OZ 生物科學公司推出 ViroMag 核酸傳遞系統

OZ生物科學公司很高興地宣布推出了一種基于磁對流TM技術的新產品，專門為病毒應用而設計： ViroMag。磁切™是一種新的革命性的核酸傳遞系統。它利用磁力將與磁性粒子相關的核酸載體和病毒驅動向并進入目標細胞。通過這種方式，完全應用劑量的病毒迅速集中在細胞表面，使100%的細胞同時接觸到所有劑量的病毒。ViroMag是一種多功能和獨特的解決方案，為許多病毒式應用。ViroMag允許科學家： " 提高轉導效率，加速轉導過程 " 感染非受納細胞 " 將病毒濃縮到細胞上或放在培養基中 " 同步細胞吸附（感染）而不修飾病毒 ViroMag是唯一可為這類應用提供解決方案的試劑。病毒染色體和要轉導的病毒一步混合；不需要分子生物學過程或生化修飾。這種試劑具有異常高的效力。

ViroMag適用于所有病毒載體，并具有獨特的特性，允許：
1.提高了被轉導細胞的百分比方面的轉導效率
2.非常迅速地將整個病毒劑量集中在細胞上，并加速轉導過程。
3.感染非受納細胞
4.用極低的載體劑量可以顯著提高病毒的傳染性。
5.同步細胞吸附/感染
6.針對特定區域的目標/限制轉導（磁靶向）

Based upon a validated and recognized magnetic drug targeting technology this innovative method is:
• Highly Efficient
• Suitable for all viruses
• Economical, Simple & Rapid
• Universal (primary cells, hard-to-transfect cells and cell lines)
• Serum compatible & Non toxic
• Amenable to high throughput automation
 
OZ Biosciences offers 4 types of ready-to-use reagents:
" ViroMag engineered to be combined with all viruses
"  PolyMag suitable for all nucleic acids and all transfection application
"  CombiMag designed to be associated with all transfection reagents
"  SilenceMag created specifically for all siRNA applications.
 

VirusType	Virusname
Adenovirus / Adeno-Associated Virus Lentivirus / Retrovirus Herpes virus Alpha virus Baculovirus Rhabdovirus Polyomavirus Paramyxovirus	Ad5 LacZ, Ad5-PEG HIV, MuLV, MLV HSV-I Sindbis virus VSV SV40 Measles

ViroMag reagent can generally be combined with any viruses. If a particular virus is not listed, this does not imply that ViroMag is not going to work. OZ Biosciences is maintaining an updated list of virus successfully tested that is available on the website: www.ozbiosciences.com.

ViroMag is applicable and has been tested successfully on a variety of immortalized cell lines (293-HEK CHO, B95a, HeLa, HT1080, K562, L, NIH3T3, VERO, BT4C...) and primary cells (PAEC, PBL…). Please consult our updated list of cells successfully tested available on the website: www.ozbiosciences.com. ViroMag is generally applicable on numerous cell types, but if a particular cell type is not listed, this does not imply that ViroMag is not going to work. OZ Biosciences is going to frequently update this list.
 
