Note: Descriptions are shown in the official language in which they were submitted.
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S014-519.572
New Conjugates for Introducing Nucleic Acid Into
Higher Eucaryotic Cells
The invention relates to the introduction of
nucleic acids into higher eucaryotic cells.
There is a need for an efficient system for S
introducing nucleic acid into live cells particularly in
gene therapy. Genes are introduced into cells in order
to achieve in ViVO synthesis of therapeutically
effective genetic products, e.g. in order to replace the
defective gene in the case of a genetic defect.
"Conventional" gene therapy is based on the principle of
achieving a lasting cure by a single treatment. -~
However, there is also a need for methods of treatment
in which the therapeutically effective DNA (or mRNA) is ~ ;~
administered like a drug ("gene therapeutic agent") once - D
or repeatedly as necessary. Examples of genetically ~
caused diseases in which gene therapy represents a ; -
promising approach are hemophilia, beta-thalassaemia and
"Severe Combined Immune Deficiency" (SCID~, a syndrome
caused by the genetically induced absence of the enzyme
adenosine deaminase. Other possible applications are in
immune regulation, in which humoral or intracellular
immunity is achieved by the administration of functional
nucleic acid which codes for a secreted protein antigen
or for a non-secreted protein antigen, by immunization.
Other examples of genetic defects in which a nucleic
acid which codes for the defective gene can be
administere~, e.g. in a form individually tailored to
the particular requirement, include muscular dystrophy
(dystrophin gene), cystic fibrosis (cystic fibrosis
transmembrane conductance regulator gene),
hypercholesterolemia (LDL receptor gene). Gene-therapy
methods of treatment are also potentially of use when
hormones, growth factors or proteins with a cytotoxic or
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immune-modulating activity are to be synthesized in the
body.
Gene therapy also appears promising for the
treatment of cancer by administering so-called "cancer
vaccines". In order to increase the immunogenicity of
tumor cells, they are altered to render them either more
antigenic or to make them produce certain im~une
modulating substances such as cytokines in order to
trigger an immune response. This is accomplished by
transfecting the cells with DNA coding for a cytokine,
e.g. IL-2, IL-4, IFN-gamma, TNF-alpha. To date, gene
transfer into autologous tumor cells has chiefly been
accomplished via retroviral vectors.
The mode of activity of antisense RNAs and DNAs as
well as ribozymes enables them to be used as therapeutic
agents for blocking the expression of certain genes
(such as deregulated oncogenes or viral genes) in vivo.
It has already been shown that short antisense
oligonucleotides can be imported into cells and exert
their inhibiting effect therein (Zamecnik et al., 1986),
even if their intracellular concentration is low,
caused, inter alia, by their restricted uptake by the
cell membrane as a result of the strong negative charge
of the nucleic acids.
Various techniques are known for gene transfer into
mammalian cells in vitro but their use in vivo is
limited (these include the introduction of DNA by means
of liposomes, electroporation, microinjection, cell
fusion, DEAE-dextran or the calcium phosphate
precipitation method).
In recent times, biological vectors have been
developed to bring about the transfer of genes by using
the efficient entry mechanisms of their parent viruses.
This strategy was used in the construction of ;
recombinant retroviral and adenoviral vectors in order
to achieve a highly efficient gene transfer in vitro and
in vivo (Berkner, 1988). For all their efficiency,
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these vectors are subj ect to restrictions in terms of
the size and construction of the DNA which is
transferred. Furthermore, these agents constitute
safety risks in view of the co-transfer of viable viral
gene elements of the original virus. Thus, for example,
the use of retroviruses is problematic because it
involves, at least to a small percentage, the danger of
side effects such as infection with the virus (by
recombination with endogenous viruses or contamination
with helper viruses and possible subsequent mutation
into the pathogenic form) or the formation of cancer.
Moreover, the stable transformation of the somatic cells
of the patient, as achieved by means of retroviruses, is
not desirable in each case because this can only make
the treatment more difficult to re~erse, e.g. if side
effects occur.
In order to circumvent these restrictions,
alternative strategies for gene transfer have been
developed, based on mechanisms which the cell uses for
the transfer of macromolecules. One example of this i~
the transfer of genes into the cell via the extremely
efficient route of receptor-mediated endocytosis (Wu and
Wu, 1987, Wagner et al., 1990 and EP-Al 0388 758). This
approach uses bifunctional molecular conjugates which
have a DNA binding domain and a domain with specificity
for a cell surface receptor (Wu and Wu, 1987, Wagner
et al., 1990). If the recognition domain (hereinafter
referred to as the "internalizing factor") is recognized
by the cell surface receptor, the conjugate is
internalized by the route of receptor-mediated
endocytosis, in which the DNA bound to the conjugate is
also transferred. Using this method, it was possible to
achieve gene transfer rates at least as good as those
achieved with the conventional methods (Zenke et al.,
1990) .
Whereas this vector system is able to transport
large quantities of DNA into cells having the suitable
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cell surface receptor, the corresponding gene expression
very often does not accord with the transfer capacity
(Cotten et al., 1990). It was assumed, inter alia, that
the reason for this phenomenon is that the DNA conveyed
into the cell by receptor-mediated endocytosis lands in
lysosomes where it undergoes degradation (Zenke
et al., 1990, Cotten et al., 1990~. Therefore, the fact
that the DNA internalized in lysosomes does not have any
specific mechanism for leaving the intracellular vesicle
system constitutes a restriction which is inherent in
this transport system.
The aim of the present invention was to reduce or
eliminate these restrictions.
A plurality of viruses effect their entry into the
eucaryotic host by means of mechanisms which correspond
in principle to the mechanism of receptor-mediated
endocytosis. Virus infection based on this mechanism
generally begins with the binding of virus particles to
receptors on the cell membrane. After this, the virus
is internalized into the cell. This internalizing
process follows a common route, corresponding to the
entrance of physiological ligands or macromolecules into
the cell: first of all, the receptors on the cell
surface arrange themselves in groups, to form a so-
called "coated pit", and the membrane is inverted
inwardly and forms a vesicle surrounded by a coating.
After this vesicle has rid itself of its clathrin coat,
acidification takes place inside it by means of a proton
pump located in the membrane. This triggers the release
of the virus from the endosome. Depending on whether
the virus has a lipid coat or not, two types of virus
release from the endosome were taken into account: in
the case of so-called "naked" viruses (e.g. adenovirus,
poliovirus, rhinovirus) it was suggested that the low pH
causes changes in conformation in virus proteins. This ~
exposes hydrophobic domains which are not accessible at ~ ~-
the physiological pH. These domains thus acquire the
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ability to interact with the endosome membrane and
thereby cause the release of the virus genome from the
endosome into the cytoplasm. As for viruses with a coat
(e.g. vesicular stomatitis virus, Semliki Forest virus,
influenza virus) it is presumed that the low pH modifies
the structure or conformation of some virus proteins,
thereby promoting the fusion of the virus membxane with
the endosome membrane. Viruses which penetrate into the
cell by means of this mechanism have certain molecular
peculiarities which enable them to break up the endosome
membrane in order to gain entry into the cytoplasm.
Other viruses, e.g. the coated viruses Sendai, HIV
and some strains of Moloney leukaemia virus, or the
uncoated viruses SV40 and polyoma, do not need a low pH-
milieu for penetration into the cell; they can either
bring about fusion with the membrane directly on the
surface of the cell (Sendai virus, possibly HIV) or they
are capable of triggering mechanisms for breaking up the
cell membrane or passing through it. It is assumed that
the viruses which are independent of pH are also capable
of using the endocytosis route (McClure et al~, 1990).
In experiments which preceded the present invention
it was established that gene transfer by means of
nucleic acid complexes in which the nucleic acid is
complexed with polycations, optionally coupled to an
internalizing factor, e.g. with transferrin-polylysine
conjugates, is significantly increased by treatment with
adenoviruses, specific retroviruses or with virus
fragments. This effect was achieved by making use of
I the phenomenon that these viruses are taken up into the
cells by endocytosis mechanisms and have a specific
mechanism for escaping from the vesicle system by
breaking open the endosomes, e.g. in the case of the
adenoviruses (Pastan et al., 1986).
Starting from these observations, the problem of
the invention was solved by developing a bioconjugate
which contains the virus as an integral part of its
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functional construct.
The invention thus relates to a conjugate which has
the ability to form complexes with nucleic acid and
which comprises an internalizing factor and a substance
having an affinity for nucleic acid, for introducing
nucleic acid into higher eucaryotic cells. The
conjugate is characterized in that the internalizing
factor is a virus which is bound to the nucleic acid-
binding substance via an antibody in such a way that it
is capable per se of penetrating into the cell as part
of the conjugate/nucleic acid complex and of releasing
the contents of the endosomes, in which the complex is
located after entering the cell, into the cytoplasm.
The invention in a further aspect relates to
complexes in which the conjugates according to the
invention are complexed with nucleic acid.
The ability of the virus to penetrate into the cell ~-
and release the content of the endosomes, in which the
conjugate/nucleic acid complex is located, into the
cytoplasm, is hereinafter referred to as the "up take
function".
The conjugates according to the invention combine
the advantages of vector systems based on internalizing
factor conjugates with the advantages which the viruses
bring into these systems.
