Modification of Baculovirus for Gene Therapy - Formatex · Modification of Baculovirus for Gene Therapy You-Kyoung Kim 1, Hu-Lin Jiang , Yeon-Ho Je1, Myung-Haing Cho2, Chong-Su Cho*,1

Modification of Baculovirus for Gene Therapy

You-Kyoung Kim1, Hu-Lin Jiang1, Yeon-Ho Je1, Myung-Haing Cho2, Chong-Su Cho*,1 1Department of Agricultural Biotechnology, Research Institute for Agriculture and Life Sciences, Seoul

National University, Seoul 151-921, South Korea 2Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742,

South Korea Gene therapy has the potential to treat devastating inherited diseases for which there is little hope of find-ing a conventional cure and is a method for the prevention, correction, or modulation of genetic and ac-quired diseases that uses genes to therapeutic proteins. Animal viral systems are by far the most effective means of gene delivery and achieve high efficiencies for both delivery and expression although they have several limitations such as toxicity, restricted targeting of specific cell types, limited DNA carrying capac-ity, production and packaging problems, recombination, and high cost. Recently, many researchers stud-ied baculovirus as another virus vector because it has several advantages such as high transduction, non-replication nature, large insertion capacity, and low cytotoxicity. In this review, modification of baculovi-rus for in vitro and in vivo gene delivery will be explained.

Keywords gene therapy; baculovirus; modification; pegylation; folate

1. Introduction

Gene therapy is a powerful approach for the treatment of devastating inherited diseases and is a method for the prevention, correction or modulation of genetic and acquired diseases by introducing genes cod-ing for therapeutic proteins [1]. The development of effective vector to overcome the barrier such as immunogenicity, potential infectivity, inflammation and non-specificity to the target cells is essential for progress in human gene therapy [2]. Generally, vectors for gene therapy are classified into nonviral vec-tor system and viral vector one. Synthetic cationic liposomes and polymers are used as nonviral vector. Although nonviral vectors have advantageous properties such as the ability to efficiently condensate DNA to protect DNA from degradation and interact with cells [3, 4], their low transfection efficiency compared with viral vector is one of the challenge in clinical trials for success of gene therapy. On the other hand, viral vectors have been commonly employed in clinical trials because of their highly evolved, specialized components, and high efficiencies for both delivery and expression.

Currently, viral vector systems, including animal viral vector such as retroviruses, lentiviruses, adeno-viruses, and adeno-associated viruses (AAV), offer the best choice for efficient gene delivery [5, 6]. Despite the intrinsic advantages of these animal viral vector systems, they have several disadvantages to be overcome. Firstly, transient gene expression and safety problem have to be overcome. Transcriptional silencing can result in transient expression in case of retrovirus [7] whereas adenoviruses show the tran-sient gene expression due to the elicitation of strong humoral and cellular immunity [8]. On the other hand, transgene integration into the host cell genome allows persistent gene expression but bears the risk of insertional oncogenesis since current viral vectors are randomly integrating [9]. Especially, lenti-viruses have capability of long-term expression. Their phathogenic nature, however, raises serious con-cerns about the safety [10]. Secondly, immune responses and pre-existing immunity of animal viral vec-tor should be concerned. In particular, adenoviruses have severe immunity [8] and AAVs show the pre-existing immunity although the immune response of AAVs is still unknown [11]. These immune re-sponses and pre-existing immunity induce the inflammatory response and results in toxicity. Thirdly, difficulty in preparation of viral vectors and restricted packaging capacity also has to be overcome as a gene carrier for efficient gene therapy [12]. * Corresponding author: e-mail: [email protected], Phone: +82 28804636

