BACKGROUND
the specific in or ex vivo transduction of cancerous, infected or malfunctioning cells with pro-apoptotic, defence or restoring genes is a promising approach for the treatment of patients.
however, gene therapy continues to present obstacles, regarding safety, transduction efficiency and specificity.
most gene delivery systems are based on animal viral vectors, which are efficient on one hand, but may include the risk of intrinsic toxicity and are costly to manufacture.
in addition to developing safer viral vectors, scientists began modifying bacteriophage particles for gene delivery purposes  <cit> .
for example, filamentous bacteriophage libraries, consist of up to  <dig> unique phage particles; each displaying a different binding ligand on its surface and encapsulating the corresponding eukaryotic cdna.
these libraries are commonly used for the selection of novel antigen-specific binding molecules <cit> .
furthermore, it is possible to select for ligand-bound phage being internalised by the target cell after receptor-binding  <cit> .
if a eukaryotic gene cassette is encoded in the phagemid vector, a small percentage of target cells will express this gene  <cit> .
thus, bacteriophage have the potential to serve as specifically targeted gene delivery vectors.
they have no natural tropism for eukaryotic cells, nor do they integrate into the cell's genome.
a filamentous bacteriophage containing a phagemid vector  encodes only the gene for a surface fusion protein, an antibiotic resistance gene and an amplification and packaging signal, but lacks the genes for all other bacteriophage proteins.
in order to produce functional phagemid particles, e. coli transformed with the phagemid vector have to be superinfected with helper phage , which contribute the missing proteins for the phagemid particle assembly in the periplasm of e. coli.
phagemid particles are easy to genetically modify in terms of targeting to a specific receptor-bearing cell population.
as phagemid vectors are relatively small in size compared to full size phage vectors, they are capable of carrying larger foreign dna sequences.
however, a major obstacle of using bacteriophage as gene transfer vehicles is their low transduction efficiency, which relies on gene transfer and expression.
gene transfer efficiency generally depends on the binding affinity of the ligand and the intracellular trafficking of the internalized receptor-bound phage, including endosomal escape and capsid breakdown.
moreover, processing of the single-stranded phage dna, including conversion into double-stranded dna and nuclear localization, is a critical factor  <cit> .
commonly, the displayed ligand is fused to the minor coat protein piii .
ligands can be displayed either monovalently or multivalently, depending on the helper phage system being used.
the importance of this factor has been demonstrated by larocca et al.
<cit>  in comparing the transduction efficiency of epidermal growth factor receptor -positive target cells with bacteriophage containing a green fluorescence protein  gene and displaying either monovalently or multivalently the egf molecule.
the multivalency of the egf, fused to the minor coat protein piii on the phage particles, increased transduction efficiency by 5-fold.
similar effects have been shown by poul and marks  using a piii-displayed anti-erbb <dig> scfv.
moreover, it has been demonstrated that phages displaying ligands fused to their major coat protein pviii  are able to bind to cell surface receptors, be internalized and processed inside mammalian cells  <cit>  leading to marker gene expression  <cit> .
it is generally believed that the wild type to fusion protein ratio of pviii displaying phage is inversely related to the size of the displayed ligand, making it difficult to functionally express larger proteins on the phage surface  <cit> .
nevertheless, it has been shown that this correlation is not so stringent.
malik et al.
demonstrated that fab fragments and enzymes can be displayed on pviii, but with a frequency of less than 1%  <cit> .
we were interested in developing a gene delivery vector for the specific transduction of hodgkin/reed-sternberg cells, the malignant cell type of hodgkin's lymphoma.
the cd <dig> receptor has been exploited as hodgkin-lymphoma associated antigen in recent experimental therapeutic strategies  <cit> .
antibodies bound to cd <dig> are internalized by clathrin-mediated endocytosis and traffic to the lysosome  <cit> .
regarding this internal trafficking of cd30-bound complexes as a prerequisite for gene transfer, we generated a phagemid vector that encodes an anti-cd <dig> scfv-pviii fusion protein and a eukaryotic cmv-egfp gene cassette.
out of this vector we produced small, anti-cd <dig> phagemid particles, capable of delivering egfp as a reporter gene for transduction.
we chose the major coat protein pviii as the fusion partner for the display of the anti-cd <dig> scfv ki- <dig>  <cit> .
fusion with pviii as an alternative to the widely used minor coat protein piii should increase the multivalency of the scfv-display and demonstrate the feasibility of displaying relatively big molecules, like scfvs  on pviii.
