Birnstiel, Max L.

Buschle, M.

Cotten, Matt

Guy, Richard H.

Plank, Christian

Wagner, Ernst

Zauner, W.

English

Open Access LMU

# 102 SYNTHETIC VIRUS-LIKE GENE DELIVERY SYSTEMS E. Wagner1»2, Μ. Buschle2, Μ. Cotten1, C. Bank1, W. Zauner1, and M.L. Birnstiel1 Research Institute of Molecular Pathology and 2Bender&Co, Vienna, Austria. Introduction Our work has been based on the concept of adopting natural transport mechanisms for the uptake of nucleic acids into cells. In order to exploit the transferrin receptor-mediated endocytosis route for import of DNA, the iron transport protein transferrin has been chemically conjugated to the DNA-binding poly cation polylysine (1,2). Polylysine upon binding to the DNA also condenses it to a donut-like particle (3). Transferrin-polylysine/DNA complexes are efficiently delivered into the endosomes of many types of ceils (see Fig.l). In some cell lines high-level gene expression has ban found, in many cell types, however, low or no expression has been observed. It became clear that the DNA is accumulating in internal vesicles and subsequent steps that deliver the DNA to the nucleus work only inefficiently. Strategies had to be developed to prevent degradation of the DNA in lysosomal compartments and promote the transport to the nucleus. DNA DNA is within cell yet separated from the cytoplasm Figure 1 Gene transfer via the transferrin (Tf) receptor-mediated endocytosis pathway. Viruses have acquired special mechanisms to release their genome from endosomes into the cytoplasm. In the case of membrane-free viruses (such as adenoviruses) the endosomal acidification process specifically activates viral coat protein domains that trigger disruption of the endosomal membrane. In the case of enveloped viruses (such as influenza virus) the viral membrane fuses with endosomal membranes. This has led us to the development of virus-like gene transfer complexes (4-7) consisting of DNA gene constructs completed with a polylysine-conjugated cell-binding ligand (such as transferrin) and polylysine-conjugated, endosome-disruption agents (such as replication-defective adenoviruses or synthetic peptides derived from viral fusion sequences) which allow cytoplasmic entry of the DNA. Transferrin-polylysine / DNA complexes linked to replication-defective and chemically (psoralen /UV) inactivated adenoviruses (6) have been delivered to and expressed at very high level in a large proportion of target cells (up to 80% in primary fibroblasts, primary myoblasts or primary human melanoma ceils). The delivered gene is carried on the exterior of the adenovirus, being therefore far less restricted in size or sequence of the DNA to be delivered. Transfer of 48 kb DNA molecules, and high level expression of the full length (8 kb) human factor V in cDNA in primary fibroblasts, myoblasts and myotubes has been demonstrated. Results One of OUT major goal has been the development of completely synthetic versions of the gene transfer system. We have replaced the whole virus by small synthetic peptides similar to sequences occurring in the hem^glutinin of influenza virus or sequences in the VP-1 protein of rhinoviruses. At neutral pH these peptides have an inactive, rather unordered structure, whereas at acidic, endosomal pH they adopt the active structure of an amphipathic helix that can interact with the lipid membrane and destabilize it. The peptides are able to disrupt liposomes, erythrocytes, or endosomal membranes triggered by the change to lower pH. We have incorporated endosome-disruptive peptides into DNA complexes either via covalent conjugation to polylysine (6,7) or by ionically binding to DNA/ polylysine complexes (see Fig.2). As a first step in assembling the complex, half of the negative charges of the plasmid DNA were saturated with transferrin-polylysine to provide a ligand for receptor-mediated endocytosis. The remaining nucleic acid charges were utilized for binding an excess of polylysine. To the resulting positively charged complex the negatively charged peptide was added to provide the endosome-disrupting activity. The complexes were used for transfection of cell lines such as murine BNLC1.2 hepatocytes, NIH 3T3 fibroblasts, Β16 melanoma cells, or primary human melanoma cells. Reporter genes (luciferase, β-galactosidase) or therapeutically more relevant genes (cytokines interleukin-2, GM-CSF; see next page) have been delivered. The (up to 10 000-fold) enhancement of gene expression mediated by the peptide strongly correlated with the capacity of peptides to lyse erythrocytes