Artificial Viral Envelops: A Novel Carrier in Gene Therapy


G. Ramani*, A. Aparna

Department of Pharmaceutics, Priyadarshini Institute of Pharmaceutical Education and Research, Pulladigunta, Guntur (Dt), Andhra Pradesh, 522017-India.

*Corresponding Author E-mail:




The use of plant viral vectors for the transient expression of heterologous proteins offers a useful tool for the large-scale production of proteins of industrial importance, such as antibodies and vaccine antigens. In the recent years, advances have been made both in the development of first generation vectors (that empoly the ‘full virus’) and second-generation (‘deconstructed virus’) vectors. Artificial viral envelopes (AVE) are lipid vesicles which mimic with fusogenic envelop of retrovirus to utilize as target selectivity along with efficiency of delivery of viruses. The major component of AVE includes phosphatidylcholine, phosphatidyl serine along with sphingomyelins. The negative charge is present on AVE’s help them for targeting the moieties such as viral binding proteins. The artificial viral envelopes show potential applications in plasmid delivery.


KEYWORDS: Artificial viral envelopes (AVE), viral vectors, full virus, deconstructed virus, fusogenic.



In comparison to classical medicines, gene therapy has the potential to mediate the highest possible level of therapeutic specificity. Every normal or diseased cell can switch on or off. A gene expression cassette in a tissue, disease, and time dependent fashion by the use of specific transcription factors that are active only in a given unique situation. Viruses have viral envelops covering their protein capsids. The envelops typically are derived from the portions of the host cell membranes( phospholipids and proteins). 1,2


Fig1: Structure of  Enveloped virus


The viral envelop when fuses with the hosts membrane, allowing the capsid and viral genome to enter and infect the host. These can cause persistant infection.3


Non envelop virus does not contain glycoprotein envelop and the capsid solely responsible for the attachment of the virus to the infected cell. These are more resistant to environmental conditions.


Polyplexes are the conjugated viral envelops that contain shields of poly ethylene glycol or other chemicals. Higer degree of PEG polycation conjugates with PH –labile linkage show longer circulation time and better accumulation at the target site.4-6


Factors to be considered in Gene therapy 7,8

·        How to deliver genes to specific cells, tissue and whole animals? (methods of delivery)

·        How much and how long the introduced gene will be expressed? The site and dose of gene delivery            

·        Is there any adverse immunological consequence of both delivery vehicle (Virus) and the gene in animals?

·        Is there any toxic effects?


Methods of gene delivery

Ø  Viral Vectors:

·        Adenovirus

·        Retrovirus

·        Lentivirus

·        Adeno-associated virus (AAV)

·        Herpes simplex virus (HSV)

Ø  Non-viral vectors:

·        Naked DNA (plasmid DNA): injection or genegun

·        Liposomes (cationic lipids): mix with genes

·        Ex-vivo

·        In vivo


Why use viral vectors 8-10

ü  Virus are obligate intracellular parasites

ü  Very efficient at transferring viral DNA into host cells

ü  Specific target cells: depending on the viral attachment proteins (capsid or glycoproteins)

ü  Gene replacement: non-essential genes of virus are deleted and exogenous genes are inserted



Generation of viral vector for gene therapy

ü  Replication-competent virus  

ü  Replication-defective virus

ü  Amplicon( doesn’t encode structural proteins .Can’t replicate beyond the first cycle of infection)


Elements needed to generate amplicon

·        Transfer Vector: plasmid (promoter, gene of interest, ori, packaging signal)

·        Packaging vector (cosmid or cell lines): provide the viral structural proteins for packaging of transfer vector

·        Helper virus (packaging of transfer vector): deleted Packaging signal sequence



Early generations of adenoviral vectors (replication defective) 11-14


Fig2: First generation adenovirus vectors are made by substituting an expression cassette for the E1 andor E3 regions. The E1 region, located at the left end of the adenovirus genome, encodes proteins necessary for the expression of the other early and late genes. The E1 region encodes proteins necessary for the expression of the other early and late genes.


The E3 region encodes products that counteract host defense mechanisms; these products are not essential for viral replication in vitro, and therefore no complementing cell line is necessary. Since adenovirus can package approximately 38 kb without affecting growth rate and viral titer. E1-deleted adenoviruses can accept insertions , while E1E3-deleted viruses allow the cloning.