RESULTS
construction of the anti-cd <dig> gene transfer phagemid vectors
the anti-cd <dig> scfv ki- <dig> has been selected from a ki- <dig> hybridoma-derived phage display library via cell panning on l <dig> hodgkin cells as previously described  <cit> .
the eukaryotic cmv-egfp gene cassette was excised from the pegfp-n <dig> expression vector and ligated into the phagemid vector pcantab6-ki- <dig>  resulting in the vector pc6-ki- <dig>  transformation of e. coli tg- <dig> and subsequent packaging of the pc6-ki- <dig> phagemid vector with m <dig> helper phage leads to m13-pc6-ki- <dig> phagemid particles with the ki- <dig> scfv monovalently displayed as a fusion protein with the minor coat protein piii .
an exchange of the piii gene of pc6-ki- <dig> with the pviii gene from the m <dig> genome and the insertion of the cmv-egfp gene cassette, resulted in the phagemid vector pak8-ki- <dig>  rescuing of phagemid particles with this phagemid vector will produce the m13-pak8-ki- <dig> phagemid particles with ki- <dig> scfv displayed in multiple copies on their surface .
as a control, the ki- <dig> scfv gene was deleted from the pak8-ki- <dig> phagemid vector, to produce the phagemid particles m13-pak <dig> displaying no anti-cd <dig> scfv on their surface, but containing a functional eukaryotic egfp expression cassette .
detection of ki- <dig> scfv fused to bacteriophage coat protein pviii
after superinfection of the phagemid vector containing e. coli clones with helper phage, the phagemid particles assemble in the bacterial periplasm and package the phagemid single-stranded dna with the major coat protein  and the minor coat proteins .
during this process, the n-terminal signal sequences of the coat proteins piii and pviii are cleaved leaving the n-terminal part of these proteins outside of the bacteriophage capsid and accessible for binding interactions.
the predicted size of the major coat protein pviii  fused with the ki- <dig> scfv  is  <dig> kda .
in western blot analysis this fusion protein was detected in m13-pak8-ki- <dig> and not in the control m13-pak <dig> phagemid particles .
specific cd <dig> binding of ki- <dig> scfv-displaying phage
to demonstrate that phagemid particles displaying the ki- <dig> scfv specifically bind cd <dig>  an enzyme-linked immuno sorbent assay  was performed, coating wells with recombinant cd <dig> and incubating them with  <dig> ×  <dig> phagemid particles.
a strong interaction of the anti-cd <dig> ki- <dig> scfv-displaying phagemid particles m13-pak8-ki- <dig> and m13-pc6-ki- <dig> with the coated cd <dig> was detected.
the control phagemid particle m13-pak <dig> and phagemid particles incubated in non-coated wells did not bind .
the binding of m13-pak8-ki- <dig> to the cd <dig> antigen was stronger than the binding of the m13-pc6-ki- <dig>
to demonstrate the cell binding specificity of the anti-cd <dig> phagemid particles, facs analyses were performed.
confirming the elisa results, the ki- <dig> scfv displaying phagemid particles bound only to the cd30-positive, hodgkin-derived cell lines l <dig>  l <dig>  l <dig> and kmh- <dig> and not to the cd30-negative multiple myeloma cell line u <dig> and the burkitt lymphoma cell line bl <dig>  the control phagemid particle m13-pak <dig> did not show binding to any cell line.
furthermore, the facs analysis revealed that m13-pak8-ki- <dig> binding to the target cells is consistently stronger than the binding of the m13-pc6-ki- <dig> phagemid particles .
the incubation of the cd30-positive cells with the anti-cd <dig> scfv-displaying phagemid particle m13-pak8-ki- <dig> leads to additional cluster formation of the cells after  <dig> hours, in contrast to the m13-pc6-ki- <dig> phagemid particles .
no cluster formation was seen with cd30-negative cells incubated with m13-pak8-ki- <dig>
specific gene transfer of cd30-positive cells by targeted bacteriophage
we performed competition assays to determine if cd30-binding phagemid particles are capable of antigen-specific gene delivery of a egfp reporter gene.
