and nonselective replication of an E1B-deleted adenovirus in hepatocellular carcinoma. Cancer Res 1999;59:4369–4374.[Abstract/Free Full Text]
  34. Ganly I, Mautner V, Balmain A. Productive replication of human adenoviruses in mouse epidermal cells. J Virol 2000;74:2895–2899.[Abstract/Free Full Text]
  35. Teramato S, Ishii T, Matsuse T. Crisis of adenoviruses in human gene therapy. Lancet 2000;355:1911–1912.[Medline]
  36. Stephenson J. Studies illuminate cause of fatal reaction in gene-therapy trial. JAMA 2001;285:2570.[Free Full Text]
  37. Wilson JM. Adenovirus-mediated gene transfer to liver. Adv Drug Deliv Rev 2001;46:205–209.[CrossRef][Medline]
  38. Barker DD, Berk AJ. Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection. Virology 1987;156:107–121.[CrossRef][Medline]
  39. Heise C, Sampson-Johannes A, Williams A et al. ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nat Med 1997;3:639–645.[CrossRef][Medline]
  40. Lowe SW. Progress of the smart bomb cancer virus. Nat Med 1997;3:606–608.[CrossRef][Medline]
  41. Habib NA, Sarraf CE, Mitry RR et al. E1B-deleted adenovirus (dl1520) gene therapy for patients with primary and secondary liver tumors. Hum Gene Ther 2001;12:219–226.[CrossRef][Medline]
  42. Dix BR, O'Carroll SJ, Myers CJ et al. Efficient induction of cell death by adenoviruses requires binding of E1B55k and p53. Cancer Res 2000;60:2666–2672.[Abstract/Free Full Text]
  43. Rothmann T, Hengstermann A, Whitaker NJ et al. Replication of ONYX-015, a potential anticancer adenovirus, is independent of p53 status in tumor cells. J Virol 1998;72:9470–9478.[Abstract/Free Full Text]
  44. Douglas JT, Kim M, Sumerel LA et al. Efficient oncolysis by a replicating adenovirus (ad) in vivo is critically dependent on tumor expression of primary ad receptors. Cancer Res 2001;61:813–817.[Abstract/Free Full Text]
  45. Morsy MA, Gu M, Motzel S et al. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene. Proc Natl Acad Sci USA 1998;95:7866–7871.[Abstract/Free Full Text]
  46. Hardy S, Kitamura M, Harris-Stansil T et al. Construction of adenovirus vectors through Cre-lox recombination. J Virol 1997;71:1842–1849.[Abstract]
  47. Morsy MA, Caskey CT. Expanded-capacity adenoviral vectors—the helper-dependent vectors. Mol Med Today 1999;5:18–24.[CrossRef][Medline]
  48. Amalfitano A, Hauser MA, Hu H et al. Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted. J Virol 1998;72:926–933.[Abstract/Free Full Text]
  49. Morral N, O'Neal W, Rice K et al. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc Natl Acad Sci USA 1999;96:12816–12821.[Abstract/Free Full Text]
  50. Balague C, Zhou J, Dai Y et al. Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood 2000;95:820–828.[Abstract/Free Full Text]
  51. Morral N, Parks RJ, Zhou H et al. High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity. Hum Gene Ther 1998;9:2709–2716.[Medline]
  52. Grimm D, Kern A, Rittner K et al. Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 1998;9:2745–2760.[Medline]
  53. Wistuba A, Kern A, Weger S et al. Subcellular compartmentalization of adeno-associated virus type 2 assembly. J Virol 1997;71:1341–1352.[Abstract]
  54. Zolotukhin S, Byrne BJ, Mason E et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 1999;6:973–985.[CrossRef][Medline]
  55. Wu N, Ataai MM. Production of viral vectors for gene therapy applications. Curr Opin Biotechnol 2000;11:205–208.[CrossRef][Medline]
  56. Fisher KJ, Kelley WM, Burda JF et al. A novel adenovirus-adeno-associated virus hybrid vector that displays efficient rescue and delivery of the AAV genome. Hum Gene Ther 1996;7:2079–2087.[Medline]
  57. Bilbao G, Feng M, Rancourt C et al. Adenoviral/retroviral vector chimeras: a novel strategy to achieve high-efficiency stable transduction in vivo. FASEB J 1997;11:624–634.[Abstract]
  58. Duisit G, Salvetti A, Moullier P et al. Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines. Hum Gene Ther 1999;10:189–200.[CrossRef][Medline]
