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ISSN: 2157-7412
Journal of Genetic Syndromes & Gene Therapy
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Hemagglutinating Virus of Japan Envelope Vectors as High-Performance Vehicles for Delivery of Small RNAs

Fuminori Kato1*, Takaharu Yagi1, Takayuki Fujieda1, Yoshitaka Kondo1, Tomona Yamaguchi1, Keizo Miyata2 and Yasufumi Kaneda3

1Central Research Institute, Ishihara Sangyo Kaisha, Ltd, Japan

2Life Science Technology Development Division, Ishihara Sangyo Kaisha, Ltd, Japan

3Division of Gene Therapy Science, Osaka University, Graduate School of Medicine, Japan

*Corresponding Author:
Fuminori Kato
Life Science Research Laboratory
Central Research Institute, Ishihara Sangyo Kaisha
Ltd., Shiga 525-0025, Japan
Tel: +08 77 562 8881
Fax: +08 77 562 9783
E-mail: [email protected]

Received date: July 26, 2013; Accepted date:September 10, 2013; Published date: September 19, 2013

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

Copyright: © 2013 Kato F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

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.

Keywords

HVJ envelope vecto; Sendai virus; Cell membrane fusion; Non-viral vector, small RNA delivery; siRNA; miRNA

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 [3-9], 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 [12-14]. 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 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. [21-23]). 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 [25-30]. 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) [31-35]. These delivery principles (Figure 1), features, procedures, and application examples have been outlined in our previous publications [21,23,27,28,31], and the vector kit is commercially available in both Japan [36] and USA [37].

Various small RNA delivery reagents are currently available globally. However, delivery to non-adherent immune cells, particularly to primary immune cells, remains extremely difficult [38]. 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 [39]. 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.

genetic-syndromes-gene-therapy-Delivery-molecules

Figure 1: Delivery of molecules (siRNAs) into cells using HVJ-E vectors.

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 [39]. 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 [40].

To address the significance of Brap2 expression in cytoplasmic p21 localization, Asada et al. used HVJ-E vectors to deliver Brap2 siRNA into U939/CB6-p21 cells [44].

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 [45].

Fichtner-Feigl et al. transfected J774 mouse macrophage-like cells with IL-13Rα2 siRNA using HVJ-E vectors, and showed blockade of TGF-β1 and collagen production in bleomycin-induced lung fibrosis [50].

Cell line*1 Target
of siRNA*2
Evaluation
method*3
HVJ-E*4 R Reagent*5 X Reagent*6
Silencing efficiency (%) Viable cells
(%)
Silencing efficiency (%) Viable cells
(%)
Silencing efficiency (%) Viable cells (%)
(Suspension)
Jurkat
U-937
THP-1
HL-60
Raji
K-562
Eg5
Eg5
Eg5
Eg5
Cdc2
Cdc2
WST-8
WST-8
WST-8
WST-8
RT-PCR
RT-PCR
81
93
94
63
73
85
84
83
100
89
100
98
31
4
45
0
12
81
91
100
100
100
94
100
6
6
3
4
30
52
80
100
100
97
100
100
(Adherent)
HeLa S3
Eg5 WST-8 80 100 76 100 53 77

Table 1: Delivery of siRNA to non-adherent immune cell strains (Comparison with lipofection reagents).

