Hemagglutinating Virus of Japan Envelope Vectors as High-Performance Vehicles for Delivery of Small RNAs

Hemagglutinating virus of Japan envelope (HVJ-E) vectors are particulate forms of the Sendai virus, and are characterized by maintained cell membrane fusion activities and completely inactivated genomes. HVJ-E vectors can be safely used as a non-viral transfection tools for laboratory research without the need for special protocols or equipment. HVJ-E particles are loaded with molecules such as DNA, proteins, antisense oligonucleotides, or small RNAs, to form HVJ-E vectors that carry these molecules into target cells by virtue of their membrane fusion activity. Interference by small RNAs such as small interfering RNA (siRNA) and microRNA (miRNA) is now established as an important biological strategy for gene silencing, and is becoming an essential method for analyzing biological processes. Various delivery reagents are currently available globally; however, delivery to non-adherent immune cells, particularly primary immune cells, remains extremely difficult. The simple and effective delivery capabilities of HVJ-E vectors overcome the above obstacle. Here we describe examples and demonstrate the utility and potential applications of HVJ-E vectors as high-performance vehicles for delivery of small RNAs. electrostatic binding-site barrier effect, inhibiting further association of delivery systems with tumor spheroids [10]. In addition, although cationic liposomes efficiently take up siRNA, limited success has been achieved with these systems in in vivo gene silencing, probably due to their intracellular stability, which results in failure to release enclosed siRNA. Also, their effectiveness is limited by their toxicity [7]. Polymeric nanoparticles are solid, biodegradable, colloidal systems that have been widely investigated as drug or gene carriers [11], and have been classified as either natural or synthetic polymers. Natural polymers for siRNA delivery include cyclodextrin, chitosan, and atelocollagen [12]. Synthetic polymers such as polyethylenimine (PEI), poly(dl-lactide-co-glycolide), and dendrimers have been extensively investigated [13]. PEI is commonly used for drug delivery because it can disrupt endosomal membranes through the proton–sponge effect [14]. Moreover, it has high transfection efficiency and has been widely studied for siRNA delivery [12-14]. PEI forms small and compact structures that spontaneously assemble polyplexes with negatively charged siRNA through a simple and rapid polycation process [12]. The resulting PEI–siRNA complexes protect siRNA from degradation by nucleases, resulting in prolonged half-lives. In addition, complete encapsulation of siRNA prevents off-target effects such as immune activation by toll-like receptor-dependent mechanisms [15]. However, the significant toxicity associated with PEI complexes has limited Citation: Kato F, Yagi T, Fujieda T, Kondo Y, Yamaguchi T, et al. (2013) Hemagglutinating Virus of Japan Envelope Vectors as High-Performance Vehicles for Delivery of Small RNAs. J Genet Syndr Gene Ther 4: 178. doi:10.4172/2157-7412.1000178


