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Description  |
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BACKGROUND OF THE INVENTION
The present invention relates to introduction of naked DNA and RNA
sequences into a vertebrate to achieve controlled expression of a
polypeptide. It is useful in gene therapy, vaccination, and any
therapeutic situation in which a polypeptide should be administered to
cells in vivo.
Current research in gene therapy has focused on "permanent" cures, in which
DNA is integrated into the genome of the patient. Viral vectors are
presently the most frequently used means for transforming the patient's
cells and introducing DNA into the genome. In an indirect method, viral
vectors, carrying new genetic information, are used to infect target cells
removed from the body, and these cells are then re-implanted. Direct in
vivo gene transfer into postnatal animals has been reported for
formulations of DNA encapsulated in liposomes and DNA entrapped in
proteoliposomes containing viral envelope receptor proteins (Nicolau et
al., Proc. Natl. Acad Sci USA 80:1068-1072 (1983); Kaneda et al., Science
243:375-378 (1989); Mannino et al., Biotechniques 6:682-690 (1988).
Positive results have also been described with calcium phosphate
co-precipitated DNA (Benvenisty and Reshef Proc. Natl. Acad Sci USA
83:9551-9555 (1986)).
The clinical application of gene therapy, as well as the utilization of
recombinant retrovirus vectors, has been delayed because of safety
considerations. Integration of exogenous DNA into the genome of a cell can
cause DNA damage and possible genetic changes in the recipient cell that
could predispose to malignancy. A method which avoids these potential
problems would be of significant benefit in making gene therapy safe and
effective.
Vaccination with immunogenic proteins has eliminated or reduced the
incidence of many diseases; however there are major difficulties in using
proteins associated with other pathogens and disease states as immunogens.
Many protein antigens are not intrinsically immunogenic. More often, they
are not effective as vaccines because of the manner in which the immune
system operates.
The immune system of vertebrates consists of several interacting
components. The best characterized and most important parts are the
humoral and cellular (cytolytic) branches. Humoral immunity involves
antibodies, proteins which are secreted into the body fluids and which
directly recognize an antigen. The cellular system, in contrast, relies on
special cells which recognize and kill other cells which are producing
foreign antigens. This basic functional division reflects two different
strategies of immune defense. Humoral immunity is mainly directed at
antigens which are exogenous to the animal whereas the cellular system
responds to antigens which are actively synthesized within the animal.
Antibody molecules., the effectors of humoral immunity, are secreted by
special B lymphoid cells, B cells, in response to antigen. Antibodies can
bind to and inactivate antigen directly (neutralizing antibodies) or
activate other cells of the immune system to destroy the antigen.
Cellular immune recognition is mediated by a special class of lymphoid
cells, the cytotoxic T cells. These cells do not recognize whole antigens
but instead they respond to degraded peptide fragments thereof which
appear on the surface of the target cell bound to proteins called class I
major histocompatibility complex (MHC) molecules. Essentially all
nucleated cells have class I molecules. It is believed that proteins
produced within the cell are continually degraded to peptides as part of
normal cellular metabolism. These fragments are bound to the MHC molecules
and are transported to the cell surface. Thus the cellular immune system
is constantly monitoring the spectra of proteins produced in all cells in
the body and is poised to eliminate any cells producing foreign antigens.
Vaccination is the process of preparing an animal to respond to an antigen.
Vaccination is more complex than immune recognition and involves not only
B cells and cytotoxic T cells but other types of lymphoid cells as well.
During vaccination, cells which recognize the antigen (B cells or
cytotoxic T cells) are clonally expanded. In addition, the population of
ancillary cells (helper T cells) specific for the antigen also increase.
Vaccination also involves specialized antigen presenting cells which can
process the antigen and display it in a form which can stimulate one of
the two pathways.
Vaccination has changed little since the time of Louis Pasteur. A foreign
antigen is introduced into an animal where it activates specific B cells
by binding to surface immunoglobulins. It is also taken up by antigen
processing cells, wherein it is degraded, and appears in fragments on the
surface of these cells bound to Class II MHC molecules. Peptides bound to
class II molecules are capable of stimulating the helper class of T cells.
Both helper T cells and activated B cells are required to produce active
humoral immunization. Cellular immunity is thought to be stimulated by a
similar but poorly understood mechanism.
Thus two different and distinct pathways of antigen processing produce
exogenous antigens bound to class II MHC molecules where they can
stimulate T helper cells, as well as endogenous proteins degraded and
bound to class I MHC molecules and recognized by the cytotoxic class of T
cells.