<dig> ×  <dig> cd30-positive l <dig> cells were incubated with  <dig> ×  <dig> m13-pak8-ki- <dig> phagemid particles in the absence or presence of  <dig> nm or  <dig> nm of the parental monoclonal antibody ki- <dig>  respectively .
in the absence of the competitor about  <dig> % of the cells show egfp expression demonstrating gene transfer.
in the presence of the parental monoclonal antibody ki- <dig>  the number of transduced cells is clearly reduced in a dose dependent manner, proving the antigen-specificity of the gene transfer.
to further demonstrate antigen-specificity and evaluate transduction efficiency,  <dig> ×  <dig> of the cd30-positive l <dig>  l <dig>  l <dig> and kmh- <dig> cells or the cd30-negative bl <dig> or u <dig> cell lines were incubated for  <dig> hours with  <dig> ×  <dig> phagemid particles m13-pak <dig>  m13-pak8-ki- <dig> or m13-pc6-ki- <dig>  respectively.
the percentage of living cells expressing the eukaryotic gene cassette cmv-egfp was determined by facs analysis after propidium iodide staining as depicted in fig.
6b.
as shown in fig.
6c the l <dig> cells remain untransduced when incubated with the control phagemid particles m13-pak <dig>  phagemid particles displaying the ki- <dig> scfv fused to the coat protein piii ) minimally transduced the l <dig> cells .
in contrast, l <dig> cells incubated with the same amount of m13-pak8-ki- <dig> phagemid particles displaying ki- <dig> scfv-pviii fusion proteins, the transduction efficiency was significantly increased to approximately 5%.
the superior transduction efficiency of the m13-pak8-ki- <dig> phagemid particles was also seen with the other cd30-positive cell lines, as summarized in fig.
6d.
no transduction could be seen with the cd30-negative cell lines bl <dig> or u <dig> using these phagemid particles.
discussion and
CONCLUSIONS
use of bacteriophage as vectors for targeted gene transfer into eukaryotic cells is an alternative method to circumvent some of the problems occurring with the established viral vectors.
bacteriophage have no natural tropism for eukaryotic cells but can be targeted towards specific cell surface receptors by displaying appropriate binding ligands, such as peptides or antibody fragments.
internalized by receptor-mediated endocytosis, their packaged single-stranded dna is further processed , leading to the expression of encoded genes under eukaryotic promoters  <cit> .
the ease with which one can target, modify, produce and purify these phage makes them a valuable tool for antigen-specific cell transduction.
here, we describe the generation of anti-cd <dig> targeted phagemid particles, specifically binding and transducing cd30-positive cells.
for this purpose, the anti-cd <dig> ki- <dig> scfv gene has been genetically fused either to the major coat protein pviii ) or the minor coat protein piii ) in a phagemid vector, which also encodes an egfp gene under the control of a cmv promoter.
the expression of the ki- <dig> scfv-pviii fusion protein was determined by western blot analysis  and elisa experiments with recombinant cd <dig> antigen demonstrating the specific binding of the ki- <dig> scfv displaying phagemid particles .
additionally, phage displaying the ki- <dig> scfv showed a specific binding to cd30-bearing cells in facs analyses.
binding affinity of the m13-pak8-ki- <dig> phagemid particles to cd <dig> antigen and cd <dig> receptor-bearing cells was always higher, compared to the m13-pc6-ki- <dig> phagemid particles.
provided that scfvs fused to the piii coat protein have the same binding affinity as pviii-fusion proteins, this difference could be explained by a multivalent display of the anti-cd <dig> ki- <dig> scfv on the m13-pak8-ki- <dig> phagemid particles indicating a binding avidity effect.
the multivalent display of the scfv on the m13-pak8-ki- <dig> phagemid particles is also supported by the fact that these phagemid particles are able to induce additional cluster formation of cd30-positive cells .
this phenomena was not seen with the supplement of m13-pc6-ki- <dig> phagemid particles or the addition of the m13-pak8-ki- <dig> phagemid particles to the cd30-negative cell line bl <dig>  but has been observed previously by the addition of monoclonal ki- <dig> igg antibody to cd30-positive cells .
the transduction experiments with different cell lines revealed that anti-cd <dig> scfv-displaying phagemid particles are able to transduce cd <dig> receptor-bearing cells.