Compared with gene transfer by receptor-mediated
endocytosis, the virus-polycation-DNA complexes
according to the invention have the advantage that they
circumvent the fundamental restriction inherent in the
known molecular conjugate systems, in that, unlike the ~
known conjugates, they have a specific mechanism which ~--
enables them to be released from the cell vesicle
system. Compared with biological vectors, the vector ~
system according to the invention constitutes a -
fundamental conceptual departure from the recombinant
viral vectors, in that the foreign DNA which is to be
transported is carried on the outside of the virion.
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Consequently, the conjugates according to the invention
can transport very large gene constructs into the cell,
with no restrictions of any kind as to the sequence.
Suitable viruses include, on the one hand, those
which are able to penetrate into the cell by receptor-
mediated endocytosis and to bring about their release -
and hence the release of the nucleic acid - from the
endosome into the cytoplasm. (The suitability of
viruses within the scope of the present invention is
further defined in that they retain this property even
when they are a component of the nucleic acid
complexes). Without wishing to be tied to this theory,
this mechanism could benefit the nucleic acid complexes
transferred into the cell in so far as the ability of
the virus to release the contents of the endosomes
prevents the fusion between the endosomes and lysosomes
and consequently prevents the enzymatic decomposition
which normally occurs in these cell organelles.
The higher eucaryotic cells are well known and do
not include yeast. (Watson et al., 1987). Examples of
higher eucaryotic cells capable of adenovirus infection
are described by Fields and Knipe, 1990.
Viruses whose uptake function, occurring at the
start of infection, occurs by receptor-mediated
endocytosis and which are suitable as part of the
conjugates according to the invention by virtue of this
property, include on the one hand viruses without a
lipid coat such as adenovirus, poliovirus, rhinovirus,
and on the other hand the enveloped viruses vesicular
stomatitis virus, Semliki Forest virus, influenza virus;
pH-dependent strains of Moloney virus are also suitable.
Particularly suitable viruses for use in the present
invention are adenovirus subgroup C, type 5, Semilki
Forest Virus, Vesicular Stomatitis Virus, Poliovirus,
Rhinoviruses and Moloney Leukemia Virus. The use of
RNA viruses for the present invention which have no
reverse transcriptase has the advantage that
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transfection in the presence of such a virus does not
lead to the formation of viral DNA in the cell.
An important advantage derived from the present
invention is that the DNA to be transferred is not
integrated into the genome of the parent virus, as in
the case with standard recombinant viral vectors (see
Berkner, 1988; Eglitis and Anderson, 19~8). Thus, the
present invention provides much greater flexibility as
to the design of the foreign gene sequence to be
expressed, as transcription is not dependent on
promoters in the parent virus gene. In addition, this
strategy allows a greatly increased size of DNA that can
be transferred, as the packaging constraints of the -~
virus do not limit the amount of DNA that can be carried
on the exterior. Over and above these practical and
immediate advantages, important potential safety
features derive from the design of the vector.
Conventional recombinant viral vectors mediate
obligatory co-delivery of genome elements of the parent
virus from which potential safety hazards derive
(Ledley, 1989; Anderson, 1984). Since the conjugates
according to the invention selectively exploit viral
entry features, the viral genome is not an essential
feature. This design allows the possibility of
modifying the present system with a functionally and/or --
structurally inactivated viral genome to minimize the
safety hazards deriving from the transfer of viable
genes from the parent virus.
Within the scope of the present invention, the term
viruses - provided that they have uptake function as
defined above - includes in addition to the wild types,
mutants which have lost certain functions of the wild
type, other than their uptake function, especially their
ability to replicate, as a result of one or more
mutations. However, mutants which have lost their
uptake function can be employed in the practice of the
invention so long as they are employed as part of a
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"ternary co~plex~ as defined herein and the mutant virus
has not lost its endosomolytic activity.
Mutants may be produced by conventional mutagenesis
processes by mutations in virus-protein regions which
are responsible for the replicative functions and the
uptake function and which may be complemented by a -
packaging line. These include, e.g. in the case of
adenovirus, ts-mutants (temperature sensitive mutants),
ElA- and ElB-mutants, mutants which exhibit mutations in
MLP-driven genes (Berkner, 1988) and mutants which
exhibit mutations in the regions of certain capsid
proteins. virus strains which have corresponding
natural mutations are also suitable. The ability of
viruses to replicate can be investigated, for example,
using plaque assays known from the literature, in which
cell cultures are covered with suspensions of various
virus concentrations and the number of lysed cells which
is visible by means of plaques is recorded (Dulbecco,
1980).
Other viruses which may be suitable for use within
the scope of the invention include so-called defective
viruses, i.e. viruses which, in one or more genes, lack
the function necessary for autonomous virus replication,
for which they require helper viruses. Examples of this
category are DI-particles (defective interfering
particles) which are derived from the infectious
standard virus, have the same structural proteins as the
standard virus, have mutations and require the standard
virus as a helper virus for replication (Huang, 1987;
Holland, 1990). Examples of this group also include the
satellite viruses (Holland, 1990). Another grou~ is the
class of parvoviruses called the adeno-associated virus
(Berns, 1990).
Since the uptake cycles of many viruses into the
cell have not yet been fully explained, it must be
assumed that there are other viruses which have the
endosomolytic activity required for their suitability
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for use in this invention.
Also suitable within the scope of this invention
may be attenuated live vaccines (Ginsberg, 1980) or
vaccination strains.
The term viruses within the scope of the present
invention also includes inactivated viruses, e.g.
viruses inactivated by chemical treatment such as
treatment with formaldehyde, by W -radiation, by
chemical treatment combined with W -radiation, e.g.
psoralen/ W-radiation, by gamma-radiation or by neutron ~-
bombardment, as well as parts of viruses, e.g. the
protein content freed from nucleic acid (the empty virus ~ -
capsid), provided that they have the uptake functions of ~--
the intact virus. ~-
Inactivated viruses that are also used for
vaccines, for example, may be prepared by standard
methods known from the literature (Davis and Dulbecco,
1980, Hearst and Thiry, 1977) and then tested to see
whether they are suitable as components of the
conjugates according to the invention.
The virus may possibly be a chimeric virus whi¢h
has a foreign epitope in a region which is not essential
for the uptake function. However, even when such
chimeric viruses have lost their uptake function, they -
may be employed within the scope of combination ~-
complexes, so long as the virus has not lost its
endosomolytic properties.
In order to select a virus, an inactivated virus or
a virus component for the particular transfection which
is to be carried out, the process used may be, for
example, to investigate the virus first of all in
preliminary tests to see whether it has an effect when
the nucleic acid/polycation complexes are taken up into
the target cell. Furthermore, its uptake functions may
be tested by using it in transfection with
bioconjugates, e.g. transferrin-polycation conjugates or
another bioconjugate with specificity for the target
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cell to be transfected, and checking its ability to
increase the gene transfer capacity by measuring the
expression of a reporter gene.
When intact viruses are used, tests are carried
out, preferably in parallel to the preliminary tests
investigating the virus for its suitability for the
proposed transfection, to see whether the virus is
capable of replicating. The investigation for ability
to replicate is carried out using plaque assays (see
above) in the case of cytopathic viruses or in the case
of viruses which significantly impair the growth of the
host cells. For other viruses, detection methods
specific to the virus in question are used, e.g. the
hemagglutination test or chemico-physical methods (using
an electron microscope).
Within the scope of this invention, the preferred
viruses are those which can be produced in a high titre,
which are stable, have low pathogenicity in their native
state and in which a targeted elimination of the ability
to replicate i5 possible, especially adenoviruses. If a
specific cell population is to be transfected, viruses
which specifically infect this cell population are
I preferred. If the transfection is intended to attack
I different cell types, viruses which are infectious for a
wide range of cell types are used.
In any case, for therapeutic use of the invention
vivo, only those viruses or virus components may be
used in which the safety risks are minimized as far as
possible, particularly the risk of replication of the
virus in the cell and recombination of virus DNA with
host DNA.
In preliminary tests, adenovirus preparations were
inactivated using a conventional W sterilizing lamp or
with formaldehyde and it was found, surprisingly, that
the extent of inactivation of the viruses was
substantially greater than the reduction in the gene
transfer effect. This is a clear indication that
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mechanisms connected with the normal infection mechanism
in the active virus can be destroyed without eliminating ;
the effect which is essential for gene transfer.
Substances with an affinity for nucleic acid which
may be used according to the invention include, for
example, homologous polycations such as polylysine,
polyarginine, polyornithine or heterologous polycations
having two or more different positively charged amino
acids, these polycations possibly having different chain
lengths, and also non-peptidic synthetic polycations
such as polyethyleneimine. Other substances with an
affinity for nucleic acid which are suitable are natural
DNA-binding proteins of a polycationic nature such as
histones or protamines or analogues or fragments
thereof.
~ he sensitivity of a given cell line to
transformation by a virus which facilitates the entry of
conjugates into the cell or constitutes a ligand for
this type of cell depends on the presence and number of
surface receptors for the virus on the target cell.
Methods of determining the number of adenovirus
receptors on the cell surface are described for HeLa and
KB cells by Svensson, 1985, and Defer 1990. It is
assumed that the adenovirus receptor is expressed fairly ~
ubiquitously. -
Therefore, many cell lines can be transformed with
a vector system which contains an adenovirus or a part
thereof. However, some higher eukaryotic cells have few
or no viral receptors. If such cells are to be
transformed, it may be necessary to use a second
conjugate of an internalising factor which is bound to a
substance having an affinity for nucleic acid, the
internalising factor being specific for a surface
receptor of the higher eukaryotic cell, the virus
conjugate and the internalising factor conjugate being
complexed with the nucleic acid. Such complexes can
successfully be used to aid the transformation of higher
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eukaryotic cells, such as epithelial respiratory tract
cells which have a relatively low cell surface
population of adenovirus receptors (e.g. the cell line
HREl).