  59. Bergelson JM, Cunningham JA, Droguett G et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997;275:1320–1323.[Abstract/Free Full Text]
  60. Curiel DT. Considerations and challenges for the achievement of targeted gene delivery. Gene Ther 1999;6:1497–1498.[CrossRef][Medline]
  61. Wickham TJ, Mathias P, Cheresh DA et al. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 1993;73:309–319.[CrossRef][Medline]
  62. Walters RW, Grunst T, Bergelson JM et al. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J Biol Chem 1999;274:10219–10226.[Abstract/Free Full Text]
  63. Wickham TJ, Roelvink PW, Brough DE et al. Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types. Nat Biotechnol 1996;14:1570–1573.[CrossRef][Medline]
  64. Wickham TJ, Segal DM, Roelvink PW et al. Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies. J Virol 1996;70:6831–6838.[Abstract/Free Full Text]
  65. Wickham TJ, Tzeng E, Shears LL et al. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins. J Virol 1997;71:8221–8229.[Abstract]
  66. Marini FC III, Yu Q, Wickham T et al. Adenovirus as a gene therapy vector for hematopoietic cells. Cancer Gene Ther 2000;7:816–825.[CrossRef][Medline]
  67. Roelvink PW, Mi LG, Einfeld DA et al. Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae. Science 1999;286:1568–1571.[Abstract/Free Full Text]
  68. Michael SI, Hong JS, Curiel DT et al. Addition of a short peptide ligand to the adenovirus fiber protein. Gene Ther 1995;2:660–668.[Medline]
  69. Von Seggern DJ, Chiu CY, Fleck SK et al. A helper-independent adenovirus vector with E1, E3, and fiber deleted: structure and infectivity of fiberless particles. J Virol 1999;73:1601–1608.[Abstract/Free Full Text]
  70. Dmitriev I, Kashentseva E, Rogers BE et al. Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells. J Virol 2000;74:6875–6884.[Abstract/Free Full Text]
  71. Huang S, Kamata T, Takada Y et al. Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells. J Virol 1996;70:4502–4508.[Abstract]
  72. Dmitriev I, Krasnykh V, Miller CR et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 1998;72:9706–9713.[Abstract/Free Full Text]
  73. Kasono K, Blackwell JL, Douglas JT et al. Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector. Clin Cancer Res 1999;5:2571–2579.[Abstract/Free Full Text]
  74. Vanderkwaak TJ, Wang M, Gomez-Navarro J et al. An advanced generation of adenoviral vectors selectively enhances gene transfer for ovarian cancer gene therapy approaches. Gynecol Oncol 1999;74:227–234.[CrossRef][Medline]
  75. Clark PR, Stopeck AT, Brailey JL et al. Polycations and cationic lipids enhance adenovirus transduction and transgene expression in tumor cells. Cancer Gene Ther 1999;6:437–446.[CrossRef][Medline]
  76. Di Nicola M, Milanesi M, Magni M et al. Recombinant adenoviral vector-lipofectAMINE complex for gene transduction into human T lymphocytes. Hum Gene Ther 1999;10:1875–1884.[CrossRef][Medline]
  77. Lanuti M, Kouri CE, Force S et al. Use of protamine to augment adenovirus-mediated cancer gene therapy. Gene Ther 1999;6:1600–1610.[CrossRef][Medline]
  78. Curiel DT. Strategies to adapt adenoviral vectors for targeted delivery. Ann N Y Acad Sci 1999;886:158–171.[CrossRef][Medline]
  79. Wickham TJ. Targeting adenovirus. Gene Ther 2000;7:110–114.[CrossRef][Medline]
  80. Parks R, Evelegh C, Graham F. Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration. Gene Ther 1999;6:1565–1573.[CrossRef][Medline]
  81. Richards CA, Austin EA, Huber BE. Transcriptional regulatory sequences of carcinoembryonic antigen: identification and use with cytosine deaminase for tumor-specific gene therapy. Hum Gene Ther 1995;6:881–893.[Medline]
  82. Spitzweg C, Zhang S, Bergert ER et al. Prostate-specific antigen (PSA) promoter-driven androgen-inducible expression of sodium iodide symporter in prostate cancer cell lines. Cancer Res 1999;59:2136–2141.[Abstract/Free Full Text]
