The first approved clinical application of gene therapy took place in the US almost three decades ago (1-3). Since then, advances in gene therapy have led to new therapeutic opportunities for once untreatable inherited genetic diseases. To date, three
The fundamental principle of gene therapy is to deliver a functional copy (a therapeutic transgene) of the mutant gene to physiologically relevant target tissues or organs of the patient to compensate for the mutant gene. A therapeutic transgene can be delivered into the patient’s body via two methods: (1) an
Safety is the highest priority in every therapeutic intervention. The vectors for gene therapy are no exception, and thus, the safety of the gene delivery vectors should be carefully and continuously monitored. Additionally, the gene delivery vectors should be able to sustain high-level and persistent transgene expression in host organisms to achieve therapeutic efficacy of the transgenes delivered.
Among all the currently available vectors for
However, despite the obvious benefits, the currently available GLAd has a notable drawback: contamination with adenovirus and RCA in its final product. Safety concerns raised by these contaminants have hindered its clinical applications. Recently, we successfully developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid. HF-GLAd, free of helper adenovirus and RCA contaminants, will facilitate its clinical applications.
In this review, we discuss the characteristics of adenovirus, the evolution of adenoviral gene delivery vectors, and the host immune responses against adenoviral vectors. Moreover, we highlight the unique features of HF-GLAd as a new platform for
The first adenovirus was isolated from the tissue culture of human adenoids in 1953 and characterized by Rowe
The capsid of adenovirus is composed of 292 capsomeres with 20 triangular facets and 12 vertices. These capsomeres consist mainly of 240 hexons (trimer of protein II) on the facet and 12 pentons on the vertices of the capsid (Fig. 1A). The penton unit consists of a penton base (pentamer of protein III) anchored in the capsid and a projecting fiber (trimer of protein VI) with a knob at its distal end (Fig. 1B). Besides, several other minor structural proteins, including IIIa, VIII, IX, vIII, terminal protein (TP) and V, are located on the internal and external surface of the capsid (Fig. 1B).
The capsid contains a relatively large adenoviral genome (30-40 kb). For example, human adenovirus type 5 (HAd5), a member of HAd serotype C, contains an approximately 36 kb genome. The HAd5 genome carries an inverted terminal repeat (ITR) (∼100 bp) at both ends, each of which is covalently attached to a TP at the 5’-terminus of each DNA strand (Fig. 1C). The ITR sequence on the left end of the adenoviral genome is followed by the ψ packaging signal that controls the encapsidation of the viral genome (Fig. 1C). In addition to the ITRs and the ψ packaging signal, there are 38 viral genes organized in 17 transcriptional units classified into early, intermediate, and late categories (Fig. 1C). The early (E) transcriptional units (E1-E4) encode proteins that regulate viral gene transcription, viral DNA replication, and the suppression of host immune responses against adenoviral infection. The intermediate transcriptional units code for two proteins, IX and IVa2. The late (L) transcriptional units (L1-L5) encode the structural proteins of adenovirus.
Most adenoviral vectors are derived from HAd5, and classified into two categories: replication-competent and replication-de-fective. The replication-competent adenovirus (RCA) has been developed typically as a tool for anti-cancer therapy. Since RCA can replicate by itself and is strongly immunogenic, it plays a role in the lysis of infected and adjacent tumor cells when injected into tumor tissues. In contrast, the replication-defective adenovirus has been primarily developed as a gene delivery vector, following the modifications by deleting viral genes partially or entirely to reduce or eliminate the expression of viral proteins. These modifications have been shown to attenuate the host immune responses. Based on the modifications, several generations of adenoviral vectors have been constructed as follows.
The first-generation adenovirus (FGAd) was constructed by deleting the E1 region (from nucleotide 400 to 3500) and the E3 region from the adenoviral genome (Fig. 2). The E1 region encodes proteins essential for the expression of other early and late genes, and thus, is crucial in initiating the life cycle of adenovirus (11). The E3 region encodes proteins that protect adenovirus from the host antiviral immune responses (12). Since these E3 proteins are dispensable for adenovirus production, the E3 region is generally deleted in the FGAd to increase its cargo capacity for the transgene.
Due to the deletion of the E1 region, FGAd cannot replicate by itself. Therefore, the production of FGAd requires packaging cell lines that express E1 proteins
Even though FGAd is devoid of the E1 region, the E1A-like factors present in many cell types can still induce the expression of other adenoviral proteins in transduced host cells (20), eliciting strong host immune responses and resulting in transient transgene expression and chronic toxicity (21). Therefore, FGAd has been recognized as a suitable platform for the delivery of transgenes in anti-cancer therapy (22) rather than a platform for delivering therapeutic transgenes to treat inherited genetic diseases, which requires high-level and persistent transgene expression.
In an attempt to attenuate the host immune responses against adenoviral proteins, the second-generation adenovirus (SGAd) was generated via additional deletions of the E2 and E4 regions (Fig. 2). The E2 region encodes three proteins related to the replication of viral DNA (23), including DNA-binding protein (DBP), terminal protein (TP), and DNA polymerase. The E4 region codes for control proteins that regulate the transcription of adenoviral DNA (24). These deletions significantly reduce the synthesis of adenoviral proteins. Nonetheless, SGAd still induces host immune responses due to the proteins expressed from the residual adenoviral genes, which results in reduced transgene expression in transduced cells (25).