In a preferred embodiment of the invention, the
complexes may therefore optionally contain, in addition
to the virus conjugate, another conjugate in which a
substance having an affinity for nucleic acid, generally
the same one as in the virus conjugate, is coupled with
an internalizing factor having an affinity for the
target cell. This embodiment of the invention is used
particularly when the target cell has no or few
receptors for the virus. In the presence of another
internalizing factor-binding factor conjugate, these
endosomolytic conjugates profit from the internalizing
ability of the second conjugate, by being complexed to
the nucleic acid together with the second conjugate and
being taken up into the cell as part of the resulting
complex, hereinafter referred to as a "combination
complex" or "ternary complex".
Specifically, preliminary tests can determine
whether the use of an (other) internalizing factor -
permits or improves the uptake of nucleic acid
complexes, by carrying out parallel transfections with
nucleic acid complexes, first without any additional
internalizing factor, i~e. with complexes consisting of
nucleic acid and virus conjugate, and then with
complexes in which the nucleic acid is conjugated with
another conjugate containing an additional internalizing
factor for which the target cells have a receptor. If
an additional internalizing factor is used, it is
defined particularly by the target cells, e.g. by
specific surface antigens or receptors specific to a ~ `
cell type which thus permit the targeted transfer of
nucleic acid into this type of cell.
The term "internalizing factor" for the purposes of
the present invention refers to ligands or frag~ents
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thereof which, after binding to the cell are
internalized by endocytosis, preferably receptor-
mediated endocytosis, or factors the binding or
internalizing of which is carried out by fusion with
elements of the cell membrane.
Suitable internalizing factors include the ligands
transferrin (Klausner et al., 1983), conalbumin (Sennett
et al., 1981), asialoglycoproteins ~such as
asialotransferrin, asialorosomucoid or asialofetuin)
(Ashwell et al., 1982), lectins (Goldstein et al., 1980
and Shardon, 1987) or substances which contain galactose
and are internalized by the asialoglycoprotein receptor,
mannosylated glycoproteins tStahl et al., 1987),
lysosomal enzymes (Sly et al., 1982), LDL (Goldstein et
al., 1982), modified LDL (Goldstein et al., 1979),
lipoproteins which are taken up into the cells via
receptors (apo B100/LDL); viral proteins such as the HIV
protein gpl20; antibodies (Mellman et al., 1984; Kuhn et
al., 1982), Abrahamson et al., 1982), or fragments
thereof against cell surface antigens, e.g. anti-CD4,
anti-CD7; cytokines such as interleukin-1 (Mizel et al.,
1987), Interleukin-2 (Smith et al., 1985), TNF (Imamure
et al, 19~7), interferons (Anderson et al., ~982~; CSF
(colony-stimulating factor), (Walker et al., 1987);
factors and growth factors such as insulin (Marshall,
1985), EGF tepidermal growth factor), (Carpenter, 1984);
PDGF (platelet-derived growth factor) (Heldin et al.,
1982); TGFB (transforming growth factor B), (Massague et
al., 1986), nerve growth factor (Hosang et al., 1987)r ;
insulin-like growth factor I (Schalch et al., 1986),~LH,
FSH, (Ascoli et al., 1978), growth hormone (Hizuka et
al., 1981), prolactin (Posner et al., 1982), glucagon
(Asada-Xubota et al., 1983), thyroid hormones (Cheng et
al., 1980); ~-2-macroglobulin protease (Kaplan et al.,
1979); and "disarmed" toxins. Other examples are ~ ~-
immunoglobulins or fragments thereof as ligands for the ~-;
Fc-receptor or anti-immunoglobulin antibodies which bind
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to SIgs (surface immunoglobulins). The ligands may be
of natural or synthetic origin (see, Trends Pharmacol.
Sci. (1989), and the references cited therein).
The following are essential requirements for the
suitability of such internalizing factors according to
the present invention,
a) that they can be internalized by the specific
cell type into which the nucleic acid is to be
introduced and their ability to be internalized is
not affected or only slightly affected if they are
conjugated with the binding factor, and
b) that, within the scope of this property, they
are capable of carrying nucleic acid "piggyback"
into the cell by the route they use.
Without being pinned down to this theory, the
combination complexes are taken up by cells either by
binding to the surface receptor which is specific to the
internalizing factor or, if a virus or virus component
is used, by binding to the virus receptor or by binding
to both receptors by receptor-mediated endocytosis.
When the endosomolytic substance is released from the
endosomes, the DNA contained in the complexes is also
released into the cytoplasm and thereby escapes the
lysosomal degradation.
The presence of viruses, virus components or non-
viral endosomolytic agents as components of
endosomolytic conjugates in the DNA complexes has the
following advantages:
1) Wider applicability of the gene transfer
technology with nucleic acid complexes, since the
¦ endosomolytic agents themselves, especially if a
virus or virus component is used, may constitute
the internalizing factor or may also be complexed -
to the DNA in conjunction with another
internalizing factor (e.g. transferrin or
asialofetuin etc.). In this way it is possible to
make use of the positive effect of the viruses even
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for cells which do not have any receptor for the
virus in question.
2) Improvement in the efficiency of gene transfer,
since the binding of the endosomolytic conjugates
to the DNA ensures that they are jointly taken up
into the cells. The coordinated uptake and release
of viruses and DNA also gives rise to the
possibility of a reduction in the quantity of DNA
and viruses required for efficient gene transfer,
which is of particular importance for use in vivo.
In the experiments carried out according to the
invention, human transferrin was used as an additional
internalizing factor; moreover, the performance of the
conjugates according to the invention was demonstrated
by means of complexes of DNA and polylysine-conjugated
virus which contained no additional internalizing
factor-binding factor conjugate.
The binding of the virus to the substance having an
affinity for nucleic acid is achieved by covalent
bonding of the substance with an affinity for nucleic
acid to an antibody. It is preferable to use an
antibody which binds to an epitope in a virus protein
region not involved in the uptake function of the virus. --~
In the tests carried out within the scope of the ~ -~
invention, the binding between an adenovirus and a ~-
polycation was achieved by covalently conjugating an
antibody with specificity for the adenovirus capsid to a
polylysine molecule. It is known that the adenovirus
fibre and penton proteins are essential for the binding
of the virus and its uptake into the cell, whereas the
main capsid protein hexon is of lesser importance in
these processes. Therefore, an antibody was used which
brings about the binding of the adenovirus to polylysine r. :~
by recognition of an epitope on the hexon protein. This
specific binding was achieved by using, on the one hand,
a chimeric adenovirus which has a foreign epitope in the
surface region of its hexon protein. On the other hand,
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a monoclonal antibody was used which is specific for the.
heterologous epitope. (This construction is
diagrammatically shown in Fig. 1). This results in a
binding of the adenovirus to polylysine without
functionally destroying the capsid proteins.
The use of a special antibody for establishing the
bond between the virus and the nucleic acid-binding
substance is not critical. The prerequisite for the
suitability of a particular antibody is that it should
not neutralize, or should only partly neutralize, the
uptake function of the virus.
Within the scope of the present invention,
antibodies against epitopes in virus protein regions
which are not essential for the uptake function are
preferred. Examples of such virus regions are the hexon
protein of the adenovirus mentioned above or influenza
neuraminidase.
However, antibodies with specificity for virus
proteins which are involved in the uptake function are
also suitable, provided that it is ensured, by
maintaining a suitable stoichiometric ratio, that the
antibody occupies only part of the cell binding regions
of the virus, so that there are still sufficient domains
free for the binding of the virus to the cell. On the
other hand, antibodies which block the uptake function
of the virus may be used so long as the complex further
comprises a second conjugate comprising an internalizing
factor and a nucleic acid-binding substance, i.e. a
combination complex.
The quantity of antibody suitable for the specific
application can be determined by titration.
Monoclonal antibodies, possibly the Fab' fragments
thereof, are preferred.
If the virus is a chimeric virus with a foreign
epitope, the antibody is directed against this epitope.
Preferably, the virus is a chimeric adenovirus where the
coding sequence for the hexon region has been modified
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to include a sequence coding for a heterologous protei~
against which an antibody can be raised. The hexon
protein is composed of a hiyhly conserved base domain
and three less conserved loops that are highly exposed
on the surface of the viron (Roberts et al., 1986).
There are several short regions in these loops where the
Ad2 and Ad5 amino acid seguences are dissimilar, with
Ad5 showing changes as well as deletions compared to
Ad2. These are potential sites for the insertion of the
heterologous gene sequences coding for the heterologous
protein which may be used to link the adenovirus
immunologically to the substance having affinity for the
nucleic acid. Preferably, the heterologous gene ~ -~
sequence is inserted in the Ad5 gene sequence at amino
acid positions 161-165, 1~8-194, 269-281 and 436-438,
referred to as sites I, II, III and IV, respectively
(see Figure 8). At each potential site, a unique
restriction site may be created by means of site~
directed mutagenesis of a subclone of the Ad5 hexon
gene. Nucleotides coding for nonconserved amino acids
may be deleted at the same time, leaving more space for
insertion of the heterologous gene sequence. In
general, because of the small numbers of amino acids
which can be inserted at sites I, II, III and IV (up to
about 65 amino acids), the heterologous gene sequence
codes for only the amino acids corresponding to the
epitope and a minimal number of flanking sequences.