Introduction
RNA interference (RNAi) is a well-known endogenous mechanism for gene regulation, and was originally identified in Caenorhabditis elegans by Fire et al. in 1998 [1]. The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double-stranded molecules containing approximately 20 nucleotides. These are known as small interfering RNAs (siRNAs). After internalization, siRNAs are incorporated into RNA-induced silencing complexes (RISC), in which the catalytic component of the RISC complex Argonaute-2 unwinds the siRNAs. The two strands of the siRNAs are referred to as passenger and guide strands; the passenger strand gets cleaved and the activated guide strand moves out and cleaves complimentary mRNA [2]. Powerful and specific gene silencing by siRNAs is becoming an essential strategy for analyzing biological processes, and has numerous applications in drug discovery, diagnosis, and medical therapy.
Numerous in vitro and in vivo siRNA delivery strategies have been developed to date [3][4][5][6][7][8][9], and their common fundamental aim is to efficiently localize siRNAs to functional sites of target cells. Generally, viral vectors are more efficient for gene delivery and expression than non-viral ones; however, viral vectors pose greater risks than non-viral vectors. Thus, viral vectors are generally not used for drug delivery, and instead non-viral vectors are used for delivering anticancer agents, synthetic oligonucleotides, antibodies, and small RNA molecules such as siRNA and microRNA (miRNA).
In the last 10 years, tremendous efforts have been made to develop therapeutic applications of gene silencing using non-viral vectors. Cationic lipids such as dioleoyl phosphatidylethanolamine (DOPE) form lipocomplexes with negatively charged siRNAs. These pH-sensitive liposomes escape from endosomes by fusing with endosomal membranes, and they are routinely used for the delivery of siRNA or plasmid DNA into mammalian cells in vitro. However, surface interactions of cationic liposomes with tumor cells produce an electrostatic binding-site barrier effect, inhibiting further association of delivery systems with tumor spheroids [10]. In addition, although cationic liposomes efficiently take up siRNA, limited success has been achieved with these systems in in vivo gene silencing, probably due to their intracellular stability, which results in failure to release enclosed siRNA. Also, their effectiveness is limited by their toxicity [7].
Polymeric nanoparticles are solid, biodegradable, colloidal systems that have been widely investigated as drug or gene carriers [11], and have been classified as either natural or synthetic polymers. Natural polymers for siRNA delivery include cyclodextrin, chitosan, and atelocollagen [12]. Synthetic polymers such as polyethylenimine (PEI), poly(dl-lactide-co-glycolide), and dendrimers have been extensively investigated [13]. PEI is commonly used for drug delivery because it can disrupt endosomal membranes through the proton-sponge effect [14]. Moreover, it has high transfection efficiency and has been widely studied for siRNA delivery [12][13][14]. PEI forms small and compact structures that spontaneously assemble polyplexes with negatively charged siRNA through a simple and rapid polycation process [12]. The resulting PEI-siRNA complexes protect siRNA from degradation by nucleases, resulting in prolonged half-lives. In addition, complete encapsulation of siRNA prevents off-target effects such as immune activation by toll-like receptor-dependent mechanisms [15]. However, the significant toxicity associated with PEI complexes has limited globally. However, delivery to non-adherent immune cells, particularly to primary immune cells, remains extremely difficult [39]. In this context, a promising feature of HVJ-E vectors is that they offer a simple and effective delivery system, even to these cells. Numerous in vitro and in vivo examples of HVJ-E-mediated delivery of small RNAs have been reported since 2003 [40]. Indeed, HVJ-E vectors have been widely used to deliver a number of different molecules to various adherent and non-adherent cells in vitro (Table 1 and 2) and to various target organs and tissues in vivo (Table 3). In this study, we describe examples of in vitro and in vivo delivery of small RNAs, and their utility and potential applications as transfection tools.