Second generation of adenoviral vectors (replication-competent) 15


Fig3: First-generation adenovirus vectors elicit a significant immune response in vivo, mainly due to the de novo synthesis of viral proteins. Therefore, additional genes necessary for viral DNA replication have been inactivated, giving rise to the second generation of adenovirus vectors. Various cell lines have been constructed that express the E2a DNA-binding protein, the E2b-encoded terminal protein and viral DNA polymerase or all or most of the E4 products. The corresponding deletions on the viral genome should allow the insertion of expression cassettes up to 14 kb.


Gutless Adenoviral vector (Amplicon) 16-23


Fig4: Generation of gutless adenovirus using the Cre/loxP system. Gutless and helper genomes are cotransfected in permissive 293 Cre-expressing cells, where both genomes are amplified and viral proteins produced. Then, packaging signal of the helper’s genome is excised bye Cre recombinase, preventing its packaging into the viral capsid, while gutless genome is still pakageable. Efficiency of the excision process allows 90-99.9% purity of the gutless vector.


Chemically modified viral vectors 24-25


Fig5: NA, Nucleic Acid, chemo-virus, chemically-modified virus. Coating of adenoviral vectors with hydrophilic polymers such as PEG  or poly-N-2-hydroxypropyl-methacrylamide (pHPMA)  shielded the virus from interaction with its native receptor or neutralizing antibodies and the incorporated ligands enabled retargeting. A very encouraging example of systemic adenovirus retargeting was recently described. PEGylated adenoviruses were retargeted using anti-E-selectin antibodies. These viruses showed longer persistence in the blood circulation compared with unmodified viruses and selectively targeted inflamed skin in mice resulting in local gene expression.


Table 1: Strengths and weaknesses of currently used vectors: 26,27


Greatest advantages

Greatest disadvantages

Retro virus

Exvivo stable transduction of blood cells

Low efficiency in vivo-risk of insertional mutagenesis

Adeno virus

High short term expression in vivo

(in liver)

Immunogenecity and inflammatory responces

Adeno-associated virus

Long term expression in vivo

(high effeciency on particle basis)

Small genome. Norepeated administration because of immunogenicity.

Naked DNA

Simple(example for vaccination)

Low efficiency

Physically enhanced delivery of DNA (elecroporation gene, gun, hydrodynamic delivery)

High expression in vivo

Limited localised area, device required.


Stragies towards specific gene therapy therapeutic nuclic acids:28

DNA (Oligo nucleotides,plamids,viruses,artificial chromosomes,bacteria)

RNA  (Oligo nucleotides,ribosomes,s-RNA,m-RNA,viruses)

Effects of theraupetic nuclic acids at the molecular genetic level:

Gain of gene function(gene substitution or replacement, trans slicing)

Loss of gene function(anti-sense, RNA interference)

Strategies to achieve specificity for the target tissue:

Targeted delivery of nuclic acids

Targeted transcription of expression cassettes

Main barriers for gene delivery and how to overcome them:29-31

1. Extracellular delivery and targetting


Fig6: Extracellular targetting of artificial viral envelops. Naked DNA is degraded by serum nucleases, condensation or encapsulation of DNA within vectors using viral capsids, cationic polymers, or lipids has greatly improved the stability of the delivered DNA. However both viral and nonviral vectors suffer from weak to nonexisting specificity to the target tissue (see previous section). To increase specificity, the concept of targeted delivery was developed, which is currently the most attractive concept to achieve specificity and, in principle, this strategy is applicable for all current vectors.


To provide a vector with the ability to distinguish between target and nontarget tissue, cell binding ligands have to be incorporated that recognize target-specific cellular receptors. In addition, vector domains with undesired binding potential to blood or nontarget cells (eg, natural receptor binding proteins in viral vectors, positive surface charge in nonviral vectors) have to be shielded or removed.