(The possible insertion sites created in the AdS hexon
gene sequence by directed mutagenesis are shown in Fig.
8; for the three-dimensional sketch of the hexon sub-
unit the representation by Roberts et al., 1986 has been
adapted.)
The epitope specificity of a particular monoclonal
antibody to a heterologous protein may be determined by
peptide scanning, see Geysen, et al., 1984, 1985, 1986,
1987, and EP-A-392,369, the disclosures of which are
fully incorporated by reference herein. According to
..
`-- 211~81~
- 19 -
this method, overlapping 8-amino acid long peptides of
the heterologous protein are prepared by methods of
solid phase synthesis. For example, peptide 1 consists
of amino acids 1-8, peptide 2 of amino acids 2-9, and so
on. The peptides remain bound to the solid carrier
after synthesis. Hybridoma cell culture supernatants or
purified monoclonal antibodies thereof are then tested
for reactivity to the immobilized peptides by ELISA.
Once the epitopic region is identified, the gene
sequence coding for the epitopic region may then be
inserted into any one of the restriction sites of
regions I, II, III or IV. There are many examples of
proteins and antibodies which are specific for the
protein. One of ordinary skill in the art can select a -~
heterologous protein-antibody combination which is
operable in the present invention with no more than
routine experimentation. For example, the known coding
sequence for a protein, for which an antibody thereto is
also known, may be inserted into the hexon region of an
adenovirus. The resulting chimeric virus can then be
tested for immunological binding, for example, to
labelled antibody in a competition, ELISA, or other
immunoassay format. Such immunoassay techniques are
well known and are practised routinely by those of
ordinary skill in the art.
I The antibody-polycation conjugates can be produced
chemically by a method known per se for the coupling of
peptides, preferably using the method described by
Wagner et al., 1990, and in EP-Al 388 758.
If monoclonal antibodies have suitable carbohydrate
side chains, particularly terminal sialic acids, in the
constant region of the heavy chain, the conjugates may
be prepared by binding the polycation to the
carbohydrate side chain, using the method described by
Wagner et al., l991bo
Another aspect of the invention relates to
complexes which are taken up into higher eucaryotic -~
, .~
. ~: :
- " 211~8~0
- 20 -
cells, containing nucleic acid and a conjugate of an
internalizing factor and a substance having an affinity
for nucleic acid. The complexes are characterized in
that the internalizing factor is a virus which is bound
to the substance having an affinity for nucleic acid via
an antibody in such a way that it has the ability to
penetrate into the cell as part of the conjugate/nucleic
acid complex and release the contents of the endosomes,
in which the co~plex is located after entering the cell,
into the cytoplasm.
As for the qualitative composition of the nucleic
acid complexes, generally the nucleic acid to be
transferred into the cell is determined first. The
nucleic acid is defined primarily by the biological
effect which is to be achieved in the cell and, in the
case of use for gene therapy, by the gene or gene
section which is to be expressed, e.g. for the purpose
of replacing a defective gene, or by the target sequence
of a gene which is to be inhibited. The nucleic acids
to be transported into the cell may be DNAs or RNAs,
whilst there are no restrictions imposed on the
nucleotide sequence.
If the invention is applied on tumour cells in
order to use them as a cancer vaccine, the DNA to be
introduced into the cell preferably codes for an
immunomodulating substance, e.g. a cytokine, such as IL-
2, IL-4, IFN-gamma, TNF-alpha. Combinations of cytokine
encoding DNAs may be particularly useful, e.g. IL-2 and
IFN-gamma. Another useful gene for insertion into
tumour cells may be the multi drug resistance gene
(mdr).
It is also possible to introduce two or more
different nucleic acid sequences into the cell, e.g. a
plasmid containing cDNAs coding for two different
proteins under the control of suitable regulatory
sequences or two different plasmid constructs containing -
different cDNAs.
2~1~800
. ~
- 21 -
Therapeutically effective inhibiting nucleic acids
for transfer into the cells in order to inhibit specific
gene sequences include gene constructs from which
antisense-RNA or ribozymes are transcribed. Furthermore,
it is also possible to introduce oligonucleotides, e.g.
antisense oligonucleotides, into the cell. Antisense
oligonucleotides comprise preferably 15 nucleotides or
more. Optionally, the oligonucleotides may be
multimerized. When ribozymes are to be introduced into
the cell, they are preferably introduced as part Oc a
gene construct which comprises stabilizing gene
elements, e.g. tRNA gene elements. Gene constructs o~
this type are disclosed in EP A O 387 775.
Apart from nucleic acid molecules which inhibit
genes, e.g. viral genes, due to their complementarity,
genes with a different mode of inhibitory action may be
employed. Examples are genes coding for viral proteins
which have so-called trans-dominant mutations
(Herskowitz, 1987). Expression o~ the genes in the cell
yields proteins which dominate the corresponding
wildtype protein and thus protect the cell, which
acquires "cellular immunity", by inhibiting viral
replication. Suitable are trans-dominant mutations of
viral proteins which are required for replication and
expression, e.g. Gag-, Tat and Rev mutants which were
shown to inhibit HIV replication (Trono et al., 1989;
Green et al., 1989; Malim et al., 1989).
Another mechanism of achieving intracellular
immunity involves expression of RNA molecules containing
the binding site for an essential viral protein, e.g.
so-called TAR decoys (Sullenger et al, 1990).
Examples of genes which can be used in somatic gene
therapy and which can be transferred into cells as -~
components of gene constructs by means of the present
invention include factor VIII (hemophilia A) (see, e.g.
Wood et al., 1984), factor IX (hemophilia B) (see, e.g.
Kurachi et al., (1982), adenosine deaminase (SCID) (see,
. .
211~8~
`
- 22 -
e.g. Valerio et al., 1984), a-l antitrypsin (emphysema
of the lungs) (see, e.g. Ciliberto et al., 1985) or the
cystic fibrosis transmembrane conductance regulator gene
(see, e.g. Riordan et al., 1989). These examples do not
constitute a restriction of any kind.
As for the size of the nucleic acids, a wide range
is possible; gene constructs of about 0.15 kb (in case
of a tRNA gene containing a ribozyme gene) to about
50 kb or more may be transferred into the cells by means
of the present invention; smaller nucleic acid molecules
may be utilised as oligonucleotides.
It is obvious that, precisely because the present
invention is not subject to any restrictions as to the
gene sequence and even very large gene constructs can be
transported with the aid of the invention, the possible
applications are extremely wide.
When determining the molar ratio of antibody-
polycation:nucleic acid it should be borne in mind that
complexing of the nucleic acid(s) takes place. In the
course of earlier inventions it had been established
that the optimum transfer of nucleic acid into the cell
can be achieved if the ratio of conjugate to nucleic
acid is selected so that the internalizing factor-
polycation/nucleic acid complexes are substantially
electroneutral. It was found that the quantity of
nucleic acid taken up into the cell is not reduced if
some of the transferrin-polycation conjugate is replaced
by non-covalently bound polycation; in certain cases
there may even be a substantial increase in DNA uptake
(Wagner et al., l991a). It had been observed that the
DNA inside the complexes is present in a form condensed
into toroidal structures with a diameter of 80 to
100 nm. The quantity of polycation is thus selected,
with respect to the two parameters of electroneutrality
and the achievement of a compact structure, whilst the
quantity of polycation which results from the charging
of the nucleic acid, with respect to achieving
;~ r~ r ~
...... . . . ~ .. .. .. . ... ...... . .... .. .. . .. .. .
~1~4~0~
- 23 -
electroneutrality of the complexes, as preferred
according ~o the invention, generally also guarantees
compacting of the DNA.
A suitable method of determining the ratio of
components contained in the complexes according to the
invention is first to define the gene construct which is
to be transferred into the cells and, as described
above, to determine a virus which is suitable for the
particular transfection. ~hen an antibody which binds
to the virus is conjugated with a polycation and
complexed with the gene construct. Starting from a
defined quantity of virus, titrations may then be
carried out by treating the target cells with this
(constant) quantity of virus and decreasing
concentrations of DNA complex (or optionally vice
versa). In this way the optimum ratio of DNA complex to
virus is determined. In a second step the cells are
treated with decreasing concentrations of the virus/DNA
complex mixture (at a constant ratio of virus to
complex) and the optimum concentration is determined.
Preferably, the virus is an adenovirus and the molar
ratio of adenovirus to substance having an affinity to
the nucleic acid is about 1:1 to about 1:100.
The length of the polycation is not critical, so
long as the complexes are substantially electroneutral, -
with respect to the preferred embodiment. The preferred
range of polylysine chain lengths is from about 20 to
about 1000 lysine monomers. However, for a given length
of DNA, there is no critical length of the polycation.
Where the DNA consists of 6,000 bp and 12,000 negative
charges, the amount of polycation per mole DNA may be,
e.g.:
60 molecules of polylysine 200 ~ ~
30 molecules of polylysine 400; or ~- -
120 molecules of polylysine 100, etc. -~
One of ordinary skill in the art can select other
combinations of polycation length and amount of
: ::
'c ~ ~ ~
~1 148~
- 24 -
polycation with no more than routine experimentation.