  83. Datta R, Rubin E, Sukhatme V et al. Ionizing radiation activates transcription of the EGR1 gene via CArG elements. Proc Natl Acad Sci USA 1992;89:10149–10153.[Abstract/Free Full Text]
  84. Weichselbaum RR, Hallahan DE, Beckett MA et al. Gene therapy targeted by radiation preferentially radiosensitizes tumor cells. Cancer Res 1994;54:4266–4269.[Abstract/Free Full Text]
  85. Lohr F, Hu K, Huang Q et al. Enhancement of radiotherapy by hyperthermia-regulated gene therapy. Int J Radiat Oncol Biol Phys 2000;48:1513–1518.[CrossRef][Medline]
  86. Gaetano C, Catalano A, Palumbo R et al. Transcriptionally active drugs improve adenovirus vector performance in vitro and in vivo. Gene Ther 2000;7:1624–1630.[CrossRef][Medline]
  87. Kornberg RD, Lorch Y. Chromatin-modifying and -remodeling complexes. Curr Opin Genet Dev 1999;9:148–151.[CrossRef][Medline]
  88. Xu L, Glass CK, Rosenfeld MG. Coactivator and corepressor complexes in nuclear receptor function. Curr Opin Genet Dev 1999;9:140–147.[CrossRef][Medline]
  89. Vanin EF, Kaloss M, Broscius C et al. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis. J Virol 1994;68:4241–4250.[Abstract/Free Full Text]
  90. Rosenberg SA, Blaese RM, Brenner MK et al. Human gene marker/therapy clinical protocols. Hum Gene Ther 2000;11:919–979.[CrossRef][Medline]
  91. Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1990;10:4239–4242.[Abstract/Free Full Text]
  92. Bonini C, Ferrari G, Verzeletti S et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 1997;276:1719–1724.[Abstract/Free Full Text]
  93. Wolfe D, Goins WF, Kaplan TJ et al. Herpesvirus-mediated systemic delivery of nerve growth factor. Mol Ther 2001;3:61–69.[CrossRef][Medline]
  94. Burton EA, Glorioso JC. Multi-modal combination gene therapy for malignant glioma using replication-defective HSV vectors. Drug Discov Today 2001;6:347–356.[CrossRef][Medline]
  95. Wolfe D, Goins WF, Yamada M et al. Engineering herpes simplex virus vectors for CNS applications. Exp Neurol 1999;159:34–46.[CrossRef][Medline]
  96. Yamada M, Oligino T, Mata M et al. Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo. Proc Natl Acad Sci USA 1999;96:4078–4083.[Abstract/Free Full Text]
  97. Hauswirth WW, Lewin AS, Zolotukhin S et al. Production and purification of recombinant adeno-associated virus. Methods Enzymol 2000;316:743–761.[Medline]
  98. Clark KR, Liu X, McGrath JP et al. Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum Gene Ther 1999;10:1031–1039.[CrossRef][Medline]
  99. Liu XL, Clark KR, Johnson PR. Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus. Gene Ther 1999;6:293–299.[CrossRef][Medline]
  100. Yan Z, Zhang Y, Duan D et al. Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc Natl Acad Sci USA 2000;97:6716–6721.[Abstract/Free Full Text]
  101. Sun L, Li J, Xiao X. Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization. Nat Med 2000;6:599–602.[CrossRef][Medline]
  102. Wagner JA, Messner AH, Moran ML et al. Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope 1999;109:266–274.[CrossRef][Medline]
  103. Takahashi M, Miyoshi H, Verma IM et al. Rescue from photoreceptor degeneration in the rd mouse by human immunodeficiency virus vector-mediated gene transfer. J Virol 1999;73:7812–7816.[Abstract/Free Full Text]
  104. Park F, Ohashi K, Chiu W et al. Efficient lentiviral transduction of liver requires cell cycling in vivo. Nat Genet 2000;24:49–52.[CrossRef][Medline]
  105. Park F, Ohashi K, Kay MA. Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver. Blood 2000;96:1173–1176.[Abstract/Free Full Text]
  106. Kafri T, Blomer U, Peterson DA et al. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 1997;17:314–317.[Medline]
  107. Blomer U, Naldini L, Kafri T et al. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol 1997;71:6641–6649.[Abstract]
Received May 24, 2001; accepted for publication December 12, 2001.




This article has been cited by other articles:


Home page
 Hum ReprodHome page