Despite the deletion of early transcriptional units (E1-E4), the early-generation adenoviral vectors still exhibit strong immunogenicity and toxicity in host organisms. These undesirable safety issues led to the development of the third-generation adenoviral vector, referred to as gutless adenovirus (GLAd). GLAd is constructed by deleting all the viral genes from an adenovirus, only leaving the ITRs and the ψ packaging signal in its genome backbone (Fig. 2). This structural characteristic eliminates the expression of viral proteins in transduced cells and only induces negligible immune responses, enabling high-level and persistent transgene expression in host organisms (26). Importantly, this large deletion also increases the cargo capacity for the transgene up to 36 kb, which allows the delivery of a large transgene or multiple transgenes.
In general, most therapeutic transgenes do not reach 36 kb. Therefore, the deleted viral genes should be replaced with a stuffer DNA to stably maintain the genome of GLAd within the size range (27-37.8 kb) for efficient encapsidation (27-29). The nature of stuffer DNA in GLAds appears to affect the expression of transgenes
Since GLAd is devoid of all the viral genes, its production requires a helper that supplies the viral proteins
The helper adenovirus and RCA contaminants are hazardous, especially to the immunocompromised patients because the adenoviral proteins expressed by these two contaminant viruses can induce toxic immune responses. Besides, the strong host immune responses against helper adenovirus and RCA contaminants can limit the expression of the therapeutic transgene delivered by GLAd, even though GLAd
Indeed, the advantages of GLAd as a gene delivery vector are enormous. However, the safety concerns raised by the helper adenovirus and RCA contaminants have hindered its clinical applications. Accordingly, no clinical data are available for GLAd. Therefore, any clinical application requires the elimination of these two contaminants from the final preparation of GLAd.
To date, the most elegant strategy to prevent helper adenovirus amplification entails the deletion of the ψ packaging signal (33). In this strategy, the ψ packaging signal flanked by two loxP sites is excised when the helper adenovirus infects Cre-recombinase-expressing cell lines, such as 293Cre (Fig. 3A). The amplification of helper adenovirus is significantly reduced (0.01-10% of total virus produced) by blocking the encapsidation of helper adenoviral genome. Based upon this Cre/LoxP production system, Palmer
A similar strategy utilizing the FLP/frt system was also developed (37). However, it was still very difficult to completely remove the contaminated helper adenovirus.
In an attempt to address the safety concerns associated with helper adenovirus contamination, researchers have developed other strategies utilizing non-adenoviral helpers, such as baculovirus-adenovirus hybrid (38) and herpes simplex virus-1 (HSV-1) (39). Unfortunately, however, these two helpers also generated undesirable RCA contaminants and were shown to be inefficient in the production of GLAd.
Taken together, it is clear that the presence of helper viruses in the GLAd production system is an unavoidable risk. Therefore, establishing a system devoid of any helper viruses is of utmost importance for the production of GLAd that is desirable for clinical applications.
To address the aforementioned safety issues of GLAd, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced in a helper virus-free manner (40). In this novel system, the helper function required for the HF-GLAd production is provided by a helper plasmid instead of a helper adenovirus. This helper plasmid does not contain the ITRs and the ψ packaging signal, both of which are essential for viral genome replication and packaging. Therefore, this helper plasmid exclusively supplies viral proteins
Briefly, the production of HF-GLAd requires two plasmids (Fig. 4A): (1) pAdBest_dITR (∼31 kb), a helper plasmid that provides adenoviral proteins
In an attempt to produce large-scale HF-GLAd, a serial amplification process was established (40). This procedure is similar to the standard amplification process used for the helper adenovirus-dependent large-scale GLAd production (34). However, the packaging cells are transfected with helper plasmid in each round of amplification (40) instead of being infected with helper adenovirus (34). This serial amplification method (P0-P3) routinely achieved large-scale production of HF-GLAd with a yield of 5 × 1010 - 1 × 1011 infectious units (ifu) (50-100 ifu/cell) in P3 (40). This yield is merely 10- to 20-fold lower than that of the helper virus-dependent method (34), indicating that the helper plasmid supplies a sufficient amount of viral proteins for HF-GLAd production, although it cannot replicate in the packaging cells.
Since the selection of stuffer DNA is pivotal for sustaining high-level and persistent expression of transgenes, we selected fragments from the second intron of the mouse
We have successfully established a two-column chromato-graphic purification method to obtain highly pure recombinant HF-GLAd for preclinical and clinical applications. We are also investigating the possible adaptation of the culture dish-based serial amplification method (40) to the multi-layer Cell Factory-based (44) approach for large-scale production of HF-GLAd.