Table 2: Extracellular targetting:

Degradation of DNA by serum nucleases

DNA condensation or encapsulation with cationic polymers or lipids

Non-specific interactions with blood components

Surface shielding with hydrophilic polymers exploit vascular abnormalities

Targetting of DNA to diseased tissue

Specific targetting ligands in combination with surface shielding(Ex: passive targetting to tumors)


2. Intracellular delivery and persistence of gene expression32,33


Fig7: Intracellular targetting of artificial viral envelops. Once the vectors have reached the target cells, vector particles are internalized via cell fusion in the case of some enveloped viruses or via receptor-mediated endocytosis, macropinocytosis, phagocytosis, or related processes in the case of lipid-free viruses and most lipoplexes and polyplexes. For successful transgene expression, several intracellular barriers then have to be overcome. vector particles need to survive in and escape from the endosome, traffic through the cytoplasm toward the nucleus, enter the nucleus, and expose the DNA to the cell's transcription and translation machinery. Especially for first-generation nonviral vectors, some of these intracellular barriers present formidable hurdles. The first step, intracellular uptake, can be taken rather easily by nonviral vectors, as appropriate receptor binding ligands may enhance binding and intracellular uptake of particles into endosomal vesicles; however, many formulations do not mediate subsequent release of particles to the cytosol. Although the “proton sponge” effect of polyethylenimine (PEI, see section “Optimizing Synthetic Viruses”) is believed to contribute to endosomal release, this effect is far from being sufficient. Nuclear entry of nonviral vectors is another big hurdle, which is currently only easily overcome in rapid-dividing cells; for example, transfection of nondividing cells with lipoplexes or PEI polyplexes was several log units less effective compared with transfection of mitotic cells where the nuclear envelope had broken down.


Table 3: Intracellular delivery and persistence ofgene expression:

DNA uptake by cells

Specific targeting ligands to enhance receptor mediated endocytosis

Endosomal escape

Membrane active peptides (eg: melittin analogs) polycationic “proton sponges” (eg: polyethylene Imines)

Transport from the cytosol in to the nuclease

Small particles to enhance cytosolic migration. Nuclear localisation signal peptides

Gene silencing

Use mini circiles (devoid of bacterial sequences)


3. Host response and toxicity34

All efforts that are made to increase the in vivo gene delivery efficiency of vectors will allow substantial reduction of the therapeutic vector dose. This obviously will strongly reduce the acute and also the long-term toxicity of vector application. Shielding the positive surface charge of lipoplexes and polyplexes with PEG greatly reduced acute toxicity of nonviral vectors.To further improve the safety profile of nonviral vectors, excess cationic reagents that are not incorporated in the nonviral vector should be removed ( “Generate Purified Vector Particles”), since these reagents exert significant cellular toxicity. In addition, preferentially biodegradable cationic DNA carriers  should be applied to reduce any long-term toxicity hazards.


The infection or transfection process of many viral and nonviral vectors can trigger host cell responses, such as inflammatory and immune responses. Although the immunogenicity of nonviral vectors can be reduced to a minimum, up to now no solution was found to prohibit host cell response after viral vector application. Strategies of immune-suppressive cotreatment, resulting in transient immune suppression during and shortly after the infection process, may help to avoid immune responses and early loss of expressing cells. In addition, chemical modification of the virus such as PEGylation may contribute to reduce immunogenicity of viral vectors.


Table 4:  Methods to avoid toxicity and immune cell response

Acute toxicity

Surface sheilding to avoid agregation in blood. Removal of contaminating poly cations, bacterial lipo polysacharides.

Cytotoxicity and long term toxicity

Biodegradable cationic gene carriers.

Immune cell responses

Transient immuno suppressant


Schematic flow chart for the encapsulation of required gene into the artificial viral envelops 35,36



Storage ( freeze drying / refregiration)

·        Polmerization of phospholipids improve the stability of liposomes.

·        Freeze drying of vesicles prevents the need for keeping vaccines refregiration.

·        Viral proteins should be enveloped on the outer side surface of envelop.

·        Whole protein / antigenic determinants / epitopes may be used for insertion.


Table 5: Composition of PBS:


137 mM


2.7 mM


8.1 mM


1.5 mM

Sodium azide 

0.5 mM


1. Used as sub unit vaccines and vectors.

2.In destruction of viruses or specific cells such as cancer cells.

3.The enevelops can be appropraitely modified to play a role in the modulation of biological pathways or reactions,especially in the endocrine and immune systems.

4.These enevelops can be targetted to T-cell lymphocytes and their sub populations.

5. Highly targetable and fusogenic drug delivery devices for delivery of antiviral agents to infected  cells.

6. Highly targetable and fusogenic drug delivery devices for delivery of highly specific cell destructing agents.

7. Highly efficient transfection device for the introduction of genetic materials including DNA, plasmids and oligodeoxynucleotides, into animal, bacterial, and plant cells.