Delivery to Transfection-resistant Non-adherent Immune Cells
Delivery of siRNAs that target Eg5 and Cdc2 (Cyclin-Dependent Kinase 1; CDK1) has been performed in various non-adherent immune cells, including Jurkat, U-937, THP-1, HL-60, Raji, and K-562 cells, for which currently available lipofection reagents are ineffective. The kinesin-like motor protein Eg5 is essential for the formation of spindle microtubules during cell division, and impaired function of this protein leads to cell cycle arrest and apoptosis. Utilizing this phenomenon, the effects of siRNA delivery were assessed quantitatively using a viable cell count method (WST-8 assay). Cdc2 is known to form a complex with cyclin B and regulates the M phase of the cell cycle. Hence, knockdown of Cdc2 mRNA was determined using real-time PCR to assess the use of the vector kit (Table 1). These experiments showed extremely efficient intracellular delivery and high knockdown effects in various non-adherent immune cells. In contrast, although lipofection products allowed effective delivery to adherent HeLa S3 cells, with few exceptions, stable delivery in non-adherent immune cells was not achieved.
Numerous other examples of HVJ-E vector-mediated delivery of small RNAs have been reported in vitro ( Table 2) and in vivo ( Table  3) since 2003 [40]. Here we describe some examples of delivery to transfection-resisitant cells such as non-adherent immune cells.
Although autoantibodies to SS-A/Ro52 (Ro52) are frequently associated with autoimmune diseases such as Sjogren's syndrome, the physiological function of Ro52 is poorly understood. In 2003, Ishii et al. used HVJ-E vectors to deliver siRNA duplexes against SS-A/Ro52 (Ro52) into Jurkat T cells overexpressing Ro52, and produced the first evidence that Ro52 is involved in CD28-mediated production of interleukin (IL)-2 [41].
Another study demonstrated essential regulation of total white blood cell numbers by the protein Bim in Bim-deficient mice. To examine whether the upregulation of Bim contributes to imatinibinduced apoptosis, K562 cells were transfected with Bim siRNA using HVJ-E vectors, resulting in effective rescue from imatinib-induced apoptosis. This result suggests that Bim plays an important apoptotic role in early hematopoietic progenitors [46].
To examine the effect of cathepsin G knockdown on the their assessment in clinical trials [7,16]. Molecular mechanisms of PEI cytotoxicity include membrane damage and activation of mitochondrial apoptotic programs following PEI-induced channel formation in outer mitochondrial membranes [7,17].
To overcome the limitations of each type of vector system, virosomes equipped with chimeric viral and non-viral vector apparatuses have been developed [18]. Initially, viral liposomes with fusogenic envelopes were constructed from hemagglutinating virus of Japan (HVJ) (Sendai virus) to compensate the limitations of one vector system with the advantages of another. This approach not only enables efficient drug delivery and gene expression but also reduces cytotoxicity of various vector components. Although virosomes have disadvantages, such as immunogenicity and instability in circulation, they have unique characteristics that may make them suitable for cancer therapy.
In this system, loaded DNA liposomes were fused with UVinactivated HVJ to form fusogenic liposomes hybrids that had higher levels of trapped DNA than reconstituted virosomes, leading to direct transmission of their cargo to the cytoplasm by bypassing endocytotic uptake [19,20]. However, fusion of liposomes with inactivated HVJ requires a complicated procedure, and fusogenic abilities are reduced (approximately 2% relative to native HVJ) due to dilution of HVJ glycoproteins [21]. To address these issues, HVJ envelope (HVJ-E) vectors have been developed, in which the cargo, such as DNA, is incorporated directly into intact, though inactivated, HVJ (reviewed by Kaneda et al. [21][22][23]). HVJ-E vectors are unique non-viral vectors that carry viral membrane components with cell membrane fusion activities [24]. HVJ-E vectors have been shown to have superior performance in transferring genes into tissues of animals as well as into various cultured cell lines [25][26][27][28][29][30][31]. The vector kit employs delivery principles that are distinctively different from those of cationic non-viral vectors, which are aggressively marketed, and it enables effective intracellular delivery of negatively charged plasmid DNAs, low molecular weight nucleic acids (siRNA, miRNA, antisense/decoy oligonucleotides), and biologically active substances such as peptide proteins (including antibodies) with various electric charges (isoelectric points) [32][33][34][35][36]. These delivery principles ( Figure 1), features, procedures, and application examples have been outlined in our previous publications [21,23,27,28,32], and the vector kit is commercially available in both Japan

Vector Delivery to Primary Immune Cells
Delivery of miRNA and siRNA to primary immune cells, to which it is considered to be more difficult to deliver, was examined using HVJ-E vectors.

Delivery of miRNA to activated T cells
Primary mouse T cells were isolated from splenocytes of 7-12-weekold female BALB/c mice using the Pan T Cell Isolation Kit II (Miltenyi Biotec), and were co-stimulated with phorbol 12-myristate 13-acetate (PMA, 5 nM) and ionomycin (1 μg/ml). After 24 h, HVJ-E vectors were   Figure 2A). Concurrent Western blot analysis corroborated this potent inhibitory effect at the protein level ( Figure 2B).

Delivery of miRNA to activated B cells
Primary mouse B cells were isolated from splenocytes of 7-12-week-old female BALB/c or C57BL/6 mice using the Pan B Cell Isolation Kit (Miltenyi Biotec), and were co-stimulated with anti CD40 antibody (1 μg/ml) and IL-4 (34.5 ng/ml). After 24 h, delivery of an miRNA mimic (Dharmacon RNAi Technologies) targeted at CyB was performed using HVJ-E vectors. Subsequent Western blot analysis at 48 h after transfection indicated a high inhibitory effect at the protein level ( Figure 3A). Moreover, after stimulation of primary B cells with anti-CD40 antibody and lipopolysaccharide (1 μg/ml) a specific knockdown effect was observed ( Figure 3B).

Delivery of siRNA to activated T cells
Delivery of CyB siRNA into primary T cells co-stimulated with PMA and ionomycin was performed using the same test system as described above, and mRNA levels were evaluated using real-time PCR. Again, mRNA knockdown was observed in a dose-dependent manner at 50 nM or less (Figure 4).