Viral vectors are the optimal gene therapy platforms because viruses have evolved to deliver their genetic material into permissive cells of other organisms. In parallel, the immune system of the host organism has also evolved to resist invasion of viral pathogens. An immune response against viral pathogens may benefit the application of vaccines (45-50) or anti-cancer therapy (51-53). However, an immune response against viral vectors used in gene therapy can eliminate the vectors and the transduced host cells. Such phenomena interfere with high-level and persistent expression of therapeutic transgenes in host organisms. Therefore, circumventing the host immune responses against viral vectors is critical for the success of
The immune responses against adenoviral vector-based gene therapy can be summarized into two main classes: a rapid and non-specific innate immune response, and a relatively slow but highly specific adaptive immune response.
The adenovirus-mediated host innate immune responses induced by virion components (
The adaptive immune responses induced by adenoviral vectors or adenovirus-based gene therapy are activated within a week (59). These immune responses can be elicited by the viral proteins expressed from the adenoviral vectors, or the products expressed by the therapeutic transgenes.
The viral proteins expressed by the early-generation adenoviral vectors induce cellular immune responses. In the early phase, the cytokines and chemokines are upregulated, leading to the infiltration of CD4+ and CD8+ T lymphocytes to the administration site of adenoviral vectors (60) and the generation of adenovirus-specific cytotoxic T lymphocytes (CTLs) (61). These cellular immune responses are initiated by antigen-presenting cells (APCs), resulting in the elimination of transduced host cells and the generation of memory immune cells against the adenoviral vectors (62).
Administration of adenoviral vectors also induces humoral adaptive immune responses via presentation of MHC-II/adeno-viral capsid antigen complexes at the surface of B lymphocytes to CD4+ T lymphocytes, which results in the activation of CD4+ T lymphocytes. Following the activation, CD4+ Th2 lymphocytes promote the proliferation of B lymphocytes and their differentiation to plasma cells that secrete antibodies against adenoviral capsid proteins. The pre-existing neutralizing antibodies (NAbs) in host organisms (63) may interfere with adenovirus infection and thereby decrease the efficacy of adenoviral vector-based gene therapy (64-66).
The products encoded by therapeutic transgenes may also be immunogenic in patients with
GLAd is devoid of all the viral genes. Thus, GLAd does not express any adenoviral proteins in transduced host cells, which minimizes the induction of adenovirus-specific adaptive immune responses, enabling high-level and persistent transgene expression in host organisms. Importantly, as HF-GLAd is produced in the absence of helper adenovirus, its final product is free of helper adenovirus and RCA contaminants. Therefore, HF-GLAd is clinically more desirable than the GLAd produced by the helper adenovirus-dependent system.
Nevertheless, HF-GLAd can still induce innate immune responses, since it shares an identical capsid structure with wild-type and early-generation adenoviruses (68). Also, pre-existing NAbs and/or adenovirus-specific CTLs present in the pa-tients previously exposed to adenoviruses can decrease the efficacy of HF-GLAd-based gene therapy (63).
Several elegant strategies have been developed to circumvent the host immune responses against adenoviral vectors. These strategies include transient immune modulation in the host organism before administrating these vectors, and selective modification of these vectors
Transient immune modulation entails either pre-deletion of immune cells or induction of immunosuppression (or immune tolerance). For example, transient depletion of specific immune cells (
As an approach for selective modification, adenoviral capsid proteins can be conjugated with chemicals, such as polyethylene glycol (PEG) (74, 75). The adenoviral vectors containing such modified capsid proteins have already been shown to improve the vector safety and transduction efficiency. Therefore, these approaches, individually or in combination, can also be adopted in HF-GLAd-based gene therapy.
Several other strategies were also investigated to minimize adenovirus-mediated host immune responses. For example, since host innate immune responses against adenoviral vectors are dose-dependent, it is crucial to establish a threshold dose to minimize the acute toxic immune responses. Also, it is preferable to select immune-privileged tissues or organs, espe-cially the eye and central nervous system (CNS), as
Recombinant adenoviral vectors have been extensively investigated in preclinical and clinical applications. However, the tragic death of Jesse Gelsinger, who was treated for ornithine transcarbamoylase (OTC) deficiency, has severely damaged the reputation of adenovirus-based gene therapy (79). Although these vectors have shown tremendous advantages and technological advances (
Gene therapy using GLAd has attracted tremendous attention in recent years. In particular, in addition to a substantial capacity for transgenes (Table 1), the safety of HF-GLAd is comparable to that of AAV, which is expected to restore the reputation of adenoviral vectors as well as facilitate its applications for
HF-GLAd is capable of delivering transgenes regardless of size because no human gene exceeds its carrying capacity. HF-GLAd can accommodate a small or a large transgene, and even multiple transgenes in a single construct. In theory, HF-GLAd might be an ideal vector to safely deliver large transgenes to treat inherited genetic diseases, such as Duchenne and Becker muscular dystrophy (with mutations in the
GLAd is one of the most promising vectors for
Currently, many recombinant HF-GLAds are under investigation in animal studies for the treatment of various inherited genetic diseases. Further, HF-GLAd also carries the potential for delivery of therapeutic transgenes to treat other diseases, such as Parkinson’s disease (96, 97), Alzheimer’s disease (98), and cancer (
This research was supported by the Chung-Ang University Young Scientist Scholarship in 2014. We thank Christine Seol for critical reading of the manuscript.
The authors have no conflicting interests.