Ref. NO Cells Origin Target genes
[40] MIN6 Mouse pancreatic ßcell GPR40
[41] Jurkat Human acute T cell leukemia SS-A/Ro52
[42] C2C12 Mouse myoblast(differentiated) Bin1
[43] primary monocyte Human monocyte Caveolin-1
[44] primary cardiac myocyte Rat cardiac myocyte phospholamban
[45] U937 Human leukemic monocyte cell Brap2
[46] K562 Human chronic myelogenous leukemia Bim
[47] HUVEC Human umbilical vein endothelial cell TSAd
[48] CMK6G3 Monkey ES cell Monkey ES cell (stably expresses EGFP) EGFP
[49] primary monocyte Human monocyte Tollip, IRAK-
[50] HuH-6, HuH-7, HepG2 Human hepatoblastoma cell Human hepatocecellular carcinoma cell ß-catenin
[51] J774 Mouse macrophage cell IL-13 Receptor a2
[52] MIN6 Mouse pancreatic ß cell GPR40
[53] primary T cell Human peripheral blood Human CARMA1
[54] Primary calvarial osteoblasts Mouse calvarial osteoblasts OPG
[55] primary mast cell Mouse bone marrow-derived GATA-1,GATA-2
[56] INS-1E, NIH-3T3 Rat ß cell, Mouse embryonic fibroblast Sox6
[57] primary macrophage C3H mouse peritoneal resident Mcl-1
[58] primary granulosa cell Mouse granulosa cell Snap25
[59] primary granulosa cell Rat granulosa cell TACE/ADAM17
[60] BASMC Bovine aortic smooth muscle cell Bovine TE(tropoelastin)
[61] U937 Human leukemic monocyte cell Human cathepsin G
[62] A549, H1299,TE13, PCNA-1, MIAPaCa-2, Du-145, ME-180 Human carcinoma (lung, esophageal, pancreas, prostate, cervical) Ku80
[63] HMVEC-dLyNeo(LEC) Human neonatal dermal lymphatic microvascular endothelial cell TLR4
[64] U251MG, D54MG Human glioma(p53 mutated at codon 273) Human glioma(p53 wild type) Survivin, p53
[65] granulosa cell Mouse granulosa cell TLR2, TLR4
[66] granulosa cell Mouse granulosa cell Mkp3
[67] COLO201 Human colon cancer FKBP51
[68] alveolar type II epithelial cells Mouse alveolar type II epithelial cells Nedd4, Nedd4-2
[69] primary alveolar type2 cell mouse lung alveolar type2 cell LPCAT1
[70] Primary neonatal cardiac ventricular myocyte Rat primary neonatal cardiac ventricular myocyte TFAM,TFB2M
[71] U937 Human leukemic monocyte cell VEGFA*1
[72] Mouse suprachiasmatic nucleus slice cultures Mouse suprachiasmatic nucleus Atf4
[73] Human alveolar epithelial type Ilike (ATI-like) cells Human alveolar epithelial Nrf2
[74] U937 Human leukemic monocyte cell PP2ACa
[39] U937 Human leukemic monocyte cell Mk2
[75] Human alveolar epithelial type Ilike (ATI-like) cells Human alveolar epithelial Nrf2
[76] U937 Human leukemic monocyte cell PP2ACa
[77] Artery smooth muscle cells Porcin e ROCK2, MLCK, ZIPL, RORa
[78] primary cardiomyocytes Neonatal rat HMGB1

Table 2: siRNA transfection studies using the HVJ-E (in vitro).

Ref. Organ/Tissue Host Target genes Delivery route Dose
[62] transplanted tumor (H1299) BALB/C nude mouse Ku80 intratumor injection day0(1shot) or
day 2, 4(2 shots)
[79] transplanted tumor (HeLa) SCID mouse Rad51 intratumor injection 35 µg,  Day 0, 2, 4
[85] transplanted tumor (B16-F10) Mouse Rad51 intratumor injection 2.5 nmol, day 6,8,10
[89] transplanted tumor (U-118MG) Mouse Eg5 intratumor injection 200 pmol, day7,10,13
[94] transplanted tumor (colon26) Mouse bcl-2 intratumor injection 40 µg/1shot
[97] transplanted tumor(A549) Mouse Nrf2 intratumor injection day 1,2,3
[80] submandibular gland Rat rCLCA(Ca2+-dependent clchannels), CFTR(cystic fibrosis transmembrane conductance regulator) retrograde ductal injection 2 nmol, day 0
[83] submandibular gland Rat CFTR (cystic fibrosis transmembrane conductance regulator) retrograde ductal injection 2 nmol day 0
[81] colon Mouse IL-13 Receptora2 intrarectal injection  
[82] colon Mouse IRF4 intrarectal injection 100 µg, day-2,1-,0,1
[87] colon Mouse Toll like receptor(TLR)-2 intrarectal injection 1 mg
[86] spinal cord Mouse GlyT1, GlyT2, GlyRa3 spinal cord injection 0.45 pmol
[88] spinal cord Mouse GlyR a3 spinal cord injection 0.45 pmol
[100] spinal cord Mouse Phospholipase C-related but catalytical inactive protein (PRIP) spinal cord injection 0.45 pmol
[92] brain Rat Ca(v)3.2 intrathecal(i.t) injection 9 µg/1shot
[98] brain Mouse CD45 intracerebroventricular (ICV) injection 5 µg
[99] brain Mouse GlyRa3 siRNA intrathecal injection  
[96] dorsal horn Rat NK1 receptor intrathecal injection 45 pmol/1shot, day0
[51] lung Mouse IL-13 Receptora2 Intratracheal injection 100 µg, day0,2,4,6,8,10, 12, 14, 16, 18, 20, 22
[84] inguinal and popliteal LN cells
(enrich in lymphoblasts)
SJL/J mouse (ex vivo ) orphan nuclear receptor NR4A2 adoptive transfer experimental
autoimmune encephalomyelitis (EAE)
model in irradiated mice/ intraperitoneal injection
of NR4A2 siRNA-treated LN cells
 
[90] spleen Mouse Histamine H4 Receptors tail vein 50 µg
[91] stomach Mouse Stat1 intraperitoneal injection 100 µg
[93] uterus Mouse RCAS1 uterine cavity  
[95] dorsal skin Mouse Int6 subcutaneous injection 40 µg

Table 3: siRNA transfection studies using the HVJ-E (in vivo).