The complexes according to the invention can be
prepared by mixing the components nucleic acid and
antibody-bound polycation, which are present in the form
of dilute solutions. The DNA complexes can be prepared
at physiological saline concentrations. Another
possibility is to use high salt concentrations (about
2 M NaCl) and subsequently adjust to physiological
conditions by slow dilution or dialysis. The best
sequence for mixing the components nucleic acid,
antibody-polycation conjugate and virus is determined by
individual preliminary tests.
The invention relates in another aspect to a
process for introducing nucleic acid into higher
eucaryotic cells, in which the cells are brought into
contact with the complexes according to the invention in
such a way that the complexes are internalized and
released from the endosomes.
The present invention relates in another aspect to
pharmaceutical preparations containing as active
component a complex consisting of therapeutically active
nucleic acid, preferably as part of a gene construct,
and an antibody coupled via a polycation. Preferably,
this preparation is in the form of a lyophilisate or in
a suitable buffer in the deep-frozen state and the virus
preparation is mixed with the complex solution shortly
before use. Possibly, the virus may already be
contained in the pharmaceutical preparation, in which
case it is in deep-frozen state. Any inert
pharmaceutically acceptable carrier can be used, e.g.
saline solution or phosphate-buffered saline solution or
any carrier in which the DNA complexes have suitable
solubility properties to allow them to be used within
the framework of the present invention. For methods of
formulating pharmaceutical preparations reference is
made to Reminington's Pharmaceutical Sciences, 1980.
Possibly, the components required for transfection,
-- 211~0~
-
- 25 -
namely DNA, virus preparation and antibody conjugate or
the conjugation partners, possibly internalising factor
conjugate and possibly free polycation, are kept
separate in a suitable buffer or kept partly separate as
components of a transfection kit, which is a further
subject of the present invention. The transfection kit
of the present invention comprises a carrier which
contains one or more containers such as test tubes,
ampoules or the like which contain the materials
required for transfection of the higher eukaryotic cells
according to the present invention. In such a
transfection kit, a first container may contain on2 or
more different DNAs. A second container may contain one
or more different internalising factor conjugates, -
allowing the transfection kit to be used as a modular
system. Whether the components are present as a ready -~
to use preparation or are kept separately to be mixed
together immediately before use will depend not only on
the specific application but on the stability of the
complexes, which can be investigated routinely using
stability tests.
For therapeutic purposes the preparations may be
administered systemically, preferably by intravenous
route. The target organs for this type of
administration may be, for example, the liver, spleen, -
lungs, bone marrow and tumours.
Recently, the feasibility of using myoblasts
(immature muscle cells) to carry genes into the muscle
fibres of mice was shown. Since the myoblasts were shown
to secrete the gene product into the blooa, this method
may have a much wider application than treatment of -
genetic defects of muscle cells like the defect involved
in muscular dystrophy. Thus, engineered myoblasts may be
used to deliver gene products which either act in the
blood or are transported by the blood.
Examples for local application are the lung tissue
(use of the complexes according to the invention as a -
`- 21~8~
- 26 -
fluid for instillation or as an aerosol for inhalation),
direct injection into the liver, muscle tissue or into a
tumour or local administration in the gastro-intestinal
tract. Another method of administering the
pharmaceutical composition is via the bile duct system.
This method of adminstration penmits direct access to
the hepatocyte membranes on the bile ducts, thereby
avoiding interaction with constituents of the blood.
Therapeutic application may also be ex vivo, in
which the treated cells, e.g. bone marrow cells,
hepatocytes or myoblasts, are returned to the body (e.g.
Ponder et al., 1991, Dhawan et al., 1~91. Another ex
vivo application of the present invention relates to so-
called cancer vaccines. The principle of this
therapeutic possibility is to isolate tumour cells from
a patient and transfect the cells with a DNA coding for
a cytokine. In a next step the cells are optionally
inactivated, e.g. by radiation, so that they no longer
replicate but still express the cytokine. Then the
genetically altered cells are administered to the
patient from whom they were taken in the form of a
vaccine. In the area surrounding the vaccination site
the cytokines secreted activate the immune system partly
by activating cytotoxic T-cells. These activated cells
are able to exert their activity in other parts of the
body and attack even untreated tumour cells. In this
way the ris~ of tumour recurrence and metastasis
development is reduced. A suitable procedure for using
cancer vaccines for gene therapy has been described by
Rosenberg et al., 1992. Instead of the retroviral
vectors proposed by Rosenberg, the gene transfer system
according to the present invention can be used.
In order to determine the capacity for gene
transfer of adenovirus-antibody-polycation/DNA
complexes, a plasmid containing the gene coding for
Photinus Pyralis Luciferase (De Wet et al., 1987) as
reporter gene was used as the DNA. HeLa cells were used
2 ~
as target cells for the complexes; these cells have a
defined population of cell surface receptors for
adenoviruses (Philipson et al., 1968). When the
components of the conjugate according to the invention
(virus, antibody-polylysine-conjugate, DNA) were used in
conjunction, high values were obtained for the
expression of the luciferase reporter gene (Fig. 3)~
comparative experiments showed that the adenovirus only
slightly increased the transfer of non-complexed
plasmid-DNA. It was also found that DNA which was
complexed with the antibody-coupled polylysine (without
binding to the virus) was not appreciably taken up in
HeLa cells. In sharp contrast to this, high gene
expression values were obtained with the complex if the
DNA was able to interact with the adenovirus by binding
via the antibody. This effect was stopped when the ~-
virions were heat treated before the complexing. Since
this treatment selectively removes the viral uptake
functions without destroying the structural integrity of ~-
the virus (Defer et al., 1990), it can be concluded from
these experiments that it is the specific uptake
functions of the adenovirus which constitute the crucial
contribution to the success of gene transfer. It was
also found that competition for the heterologous epitope
on the surface of the chimeric adenovirus by a specific,
non-polylysine-bound monoclonal antibody also brings
about a reduction in the net gene expression. This
effect did not occur when a non-specific antibody was
used. It is therefore the specific interaction between
the antibody-bound DNA and the corresponding adenovirus
surface epitope which is essential to the achievement of
functional gene transfer by means of the complex. It
was also found that polylysine-complexed DNA was not
appreciably transferred into the target cells by the
adenovirus. This is an indication that the gene
transfer capacity of the complexes is not based on the
condensing of DNA but depends on the antibody-mediated
- -`` 211~800 :
- 28 -
binding of the reporter gene to the virion~
In accordance with this, the use of a virus which
did not have the epitope recognized by the polylysine-
coupled antibody could not achieve the high gene
expression values achieved by a virus which did have
this epitope. However, this virus was able to increase
the extent of gene transfer above the background level.
Since it is known that adenoviruses are capable of non-
specifically augmenting the cellular uptake of
macromolecules through the liquid phase (Defer et al.,
1990), this result was not unexpected. The fact that
this non-specific transport brought about a
significantly lower expression of the reporter gene than
the specific virus which was able to bind to the
antibody-polylysine/DNA complex demonstrates the
importance of specific binding of the components of the
complex.
The interaction of plasmid DN~ with polylysine
conjugates results in significant structural changes in
the DNA molecule, which are most clearly characterized
by striking condensation into a toroidal structure of 80
to 100 nm (Wagner et al., l991a). The diameter of the
virus is of the order of 70 to 80 nm (Philipson, 1983).
It was therefore assumed, on the basis of steric
considerations, that the optimum ratio of adenovirus to
antibody-polylysine-complexed DNA should be no more than
1:1. Furthermore, the diameter of the coated pits by
means of which the initial uptake step of receptor-
mediated endocytosis is carried out, is about 100 nm
(Darnell et al., 1975). On the basis of this fact it
was assumed that multimers exceeding this size would be
restricted in their uptake capacity. Within the scope
of the present invention these correlations were
analyzed, whilst the use of adenovirus in molar excess
relative to the antibody-polylysine-complexed DNA showed
that the maximum expression of reporter gene was
achieved at a ratio of 1:1 (Fig. 4). The optimum
, ~
4 8 ~
- 29 -
conjugate, within the scope of the experiments carried
out, was therefore found to be one which consists of a
single adenovirus internalizing domain in conjunction
with a single antibody-polylysine/DNA binding domain.
Next, the gene transfer efficiency of adenovirus-
antibody-polycation conjugates having this optimum ratio
was investigated. If logarithmic dilutions of the
complex were added to the target cells, there was a
corresponding logarithmic reduction in expression of the
reporter gene (Fig. 5), whilst it was noticeable that 107 ~-
DNA molecules, applied to lo6 HeLa cells using this
vector system, resulted in the detectable expression of
the reporter gene. Surprisingly, therefore, efficient
expression of a foreign gene was achieved with as few as
10 DNA molecules per cell in the form of adenovirus-
polycation-DNA complexes.
Therefore, with regard to the magnitude of DNA
uptake, the conjugates according to the invention show
clear superiority over the DNA gene transfer vectors,
which are required in numbers of approximately 500,000
DNA molecules per cell (Felgner et al., 1987, Felgner
et al., 1989, Maurer, 1989). Since these methods
efficiently convey the majority of the DNA into the
target area of the cells, namely the cytosol (Felgner
et al., 1989, Malone et al., 1989, Loyter et al., 1982),
the efficiency of the conjugates according to the
invention may possibly not be based exclusively on the
increase in release of the foreign DNA into the
cytoplasm; other mechanisms on the route of gene
transfer may also be enhanced.