8. Highly efficient device for the intra cellular delivery of macro molecules including peptides and proteins.

9. Non-biohazard in vitro model systems for viral infectivity.

10. Basic research:

·        An alternative to transfection of naked DNA.

·        High transfection rate (nearly 100% of cells).

·         Construction of a viral vector is a much more laborious process.

11. Gene therapy:

Several gene therapy trials & a huge number of laboratory successes.

·        Immune response to viruses.

      (a mortality case in 1999: an adenoviral vector)

·        Insert genomes on host chromosome: possibility of cancer formation.

      (two cases with leukemia in 2002: SCID retroviral gene therapy)

·        AAV (adeno-associated virus)-based vectors are much safer:

      they always integrate at the same site in the human genome.

12. Vaccines:

·        Viruses expressing pathogen proteins.

·        A kind of DNA vaccines which activate T cells.

·        Adenoviruses are being actively developed as vaccines.



Artificial viral enevelops are similar to enveloped virus particle and apply as a synthetic vesicular DNA carrier which consists of reverse process of preparation of cationic lipid –DNA complexes for high degree of encapsulation eficiency.The condensed DNA interaccts spontaneously with a liposome containing one or more anionic components by using certain critical concentration of condensing agent.



1.       Kircheis R, Schuller S, Brunner S, et al.  Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. J Gene Med. 1999;1:111-120.

2.       Goncalves C, Mennesson E, Fuchs R, Gorvel JP, Midoux P, Pichon C.  Macropinocytosis of polyplexes and recycling of plasmid via the clathrin-dependent pathway impair the transfection efficiency of human hepatocarcinoma cells. Mol Ther. 2004;10:373-385.

3.       Kopatz I, Remy JS, Behr JP.  A model for non-viral gene delivery: through syndecan adhesion molecules and powered by  actin. J  Gene Med. 2004;6:769-776.

4.       Rejman J, Bragonzi A, Conese M.  Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther. 2005;12:468-474.

5.       Sonawane ND, Jr, Szoka FC, Jr, Verkman AS.  Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol Chem. 2003;278:44826-44831.

6.       Brunner S, Sauer T, Carotta S, Cotten M, Saltik M, Wagner E.  Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 2000;7:401-407.

7.       Fischer A, Hacein-Bey-Abina S, Lagresle C, Garrigue A, Cavazana-Calvo M.  Gene therapy of severe combined immunodeficiency disease: proof of principle of efficiency and safety issues. Gene therapy, primary immunodeficiencies, retrovirus, lentivirus, genome. Bull Acad Natl Med. 2005;189:779-785. PubMed

8.       Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ.  Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem. 1995;270:18997-19007.

9.       Lim YB, Kim SM, Suh H, Park JS.  Biodegradable, endosome disruptive, and cationic network-type polymer as a highly efficient and nontoxic gene delivery carrier. Bioconjug Chem. 2002;13:952-957.

10.     Kim YH, Park JH, Lee M, Kim YH, Park TG, Kim SW.  Polyethylenimine with acid-labile linkages as a biodegradable gene carrier. J Control Release. 2005;103:209-219.

11.     Gilgenkrantz H et al. Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: role of the immune response. Hum Gene Ther 1995; 6: 1265-1274.

12.     Amalfitano A, Begy C, Chamberlain J. Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors. Proc Natl Acad Sci USA 1996; 93: 3352-3356.

13.     Zhou H, O'Neal W, Morral N, Beaudet A. Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted. J Virol 1996; 70: 7030-7038.

14.     Gorziglia M et al. Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy. J Virol 1996; 70: 4173-4178,

15.     Lusky M et al. In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted. J Virol 1998; 72: 20222032,

16.     Langer S, Schaack J. 293 cell lines that inducibly express high levels of adenovirus type 5 precursor terminal protein. Virology 1996; 221: 172-179,

17.     Gorziglia M et al. Generation of an adenovirus vector lacking E1, E2a, E3, and all of E4 except open reading frame 3. J Virol 1999; 73: 6048-6055

18.     He T-C et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998; 95: 2509-2514,

19.     Brough D et al. A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions E1 and E4. J Virol 1996; 70: 6497-6501

20.     Gao G, Yang Y, Wilson J. Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy. J Virol 1996; 70: 8934-8943,

21.     Yeh P et al. Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit. J Virol 1996; 70: 559-565,

22.     Krougliak V, Graham F. Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants. Hum Gene Ther 1995; 6: 1575-1586,

23.     Wang Q, Jia X, Finer M. A packaging cell line for propagation of recombinant adenovirus vectors containing two lethal gene-region deletions. Gene Therapy 1995; 2: 775-783,

24.     Croyle MA, Chirmule N, Zhang Y, Wilson JM.  “Stealth” adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. J Virol. 2001;75:4792-4801.
PubMed  DOI: 10.1128/JVI.75.10.4792-4801.2001.