1) Adenovirus
a) The combination of paramagnetic nanoparticles with adenovirus has shown up to 500-fold enhancement of gene expression compared with standard infection 1,  2 . Transduction of suspension cells (K562 and human peripheral blood lymphocytes) has been seen only with magnetic nanoparticules 1-3. Enhancement of Adenovirus (Ad5 vector) transduction  has also  been  reported on CHO cells 4 . In addition,  magnetic field-guided  local transduction was demonstrated in vivo (stomach) with an adenovirus combines to magnetic nanoparticles 1 .
b) In the same way, Pandori et al. have reported that  nanoparticles conjugate to Adenovirus significantly enhance their ability to transduce target cells in vitro 5 . This approach required a chemical modification of viral envelopes or viral surface proteins to contain the binding moiety to the magnetic nanoparticles. This lengthy approach and the genetic modification of viral envelopes strategy have shown limited success in many occasions and a more straightforward method is preferable. In contrast,  ViroMag allows you to achieve identical results, in one step procedure to associate virus and paramagnetic nanoparticles, without the requirement of genetically or biochemically modifying your virus.
2) Adeno-associatedvirus(AAV). The transduction efficiency of cells infected with AAV bound to magnetic micropsheres has been shown to be 10-fold higher than unbound vectors in HeLa cells 6 . In this report, higher and localized transduction efficiency was also achieved invivowhen AAV bounds to magnetic particles were administered either intramuscularly or intravenously.
3) Retrovirus/Lentivirus
a) Pseudo-typed HIV-I viruses carrying a luciferase reporter gene were produced in 293 cells. Supernatants containing the recombinant HIV-1-Luc viruses (rHIV-Luc) were associated with ViroMagor not at a ratio of 1 µL of ViroMag per mL of rHIV-Luc supernatant. Mixtures were added to U87-CD4-CCR5 cells and luciferase activity was measured at 72 hours. ViroMagclearly increased the HIV-1 infection efficiency as shown in figure below.
b) The infectivity of lentiviruses (HIV-1 and a pseudotype lenti-VSVG) has been shown to be increased by about 100-fold when the virus were adsorbed on magnetic nanoparticles 7 .
c) A magnetic retroviral vectors formed by the combination of paramagnetic nanoparticles and a Retrovirus (Moloney Leukemia Virus) demonstrated major higher gene transduction efficiency 8 .
4) MeaslesVirus. Kadota, S.I., et al. 2005. J. Virol. Methods10
Magnetofection enhanced the infection of adenoviruses and retroviruses. It is also shown that Magnetofection enhances the infection of measles virus, a paramyxovirus 10 . When cells expressing a measles virus receptor human SLAM were infected with a measles virus that encodes the green fluorescent protein, Magnetofection enhanced measles virus infection by 30- to 70-fold. The infection of SLAM-negative cells with measles virus was also enhanced by Magnetofection, but to a lesser extent. These results indicate that Magnetofection could be useful for isolation of measles virus from clinical specimens.
5) Baculovirus invitrotransduction efficiency has been significantly increased with magnetic particles 11 
" concentrate viral dose and accelerate the infection process 1-12
1) Retrovirus/Lentivirus.
a) Concentration of viruses from cell culture supernatants has been reported wherein retroviral titers could be increased by 1000 to 4000 fold 9 .
b) The rate of retroviral infection is primarily limited by diffusion-dependent cell association. Association of a Lentivirus (HIV-I) with magnetic nanoparticles has led to a considerable concentration of the viral dose on cell surface 7 . Cellular uptake of HIV-1 was increased by 70-fold.
c) Concentration and acceleration of the infection process has also been demonstrated with a pseudo-typed HIV-I virus carrying a luciferase reporter gene. Pseudo-typed HIV-I viruses carrying a luciferase reporter gene were  produced  in  293  cells.  Supernatants  containing  the  recombinant  HIV-1-Luc  viruses  (rHIV-Luc)  were associated or not with  ViroMag at a ratio of 1 µL of ViroMag per mL of rHIV-Luc supernatant. Mixtures were added to U87-CD4-CCR5 cells with or without magnetic field. In order to monitor the time course of infection, virus supernatant was washed out after 0.5, 1, 2, 4 and 6 hours and luciferase activity measured at 72 h.
2) Adeno-associatedvirus (AAV). 1% of AAV vector bound to magnetic particles resulted in same level of transduction than 100% of the free vector due mainly to their concentration on cell surfaces 6 .
" Isolate virus from low virus containing samples with ViroMag 10, 12
1) Concentration of measlesvirus10 . Magnetofection allow the detection of transduction when measles virus stock solution (5 x 102 TCID50) was diluted up to 125-fold
2) Non enveloped virus (SV40) and enveloped virus such as Sindbisvirus,HSVtypeIandVSV have also been successfully concentrated with magnetic nanoparticules (up to 100 times for DNA viruses and up to 1000 fold for RNA viruses) to enhance the sensitivity of virus detection by polymerase chain reaction 12 . Satohetal. have reported a reduced infectivity of the viruses associated with magnetic nanoparticles having a size of 0.8µm in diameter and coupled to a very  large  polymer. This  is  not surprising since the  beads size  preclude to internalization and infection and the polymer biological activity is extremely low. In contrast, ViroMag small nanoparticles formulation  is concentrating viruses  in the same way and  is  improving viruses’infectivity as demonstrated for various types of viruses due to their small size and the particularly active polymers associated
1-4, 10, 11.
" Magnetic nanoparticles can restore transduction efficiency
Transduction efficiency of PEGylated adenovirushas been restored by the use of paramagnetic nanoparticles 4 . Polyethylene Glycol (PEG) conjugate to adenoviral capsid can protect the vectors from neutralizing antibodies, vector pharmacokinetics and reduce innate immune response. However, when the adenoviral vector PEGylation provides safe features it also blocks invitrotransduction. Association of PEGylated adenovirus with magnetic particles can restore and even increase transduction efficiency in comparison to unmodified adenovirus.
 