In the configuration of the adenovirus-polylysine-
DNA complexes, the adenovirus part acts both as an agent
for breaking up endosomes and also as the ligand domain
of the complex. Therefore, the gene transfer efficiency
of the complexes for a givPn target cell should reflect
the relative number of adenovirus cell surface
receptors. Both the cell line HeLa and the cell line
.
`- 21~48~
- 30 -
Ks, which have large amounts of adenovirus receptors
(Philipson et al. 1968) exhibited a correspondingly high
degree of accessibility for gene transfer by means of
adenovirus-polylysine-DNA complexes (see Fig. 6). By
contrast, the relatively low number of adenovirus
receptors which characterises the cell lines MRC-5
(Precious and Russell 1985) and HBE1, is reflected in a
low gene transfer level by means of the complexes into
these cells.
Ternary complexes which contained another cell
surface ligand (internalising factor) in addition to the
adenovirus, showed significantly higher levels than the
conjugates which had only transferrin or adenovirus
ligand domains (Fig. 7A). The magnitude of this
increase was clearly not based on an additive effect of
transferrin-polylysine-DNA complexes plus adenovirus-
polylysine-DNA complexes. Since the ternary complexes
can be internalised by means of the adenovirus or
transferrin uptake mechanism, this obvious interaction
leads one to suppose that the adenovirus domain
facilitates entry into the cell by both routes,
presumably on the basis of endosomolysis brought about
by adenovirus. In order to demonstrate the selective
co-operation of the adenoviral domain of the ternary
complex when endosomes are broken up, the combination
complexes were applied to cell lines which have
different degrees of receptiveness for adenovirus-
polylysine-DNA complexes (Fig. 7B). The epithelial
respiratory tract cell line exhibits very low gene
transfer values, compared with HeLa cells, achieved by
AdpL-DNA complexes (Fig. 6), reflecting the relatively ~ -
low cell surface population of adenovirus receptors,
characteristic of this cell line. By clear contrast,
the use of ternary AdpL/TfpL complexes resulted in
levels of gene expression which were comparable with
those seen in HeLa cells. The receptiveness of this
cell line for gene transfer by the ternary complexes
- 2~8~ ~
- 31 -
agrees with the concept that the adenovirus domain is
taken up by means of the transferrin mechanism, whilst
intensifying the gene transfer by causing endosomes to
break up. From this arises the possibility of using the
endosomolytic property of adenovirus and other viruses
in the construction of conjugates which are thereby
enabled to escape from the cell vesicle system.
Within the scope of the present invention, the
direct in vivo transfer of a gene into the respiratory
tract epithelium by means of the complexes according to
the invention was demonstrated in a rodent model. This ~-
supports the possibility of using the present invention
to achieve transient gene expression in the respiratory
tract epithelium. The possibility of achieving genetic
modification of respiratory tract cells in situ is a
possible strategy of gene therapy for diseases of the
respiratory tract epithelium. In the tests carried o~t,
the transferrin-polylysine-DNA complexes yielded a low
level of reporter gene expression. This agrees with the
fact that this type of conjugate should be enclosed in
the endosomes. The binary adenovirus polylysine
complexes brought about a significantly higher gene
expression. This was further increased by using a
second internalising factor in the combination complex
hTfpL/AdpL. To find out whether the net gene expression
agrees with the transduction frequency, the proportion
of cells which had been transduced with the various
types of complex was determined. It was discovered that
there is such an agreement; the respiratory tract
epithelium modified with hTfpL in the primary culture
showed less than 1% transduction frequency, the AdpL
-complexes showed frequencies in the range from 20 to 30
and the combination complex showed more than 50%
modified cells.
The experiments carried out in vivo on rodent
models agreed with the results obtained in the primary -
culture. The examination of histological lung sections
. '~'
211~80~
- 32 -
from rats which had been treated with lacZ combination
complexes showed uneven zones of ~-galactosidase
activity which contained numerous positive cells. The
positive regions were associated with the bronchiolar
and distal regions of the respiratory tract.
Summary of the Fiaures
Fig. 1: Diagrammatic representation of adenovirus-
polycation-DNA complexes containing a foreign epitope on
the adenovirus capsid.
Fig. 2: Preparation of the chimeric adenovirus
Ad5-P202.
Fig. 3: Gene transfer to HeLa cells using
adenovirus-polycation-DNA complexes.
Fig. 4: Determining the optimum ratio of
adenovirus and polylysine-antibody-complexed DNA.
Fig. 5: Determin~ng the gene transfer achieved by
means of adenovirus-polycation-DNA complexes.
Fig. 6: Gene transfer to various eukaryotic cell
mediated by adenovirus-polycation-DNA complexes.
Fig. 7: Gene transfer mediated by combination
complexes containing adenovirus and human transferrin
conjugates.
A: Comparison of the efficiency of combination
complexes with binary complexes in HeLa cells.
B: Comparison of HeLa cells and HBEl cells with
regard to the efficiency of gene transfer by combination
complexes.
Fig. 8: Three dimensional representation of the
hexon sub-unit; possible insertions sites in the AD5
hexon gene sequence, obtained by site directed ~ -
mutagenesis;
Fig. 9: Transfection of primary respiratory tract
epithelial cell cultures. Relative level of net gene
transfer;
Fig. 10: Transfection of primary respiratory tract
epithelial cells cultures. Relative frequency of
' ~ '
'~ :
211~80~
- 33 -
transduction; ~ -
Fig. 11: Gene transfer via the intra-tracheal
route in vivo. Relative level of net gene transfer in
vivo;
Fig. 12: Gene transfer through the intra-tracheal
route in vivo. Localisation of heterologous gene
expression in the respiratory tract epithelium;
Fig. 13: In vivo application of chimeric
adenovirus-lectin-polylysine DN~ complexes.
2~14800
- 34 -
Examples
The invention is illustrated by means of the
following Examples:
Example 1
Preparation of antibody-polylysine coniuqates
1) Preparation of the chimeric adenovirus Ad-P202
In order to make changes in the Ad5 hexon gene it
was first necessary to subclone the gene. The plasmid
pEcoRIA-Ad5 (Berkner and Sharp, 1983) contains the left-
hand part of the adenovirus genome of map unit (m.u.) 0
to 76. The hexon gene is between m.u.52 and m.u.60. A
2.3 kbp HindIII/SstI-fragment contains that part of the
hexon gene in which the change is to be made. Since a
plurality of HindIII and SstI sites are contained in
pEcoRIA-Ad5 it was necessary to construct several
intermediate plasmids in order to be able to assemble
the altered hexon gene in the original plasmid. A
SalI/BamHI fragment (m.u. 46 to 60) contains the hexon
gene without any additional HindIII or SstI sites. ~-
First of all, the adenovirus DNA was recloned from m.u.
0 to 76 by using a vector designated pl42 (derived from -
the commercially obtainable plasmid pIBI24 (IBI, Inc.)
by restriction digestion with PvuII, followed by the
insertion of an EcoRI linker) which contains no SstI or
BamHI sites. Then the SalI sites at m.u. 26 were
eliminated ~y deleting the XbaI fragment (m.u. 3.7 to
29); the resulting vector was designated pl41-12.
Finally, the desired HindIII/SstI-fragment was cloned in
M13mpl8 and was therefore ready for mutagenesis. Site
directed mutagenesis was carried out with one of the
resulting clones using the method described by Kunkel,
1985. The codons 188 to 194 of the hexon gene were
removed and at this position a unique PmlI-site
occurring only once was introduced. The resulting clone
~ A~','!`'`'`' `~ ~ ~
2i~4800
- 35 -
(167-1) was then cut with PmlI and a double stranded
oligonucleotide coding for the amino acids 914-928 of
the mycoplasma pneumoniae Pl-protein was inserted
(Inamine et al., 1988). The Pl-sequence contains an
epitope which is recognized by the monoclonal antibody
301, the preparation of which is described hereinafter.
The modified HindIII/SstI-fragment was isolated ~rom
pl67-1 and ligated back into the original plasmid
pEcoRIA-Ad5. The preparation of Ad-P202 is shown in
Fig. 2.
2) Preparation of a monoclonal antibody with
specificity for the chimeric adenovirus (MP301)
a) Immunization
The monoclonal antibody was prepared by standard
methods.
The Mycoplasma pneumoniae strain M-129 ~ATCC#29342)
was used as the antigen. After cultivation in a culture
flask (~u et al., 1977) it was washed 3 times with PBS,
Mycoplasma pneumoniae was harvested and taken up in
0.5 ml of PBS. 10 ~g of the antigen were used for
immunization: 3 female BALB/c mice about six weeks old
were immunized in accordance with the following
protocol:
1st immunization: about 10 ~g of antigen per mouse in
complete Freund's adjuvant by intraperitoneal route.
2nd immunization: about 10 ~g of antigen per mouse in
incomplete Freund's adjuvant by subcutaneous route, 3
weeks after the first immunization.
3rd immunization: about 10 ~g of antigen per mouse in
incomplete Freund's adjuvant by intraperitoneal route, 2
weeks after the second immunization.
4 8 1~ 0
- 36 -
One week later, samples of serum were taken from
the mice and the serum titres were measured. The mouse
with the hi~hest titre was boos~ed by i.v. injection of
10 ~g antigen into the tail; the spleen cells of this
mouse were taken out after 3 days for fusion with
hybridoma cells.
b) Fusion:
About lo8 spleen cells were fused with about 1O8
myeloma cells of the line SP2/0 Agl4 (ATCC CRL-1581) in
the presence of PEG 4000 (50% in serum-free culture
medium) using the method of Kohler and Milstein, 1975.