25.     Lanciotti J, Song A, Doukas J, et al.  Targeting adenoviral vectors using heterofunctional polyethylene glycol FGF2 conjugates. Mol Ther. 2003;8:99-107. PubMed  DOI: 10.1016/S1525-0016(03)00139-4.

26.     Niidome T, Huang L.  Gene therapy progress and prospects: nonviral vectors. Gene Ther. 2002;9:1647-1652.PubMed  DOI: 10.1038/

27.     McCormick F.  Cancer gene therapy: fringe or cutting edge? Nat Rev Cancer. 2001;1:130-141.PubMed  DOI: 10.1038/35101008.

28.     Wu GY, Wu CH.  Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem. 1987;262:4429-4432.

29.     Nettelbeck DM, Miller DW, Jerome V, et al.  Targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105). Mol Ther. 2001;3:882-891.PubMed  DOI: 10.1006/mthe.2001.0342.

30.     Kircheis R, Schuller S, Brunner S, et al.  Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. J Gene Med. 1999;1:111-120. PubMed  DOI: 10.1002/(SICI)1521-2254(199903/04)1:2<111::AID-JGM22>3.0.CO_2-Y

31.     Chollet P, Favrot MC, Hurbin A, Coll JL.  Side-effects of a systemic injection of linear polyethylenimine-DNA complexes. J Gene Med. 2002;4:84-91.PubMed  DOI: 10.1002/jgm.237.

32.     Goncalves C, Mennesson E, Fuchs R, Gorvel JP, Midoux P, Pichon C.  Macropinocytosis of polyplexes and recycling of plasmid via the clathrin-dependent pathway impair the transfection efficiency of human hepatocarcinoma cells. Mol Ther. 2004; 10: 373-385. PubMed  DOI: 10.1016/j. ymthe.2004.05.023

33.     Rejman J, Bragonzi A, Conese M.  Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther. 2005;12:468-474. PubMed  DOI: 10.1016/j.ymthe.2005.03.038.

34.     Lim YB, Kim SM, Suh H, Park JS.  Biodegradable, endosome disruptive, and cationic network-type polymer as a highly efficient and nontoxic gene delivery carrier. Bioconjug Chem. 2002;13:952-957.PubMed  DOI: 10.1021/bc025541n.

35.     Weder, H.G. Zumbuehl(1984), The preparation of variably sized homogenous liposomes for laboratory, clinical and industrial use by controlled detergent dialysis, Liposome technology1:79-107.

36.     Deamer, D.W.P.S. Ulster(1983) “Liposomes preparation: Methods and mechanisms”. Pp.27-51.

37.     R. G. Crystal. The gene as the drug. Nature Med., 1:15–17, 1995.

38.     F. L. Sorgi and H. Schreier. Non-viral vectors for gene delivery, Biopharmaceutical Drug Design and Development (S. Wu-Pong, and Y. Rojanasakul, eds.), The Humana Press, Totowa, N.J., p. 107–142, 1999.

39.     X. Gao and L. Huang. Cationic liposome-mediated gene transfer. Gene Therapy, 2: 710–722 1995.

40.     H. Schreier, and S. M. Sawyer. Liposomal DNA vectors for cystic fibrosis gene therapy. Current applications, limitations, and future directions. Adv. Drug Del. Rev., 19:73–87, 1996.

41.     H. Schreier, M. Ausborn, S. Gu¨nther, V. Weissig, and R. Chander. (Patho) physiologic pathways to drug targeting: artificial viral envelopes. J. Mol. Recognit., 8:59– 62, 1995.

42.     R. Chander and H. Schreier. Artificial viral envelopes containing recombinant HIV gp160. Life Sci., 50:481–489, 1992.




Received on 27.02.2013       Modified on 04.04.2013

Accepted on 13.04.2013      © RJPT All right reserved

Research J. Pharm. and Tech. 6(5): May 2013; Page 477-485