the addition of the parental monoclonal antibody ki- <dig> significantly reduced the amount of cd30-positive l <dig> cells transduced by the m13-pak8-ki- <dig> phagemid particles .
there was no unspecific transduction of cd30-negative cells, nor did control phagemid particles m13-pak <dig> transduce the cd30-positive cells .
this indicates that cd <dig> antigen on the targeting cell lines is required for transduction with anti-cd <dig> phagemid particles and establishes the high specificity of these gene transfer particles.
incubation of the target cells with the m13-pak8-ki- <dig> phagemid particles led to a maximum transduction rate of 5%, a rate approximately 10-fold higher compared to the m13-pc6-ki- <dig> phagemid particles.
this enabled a direct comparison of pviii- and piii- fusion protein display for receptor-specific gene transfer.
although there is a postulated avidity effect responsible for the stronger binding of the m13-pak8-ki- <dig> phagemid particles to recombinant cd <dig> and cd30-positive cells compared to the m13-pc6-ki- <dig> phagemid particles, this significant difference in transduction efficiency was somewhat surprising.
other groups using monovalent piii-display with transduction efficiencies of 1–4% were able to increase the efficiencies up to 10% by using multivalent piii-display  <cit> .
it has been described that cd <dig> receptor is being internalized by clathrin-mediated endocytosis, maturing to lysosomes and that cross-linking of cd <dig> receptors by monoclonal antibodies enhances this trafficking at least by 3-fold  <cit> .
in accordance with our results, this supports our hypothesis that pviii-based display of scfvs is multivalent, leading to increased internalization and transduction efficiency of cd30-bearing cells.
although it has been shown that multivalent scfv-piii display on bacteriophage is possible and increases transduction efficiency  <cit> , our approach serves as an alternative or even superior system, especially if the internalization depends on multipoint 'zipper' contacts of the phage with the cell surface receptors.
a multivalent display on pviii may provide an increased binding and phage uptake because pviii fusion proteins are spread over the surface of the phage, in contrast to piii fusion proteins, which are focused on the tip of the phage.
this so-called 'disseminated display' would also explain why m13-pak8-ki- <dig>  in contrast to the m13-pc6-ki- <dig> phagemid particles, are able to connect cd30-positive cells leading to cluster formation .
this also explains the stronger binding activity of the m13-pak8-ki- <dig> phagemid particles  and the higher transduction rate .
however, this potential cross-linking of cells by single m13-pak8-ki- <dig> phagemid particles does not prevent internalization of other m13-pak8-ki- <dig> phagemid particles, because simultaneous binding of receptors on one cell is even more likely than cross linking of receptors on separate cells.
the feasibility of displaying molecules as large as scfvs  on the major coat protein raises the possibility of generating bispecific phagemid particles rescued from e. coli, encoding an additional piii- or pviii-fusion protein, thereby increasing specificity and avidity of the cell-targeting phagemid particle.
the unequal transduction rates of the different cd30-positive cell lines with the m13-pak8-ki- <dig> phagemid particles are not directly explainable by their individual cd <dig> receptor densities.
for example, cell line l <dig> showed only a moderate binding of the anti-cd <dig> phagemid particles in facs analysis compared to the l <dig> cell line, but revealed the highest egfp-expression rate .
this demonstrates that the efficiency of the gene transfer seems to be dependent on cell line-specific trafficking of the bacteriophage after binding.
because the gene transfer efficiency of targeted bacteriophage is in general still very low, it seems to be necessary to further optimize trafficking, dna processing and nuclear localization of the phagemid dna.
so far, satisfying transduction rates  with bacteriophage have been reached only in vitro by genotoxic treatment  <cit> .
to our knowledge, this is the first approach demonstrating a potential advantage of the major coat protein pviii as fusion partner for the display of scfvs in gene transfer experiments, compared to the commonly used minor coat protein piii.
furthermore, it is the first approach generating a cd30-specific gene transfer vector to be used for the transduction of hodgkin-, reed sternberg cells.
a possible application of this system would be the specific induction of apoptosis in these cells.
it is believed that cancer cells are on the verge of apoptosis, containing processed effector caspases.
at the same time, they also express inhibitor of apoptosis proteins , which can not be neutralized due to malfunctioning apoptotic pathways  <cit> .
it has been demonstrated that overexpression of second mitochondrial activator of caspases  in hodgkin-derived cell lines blocks x-linked iaps activation  <cit> , and therefore would make a promising candidate gene for restoration of the apoptotic pathway in hodgkin's disease via targeted gene therapy.
provided that the transduction efficiency of cd30-specific bacteriophage is further increased by additional molecular modifications, they can potentially serve as specific, safe, cheap and efficient gene therapy vectors for in and ex vivo applications.
this is supported by the fact that tumor treatment with targeted bacteriophage in vivo has been demonstrated recently  <cit> .