Then the cells were grown for 2 weeks in HAT-selection
medium, then for one week in HT-medium and finally in
normal culture medium (DMEM plus 10% FCS plus
penicillin, streptomycin). By means of radioimmuno-
sorbent assay (RIA) screening was carried out for
antibody-producing clones and specificity for the
Mycoplasma pneumoniae Pl protein was determined using -~
Western blot. The "soft agar" method was used to obtain
monoclones.
c) Investiaation of the monoclonal antibody MP301 for
neu~tralizina effect o~ adenovirus Ad-P202
In order to determine whether the monoclonal
antibody MP301 neutralizes the ability of the virus to
infect cells, the titre of Ad-P202 was determined once
with and once without the addition of antibody
(7 ~g/ml), using HeLa-cells (approximately 50% confluent
in 2% FCS/DMEM on 96-well plates) as the target cells.
Serial dilutions were prepared of Ad-P20~ which were
applied to the HeLa-titre plates with or without
antibody. The plates were incubated for 48 hours at
37~, 5% CO2, stained with crystal violet and
investigated for IC 50 (inhibition concentration, about
50% cell lysis). The titre of 1:2048 was obtained with
and without antibody.
2il~
- 37 -
d) Preparation of MP301-Dolylvsine coniuqates
Coupling of the monoclonal antibody to polylysine
was carried out using the method described by Wagner
et al., 1990, and in EP-Al 388 758.
20.6 nmol (3.3 mg) of the monoclonal antibody MP301
in 1 ml of 200 mM HEPES pH 7.9 were treated with a 5 mM
ethanolic solution of SPDP (loo nmol). After 3 hours at
ambient temperature the modified antibody was gel-
filtered over a Sephadex G-25 column, thereby obtaining
19 nmol of antibody modified with 62 nmol of
dithiopyridine linker. The modified antibody was
allowed to react with 3-mercaptopropionate-modified
polylysine (22 nmol, average chain length 300 lysine
monomers, FITC-labelled, modified with 56 nmol mercapto-
propionate linker~ in 100 mM HEPES pH 7.9 under an argon
atmosphere. Conjugates were isolated by cation exchange
chromatography on a Mono S HR5 column (Pharmacia).
(Gradient: 20 to 100% buffer. Buffer A: 50 mM HEPES pH
7.9; buffer B: buffer A plus 3 M sodium chloride. The
product fraction eluted at a salt concentration of
between 1.65 M and 2 M. Dialysis against HBS (20mM
HEPES pH 7.3, 150 mM NaCl) produced a conjugate
consisting of 9.1 nmol MP301 and 9.8 nmol polylysine.
Example 2
Gene transfer bY means of adenovirus-polycation-DNA-
complexes in EucarYotic cells
In the course of the experiments carried out in
this Example, various combinations of specific and non-
specific complex components were examined for their
ability to transport a reporter gene into HeLa and other
cells.
Complexing of DNA with the antibody-coupled
polylysine was carried out by diluting 6 ~g of purified
pRSVL-DNA in HBS (150 mM NaCl, 20 mM HEPES, pH 7.3) to a
total volume of 350 ~1 and purifying it with 9.5 ~g of
2~148~1D
- 38 -
MP301pL in 150 ~1 of total volume of the same buffer.
(pRSVL contains the Photinus pyralis luciferase gene
under the control of the Rous Sarcoma virus LTR
enhancer/promoter (Uchida et al., 1977, De Wet et al.,
1987), prepared by Triton X Lysis Standard Method
(Maniatis), followed by CsCl/EtBr equilibrium density
gradient centrifugation, decolorizing with butanol-l and
dialysis against 10 mM tris/HCl pH 7.5, 1 mM EDTA in
350 ~1 HBS (150 mM NaCl, 20 mM HEPES, pH 7.3).) The
quantity of antibody-coupled polylysine is based on a
calculation of the guantity required to achieve
electroneutrality of the imported DNA. The polylysine-
antibody-complexed DNA was diluted in HBS to a final
concentration of 2 x 1011 DNA molecules per ml. The
adenovirus P202-Ad5 was diluted in ice~cold DMEM,
supplemented with 2% FCS, to a final concentration of
2 x 10l1 virus particles per ml. Equal volumes of
antibody-polylysine DNA and virus were combined and
incubated for 30 minutes at ambient temperature. The
target cells used for the gene transfer were HeLa cells
which had been grown in DMEM medium supplemented with 5% ~-
FCS, 100 I.U. penicillin/ml and 100 ~g streptomycin/ml,
in 60 mm tissue culture dishes (300,000 cells~. For
comparison to HeLa cells, the cell lines HBEl, KB (ATCC
No. CCL 173 and MRC-5 (ATCC No. CCL 171) were evaluated.
HBEl, a respiratory cell line, was grown in F12-7X
medium as described by Willumson et al., 19~9. KB and
MRC-5 were grown in Eagle's minimal essential medium/10%
heat-inactivated FCS/penicillin at 100 international
units per ml/streptomycin at 100 ~g per ml/10 mM
nonessential amino acids/2 mM glutamine.
Before application of the transfection medium, the ; -
plates were cooled at 4C for 30 minutes, the medium was
removed, 1 ml of transfection medium was added and the
cells were incubated for 2 hours at 4C. This step was
carried out in order to bring about binding of the DNA
complexes to the cells without them being internalized. -~
, . ~: -. .
.. . ~
211~
-
- 39 -
After this binding step, the plates were washed three
times with ice-cold 2% FCS/DMEM in order to eliminate
any non-bound reaction components in the liquid phase.
After the addition of 2 ml of ice-cold 2% FCS/DMEM the
plates were slowly heated. Then the plates were placed
in an incubator for 16 hours (37C, 5% C02). In order to
measure the expression of reporter gene, cell lysates -~
were prepared, standardized in terms of their total
protein content and investigated for luciferase activity
exactly as described by Zenke et al., 1990. (The
luminometer was calibrated so that one picogram of
luciferase yields 50,000 light units.)
pRSVL reporter plasmid DNA was combined with
adenovirus P202-Ad5 without having been previously
complexed with the polylysine antibody conjugate ~DNA +
P202-Ad5). Furthermore, pRSVL-DNA, complexed with the
antibody-coupled polylysine, was investigated in the
absence of the specific virus (DNA + MP301pL) and these
two reaction media were compared with a reaction medium
containing the total combination of the complex
components (DNA + MP301pL + P202Ad5). Analogously, the
complexes were investigated for their ability to perform
gene transfer by using a specific antibody which had
been heat inactivated before complexing (50-C, 30 min)
(DNA + M~301pL + P202-Ad5). Competition experiments
were carried out with the specific adenovirus in the
presence of the polylysine-coupled antibody MP301 plus a
ten-fold molar excess of non-polylysine-coupled MP301
(DNA + MP301pL + MP301 + P202-Ad5) or in the presence of
MP301pL and a ten-fold molar excess of non-coupled
irrelevant monoclonal antibody, anti-rat-IgG (DNA +
MP301pL + anti-rat IgG + P202-AD5). Furthermore, before
incubation with the specific virus, the reporter plasmid
DNA was complexed with non-conjugated polylysine (4 ~g)
in an amount equimolar to the antibody-coupled
polylysine (DNA + pL + P202-Ad5). The complex forming
reactions using the adenovirus WT300, which lacks the
sa~
: ::
- 40 -
epitope recognized by MP301, were carried out exactly as
for the specific virus P202-ADs. The experiments were
carried out three times in all. The results are shown
in Fig. 3; the data represent mean values + SEM. The
dotted horizontal line shows the background signal oP
untreated HeLa cells. The results obtained with the
cell lines HBEl, KB and MRC-5 compared with He~a cells
are shown in Fig. 6.
Example 3
Determination of oPtimum ratio of adenoyirus and
antibodv-polvlYsine/DNA for aene transfer
In the experiments carried out, the results of
which are given in Fig. 4, adenovirus-antibody-
polylysine/DNA complexes with the complex components in
various proportions were examined for their ability to
permit gene transfer into HeLa cells. The complex
forming reactions were carried out as given in
Example 2, except that 2.5 x 101 DNA molecules complexed
with the antibody-polylysine conjugate were used, with
different amounts of the specific adenovirus P202-Ad5.
The cultivation of the cells, the application of the ;
complexes to the cells, incubation of the cells and
measurement of the reporter gene expression were as in
Example 2. The data shown represent mean + SEM from
four different experiments.
:~,~" ',
Example 4
The measurement of the qene transfer performance of
adenovirus-~olvcation-DNA complexes
Limiting dilutions of the complex, prepared exactly
as in Example 2, were investigated to see how effective
they are at enabling the detectable expression of the
reporter gene in HeLa cells. After complex formation,
logarithmic dilutions of the complex in 2% FCS/DMEM were
. :
'~
: ::
,~
:', ~ :
~ ; } ~ i 2 ,:
21~8~0
- 41 -
prepared. 1 ml aliquots of the various dilutions were
applied to 60 mm tissue culture dishes which contained
5 x 105 HeLa cells. After one hour incubation (37~, 5
Co2), 3 ml of 5% FCS/DMEM were added and the plates were
incubated for a further 16 hours under the same
conditions. The reporter gene expression was measured
as in Example 2. The values for luciferase expre~sion
given in Fig. 5 corxespond to the mean values + SEM from
3 or 4 experiments. The dotted horizontal line shows
the background signal of untreated HeLa cells.