in an acidic environment Proceed. Intern. Symp. Control. Ref. β/oact Mater., 21 (1994), Controlled Release Society, tnc. -3-# 102 Figure 2 Synthetic virus-like gene transfer complexes. Complexes consisting of DNA, transferrin-polylysine conjugates (TfpLys) and membrane-disruptive peptides ionically (pLys, Peptide) bound to poiylysine. Conclusions What are the future options? The delivery of genes to tumor cells is considered to be an exceptionally well-suited application of transferrin receptor-mediated gene delivery. Rapidly proliferating tumor cells can be transfected with highest efficiency, but also slowly-dividing or non-dividing cells are accessible for receptor-mediated gene transfer. One might consider using the system to deliver toxin genes directly into the tumor mass for elimination of these cells; however, the poor survival of our complexes in the blood stream coupled with the requirement to introduce the gene into most cells of the tumor limits this application. We favor the concept of generating cytokine gene-modified melanoma vaccines (8). These are supposed for application in patients with minimum residual disease, where most of the tumor mass has been removed by surgery. A tumor cell culture has to be established, transfected with a immunstimulatory agent such as a cytokine gene, irradiated with 100 Gy in order to block replication. Such a cytokine-gene modified irradiated vaccine when injected s.c. into the patient is supposed to induce a systemic immune response against residual tumor cells that otherwise lead to metastasis formation. Cytokines are important actors in the activation of an immune response. Interleukin 2 (IL-2) is produced by stimulated T-helper cells that have seen e.g. tumor-antigen presented by cells such as macrophages or dendritic cells. EL-2 also stimulates effector cells such as CTLs that are able to eliminate residual tumor cells in the patients1 body. IL-2 expressing tumor cells should have the ability to directly activate a cellular immune response. In mouse models using syngeneic melanoma cells (see ref. (8) and our unpublished results) the vaccination with gene-modified tumor cells prevented the development of tumor in the prophylactic setting (vaccination before -4-challenge with unmodified tumor cells) or the therapeutic setting (vaccination of animals bearing small tumors). Transfection of human melanoma cultures (preferably with low passage numbers, ^10 passages) by adenovirus dll014 / transferrin-polylysine / DNA complexes resulted in high interieukin-2 expression levels between 1 000 and 190000 units IL-2. Currently we are also evaluating peptide-transfection complexes as alternative to the use of adenovirus complexes. Murine B16 melanoma cells and primary human melanoma cells were transfected efficiently using the peptide method, resulting in high levels of the cytokines GM-CSF (up to 6 000 ng / 24 hrs / ΙΟ6 Β16 cells) or IL-2 ( up to 12 000 units / 24 hrs / 106 human melanoma cells) secreted by the cells transfected with the corresponding genes. These results suggest that the synthetic virus-like DNA complexes may be useful for gene therapeutic application. References 1 Wagner, E . , Zenke, M., Cotten, M., Beug, Η. and Birnstiel, MX., Proc. Natl. Acad. Sei. USA 87, 3410-3414(1990). 2 Wagner, E., Cotten, M., Meditier, Κ., Kirlappos, Η. and Birnstiel, M i . , Bioconjugate Chem. 2, 226-231 (1991). 3 Wagner, E., Cotten, M., Foisner, R. and Birnstiel, M.L., Proc. Natl. Acad. Sei. USA 88, 4255-4259 (1991) . 4 Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K., Curiel, D.T. and Birnstiel, M.L., Proc. Natl. Acad. Sei. USA 89, 6099-6103 (1992). 5 Cotten, M., Wagner, E., Zatloukal, K., Phillips, S., CurieUD.T. and Birnstiel, M.L., Proc. Natl. Acad. Sei. USA 89, 6094-6098 (1992). 6 Wagner, E., Plank, C, Zatloukal, K„ Cotten, M. and Birnstiel, MX., Proc. Natl. Acad. Sei. USA 89, 7934-7938 (1992). 7 Plank, C, Zatloukal, K., Cotten, M.. Mechtler. K. and Wagner, E„ Bioconjugate Chem. 3, 533-539 (1992) . 8 Zatloukal, K., Schmidt, W., Cotten, M., Wagner, E., StingI, G. and Birnstiel, M.L., Gene 135, 199-207 (1993) . Acknowledgements We thank Claudia Denk, Helen Kirlappos, Margareta Kursa, Karl Mechtler, Medhya Saltik, Ursula Scheer and Alexandra Sinski for their excellent technical assistance. We are grateful to Dr. Gotthold Schaffner for providing the Plasmid pGShIL-2tetr. We thank Dr. Susanne Schreiber and Dr. Georg StingI for providing melanoma isolates. Proceed. Intern. Symp. Control. Reh Bioact Mater., 21 (1994), Controlled Release Society, Inc. 