There is little or no difference in the distribution of MHC molecules.
Essentially all nucleated cells express class I molecules whereas class II
MHC proteins are restricted to some few types of lymphoid cells.
Normal vaccination schemes will always produce a humoral immune response.
They may also provide cytotoxic immunity. The humoral system protects a
vaccinated individual from subsequent challenge from a pathogen and can
prevent the spread of an intracellular infection if the pathogen goes
through an extracellular phase during its life cycle; however, it can do
relatively little to eliminate intracellular pathogens. Cytotoxic immunity
complements the humoral system by eliminating the infected cells. Thus
effective vaccination should activate both types of immunity.
A cytotoxic T cell response is necessary to remove intracellular pathogens
such as viruses as well as malignant cells. It has proven difficult to
present an exogenously administered antigen in adequate concentrations in
conjunction with Class I molecules to assure an adequate response. This
has severely hindered the development of vaccines against tumor-specific
antigens (e.g., on breast or colon cancer cells), and against weakly
immunogenic viral proteins (e.g., HIV, Herpes, non-A, non-B hepatitis, CMV
and EBV).
It would be desirable to provide a cellular immune response alone in
immunizing against agents such as viruses for which antibodies have been
shown to enhance infectivity. It would also be useful to provide such a
response against both chronic and latent viral infections and against
malignant cells.
The use of synthetic peptide vaccines does not solve these problems because
either the peptides do not readily associate with histocompatibility
molecules, have a short serum half-life, are rapidly proteolyzed, or do
not specifically localize to antigen-presenting monocytes and macrophages.
At best, all exogenously administered antigens must compete with the
universe of self-proteins for binding to antigen-presenting macrophages.
Major efforts have been mounted to elicit immune responses to poorly
immunogenic viral proteins from the herpes viruses, non-A, non-B
hepatitis, HIV, and the like. These pathogens are difficult and hazardous
to propagate in vitro. As mentioned above, synthetic peptide vaccines
corresponding to viral-encoded proteins have been made, but have severe
pitfalls. Attempts have also been made to use vaccinia virus vectors to
express proteins from other viruses. However, the results have been
disappointing, since (a) recombinant vaccinia viruses may be rapidly
eliminated from the circulation in already immune individuals, and (b) the
administration of complex viral antigens may induce a phenomenon known as
"antigenic competition," in which weakly immunogenic portions of the virus
fail to elicit an immune response because they are out-competed by other
more potent regions of the administered antigen.
Another major problem with protein or peptide vaccines is anaphylactic
reaction which can occur when injections of antigen are repeated in
efforts to produce a potent immune response. In this phenomenon, IgE
antibodies formed in response to the antigen cause severe and sometimes
fatal allergic reactions.
Accordingly, there is a need for a method for invoking a safe and effective
immune response to this type of protein or polypeptide. Moreover, there is
a great need for a method that will associate these antigens with Class I
histocompatibility antigens on the cell surface to elicit a cytotoxic T
cell response, avoid anaphylaxis and proteolysis of the material in the
serum, and facilitate localization of the material to monocytes and
macrophages.
A large number of disease states can benefit from the administration of
therapeutic peptides. Such peptides include lymphokines, such as
interleukin-2, tumor necrosis factor, and the interferons; growth factors,
such as nerve growth factor, epidermal growth factor, and human growth
hormone; tissue plasminogen activator; factor VIII:C;
granulocyte-macrophage colony-stimulating factor; erythropoietin; insulin;
calcitonin; thymidine kinase; and the like. Moreover, selective delivery
of toxic peptides (such as ricin, diphtheria toxin, or cobra venom factor)
to diseased or neoplastic cells can have major therapeutic benefits.
Current peptide delivery systems suffer from significant problems,
including the inability to effectively incorporate functional cell surface
receptors onto cell membranes, and the necessity of systemically
administering large quantities of the peptide (with resultant undesirable
systemic side effects) in order to deliver a therapeutic amount of the
peptide into or onto the target cell.
These above-described problems associated with gene therapy, immunization,
and delivery of therapeutic peptides to cells are addressed by the present
invention.