ViroMagimprovesviralinfectiouscapacity
Retrovirus/Lentivirus
a) Enhancement of infectivity with ViroMag. Low retroviral titer preparation was associated to ViroMag and used to transduce NIH-3T3 cells 1-3 . Whereas no transductions were observed with virus alone, ViroMag led in a 20-   fold enhancement over a standard transduction approach consisting of Virus plus polybrene (see figure below).
b) Improvement  of  retrovirus  infectivity  was  also  demonstrated  by  Hughes et al.  20  times  infectivity enhancement was achieved with a high retroviral titer when combined to paramagnetic particles 9 .
c) The  infectivity  of  a  lentivirus  was  shown  to  be  clearly  increased  when  associated  with  paramagnetic nanoparticles in comparison to the virus alone 7 . This enhancement of infectivity was seen even at low MOI.
NIH 3T3 cells were infected with a low titer preparation   of   MuLV (Murine  Leukemia Virus) +/- ViroMag  in the  presence and  in the absence of magnetic field. Conditions:
1-  Standard Transduction
2-  + Polybrene
3-  +  Polybrene  +  ViroMag (no  magnetic
field)
4-  + ViroMag(no magnetic field)
5-  + Polybrene + ViroMag + magnetic field 6-   + ViroMag + magnetic field
 