 the specific in or ex vivo transduction of cancerous, infected or malfunctioning cells with pro-apoptotic, defence or restoring genes is a promising approach for the treatment of patients. however, gene therapy continues to present obstacles, regarding safety, transduction efficiency and specificity. most gene delivery systems are based on animal viral vectors, which are efficient on one hand, but may include the risk of intrinsic toxicity and are costly to manufacture. in addition to developing safer viral vectors, scientists began modifying bacteriophage particles for gene delivery purposes. for example, filamentous bacteriophage libraries, consist of up to unique phage particles; each displaying a different binding ligand on its surface and encapsulating the corresponding eukaryotic cdna. these libraries are commonly used for the selection of novel antigen-specific binding molecules. furthermore, it is possible to select for ligand-bound phage being internalised by the target cell after receptor-binding. if a eukaryotic gene cassette is encoded in the phagemid vector, a small percentage of target cells will express this gene. thus, bacteriophage have the potential to serve as specifically targeted gene delivery vectors. they have no natural tropism for eukaryotic cells, nor do they integrate into the cell's genome. a filamentous bacteriophage containing a phagemid vector encodes only the gene for a surface fusion protein, an antibiotic resistance gene and an amplification and packaging signal, but lacks the genes for all other bacteriophage proteins. in order to produce functional phagemid particles, e. coli transformed with the phagemid vector have to be superinfected with helper phage, which contribute the missing proteins for the phagemid particle assembly in the periplasm of e. coli. phagemid particles are easy to genetically modify in terms of targeting to a specific receptor-bearing cell population. as phagemid vectors are relatively small in size compared to full size phage vectors, they are capable of carrying larger foreign dna sequences. however, a major obstacle of using bacteriophage as gene transfer vehicles is their low transduction efficiency, which relies on gene transfer and expression. gene transfer efficiency generally depends on the binding affinity of the ligand and the intracellular trafficking of the internalized receptor-bound phage, including endosomal escape and capsid breakdown. moreover, processing of the single-stranded phage dna, including conversion into double-stranded dna and nuclear localization, is a critical factor. commonly, the displayed ligand is fused to the minor coat protein piii. ligands can be displayed either monovalently or multivalently, depending on the helper phage system being used. the importance of this factor has been demonstrated by larocca et al. in comparing the transduction efficiency of epidermal growth factor receptor -positive target cells with bacteriophage containing a green fluorescence protein gene and displaying either monovalently or multivalently the egf molecule. the multivalency of the egf, fused to the minor coat protein piii on the phage particles, increased transduction efficiency by 5-fold. similar effects have been shown by poul and marks using a piii-displayed anti-erbb scfv. moreover, it has been demonstrated that phages displaying ligands fused to their major coat protein pviii are able to bind to cell surface receptors, be internalized and processed inside mammalian cells leading to marker gene expression. it is generally believed that the wild type to fusion protein ratio of pviii displaying phage is inversely related to the size of the displayed ligand, making it difficult to functionally express larger proteins on the phage surface. nevertheless, it has been shown that this correlation is not so stringent. malik et al. demonstrated that fab fragments and enzymes can be displayed on pviii, but with a frequency of less than 1%. we were interested in developing a gene delivery vector for the specific transduction of hodgkin/reed-sternberg cells, the malignant cell type of hodgkin's lymphoma. the cd receptor has been exploited as hodgkin-lymphoma associated antigen in recent experimental therapeutic strategies. antibodies bound to cd are internalized by clathrin-mediated endocytosis and traffic to the lysosome. regarding this internal trafficking of cd30-bound complexes as a prerequisite for gene transfer, we generated a phagemid vector that encodes an anti-cd scfv-pviii fusion protein and a eukaryotic cmv-egfp gene cassette. out of this vector we produced small, anti-cd phagemid particles, capable of delivering egfp as a reporter gene for transduction. we chose the major coat protein pviii as the fusion partner for the display of the anti-cd scfv ki-. fusion with pviii as an alternative to the widely used minor coat protein piii should increase the multivalency of the scfv-display and demonstrate the feasibility of displaying relatively big molecules, like scfvs on pviii.  