SUMMARY OF THE INVENTION
The present invention provides a method for delivering a pharmaceutical or
immunogenic polypeptide to the interior of a cell of a vertebrate in vivo
comprising the step of introducing a preparation comprising a
pharmaceutically acceptable injectable carrier and a naked polynucleotide
operatively coding for the polypeptide into the interstitial space of a
tissue comprising the cell, whereby the naked polynucleotide is taken up
into the interior of the cell and has an immunogenic or pharmacological
effect on the vertebrate. Also provided is a method for introducing a
polynucleotide into muscle cells in vivo, comprising the steps of
providing a composition comprising a naked polynucleotide in a
pharmaceutically acceptable carrier, and contacting the composition with
muscle tissue of a vertebrate in vivo, whereby the polynucleotide is
introduced into muscle cells of the tissue. The polynucleotide may be an
antisense polynucleotide. Alternatively, the polynucleotide may code for a
therapeutic peptide that is expressed by the muscle cells after the
contacting step to provide therapy to the vertebrate. Similarly, it may
code for an immunogenic peptide that is expressed by the muscle cells
after the contacting step and which generates an immune response, thereby
immunizing the vertebrate.
One particularly attractive aspect of the invention is a method for
obtaining long term administration of a polypeptide to a vertebrate,
comprising the step of introducing a naked DNA sequence operatively coding
for the polypeptide interstitially into tissue of the vertebrate, whereby
cells of the tissue produce the polypeptide for at least one month or at
least 3 months, more preferably at least 6 months. In this embodiment of
the invention, the cells producing the polypeptide are nonproliferating
cells, such as muscle cells.
Another method according to the invention is a method for obtaining
transitory expression of a polypeptide in a vertebrate, comprising the
step of introducing a naked mRNA sequence operatively coding for the
polypeptide interstitially into tissue of the vertebrate, whereby cells of
the tissue produce the polypeptide for less than about 20 days, usually
less than about 10 days, and often less than 3 or 5 days. For many of the
methods of the invention, administration into solid tissue is preferred.
One important aspect of the invention is a method for treatment of muscular
dystrophy, comprising the steps of introducing a therapeutic amount of a
composition comprising a polynucleotide operatively coding for dystrophin
in a pharmaceutically acceptable injectable carrier in vivo into muscle
tissue of an animal suffering from muscular dystrophy, whereby the
polynucleotide is taken up into the cells and dystrophin is produced in
vivo. Preferably, the polynucleotide is a naked polynucleotide and the
composition is introduced interstitially into the muscle tissue.
The present invention also includes pharmaceutical products for all of the
uses contemplated in the methods described herein. For example, there is a
pharmaceutical product, comprising naked polynucleotide, operatively
coding for a biologically active polypeptide, in physiologically
acceptable administrable form, in a container, and a notice associated
with the container in form prescribed by a governmental agency regulating
the manufacture, use, or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the polynucleotide for
human or veterinary administration. Such notice, for example, may be the
labeling approved by the U.S. Food and Drug Administration for
prescription drugs, or the approved product insert.
In another embodiment, the invention provides a pharmaceutical product,
comprising naked polynucleotide, operatively coding for a biologically
active peptide, in solution in a physiologically acceptable injectable
carrier and suitable for introduction interstitially into a tissue to
cause cells of the tissue to express the polypeptide, a container
enclosing the solution, and a notice associated with the container in form
prescribed by a governmental agency regulating the manufacture, use, or
sale of pharmaceuticals, which notice is reflective of approval by the
agency of manufacture, use, or sale of the solution of polynucleotide for
human or veterinary administration. The peptide may be immunogenic and
administration of the solution to a human may serve to vaccinate the
human, or an animal. Similarly, the peptide may be therapeutic and
administration of the solution to a vertebrate in need of therapy relating
to the polypeptide will have a therapeutic effect.
Also provided by the present invention is a pharmaceutical product,
comprising naked antisense polynucleotide, in solution in a
physiologically acceptable injectable carrier and suitable for
introduction interstitially into a tissue to cause cells of the tissue to
take up the polynucleotide and provide a therapeutic effect, a container
enclosing the solution, and a notice associated with the container in form
prescribed by a governmental agency regulating the manufacture, use, or
sale of pharmaceuticals, which notice is reflective of approval by the
agency of manufacture, use, or sale of the solution of polynucleotide for
human or veterinary administration.
One particularly important aspect of the invention relates to a
pharmaceutical product for treatment of muscular dystrophy, comprising a
sterile, pharmaceutically acceptable carrier, a pharmaceutically effective
amount of a naked polynucleotide operatively coding for dystrophin in the
carrier, and a container enclosing the carrier and the polynucleotide in
sterile fashion. Preferably, the polynucleotide is DNA.