We are grateful to Dr.  A.  Kruger and C.  Plank (Institute of Experimental Oncology, Munich) for kindlysharingthesedata.
a) Adenoviral infections are dependent on the presence of CAR receptor on the cells surface. Unfortunately, many important and interesting target tissues for fundamental research and gene therapy are non-permissive to viral gene delivery (tumor tissues and apical surface of lung epithelium may express variable, little or none of the required receptors). The association of viral vectors with ViroMag is sufficient to force infection of non- permissive cells (lacking CAR) as shown with adenovirus in NIH 3T3, K562 cells and human peripheral blood lymphocytes 1, 2 .
Without Magnetic field        With Magnetic field
3 x 105  NIH 3T3 cells (lacking CAR) were infected with a constant dose  (200  MOI) of recombinant  adenovirus  (coding for  LacZ)  +/- ViroMag in  the  presence  (right) and  in  the  absence  (left)  of permanent magnets positioned under the culture plates in a 6 well- plate.
1) Virus alone
2) PBS plus ViroMag(6µl)
3) & 4) Virus plus ViroMag(3µl)
5) & 6) Virus plus ViroMag(6µl
WearegratefultoDr. C. PlankandDr. M. Anton(Technical University, Munich)forkindlyprovidingthesedata.
b) Likewise, Pandoriet al. have shown that nanoparticles conjugate to Adenovirusallow transduction of less permissive cell line (C6) with a 20-fold transduction improvement over the free virus 5 . Most importantly as described above, they also demonstrated the possibility to infect cells markedly non-permissive (COLO 205) to adenovirus only after association with nanoparticles. ViroMagallows you to achieve identical results without the requirement of biochemical modification of your virus.
2) Measlesvirus. Infection of SLAM-negative cells (VERO, HeLa, CHO and L cells) with the measles virus has been described with very low dose of virus (5 x 102 TCID50) 10 .
Synchronized infection by HIV-1 of primary endothelial cells was reported with the use of magnetic nanoparticles 7 . The  number  of  productive  adsorption  events  by virus  alone  reached  a  maximal  level  after  20h  which corresponds to a decrease  in  residual  infectivity  left  in the culture  medium.  In  contrast, when virus was complexed  onto  paramagnetic  nanoparticles  optimum  cellular  uptake  was  reached  after  1  minute.  This magnetically controlled viral adsorption is advantageous to synchronize infection and to accurately monitor the kinetics of viral replication cycle.
1) A recombinant adenovirus carrying the LacZ gene (200 MOI) was combined with ViroMagand incubated on NIH 3T3 cells (lacking CAR) in the presence (left) and in the absence (right) of a magnetic field  for  5  minutes.  High  transduction  can  be  achieved  under magnetic  influence  and  can  be  confined  to  the  area  where  the magnet  has  been  positioned.  Similar  results  have  been  published with the same technology 1 .
2) In another study, a specific targeting to define area has been shown with a recombinant adenovirus carrying the LacZgene coupled to microbeads or to paramagnetic microparticles 5 . This paper has used a stylish design to clearly demonstrate the magnetically targeted transduction.
3) Targeting the region of transduction has also been reported with an adeno-associatedviruscomplexed to magnetic microsphere 6 .
4) In the same way, magnetic targeting confine to specific area has been reported with an avidin modified Baculovirus 11 . ViroMag allows you to achieve identical results without the requirement of genetically or biochemically changing your virus.
5) In vitro magnetic targeting has also been demonstrated with Retrovirus 9 . In this manuscript the elegant design of the magnet shape strongly show a confine and specific targeting to the area dictated solely by the presence of the magnet. In another report, site specific delivery was obtained with a magnetic retroviral vector (MLV) 8 .
Please consult our list of references available on the website: www.ozbiosciences.com.
Adenovirus:
1. Scherer F, et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. GeneTher. 2002; 9(2):102-9.
2. Plank C, et al. Enhancing and targeting nucleic acid delivery by magnetic force. Expert Opin BiolTher. 2003; 3(5):745-58.
3. Schillinger, U., et al. Advances in Magnetofection – magnetically guided nucleic acid delivery. 2005. J. Magn. Magn. Mat. 293: 501-508.
4. Mok,  H., et al.  Evaluation of polyethylene glycol  modification of first-generation and  helper-dependent adenoviral vectors to reduce innate immune responses. 2005. Mol. Ther. 11(1): 66-79.
5. Pandori, M.W., et al. Adenovirus-Microbead Conjugates Possess Enhanced Infectivity: A New Strategy to Localized Gene Delivery. 2002. Virology299: 204-212.
Adeno-Associated Virus:
6. Mah, C., et al. Improved Method of Recombinant AAV2 Delivery for Systemic Targeted Gene Therapy. 2002. Mol. Ther. 6(1): 106-112.
Retrovirus / Lentivirus:
7. Haim, H., et al. Synchronized infection of cell cultures by magnetically controlled virus. 2005. J. Virol. 79(1): 622-5.
1. Scherer F, et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. GeneTher. 2002; 9(2):102-9.
8. Tail et al. Generation of magnetic retroviral vectors with magnetic nanoparticles. 2003. Rev. Adv. Mater. Sci. 5:319-323
9. Hughes, C., et al. Streptavidin paramagnetic particles provide a choice of three affinity- based capture and magnetic concentration strategies for retroviral vectors. 2001. Mol. Ther. 3(4): 623-30.
Measles Virus:
10. Kadota, S.I., et al. Enhancing of measles virus infection by magnetofection. 2005. J. Virol. Methods.
Baculovirus:
11. Raty, J.K., et al. Enhanced gene delivery by avidin-displaying baculovirus. 2004. Mol. Ther. 9(2): 282-91.
Alpha virus, Herpes virus, Polyomavirus (SV40)
12. Satoh et al. Virus concentration  using  polyethyleneimine-conjugated  magnetic  beads for  improving the sensitivity of nucleic acid amplification tests. J. Virol. Methods 114: 11- 19