construction of the anti-cd gene transfer phagemid vectors the anti-cd scfv ki- has been selected from a ki- hybridoma-derived phage display library via cell panning on l hodgkin cells as previously described. the eukaryotic cmv-egfp gene cassette was excised from the pegfp-n expression vector and ligated into the phagemid vector pcantab6-ki- resulting in the vector pc6-ki- transformation of e. coli tg- and subsequent packaging of the pc6-ki- phagemid vector with m helper phage leads to m13-pc6-ki- phagemid particles with the ki- scfv monovalently displayed as a fusion protein with the minor coat protein piii. an exchange of the piii gene of pc6-ki- with the pviii gene from the m genome and the insertion of the cmv-egfp gene cassette, resulted in the phagemid vector pak8-ki- rescuing of phagemid particles with this phagemid vector will produce the m13-pak8-ki- phagemid particles with ki- scfv displayed in multiple copies on their surface. as a control, the ki- scfv gene was deleted from the pak8-ki- phagemid vector, to produce the phagemid particles m13-pak displaying no anti-cd scfv on their surface, but containing a functional eukaryotic egfp expression cassette. detection of ki- scfv fused to bacteriophage coat protein pviii after superinfection of the phagemid vector containing e. coli clones with helper phage, the phagemid particles assemble in the bacterial periplasm and package the phagemid single-stranded dna with the major coat protein and the minor coat proteins. during this process, the n-terminal signal sequences of the coat proteins piii and pviii are cleaved leaving the n-terminal part of these proteins outside of the bacteriophage capsid and accessible for binding interactions. the predicted size of the major coat protein pviii fused with the ki- scfv is kda. in western blot analysis this fusion protein was detected in m13-pak8-ki- and not in the control m13-pak phagemid particles. specific cd binding of ki- scfv-displaying phage to demonstrate that phagemid particles displaying the ki- scfv specifically bind cd an enzyme-linked immuno sorbent assay was performed, coating wells with recombinant cd and incubating them with × phagemid particles. a strong interaction of the anti-cd ki- scfv-displaying phagemid particles m13-pak8-ki- and m13-pc6-ki- with the coated cd was detected. the control phagemid particle m13-pak and phagemid particles incubated in non-coated wells did not bind. the binding of m13-pak8-ki- to the cd antigen was stronger than the binding of the m13-pc6-ki- to demonstrate the cell binding specificity of the anti-cd phagemid particles, facs analyses were performed. confirming the elisa results, the ki- scfv displaying phagemid particles bound only to the cd30-positive, hodgkin-derived cell lines l l l and kmh- and not to the cd30-negative multiple myeloma cell line u and the burkitt lymphoma cell line bl the control phagemid particle m13-pak did not show binding to any cell line. furthermore, the facs analysis revealed that m13-pak8-ki- binding to the target cells is consistently stronger than the binding of the m13-pc6-ki- phagemid particles. the incubation of the cd30-positive cells with the anti-cd scfv-displaying phagemid particle m13-pak8-ki- leads to additional cluster formation of the cells after hours, in contrast to the m13-pc6-ki- phagemid particles. no cluster formation was seen with cd30-negative cells incubated with m13-pak8-ki- specific gene transfer of cd30-positive cells by targeted bacteriophage we performed competition assays to determine if cd30-binding phagemid particles are capable of antigen-specific gene delivery of a egfp reporter gene. × cd30-positive l cells were incubated with × m13-pak8-ki- phagemid particles in the absence or presence of nm or nm of the parental monoclonal antibody ki- respectively. in the absence of the competitor about % of the cells show egfp expression demonstrating gene transfer. in the presence of the parental monoclonal antibody ki- the number of transduced cells is clearly reduced in a dose dependent manner, proving the antigen-specificity of the gene transfer. to further demonstrate antigen-specificity and evaluate transduction efficiency, × of the cd30-positive l l l and kmh- cells or the cd30-negative bl or u cell lines were incubated for hours with × phagemid particles m13-pak m13-pak8-ki- or m13-pc6-ki- respectively. the percentage of living cells expressing the eukaryotic gene cassette cmv-egfp was determined by facs analysis after propidium iodide staining as depicted in fig. 6b. as shown in fig. 6c the l cells remain untransduced when incubated with the control phagemid particles m13-pak phagemid particles displaying the ki- scfv fused to the coat protein piii ) minimally transduced the l cells. in contrast, l cells incubated with the same amount of m13-pak8-ki- phagemid particles displaying ki- scfv-pviii fusion proteins, the transduction efficiency was significantly increased to approximately 5%. the superior