From yet another perspective, the invention includes a pharmaceutical
product for use in supplying a biologically active polypeptide to a
vertebrate, comprising a pharmaceutically effective amount of a naked
polynucleotide operatively coding for the polypeptide, a container
enclosing the carrier and the polynucleotide in a sterile fashion, and
means associated with the container for permitting transfer of the
polynucleotide from the container to the interstitial space of a tissue,
whereby cells of the tissue can take up and express the polynucleotide.
The means for permitting such transfer can include a conventional septum
that can be penetrated, e.g., by a needle. Alternatively, when the
container is a syringe, the means may be considered to comprise the
plunger of the syringe or a needle attached to the syringe. Containers
used in the present invention will usually have at least 1, preferably at
least 5 or 10, and more preferably at least 50 or 100 micrograms of
polynucleotide, to provide one or more unit dosages. For many
applications, the container will have at least 500 micrograms or 1
milligram, and often will contain at least 50 or 100 milligrams of
polynucleotide.
Another aspect of the invention provides a pharmaceutical product for use
in immunizing a vertebrate, comprising a pharmaceutically effective amount
of a naked polynucleotide operatively coding for an immunogenic
polypeptide, a sealed container enclosing the polynucleotide in a sterile
fashion, and means associated with the container for permitting transfer
of the polynucleotide from the container to the interstitial space of a
tissue, whereby cells of the tissue can take up and express the
polynucleotide.
Still another aspect of the present invention is the use of naked
polynucleotide operatively coding for a physiologically active polypeptide
in the preparation of a pharmaceutical for introduction interstitially
into tissue to cause cells comprising the tissue to produce the
polypeptide. The pharmaceutical, for example, may be for introduction into
muscle tissue whereby muscle cells produce the polypeptide. Also
contemplated is such use, wherein the peptide is dystrophin and the
pharmaceutical is for treatment of muscular dystrophy.
Another use according to the invention is use of naked antisense
polynucleotide in the preparation of a pharmaceutical for introduction
interstitially into tissue of a vertebrate to inhibit translation of
polynucleotide in cells of the vertebrate.
The tissue into which the polynucleotide is introduced can be a persistent,
non-dividing cell. The polynucleotide may be either a DNA or RNA sequence.
When the polynucleotide is DNA, it can also be a DNA sequence which is
itself non-replicating, but is inserted into a plasmid, and the plasmid
further comprises a replicator. The DNA may be a sequence engineered so as
not to integrate into the host cell genome. The polynucleotide sequences
may code for a polypeptide which is either contained within the cells or
secreted therefrom, or may comprise a sequence which directs the secretion
of the peptide.
The DNA sequence may also include a promoter sequence. In one preferred
embodiment, the DNA sequence includes a cell-specific promoter that
permits substantial transcription of the DNA only in predetermined cells.
The DNA may also code for a polymerase for transcribing the DNA, and may
comprise recognition sites for the polymerase and the injectable
preparation may include an initial quantity of the polymerase.
In many instances, it is preferred that the polynucleotide is translated
for a limited period of time so that the polypeptide delivery is
transitory. The polypeptide may advantageously be a therapeutic
polypeptide, and may comprise an enzyme, a hormone, a lymphokine, a
receptor, particularly a cell surface receptor, a regulatory protein, such
as a growth factor or other regulatory agent, or any other protein or
peptide that one desires to deliver to a cell in a living vertebrate and
for which corresponding DNA or mRNA can be obtained.
In preferred embodiments, the polynucleotide is introduced into muscle
tissue; in other embodiments the polynucleotide is incorporated into
tissuess of skin, brain, lung, liver, spleen or blood. The preparation is
injected into the vertebrate by a variety of routes, which may be
intradermally, subdermally, intrathecally, or intravenously, or it may be
placed within cavities of the body. In a preferred embodiment, the
polynucleotide is injected intramuscularly. In still other embodiments,
the preparation comprising the polynucleotide is impressed into the skin.
Transdermal administration is also contemplated, as is inhalation.
In one preferred embodiment, the polynucleotide is DNA coding for both a
polypeptide and a polymerase for transcribing the DNA, and the DNA
includes recognition sites for the polymerase and the injectable
preparation further includes a means for providing an initial quantity of
the polymerase in the cell. The initial quantity of polymerase may be
physically present together with the DNA. Alternatively, it may be
provided by including mRNA coding therefor, which mRNA is translated by
the cell. In this embodiment of the invention, the DNA is preferably a
plasmid. Preferably, the polymerase is phage T7 polymerase and the
recognition site is a T7 origin of replication sequence.