Delivery of siRNA to naive T cells
The effects of ZAP70 siRNA in mouse naïve thymocytes were evaluated using Western blot analysis. ZAP70 is a Syk family protein tyrosine kinase that forms a complex with T-cell antigen receptors and plays an important role in transducing downstream signals. Of 3 delivered ZAP70 siRNAs, 2 exerted inhibitory effects at the protein level ( Figure 5). T cells isolated from murine splenocytes using the T Cell isolation kit were co-stimulated with PMA (5nM) and ionomycin (1 μg/ml). After 24 hrs, miRNA mimic targeted at cyclophilin B was transfected into the activated primary T cells using HVJ-E vectors. Real-time PCR evaluation (A) and western blot analysis (B) were performed at 48 hrs after the transfection T cells isolated from murine splenocytes using the T Cell isolation kit were co-stimulated with PMA (5nM) and ionomycin (1 μg/ml). After 24 hrs, siRNA targeted at cyclophilin B was transfected into the activated primary T cells using the HVJ-E. Real-time PCR evaluation was performed at 48 hrs after the transfection Their results suggested that the CD26-caveolin-1 interaction plays an important role in the up-regulation of CD86 in TT-loaded monocytes, and indicated subsequent engagement with CD28 on T cells [43]. They also identified Toll-interacting protein-and IL-1 receptor-associated serine/threonine kinase 1 (IRAK-1) as caveolin-1-interacting proteins in monocytes. Subsequent transfection of siRNAs against each target molecule into human peripheral monocytes demonstrated their function in signaling pathways of antigen-presenting cells [49]. Moreover, transfection of siRNAs into freshly isolated human T cells from healthy adult peripheral blood mononuclear cells demonstrated the role of CARD-containing MAGUK protein 1 on CD26-mediated T-cell co-stimulation. This study provided novel insights into the regulation of T-cell co-stimulation via CD26 [53].

Other important studies in primary immune cells
Using HVJ-E vectors, Masuda et al. transfected GATA-1 and GATA-2 siRNAs into bone marrow-derived mast cells (BMMCs) that were isolated from femoral BM cells of BALB/c mice, and examined the role of GATA transcriptional factors in differentiated MCs. Delivery of these siRNAs into BMMCs resulted in markedly decreased levels of GATA-1 and GATA-2 proteins. Subsequently, they showed that repression of GATA activity leads to impaired cell survival and IgEinduced degranulation and cytokine production, indicating that GATA plays a critical role in the activation of MCs [55].

Delivery of siRNA using HVJ-E Vectors in vivo
Many in vivo examples of HVJ-E vector-mediated siRNA delivery have been reported since 2005 (Table 3).
Ito et al. [79] used HVJ-E vectors to deliver siRNAs against Rad51, which mediates homologous recombination in dsDNA break repair, and showed enhanced sensitivity to cisplatin (CDDP) in vitro and in vivo. Indeed, over expression of the human Rad51 gene in HeLa cells renders HeLa cells resistant to CDDP. When Rad51 siRNA was delivered into HeLa cells using HVJ-E, Rad51 expression was completely suppressed.
In another notable study, 3 days after bleomycin challenge, Fichtner-Feigl et al. [81] administered HVJ-E-encapsulated IL-13Rα2specific siRNAs by intra-rectal instillation (0.1 mg/instillation) into mice every second day for 24 days. On days 13 and 24, expression of IL-13Rα2 in lung tissue was greatly diminished, and TGF-β1 secretion and collagen formation were also significantly decreased. These data suggest that IL-13Rα2 signaling during prolonged inflammation is an important therapeutic target for prevention of TGF-β1-mediated fibrosis [51]. Additional studies of chronic 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis showed that fibrosis was dependent on the development of IL-13 responses that induced TGF-β1 via a novel cell surface-expressed IL-13 receptor [81].
Ishibashi et al. injected rCLCA siRNA (2 nmol) into submandibular glands of rats in a retrograde manner using HVJ-E vectors. After 48 hrs, significantly higher Cl − levels and suppression of Cl − channel proteins were observed on siRNA-injected sides. This report provided the first in vivo evidence for the physiological significance of rCLCA and CFTR in transepithelial Cl − transport in ductal systems of rat submandibular glands [80]. They also, using HVJ-E vector-mediated siRNA delivery, revealed a significant involvement of apical P2Y2 receptor-regulated CFTR activity in rat submandibular gland [83].
Morita et al. [86][87][88], Kitayama et al. [100] used HVJ-E vectors to deliver siRNAs against Glycine transporter (GlyT) 1, GlyT2, glycine receptorα3 subunit (GlyRα3), protein phospholipase C-related but catalytically inactive protein (PRIP)-1 and PRIP-2 by injection into the subarachnoid space between the L 5 and L 6 vertebrate of mice. A peak of gene suppression occurred at 2-3 days post-injection, and this recovered to original levels approximately 8 days after injection. The results suggested that suppressed expression of PRIPs induced an elevated expression of K + -Cl --cotransporter-2 in the spinal cord, resulting in inhibition of nociception and amelioration of neuropathic pain in PRIP-1 and PRIP-2 double knockout mice.