Synthetic virus-like gene delivery systems

Universität München: Elektronischen Publikationen

# 102 
SYNTHETIC VIRUS-LIKE GENE DELIVERY SYSTEMS 
E. Wagner1»2, Μ. Buschle2, Μ. Cotten1, C. Bank1, W. Zauner1, and M.L. Birnstiel1 
Research Institute of Molecular Pathology and 2Bender&Co, Vienna, Austria. 
Introduction 
Our work has been based on the concept of adopting 
natural transport mechanisms for the uptake of nucleic 
acids into cells. In order to exploit the transferrin receptor-
mediated endocytosis route for import of DNA, the iron 
transport protein transferrin has been chemically 
conjugated to the DNA-binding poly cation polylysine 
(1,2). Polylysine upon binding to the DNA also condenses 
it to a donut-like particle (3). Transferrin-polylysine/DNA 
complexes are efficiently delivered into the endosomes of 
many types of ceils (see Fig.l). In some cell lines high-
level gene expression has ban found, in many cell types, 
however, low or no expression has been observed. It 
became clear that the DNA is accumulating in internal 
vesicles and subsequent steps that deliver the DNA to the 
nucleus work only inefficiently. Strategies had to be 
developed to prevent degradation of the DNA in lysosomal 
compartments and promote the transport to the nucleus. 
DNA 
DNA is within cell yet 
separated from the cytoplasm 
Figure 1 Gene transfer via the transferrin (Tf) 
receptor-mediated endocytosis pathway. 
Viruses have acquired special mechanisms to release 
their genome from endosomes into the cytoplasm. In the 
case of membrane-free viruses (such as adenoviruses) the 
endosomal acidification process specifically activates viral 
coat protein domains that trigger disruption of the 
endosomal membrane. In the case of enveloped viruses 
(such as influenza virus) the viral membrane fuses with 
endosomal membranes. This has led us to the development 
of virus-like gene transfer complexes (4-7) consisting of 
DNA gene constructs completed with a polylysine-
conjugated cell-binding ligand (such as transferrin) and 
polylysine-conjugated, endosome-disruption agents (such 
as replication-defective adenoviruses or synthetic peptides 
derived from viral fusion sequences) which allow 
cytoplasmic entry of the DNA. 
Transferrin-polylysine / DNA complexes linked to 
replication-defective and chemically (psoralen /UV) 
inactivated adenoviruses (6) have been delivered to and 
expressed at very high level in a large proportion of target 
cells (up to 80% in primary fibroblasts, primary myoblasts 
or primary human melanoma ceils). The delivered gene is 
carried on the exterior of the adenovirus, being therefore 
far less restricted in size or sequence of the DNA to be 
delivered. Transfer of 48 kb DNA molecules, and high 
level expression of the full length (8 kb) human factor V in 
cDNA in primary fibroblasts, myoblasts and myotubes has 
been demonstrated. 
Results 
One of OUT major goal has been the development of 
completely synthetic versions of the gene transfer system. 
We have replaced the whole virus by small synthetic 
peptides similar to sequences occurring in the 
hem^glutinin of influenza virus or sequences in the VP-1 
protein of rhinoviruses. At neutral pH these peptides have 
an inactive, rather unordered structure, whereas at acidic, 
endosomal pH they adopt the active structure of an 
amphipathic helix that can interact with the lipid membrane 
and destabilize it. The peptides are able to disrupt 
liposomes, erythrocytes, or endosomal membranes 
triggered by the change to lower pH. 
We have incorporated endosome-disruptive peptides 
into DNA complexes either via covalent conjugation to 
polylysine (6,7) or by ionically binding to DNA/ 
polylysine complexes (see Fig.2). As a first step in 
assembling the complex, half of the negative charges of the 
plasmid DNA were saturated with transferrin-polylysine to 
provide a ligand for receptor-mediated endocytosis. The 
remaining nucleic acid charges were utilized for binding an 
excess of polylysine. To the resulting positively charged 
complex the negatively charged peptide was added to 
provide the endosome-disrupting activity. The complexes 
were used for transfection of cell lines such as murine 
BNLC1.2 hepatocytes, NIH 3T3 fibroblasts, Β16 
melanoma cells, or primary human melanoma cells. 
Reporter genes (luciferase, β-galactosidase) or 
therapeutically more relevant genes (cytokines interleukin-
2, GM-CSF; see next page) have been delivered. The (up 
to 10 000-fold) enhancement of gene expression mediated 
by the peptide strongly correlated with the capacity of 
peptides to lyse erythrocytes in an acidic environment 
Proceed. Intern. Symp. Control. Ref. β/oact Mater., 21 (1994), Controlled Release Society, tnc. -3-