Example 5
Gene Transfer bY means of Combination Complexes
Containing Adenovirus and Human Transferrin
To prepare ternary complexes containing a
combination of adenovirus and human transferrin domains,
the epitope-tagged adenovirus P202-Ad5 (2.5 x 101
particles) was diluted in 750 ~1 2% FCS/DMEM and
combined with polylysine monoclonal antibody MP301pLys
(2 ~g) diluted in 250 ~1 HBS. Incubation was performed
for 30 minutes at room temperature. Plasmid DNA pRSVL
(6 ~g~ diluted in 250 ~1 HBS was then added to the
mixture and incubated for an additional 30 minutes at
room temperature. The resulting adenovirus-polylysine-
DNA complexes were predicted to possess incompletely
condensed DNA based upon total polylysine content. To
complete DNA condensation and contribute a human
transferrin moiety to the complexes, human transferrin
polylysine conjugates ~Wagner et al., 1990) (9 ~g)
diluted in 250 ~1 HBS were added to the adenovirus-
polylysine-DNA complexes. A final incubation of 30
minutes at room temperature was performed. The
resulting combination complexes were incubated with
tissue culture cells to achieve specific binding of the
formed complexes (4~C, 2 hours). The plates were then
washed three times with ice-cold 2% FCS/DMEM and
r ~ ' A~
8 0 ~
- 42 -
returned to the in_ubator (37C, 5% co2) for 16 hours
after the addition of 2 ml 2% FCS/DMEM. Evaluation of
reporter gene expression was as before.
Fig. 7A shows the relative values for gene
expression brought about by human transferrin-
polylysine-DNA complexes (hTfpL), adenovirus-polylysine-
DNA complexes (AdpL) and ternary complexes, in HeLa
cells. Fig. 7B shows the relative accessibility for
gene transfer by ternary complexes (AdpL/hTfpL) of HeLa
and HBEl cells.
Example 6
Gene Transfer in Respiratory Tract Epithelial Cells
usinq Binary and TernarY Adenovirus Com~lexes
For these experiments the rat Sigmodon hispidus
("Cotton Rat") was used which has been found to be a
suitable animal model for human adenoviral lung diseases
(Pacini et al., 1984). In addition, the binary and -~
ternary complexes described in the preceding example
were used.
a) Transfection of Primary ResPiratory Tract
EPithelial Cell Cultures
The primary cultures were prepared using known
methods (Van Scott et al., 1986). The dissociated cells
were harvested, washed three times with F12-7X medium
and plated out into 3cm tissue culture dishes at a
density of 5 x 105 cells per dish. The cells were kept
in F12-7X medium and when 50 to 75% confluence was ~-
achieved they were used for the gene transfer
experiments, and this normally took 2-3 days. For the
gene transfer experiments the complexes were applied
directly to the cells and incubated for 24 hours. For
these experiments, pCMV DNA was used. The plasmid pCMV
was prepared by removing the BAMHl insert of the plasmid
pSTCX556 (Severne et al., 19~8), treating the plasmid
211~0
- 43 -
with klenow fragment and using the HindIII/SspI and
klenow-treated fragment from the plasmid pRSVL, which
contains the sequence coding for luciferase, or the
sequence coding for ~-galactosidase (MacGregor and
Caskey, 1989), and the resulting plasmids were
designated pCMVL and pCMV~-gal. Complex formation was
carried out analogously to pRSVL.
i) Relative Level of Net Gene Transfer
For these experiments the reporter plasmid pCMVL
was used. The cells were investigated for luciferase
gene expression after 24 hours; the results are given in
Fig. 9. The bottom axis shows the measurement of the
unmodified cells whilst the Y axis shows the luciferase
gene expression as light units per 25 ~g of total
protein, obtained from cell lysates. The experiments
were each carried out 3-4 times and the results are mean
+ SEM.
ii) Relative Transduction Frequency
In these experiments the plasmid pCMV~-gal was used
as reporter DNA. The cells were transfected as
described above and after 24 hours the reporter gene
expression was determined by staining using the method
described by MacGregor et al., 1989. The results are
shown in Fig. 10 (magnification: 320 x). A: hTfpL, B:
AdpL, C: hTfpL/AdpL.
¦ b) Gene Transfer via the Intra-tracheal Route in vivo
I The animals were anaesthetised with methoxyflurane.
After a vertical cut had been made in the ventral side
of the neck the wind pipe was cut off squarely. The
complexes (250-300 ~1: 3 ~g of plasmid DNA) were
injected directly into the wind pipe in full view in the
animals which had been positioned at an angle of 45.
The animals were killed with C02 and the wind pipe and
lungs were harvested en bloc after in situ rinsing with
s o n
- 44 -
cold phosphate buffered saline solution (PBS). For the
luciferase test the lung tissue was homogenised in
extraction buffer, the lysates were standardised to a
total protein content and the luciferase gene expression
was measured as described.
i) Relative Level of Net Gene Transfer in vivo
24 hours after transfection the luciferase
expression was measured. The results are shown in Fig.
11. The light units specified relate to 1250 ~g of
total protein, obtained from the lung lysates. The ;~
experiments were each carried out 3-4 times and the
results are given as mean values + SEM.
ii) Localisation of Heteroloqous Gene Expression in the
Res~iratorv Tract E~ithelium
For these tests, the plasmid pCMV~-gal was used as
reporter DNA; hTfpL/AdpL combination complexes were
used. 24 hours after the injection, 14 ~g thick frozen
sections of the harvested lungs were investigated for
expression of the reporter gene by staining with X-gal
and counter-staining with Nuclear Fast Red. The
stainings are shown in Fig. 12 (magnification: 600 x);
they show the results of transfection of rats treated
with hTfpL/AdpL complexes, containing an irrelevant non-
lacZ plasmid designated pRc/RSV or pCMV~-gal, containing ~ ~
the lacZ reporter plasmid. A: Example of a bronchiole ~ ~;
treated with complexes containing pRc/RSV; B: Example of
a bronchus, treated with complexes containing pCMB~-gal;
C: Example of the distal respiratory tract region
treated with complexes containing prC/RSV; D: Example of
the distal respiratory tract region treated with
complexes containing pCMV~-gal; E: Enlargement of the ~-
galatosidase-positive region from lungs, treated with
complexes containing pCMV~-gal (magnification: 1.000 x).
~148~Q
- 45 -
Example 7
Construction of Coniuqates with Specificity for Lunq
Reaions
conjugates were constructed with a view to
selective binding to the ciliated section of the
respiratory tract epithelium. The construction of such
conjugates demands (generally as well as in this
particular instance) confirmation of the binding
properties of the ligand candidates in the conjugate
configuration. SNA lectin was selected as a candidate.
a) Pre~aration of Ternarv Adenovirus-Polvlvsine/
Lectin-PolylYsine/DNA ComDlexes
As in the preceding examples the chimeric
adenovirus P202 (2 x 101 particles) was combined with
the antibody polylysine conjugate MP301pL (1.2S ~g) in
250 ~1 HBS and incubated for 30 minutes at ambient
temperature. Then the reporter plasmid pCMVL was added
(6 ~g in 125 ~1 of HBS) and incubation was continued for
a further 30 minutes at ambient temperature. A
commercially available biotinylated lectin SNA (E-Y Lab,
San Mateo, CA: 2.8 ~g) in 62.5 ~1 of HBS was combined
with streptavidin-polylysine (1.35 ~g in 62.5 ~1 HBS)
and left to stand for 30 minutes at ambient temperature
in order to form SNA-polylysine. The SNA-polylysine was
combined with the above reaction mixture in order to
form SNA-adenovirus-polylysine DNA complexes. As a
comparison, complexes were prepared without the SNA
ligand as described in the preceding examples.
b) In vivo use of Lectin Com~lexes in the Lunqs
The cell-specific tropism of lectin SNA for the
ciliated human respiratory tract epithelium has its
counterpart in the ferret, which was used as animal
model in the experiments. Male animals weighing about
1.5 kg were used. For each animal the complexes
. ~
2114800
- 46 -
prepared in a) were used in four-fold amounts. The
animals were anaesthetised and the complexes were
introduced into the central lobe of the right lung by
means of a bronchus scope. After 24 hours the different
lung regions were harvested, homogenised and ~-
investigated for luciferase activity. The lung regions
investigated included parts which were not in contact
with the complexes when they were administered (left
hand upper lung section, parenchyme of the left hand
upper lobe, lower part of the wind pipe) and parts which
were in contact with the complexes (right hand central
lobe, parenchyme of the right hand central lobe). As
can also be seen from Fig. 13, the regions which were in
contact with the complexes exhibited luciferase ;
expression (right hand central lobe, parenchyme of the
right hand central lobe; second and third bars), wh~reas
the other regions (lower part of the wind pipe, first
bar; parenchyme of the left hand upper lobe, fourth bar;
left hand upper lung section, fifth bar) showed no
expression.
c) Investiaating the_Specificitv of the Liqand
In parallel thereto, the specificity of binding of
lectin conjugates was investigated. Since no anti~
lectin antibody was available, transferrin/lectin-
polylysine/DNA complexes were prepared and binding was
carried out with a primary anti~transferrin antibody,
backed up by a secondary horse-raddish peroxidase-
coupled anti-mouse antibody. The conjugates were
detected in the apical part of the ciliated cell
population, but the test design did not allow of any
clear conclusions as to specific binding.
-- 21~8~
- 47 -
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