In accordance with another aspect of the invention, there is provided a
method for treating a disease associated with the deficiency or absence of
a specific polypeptide in a vertebrate, comprising the steps of obtaining
an injectable preparation comprising a pharmaceutically acceptable
injectable carrier containing a naked polynucleotide coding for the
specific polypeptide; introducing the injectable preparation into a
vertebrate and permitting the polynucleotide to be incorporated into a
cell, wherein the polypeptide is formed as the translation product of the
polynucleotide, and whereby the deficiency or absence of the polypeptide
is compensated for. In preferred embodiments, the preparation is
introduced into muscle tissue and the method is applied repetitively. The
method is advantageously applied where the deficiency or absence is due to
a genetic defect. The polynucleotide is preferably a non-replicating DNA
sequence; the DNA sequence may also be incorporated into a plasmid vector
which comprises an origin of replication.
In one of the preferred embodiments, the polynucleotide codes for a
non-secreted polypeptide, and the polypeptide remains in situ. According
to this embodiment, when the polynucleotide codes for the polypeptide
dystrophin, the method provides a therapy for Duchenne's syndrome;
alternatively, when the polynucleotide codes for the polypeptide
phenylalanine hydroxylase, the method comprises a therapy for
phenylketonuria. In another preferred embodiment of the method, the
polynucleotide codes for a polypeptide which is secreted by the cell and
released into the circulation of the vertebrate; in a particularly
preferred embodiment the polynucleotide codes for human growth hormone.
In yet another embodiment of the method, there is provided a therapy for
hypercholesterolemia wherein a polynucleotide coding for a receptor
associated with cholesterol homeostasis is introduced into a liver cell,
and the receptor is expressed by the cell.
In accordance with another aspect of the present invention, there is
provided a method for immunizing a vertebrate, comprising the steps of
obtaining a preparation comprising an expressible polynucleotide coding
for an immunogenic translation product, and introducing the preparation
into a vertebrate wherein the translation product of the polynucleotide is
formed by a cell of the vertebrate, which elicits an immune response
against the immunogen. In one embodiment of the method, the injectable
preparation comprises a pharmaceutically acceptable carrier containing an
expressible polynucleotide coding for an immunogenic peptide, and on the
introduction of the preparation into the vertebrate, the polynucleotide is
incorporated into a cell of the vertebrate wherein an immunogenic
translation product of the polynucleotide is formed, which elicits an
immune response against the immunogen.
In an alternative embodiment, the preparation comprises one or more cells
obtained from the vertebrate and transfected in vitro with the
polynucleotide, whereby the polynucleotide is incorporated into said
cells, where an immunogenic translation product of the polynucleotide is
formed, and whereby on the introduction of the preparation into the
vertebrate, an immune response against the immunogen is elicited. In any
of the embodiments of the invention, the immunogenic product may be
secreted by the cells, or it may be presented by a cell of the vertebrate
in the context of the major histocompatibility antigens, thereby eliciting
an immune response against the immunogen. The method may be practiced
using non-dividing, differentiated cells from the vertebrates, which cells
may be lymphocytes, obtained from a blood sample; alternatively, it may be
practiced using partially differentiated skin fibroblasts which are
capable of dividing. In a preferred embodiment, the method is practiced by
incorporating the polynucleotide coding for an immunogenic translation
product into muscle tissue.
The polynucleotide used for immunization is preferably an mRNA sequence,
although a non-replicating DNA sequence may be used. The polynucleotide
may be introduced into tissues of the body using the injectable carrier
alone; liposomal preparations are preferred for methods in which in vitro
transfections of cells obtained from the vertebrate are carried out.
The carrier preferably is isotonic, hypotonic, or weakly hypertonic, and
has a relatively low ionic strength, such as provided by a sucrose
solution. The preparation may further advantageously comprise a source of
a cytokine which is incorporated into liposomes in the form of a
polypeptide or as a polynucleotide.
The method may be used to selectively elicit a humoral immune response, a
cellular immune response, or a mixture of these. In embodiments wherein
the cell expresses major histocompatibility complex of Class I, and the
immunogenic peptide is presented in the context of the Class I complex,
the immune response is cellular and comprises the production of cytotoxic
T-cells.
In one such embodiment, the immunogenic peptide is associated with a virus,
is presented in the context of Class I antigens, and stimulates cytotoxic
T-cells which are capable of destroying cells infected with the virus. A
cytotoxic T-cell response may also be produced according the method where
the polynucleotide codes for a truncated viral antigen lacking humoral
epitopes.