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Unlike these animal viral vectors, baculoviruses is a family of insect virus, which are classified mainly into nuclear polyhedrosis viruses (NPV) and granulosis viruses. Among the numerous baculoviruses, the Autographa califarnica multiple nuclear polyhedrosis virus (AcMNPV) is widely studied prototype of the baculoviridae family consists of a large, enveloped virus with a double-stranded, circular DNA ge-nome (~130kb) which is condensed with a protamine-like protein into the core and packed into the nu-cleocapsids [13]. Initially baculovirus systems were developed for the production of numerous recombi-nant proteins such as interferon β in insect cells [14, 15] or used as a biopesticides [16]. However, after first finding of internalization by baculoviruses into mammalian cells [17], baculoviruses have been researched as a viral vector for gene therapy. In particular, the transduction of baculoviruses and efficient expression of target genes in several mammalian cell types in vitro and in vivo can be improved by con-struction of baculovirus vectors containing an appropriate eukaryotic promoter [18]. Moreover, bacu-loviruses have numerous significant properties as a gene carrier for gene therapy compared with animal viral vectors. Most important advantages are nonreplicative and nontoxic attributes of baculovirus. High species specificity, lack of replication in mammalian cells, and comparatively small cytotoxic effects provide baculovirus vector system with safety as a gene vehicle [19]. In addition, baculoviruses can be produced easily and have a large cloning capacity compared with animal viral vector along with a prop-erty to avoid the problem of pre-existing immunity [12]. Therefore, baculovirus system is regarded as a fresh and attractive candidate for gene therapy application [19]. However, its use as a gene therapy vec-tor has some limitations such as its inactivation in human serum and whole blood thereby limiting its in vivo application via systemically or intraportally [20]. In addition to inactivation of baculoviruses by complement system, lack of specificity in targeted delivery is one of obstacles of baculoviruses. Al-though several studies reported that baculoviruses enter into variety of susceptible cells with high trans-duction efficiency [21-23], which does not clearly indicates the targeted delivery by baculoviruses with high specificity for target organ or tissue.

This review specifically highlights the recent progress in application of baculoviruses as a gene deliv-ery vector along with its mechanism to overcome various barriers by genetic and/or chemical modifica-tions.

2. Application of baculovirus for gene therapy

2.1 Mechanism of baculovirus entry into mammalian cells

Although baculoviruses can be used for the transduction of large variety of mammalian cells, mecha-nisms involved in the baculovirus entry into vertebrate cells are still unknown [12]. Entry of baculovi-ruses into insect cells occurs via endocytosis followed by low pH-induced fusion between a viral enve-lope protein and the endosomal membrane, thus allowing viral entry into the cytoplasm and nucleus [24]. Gp64 glycoprotein of baculovirus, a major component of the budded virus envelope, plays an important role in virus attachment and endosomal escape in insect cells [25, 26]. Generally, it is considered that the entry of baculoviruses in mammalian cells follows the same route as that of in insect cells, where gp64 palys an important role [27].

Interaction between component of baculovirus envelope such as gp64 and surface molecule of mam-malian cell is essential for baculovirus to enter via endocytosis. To date, many researches have been reported that specific surface molecules of mammalian cells as docking point are involved in attachment of baculovirus on mammalian cell membrane [28-30]. Duisit et al. reported that electrostatic interaction between heparan sulfate on cell membrane and gp64 on baculovirus envelope facilitates viral entry into mammalian cells. It was found that the decrease in transgene expression after removal of heparin sul-phate from cell membrane, indicated that heparan sulfate acts as docking motif for entry of baculoviruses by interaction with gp64 [28]. Also, another groups reported that phospholipids [29] and asialoglycopro-tein receptors (ASGPR) [21] on the cell membrane might serve as important docking points for gp64. However, van Loo et al. contradictorily reported that ASGPR is not a key molecule for baculovirus bind-

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ing because of its successful transduction in ASGPR deficient cell, Pk1 and glycoprotein receptor as a docking point is not essential for entry of baculovirus [30]. According to these reports, docking motif, plays a key role in attachment of baculoviruses and entry via endocytosis, which is still unclear in both insect and mammalian cells [31].

Intracellular trafficking processes such as endosomal escape, cytoplasmic trafficking, and nuclear im-port are also significant steps for efficient transgene expression. Stanbridge et al. reported that NIH-3T3, a cell line less susceptible to baculovirus transduction, has no limitation of attachment with baculoviruses [32]. Similarly another group reported that inefficient gene expression in less susceptible cells is not due to a blocking in entry of the baculovirus into a cell, but rather due to the inability of the virus to escape endosomes [33]. Moreover, Kukkonen et al. suggested that the blocking of transgene expression may not depend on the escape from the endosome, but rather on cytoplasmic trafficking or nuclear import of the nucleocapsids [31]. van Loo et al. clearly emphasized the role of actin filaments as a cytoplasmic trans-porters in intracellular trafficking followed by transportation of nucleocapsid via nuclear pore [30]. These studies highlight both attachment of baculoviruses on mammalian cell membrane in extracellular aspect and importance of intracellular trafficking for efficient transgene expression.