To examine the effect of cathepsin G knockdown on the degradation of 4-hydroxy-2-nonenal (HNE)-modified glyceraldehyde- 3-phosphate (GAPDH), U937 cells were transfected with a cathepsin G-specific siRNA (CTSG siRNA) using HVJ-E vectors. The resulting specific knockdown of cathepsin G successfully demonstrated its role in the degradation of HNE-modified GAPDH [60].

Jafarifar et al. transfected miRNAs into U937 cells using HVJ-E vectors, and demonstrated roles of their targets in hypoxia-induced expression of vascular endothelial growth factor-A (VEGFA). These experiments demonstrated that miR-297 and -299 were endogenous negative regulators of VEGFA expression in human monocytic cells, and elucidated a new mechanism of VEGFA regulation by hypoxia [70].

Using HVJ-E vectors, Kobayashi et al. transfected siRNA against the serine/threonine phosphatase protein phosphatase 2A (PP2A) into U937 cells to investigate the role of PP2A on corticosteroid sensitivity in severe asthma. Knockdown of PP2A significantly blocked the inhibitory effects of dexamethasone on TNFα-induced IL-8 release in U937 cells [73,75].

Tietz and Berghoff failed to create an efficient knockdown of MAPK-activated kinase 2 (MK2) in U937 cells, despite transfecting siRNA and shRNA using various delivery reagents and methods [38]. Subsequently, with the use of HVJ-E vectors, these investigators successfully transfected siRNA into U937 cells and showed >90% reduction in MK2 expression. Moreover, analyses of cell viability showed 85% survival 48 h after transfection. These data lead to the conclusion that knockdown of MK2 using HVJ-E vectors is a simple and rapid method for transfection without the use of harsh conditions [38].

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 used to deliver an miRNA mimic (Dharmacon RNAi Technologies) targeted at the housekeeping gene cyclophilin B (CyB) in primary T cells. After 48 h, real-time PCR showed high knockdown efficacy with extremely low miRNA doses (1 nM or less; 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).

genetic-syndromes-gene-therapy-Delivery-miRNA

Figure 2: Delivery of miRNA into activated T cells using HVJ-E vectors.

genetic-syndromes-gene-therapy-activated-cells

Figure 3:Delivery of miRNA into activated B cells using HVJ-E vectors

genetic-syndromes-gene-therapy-HVJ-vectors

Figure 4:Delivery of siRNA into activated T cells using HVJ-E vectors.

Other important studies in primary immune cells

Using HVJ-E vectors, Ohnuma et al. transferred caveolin-1 siRNAs into human peripheral monocytes to study the role of CD26-caveolin-1 interactions in tetanus toxoid (TT)-loaded monocytes. 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 [42]. 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 [48]. 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 [52].

genetic-syndromes-gene-therapy-naive-cells

Figure 5:Delivery of siRNA into naive T cells using HVJ-E vectors.

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 [54].

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. [80] administered HVJ-E-encapsulated IL-13Rα2- specific 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 [50]. 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 [79]. They also, using HVJ-E vector-mediated siRNA delivery, revealed a significant involvement of apical P2Y2 receptor-regulated CFTR activity in rat submandibular gland [82].

Morita et al. [86] and Morita et al. [87,99] 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 L5 and L6 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 [100]. 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 1010 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].

Acknowledgment

We thank Mr. Hirotaka Fukushima for great support, and thank Mr. Kyuya Morino for reliability survey of our experiments in this article. We also gratefully acknowledge Ms. Tomoko Kawagoe for experimental support.

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Immunology & Microbiology Journals

David Gorantl

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1-702-714-7001Extn: 9014

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Stephanie Skinner

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1-702-714-7001Extn: 9039

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Rachle Green

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Jim Willison

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1-702-714-7001 Extn: 9042

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Nimmi Anna

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1-702-714-7001 Extn: 9038

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1-702-714-7001 Extn: 9042

 
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