# 102 
Figure 2 Synthetic virus-like gene transfer 
complexes. Complexes consisting of DNA, transferrin-
polylysine conjugates (TfpLys) and membrane-disruptive 
peptides ionically (pLys, Peptide) bound to poiylysine. 
Conclusions 
What are the future options? The delivery of genes to 
tumor cells is considered to be an exceptionally well-suited 
application of transferrin receptor-mediated gene delivery. 
Rapidly proliferating tumor cells can be transfected with 
highest efficiency, but also slowly-dividing or non-
dividing cells are accessible for receptor-mediated gene 
transfer. One might consider using the system to deliver 
toxin genes directly into the tumor mass for elimination of 
these cells; however, the poor survival of our complexes in 
the blood stream coupled with the requirement to introduce 
the gene into most cells of the tumor limits this application. 
We favor the concept of generating cytokine gene-
modified melanoma vaccines (8). These are supposed for 
application in patients with minimum residual disease, 
where most of the tumor mass has been removed by 
surgery. A tumor cell culture has to be established, 
transfected with a immunstimulatory agent such as a 
cytokine gene, irradiated with 100 Gy in order to block 
replication. Such a cytokine-gene modified irradiated 
vaccine when injected s.c. into the patient is supposed to 
induce a systemic immune response against residual tumor 
cells that otherwise lead to metastasis formation. Cytokines 
are important actors in the activation of an immune 
response. Interleukin 2 (IL-2) is produced by stimulated T-
helper cells that have seen e.g. tumor-antigen presented by 
cells such as macrophages or dendritic cells. EL-2 also 
stimulates effector cells such as CTLs that are able to 
eliminate residual tumor cells in the patients1 body. IL-2 
expressing tumor cells should have the ability to directly 
activate a cellular immune response. 
In mouse models using syngeneic melanoma cells (see 
ref. (8) and our unpublished results) the vaccination with 
gene-modified tumor cells prevented the development of 
tumor in the prophylactic setting (vaccination before 
-4-
challenge with unmodified tumor cells) or the therapeutic 
setting (vaccination of animals bearing small tumors). 
Transfection of human melanoma cultures (preferably with 
low passage numbers, ^10 passages) by adenovirus 
dll014 / transferrin-polylysine / DNA complexes resulted 
in high interieukin-2 expression levels between 1 000 and 
190000 units IL-2. Currently we are also evaluating 
peptide-transfection complexes as alternative to the use of 
adenovirus complexes. Murine B16 melanoma cells and 
primary human melanoma cells were transfected efficiently 
using the peptide method, resulting in high levels of the 
cytokines GM-CSF (up to 6 000 ng / 24 hrs / ΙΟ6 Β16 
cells) or IL-2 ( up to 12 000 units / 24 hrs / 106 human 
melanoma cells) secreted by the cells transfected with the 
corresponding genes. These results suggest that the 
synthetic virus-like DNA complexes may be useful for 
gene therapeutic application. 
References 
1 Wagner, E . , Zenke, M., Cotten, M., Beug, Η. and 
Birnstiel, MX., Proc. Natl. Acad. Sei. USA 87, 
3410-3414(1990). 
2 Wagner, E., Cotten, M., Meditier, Κ., Kirlappos, Η. 
and Birnstiel, M i . , Bioconjugate Chem. 2, 226-231 
(1991). 
3 Wagner, E., Cotten, M., Foisner, R. and Birnstiel, 
M.L., Proc. Natl. Acad. Sei. USA 88, 4255-4259 
(1991) . 
4 Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, 
H., Mechtler, K., Curiel, D.T. and Birnstiel, M.L., 
Proc. Natl. Acad. Sei. USA 89, 6099-6103 (1992). 
5 Cotten, M., Wagner, E., Zatloukal, K., Phillips, S., 
CurieUD.T. and Birnstiel, M.L., Proc. Natl. Acad. 
Sei. USA 89, 6094-6098 (1992). 
6 Wagner, E., Plank, C, Zatloukal, K„ Cotten, M. and 
Birnstiel, MX., Proc. Natl. Acad. Sei. USA 89, 
7934-7938 (1992). 
7 Plank, C, Zatloukal, K., Cotten, M.. Mechtler. K. 
and Wagner, E„ Bioconjugate Chem. 3, 533-539 
(1992) . 
8 Zatloukal, K., Schmidt, W., Cotten, M., Wagner, E., 
StingI, G. and Birnstiel, M.L., Gene 135, 199-207 
(1993) . 
Acknowledgements 
We thank Claudia Denk, Helen Kirlappos, Margareta 
Kursa, Karl Mechtler, Medhya Saltik, Ursula Scheer and 
Alexandra Sinski for their excellent technical assistance. 
We are grateful to Dr. Gotthold Schaffner for providing the 
Plasmid pGShIL-2tetr. We thank Dr. Susanne Schreiber 
and Dr. Georg StingI for providing melanoma isolates. 
Proceed. Intern. Symp. Control. Reh Bioact Mater., 21 (1994), Controlled Release Society, Inc. 


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Synthetic virus-like gene delivery systems

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