2.2 In vitro application

The first issue of baculovirus as a gene carrier for human gene therapy was whether the virus can efficiently transfer the gene to the mammalian cells. Although Carbonell et al. confirmed the entry of baculovirus into mammalian cells in 1985, very low level reporter gene expression was observed by them [34]. This low level transgene expression was overcome using recombinant baculoviruses harboring a cytomegalovirus (CMV) promoter-luciferase gene cassette [35] or an Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter-β galactosidase (β-gal) gene cassette [21]. These results demonstrated that promoter strength is one of essential factors for stable gene expression in mammalian cells. After these findings, another promoters such as hybrid chicken β-actin promoter (CAG) [22, 32], Simian virus 40 (SV40) promoter [36], and hybrid neuronal promoter [37] were evaluated. These studies confirmed that CMV and RSV promoters were the most active in all cell lines tested and SV40 promoter was the weakest among these promoters, indicating that selection of proper promoter is directly related to successful transgene expression for gene therapy using baculovirus system.

Firstly, it was reported that the highly efficinet baculovirus-mediated gene delivery were observed in limited cell lines such as HepG2 [38]. Following the findings about promoter strength for gene expression, list of susceptible cells were rapidly expanded through subsequent studies. Baculovirus vector-delivered expression of several genes has been observed in these permissive cells which are included in numerous cell types of human (e.g. HepG2, Huh-7, HeLa and KB), rodent (e.g. RGM1 and C2C12) [39], and bovine (e.g. MDBK ant BT) [12]. Although baculovirus vector system can be utilized in numerous cell lines from various origin, there is another factor to consider for in vitro application of baculoviruses. Addition of histone deacetylase inhibitors such as sodium butyrate [40], trichostatin A [41], and valproic acid [23] improved the transduction efficiency, indicating that chromatin state of baculovirus genome is important in the transduced cells for transgene expression because these compounds induce a hyperacetylation of the chromatin and enhancement of transcription despite of their undesired cytotoxicity [23].

2.3 In vivo application

Baculoviruses are considered as attractive candidates for in vivo appications due to their inherent inability to replicate in mammalian cells[42]. Initial attempts for its in vivo application were performed in liver tissue [20, 27] because of its high transduction efficiency in liver cell lines and primary hepatocyte cells as revealed in in vitro studies [38]. Unexpectedly, transgene expression was undetectable when a baculovirus vector was introduced into mice and rats by a variety of methods, including direct injection into liver parenchyma. This failure suggested that baculoviruses in plasma and

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whole body is inactivated by complement system [20]. As the results, complement system appears to be a potent barrier for in vivo application of baculoviruses.

To overcome this obstacle, cobra venom factor (CVF), a complement-activating protein from cobra venom, was pretreated, thereby preventing the inactivation of baculovirses in the presence of serum [20]. In addition approach, baculoviruses were applied for in vivo gene transfer in the areas where baculovirus should not be exposed to complement system such as delivery in brain tissue. Sarkis et al. [40] reported that baculovirus-derived vector was able to transduce neural cells in vivo in the mouse and the rat after direct injection of the vector into the brain and the level of gene expression was found similar between CVF treated and untreated animals. These results indicated that the particular immunological characteristics existed in brain due to blood-brain barrier and/or the complement level in the brain is insufficient to affect the gene transfer [40]. These approaches can expand the possibility of baculoviruses application for gene therapy both in vitro and in vivo.

3. Modification of baculovirus for improved gene delivery

3.1 Key steps affecting transduction efficiency of baculovirus

Generally, there are three main barriers such as extracellular barrier, intracellular barrier, and toxicity along with host cell response for gene delivery. Thus gene carriers including viral vectors should over-come these barriers for obtaining efficacy [9]. First of all, complement inactivation of baculoviruses is main extracellular obstacle for efficient gene delivery in vivo application as mentioned earlier. Before entry into cells, viability of baculoviruses is abruptly decreased by complement system, indicating that baculoviruses have no chance to enter into cells in the presence of serum, especially complement system. Another extracellular barrier is non-specific transduction of baculoviruses. Likewise other viral vectors, baculoviruses do not have specificity to target desired tissues or organs. Although unknown docking points on the cell membrane exist for entry of baculoviruses [28-30], they cannot provide baculoviruses with the ability to distinguish between target and nontarget cells. To provide a vector with the specificity, cell binding ligands have to be incorporated to recognize target-specific cellular receptors and vector domains with undesired binding potential to blood or nontarget cells have to be shielded or removed [9].