Conclusions and Future Outlook
With the advent of next-generation sequencers, identification and detection of small RNAs undetectable using conventional methods have now become possible, and these small RNAs are being sequenced in various tissues from a variety of organisms. Although small RNAs that function in RNA silencing are roughly classified into 3 groups (siRNA, miRNA, and piRNA), novel small RNAs are expected to be discovered in the future. Hence, targeted delivery of small RNAs is essential for analyzing biological functions and screening their potential therapeutic targets. In this study, we showed that delivery of small RNAs using HVJ-E vectors resulted in high-performance knockdown in various cell types, including transfection-resistant cells such as non-adherent immune and primary immune cells. Numerous in vivo studies corroborate the utility of HVJ-E vectors as high-performance vehicles for delivery of small RNAs. HVJ-E vectors are expected to contribute profoundly to biological research, and promise applications in drug discovery and diagnosis as quick functional assessment tools for small RNAs.
HVJ-E vector itself induces anti-tumor immunity, including T cell-mediated and non-T cell-mediated immunity through multiple pathways (activation of natural killer cells, and dendritic cells, suppression of regulatory T cells through interleukin-6 secretion from dendritic cells). These activities may be desirable in the treatment of cancer, although these phenomena may adversely affect outcomes in other applications of HVJ-E, including siRNA delivery. Interestingly, the production of cytokines and chemokines in dendritic cells by HVJ-E vector was abolished by the treatment of HVJ-E with Triton-X100, but maintained by Tween 80-treated HVJ-E (unpublished data by Y. Kaneda). Both detergents were available for the incorporation of therapeutic molecules into HVJ-E vectors. Using two different detergents at the incorporation step, it is possible to construct HVJ-E vectors with or without anti-tumor immunity [28]. However, HVJ-E vectors have HN protein in addition to F protein on their envelope membrane. The HN protein binds to its receptor (acetylated sialic acid) on the cell membrane. This protein is also involved in the agglutination of red blood cells. Namely, systemic administration of HVJ-E vector results in functional inactivation with hemagglutination in the blood. Therefore, it is advisable to select a route of administration involving less exposure to blood (direct injection to organs or tissue is recommended) or to perform perfusion of the animal prior to administration. To overcome the limitations of HVJ-E vector, HN-depleted HVJ was developed using HN-specific siRNA. LLCMK2 cells were transformed with HN-specific siRNA. The HVJ progeny produced from these cells lost HN but retained F protein.
No hemagglutination was detected by the HN-depleted HVJ. Finally, by combining the construction of chimera containing the F protein and transferrin with the use of HN-specific siRNA, tumor-targeting HVJ-E vector without HN was developed [101]. Final goal is the systemic injection of tumor-targeting HN-depleted HVJ-E incorporating therapeutic molecules to various refractory cancers [28].
A clinical grade HVJ-E vector is currently being produced for use in clinical trials. Thus far, the virus has only been produced in chick eggs [24], however, egg-derived HVJ is difficult to use in clinical trials. It is also difficult to produce large amount of the virus in cultured cells. However, we recently succeeded in producing large amount of HVJ in human cells using animal product-free medium. Now we can produce more than 10 10 particles/ml of culture medium of human cell-derived HVJ. A pilot plant to commercially produce clinical grade HVJ-E vector has already been established. Thus, a human cell-derived HVJ-E vector is now ready for clinical use [26,28].