transduction efficiency of the m13-pak8-ki- phagemid particles was also seen with the other cd30-positive cell lines, as summarized in fig. 6d. no transduction could be seen with the cd30-negative cell lines bl or u using these phagemid particles. discussion and  use of bacteriophage as vectors for targeted gene transfer into eukaryotic cells is an alternative method to circumvent some of the problems occurring with the established viral vectors. bacteriophage have no natural tropism for eukaryotic cells but can be targeted towards specific cell surface receptors by displaying appropriate binding ligands, such as peptides or antibody fragments. internalized by receptor-mediated endocytosis, their packaged single-stranded dna is further processed, leading to the expression of encoded genes under eukaryotic promoters. the ease with which one can target, modify, produce and purify these phage makes them a valuable tool for antigen-specific cell transduction. here, we describe the generation of anti-cd targeted phagemid particles, specifically binding and transducing cd30-positive cells. for this purpose, the anti-cd ki- scfv gene has been genetically fused either to the major coat protein pviii ) or the minor coat protein piii ) in a phagemid vector, which also encodes an egfp gene under the control of a cmv promoter. the expression of the ki- scfv-pviii fusion protein was determined by western blot analysis and elisa experiments with recombinant cd antigen demonstrating the specific binding of the ki- scfv displaying phagemid particles. additionally, phage displaying the ki- scfv showed a specific binding to cd30-bearing cells in facs analyses. binding affinity of the m13-pak8-ki- phagemid particles to cd antigen and cd receptor-bearing cells was always higher, compared to the m13-pc6-ki- phagemid particles. provided that scfvs fused to the piii coat protein have the same binding affinity as pviii-fusion proteins, this difference could be explained by a multivalent display of the anti-cd ki- scfv on the m13-pak8-ki- phagemid particles indicating a binding avidity effect. the multivalent display of the scfv on the m13-pak8-ki- phagemid particles is also supported by the fact that these phagemid particles are able to induce additional cluster formation of cd30-positive cells. this phenomena was not seen with the supplement of m13-pc6-ki- phagemid particles or the addition of the m13-pak8-ki- phagemid particles to the cd30-negative cell line bl but has been observed previously by the addition of monoclonal ki- igg antibody to cd30-positive cells. the transduction experiments with different cell lines revealed that anti-cd scfv-displaying phagemid particles are able to transduce cd receptor-bearing cells. the addition of the parental monoclonal antibody ki- significantly reduced the amount of cd30-positive l cells transduced by the m13-pak8-ki- phagemid particles. there was no unspecific transduction of cd30-negative cells, nor did control phagemid particles m13-pak transduce the cd30-positive cells. this indicates that cd antigen on the targeting cell lines is required for transduction with anti-cd phagemid particles and establishes the high specificity of these gene transfer particles. incubation of the target cells with the m13-pak8-ki- phagemid particles led to a maximum transduction rate of 5%, a rate approximately 10-fold higher compared to the m13-pc6-ki- phagemid particles. this enabled a direct comparison of pviii- and piii- fusion protein display for receptor-specific gene transfer. although there is a postulated avidity effect responsible for the stronger binding of the m13-pak8-ki- phagemid particles to recombinant cd and cd30-positive cells compared to the m13-pc6-ki- phagemid particles, this significant difference in transduction efficiency was somewhat surprising. other groups using monovalent piii-display with transduction efficiencies of 1–4% were able to increase the efficiencies up to 10% by using multivalent piii-display. it has been described that cd receptor is being internalized by clathrin-mediated endocytosis, maturing to lysosomes and that cross-linking of cd receptors by monoclonal antibodies enhances this trafficking at least by 3-fold. in accordance with our results, this supports our hypothesis that pviii-based display of scfvs is multivalent, leading to increased internalization and transduction efficiency of cd30-bearing cells. although it has been shown that multivalent scfv-piii display on bacteriophage is possible and increases transduction efficiency, our approach serves as an alternative or even superior system, especially if the internalization depends on multipoint'zipper' contacts of the phage with the cell surface receptors. a multivalent display on pviii may provide an increased binding and phage uptake because pviii fusion proteins are spread over the surface of the phage, in contrast to piii fusion proteins, which are focused on the tip of the phage. this so-called'disseminated display' would also