After entry of baculoviruses via endocytosis, intracellular delivery follows: (1) endosomal escape, (2) transport from the cytosol into the nucleus, and (3) persistent gene expression. Each step is important for our final goal, the stable gene expression. However, mechanism of intracellular delivery of baculoviruses is still not well-known. Some studies reported that low pH-induced fusion between a viral envelope protein and the endosomal membrane can allow baculoviruses to escape from endosome [27] and actin filaments act as cytoplasmic transporters in intracellular trafficking [30]. In addition, gene expression duration of baculoviruses is significantly shorter than expression mediated by retroviral, lentiviral, and AAV vectors. Transgenes carried by another viral vectors can persist in the host nucleus, either in an integrated or episomal form, for a longer period [43, 44]. On the other hands, baculoviral DNA persists in the nuclei of transduced mammalian cells for only 24-48h [17].

Final barriers for gene delivery are cytotoxicity and immune responses. In comparison with other viral vectors, baculoviruses have no concern with these obstacles because it was reported that baculoviruses are nontoxic to mammalian cells [45], do not replicate in transduced cells [42, 46], and may avoid the problem of preexisting immunity [12]. These advantages make baculoviruses an attractive candidate as a viral vector for gene therapy.

Modification of baculoviruses by genetic and/or chemical method is a potential approach to overcome these main barriers and acheive the efficient gene expression which is disccussed in next part (sectons 3.2 and 3.3)

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3.2 Genetic modification

Genetic modification of baculoviruses has been studied for improving the ability of binding on the mammalian cell surface of native baculoviruses, thereby enhancing the transduction efficiency, and overcoming drawbacks of baculoviruses as a gene carrier. As mentioned above, viral surface characteris-tics play a significant role in viral binding and entry into mammalian cells, and results in enhanced gene expression. Figs. 1-A, 1-B, and 1-C show general genetic manipulation scheme. Genetic manipulations are mainly performed by mutation of the major viral envelope protein (gp64), results in foreign peptides or proteins display on the baculovirus envelope as N-terminal or internal fusions to gp64 shown in Fig. 1-A. As incorporation of the fusion protein onto the viral surface usually represents only a small propor-tion by fusion to gp64 [47], the coat protein of a different virus, vesicular stomatitis virus (VSV) or its membrane anchor domain have been used as an alternative baculovirus surface display strategies shown in Fig. 1-B [48]. Also, as shown in Fig. 1-C, desired foreign peptides or proteins can be displayed by fusion to major viral capsid protein vp39 instead of viral envelope.

Fig. 1 A generalized baculovirus display strategies by genetic and chemical modifications. Genetic modifications display foreign peptides or proteins by fusion to either gp64 (A) or heterologous membrane anchor derived from VSV-G (B) on the baculovirus envelope or by fusion to vp39 on the baculovirus capsid (C). Targeting ligands can be introduced on the baculovirus envelope by chemical attachment with viral envelope protein (D).

The major baculoviral envelope glycoprotein, gp64, has been considered as a good fusion partner for

genetic manipulation because gp64 plays an important role in entry into mammalian cells. Therefore, incorporation of targeting and/or functional moiety by fusion with gp64 has been widely studied for improving the efficacy of baculovirus in gene therapy application. Ojala et al. constructed the recombinant baculoviruses displaying a functional single chain antibody fragment (scFv) specific for the cacinoembryonic antigen (CEA) (pCEAscFvgp64) and synthetic IgG binding domains (ZZ) (pZZgp64) [49]. As the results, although improved binding cannot enhance the subsequent gene transfer and gene expression, binding of recombinant baculoviruses to the surface of mammalian cells can be significantly improved by modification of gp64 to display an appropriate targeting moiety,. Based on their results, the avidin-biotin technology was used for baculovirus targeting, resulting in both enhanced and targeted transduction [50]. Avidin display by genetic fusion to the N-terminaus of the baculovirus major envelope glycoprotein gp64 significantly enhanced the transduction efficacy and chimeric avidin-gp64 enabled viral targeting and efficient gene transfer to biotinylated cell. Also, RGD-motif, recognized by αV integrin, was also incorporated by fusion with gp64 for enhancing their targeting ability [51]. This tropism-modification by RGD-motif enhanced the transduction efficiency as well as enhanced binding of modified virus with the surface of human lung carcinoma cells (A549), known to contain αV integrins. Furthermore, Huser et al. reported complemet resistant baculovirus by incorporation of decay-accelerating factor (DAF) into the baculovirus envelope [52]. DAF acts as an important regulator to

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control the activation of complement cascade [53] and blocks C at the central step of formation of both the classical and alternative pathway convertases by increasing the rate of decay of the C3bBb and C4b2a complexes [54]. As the results, DAF-gp64 displayed baculoviruses on viral envelope can rescue from complement system, indicating that these complement-resistant baculoviruses have a great potential for gene therapy application in vivo. Gp64 fused peptides or proteins thereby providing baculovirus with specificity for desired cells or tissues and complement resistance, critical factors for success gene therapy application both in vitro and in vivo.

As gp64 based fusion protein must compete with wild type gp64 for access to limited areas of the as-sembling virion, the level of incorporation is relatively low [55] even though genetic modification by fusion to gp64 has many advantages. Therefore alternative baculovirus surface display strategies have been applied using heterologous viral glycoproteins, which are able to access the virion assembly path-way without competition with gp64, are other methods. Vesicular stomatitis virus glycoprotein (VSV-G) based on EGFP as a fusion protein, was confirmed to be distributed over the viral envelope of the bacu-lovirus [48]. Makela et al. constructed tumor-targeting ligands such as LyP-1, F3, and CGKRK intro-duced baculoviruses on their envelope by VSV-G based fusion strategies [19]. It was found that intro-duction of tumor-targeting peptides into the viral envelope of baculovirus facilitated the modification of the tropism and these modifications further resulted in enhanced binding and transduction of certain human carcinoma cell lines.

Viral capsid display has been developed as an alternative to viral envelope display. In particular, vp39, the major component of the capsid structure with a random distribution over the capsid surface, was considered as a good candidate to anchor desired proteins onto the surface of the baculoviruses capsid [47] because it has been found to associate with actin polymerisation during entry into the cytoplasm and the nucleus of infected insect cells [56]. Kukkonen et al. reported the successful fusion of EGFP with either N- or C-terminus of vp39 without compromising the viral titer or functionality [31]. Vp39 based fusion system offered the possibilities for nuclear targeting, thus facilitating the nuclear import studies and gene therapy applications [57].

Since first attempt of baculovirus in gene therapy, genetic modification has been evaluated and devel-oped for enhancing the efficacy. Through these studies, genetic manipulations provide the ability for cellular and nuclear targeting by tissue targeting and nuclear localization signals display in different combinations on the viral envelope and nucleocapsid, respectively [57].

3.3 Chemical modification

Fig. 2 Effect of pegylation on gene transduction into A549, 293T, and HepG2 cells with pegylated baculovirus. GFP expression was determined by folw cytometry at 24h after infection [59].

Chemical modification of baculoviruses also provides the possibility of cellular binding and targeting by improving the surface characteristics similar to genetic manipulation. Fig. 1-D shows the scheme of chemical modification. Although diagrammatic representation of chemical modification seems to be same with VSV-G based genetic modification as shown in Fig. 1-B, their processes are totally different. As viral envelopes are composed of numerous proteins, they offer a great platform for chemical binding

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with targeting ligands. Moreover, chemical attachment of the targeting ligands or peptides to virus gives some advantages such as easy modification and flexibility [58] compared with time-consuming genetic means. However, batch-to-batch variation in the characteristics of chemical reaction and degeneration of virus due to the possibility of attachment with envelope protein involved in recognition and entry into the mammalian cells should be overcome to increase the utility of chemical approach [50].

Fig. 3 GFP expression in mouse organ after inoculation of baculoviruses. BALB/c (6 weeks) mice were inoculated with pegylated baculoviruses by I.V. injection through tail vein. At 48h after injection, dissect the mice and GFP expression was determined according to organ and degree of pegylaion [59].