explain why m13-pak8-ki- in contrast to the m13-pc6-ki- phagemid particles, are able to connect cd30-positive cells leading to cluster formation. this also explains the stronger binding activity of the m13-pak8-ki- phagemid particles and the higher transduction rate. however, this potential cross-linking of cells by single m13-pak8-ki- phagemid particles does not prevent internalization of other m13-pak8-ki- phagemid particles, because simultaneous binding of receptors on one cell is even more likely than cross linking of receptors on separate cells. the feasibility of displaying molecules as large as scfvs on the major coat protein raises the possibility of generating bispecific phagemid particles rescued from e. coli, encoding an additional piii- or pviii-fusion protein, thereby increasing specificity and avidity of the cell-targeting phagemid particle. the unequal transduction rates of the different cd30-positive cell lines with the m13-pak8-ki- phagemid particles are not directly explainable by their individual cd receptor densities. for example, cell line l showed only a moderate binding of the anti-cd phagemid particles in facs analysis compared to the l cell line, but revealed the highest egfp-expression rate. this demonstrates that the efficiency of the gene transfer seems to be dependent on cell line-specific trafficking of the bacteriophage after binding. because the gene transfer efficiency of targeted bacteriophage is in general still very low, it seems to be necessary to further optimize trafficking, dna processing and nuclear localization of the phagemid dna. so far, satisfying transduction rates with bacteriophage have been reached only in vitro by genotoxic treatment. to our knowledge, this is the first approach demonstrating a potential advantage of the major coat protein pviii as fusion partner for the display of scfvs in gene transfer experiments, compared to the commonly used minor coat protein piii. furthermore, it is the first approach generating a cd30-specific gene transfer vector to be used for the transduction of hodgkin-, reed sternberg cells. a possible application of this system would be the specific induction of apoptosis in these cells. it is believed that cancer cells are on the verge of apoptosis, containing processed effector caspases. at the same time, they also express inhibitor of apoptosis proteins, which can not be neutralized due to malfunctioning apoptotic pathways. it has been demonstrated that overexpression of second mitochondrial activator of caspases in hodgkin-derived cell lines blocks x-linked iaps activation, and therefore would make a promising candidate gene for restoration of the apoptotic pathway in hodgkin's disease via targeted gene therapy. provided that the transduction efficiency of cd30-specific bacteriophage is further increased by additional molecular modifications, they can potentially serve as specific, safe, cheap and efficient gene therapy vectors for in and ex vivo applications. this is supported by the fact that tumor treatment with targeted bacteriophage in vivo has been demonstrated recently. 
this opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low. these phage particles showed an improved binding and transduction efficiency of cd positive hodgkin-lymphoma cells, compared to bacteriophage with the anti-cd single chain variable fragment fused to the minor coat protein piii. the display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. these results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors. we can conclude from the results that the postulated multivalency of the anti-cd30-pviii displaying bacteriophage combined with disseminated display of the anti-cd scfv on the whole particle surface is responsible for the improved gene transfer rate. here we demonstrate the display of an anti-cd single chain variable fragment fused to the major coat protein pviii on the surface of bacteriophage. after uptake of the phage, their internal single-stranded dna is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette.
 the display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. after uptake of the phage, their internal single-stranded dna is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette. this opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low.  here we demonstrate the display of an anti-cd single chain variable fragment fused to the major coat protein pviii on the surface of bacteriophage. these phage particles showed an improved binding and transduction efficiency of cd positive hodgkin-lymphoma cells, compared to bacteriophage with the anti-cd single chain variable fragment fused to the minor coat protein piii.  we can conclude from the results that the postulated multivalency of the anti-cd30-pviii displaying bacteriophage combined with disseminated display of the anti-cd scfv on the whole particle surface is responsible for the improved gene transfer rate. these results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors. 