Although chemical modified viral vector has a great potential for gene therapy application, develop-

ment of chemo-baculoviruses is still not well-studied. Chemical modification of baculovirus envelope by poly (ethylene glycol) (PEG) was reported in our previous study [59]. PEG-introduced baculovirus (PEG-Bac) was easily produced by chemical reaction between primary amine groups of viral envelope and carboxyl one of activated methoxypolyethylene glycol succinimidyl succinate (mPEG-SS, MW 5000). The pegylation of baculovirus did not affect the titers of baculovirus, however virus titers in in-sect cells was slowly decreased with increase of pegylation due to shielding of gp64 involved in entry of baculovirus into insect cells. This result indicated that chemical attachment of PEG does not induce the

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degeneration of baculovirus itself. Rather, pegylation improved the stability of baculoviruses, thus en-hancing the transduction efficiency in vitro despite of decrease in transduction efficiency shown by high concentration of PEG by shielding effect as shown in Fig. 2. Fig. 3 shows high transduction efficiency of pegylated baculovirus in vivo in lung and brain along with the GFP expression in other organs in agree-ment with in vitro results.

Chemical modification of baculovirus provides the specificity by attaching the targeting ligands. Al-though the introduction of PEG on viral envelope of baculovirus showed improvement in stability, regu-lation of transduction efficiency, and enhancement of transduction efficiency, targeting to desired tissue or organ was still lacking due to absence of specificity. Kepping in mind above results, we attached folate-PEG (F-PEG) on viral envelope [60]. Folate, one of tumor-targeting ligands, has many unique advantages such as presumed lack of immunogenicity, unlimited availability, functional stability, defined conjugation chemistry, and a favourable non-destructive cellular internalization pathway [61]. Moreover, the selective amplification of FR expression among human malignancies suggests its potential utility as a cellular marker that can be exploited in targeted drug and gene delivery [62]. Therefore, introduction F-PEG resulted in improving the surface characteristics of baculoviruses and enhancing transduction effi-ciency significantly in folate receptor (FR)-positive KB cells compared to that of pegylated baculovi-ruses because folate served as a targeting ligand as shown in Fig. 4-A [60]. Moreover, blocking of the transduction of F-P-Bac on the folate-enriched regular medium was also confirmed whereas there was little difference in luciferase activity of P-Bac between regular and folate-free medium as shown in Fig. 4-B [60].

Fig. 4 Transduction efficiency of F-PEG introduced baculovirus (F-P-Bac) and PEG introduced baculovirus (P-Bac) in the KB cell lines (A) and competition assay of F-P-Bac and P-Bac on folate-enriched regular medium (B). Both were measured by luciferase assay at 24h after transduction [60].

Chemical modifications of viral envelope can be easily controlled by introduction a variety of ligands

to give specificity although these are still not well-developed compared with genetic modification. Therefore, modified baculoviruses by chemical means has a great potential for gene therapy applications.

4. Conclusions and Future prospects

Baculoviruses have been considered as a promising tool for gene carriers due to their promising charac-teristics such as transduction to broad range of mammalian cells, the non-replicative nature, low cyto-pathic effect upon infected cells, ability to package large DNA inserts, and ease of production for gene therapy applications. However, unclear mechanism of entry, inactivation of baculovirus in human serum and whole blood by complement system, and non-specific transduction restrict the application of bacu-lovirus vectors. Especially, specificity of viral vectors in recognizing the target cells only and exploiting the proper intracellular trafficking routes are the key issues in viral vector design. To reach this goal,

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baculoviruses have to be modified by genetic or chemical manipulations. The genetic or chemical modi-fied baculviruses greatly improved the potential for gene therapy applications. However, genetic or chemical modifications still have limitations such as the time-consuming process for insertion of desired moieties or degeneration of baculovirus owing to chemical attachment on viral envelope, respectively. Hence, the combination of genetic and chemical manipulation may hold great promise. Moreover, gene therapy based on baculoviral vector will be further developed by the construction of modified baculovi-ruses containing therapeutic gene or siRNA.

Acknowledgement This work was supported by the grants from Ministry of Science and Technology in Korea (M10414030002-05N1403-00210).

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Modification of Baculovirus for Gene Therapy - Formatex · Modification of Baculovirus for Gene Therapy You-Kyoung Kim 1, Hu-Lin Jiang , Yeon-Ho Je1, Myung-Haing Cho2, Chong-Su Cho*,1