In another of these embodiments, the immunogenic peptide is associated with
a tumor, is presented in the context of Class I antigens, and stimulates
cytotoxic T cells which are capable of destroying tumor cells. In yet
another embodiment wherein the injectable preparation comprises cells
taken from the animal and transfected in vitro, the cells expressing major
histocompatibility antigen of class I and class II, and the immune
response is both humoral and cellular and comprises the production of both
antibody and cytotoxic T-cells.
In another embodiment, there is provided a method of immunizing a
vertebrate, comprising the steps of obtaining a positively charged
liposome containing an expressible polynucleotide coding for an
immunogenic peptide, and introducing the liposome into a vertebrate,
whereby the liposome is incorporated into a monocyte, a macrophage, or
another cell, where an immunogenic translation product of the
polynucleotide is formed, and the product is processed and presented by
the cell in the context of the major histocompatibility complex, thereby
eliciting an immune response against the immunogen. Again, the
polynucleotide is preferably mRNA, although DNA may also be used. And as
before, the method may be practiced without the liposome, utilizing just
the polynucleotide in an injectable carrier.
The present invention also encompasses the use of DNA coding for a
polypeptide and for a polymerase for transcribing the DNA, and wherein the
DNA includes recognition sites for the polymerase. The initial quantity of
polymerase is provided by including mRNA coding therefor in the
preparation, which mRNA is translated by the cell. The mRNA preferably is
provided with means for retarding its degradation in the cell. This can
include capping the mRNA, circularizing the mRNA, or chemically blocking
the 5' end of the mRNA. The DNA used in the invention may be in the form
of linear DNA or may be a plasmid. Episomal DNA is also contemplated. One
preferred polymerase is phage T7 RNA polymerase and a preferred
recognition site is a T7 RNA polymerase promoter.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention requires obtaining naked
polynucleotide operatively coding for a polypeptide for incorporation into
vertebrate cells. A polynucleotide operatively codes for a polypeptide
when it has all the genetic information necessary for expression by a
target cell, such as promoters and the like. These polynucleotides can be
administered to the vertebrate by any method that delivers injectable
materials to cells of the vertebrate, such as by injection into the
interstitial space of tissues such as muscles or skin, introduction into
the circulation or into body cavities or by inhalation or insufflation. A
naked polynucleotide is injected or otherwise delivered to the animal with
a pharmaceutically acceptable liquid carrier. For all applications, the
liquid carrier is aqueous or partly aqueous, comprising sterile,
pyrogen-free water. The pH of the preparation is suitably adjusted and
buffered.
In the embodiments of the invention that require use of liposomes, for
example, when the polynucleotide is to be associated with a liposome, it
requires a material for forming liposomes, preferably cationic or
positively charged liposomes, and requires that liposomal preparations be
made from these materials. With the liposomal material in hand, the
polynucleotide may advantageously be used to transfect cells in vitro for
use as immunizing agents, or to administer polynucleotides into bodily
sites where liposomes may be taken up by phagocytic cells.
Polynucleotide Materials
The naked polynucleotide materials used according to the methods of the
invention comprise DNA and RNA sequences or DNA and RNA sequences coding
for polypeptides that have useful therapeutic applications. These
polynucleotide sequences are naked in the sense that they are free from
any delivery vehicle that can act to facilitate entry into the cell, for
example, the polynucleotide sequences are free of viral sequences,
particularly any viral particles which may carry genetic information. They
are similarly free from, or naked with respect to, any material which
promotes transfection, such as liposomal formulations, charged lipids such
as Lipofectin.TM. or precipitating agents such as CaPO.sub.4.
The DNA sequences used in these methods can be those sequences which do not
integrate into the genome of the host cell. These may be non-replicating
DNA sequences, or specific replicating sequences genetically engineered to
lack the genome-integration ability.
The polynucleotide sequences of the invention are DNA or RNA sequences
having a therapeutic effect after being taken up by a cell. Examples of
polynucleotides that are themselves therapeutic are anti-sense DNA and
RNA; DNA coding for an anti-sense RNA; or DNA coding for tRNA or rRNA to
replace defective or deficient endogenous molecules. The polynucleotides
of the invention can also code for therapeutic polypeptides. A polypeptide
is understood to be any translation product of a polynucleotide regardless
of size, and whether glycosylated or not. Therapeutic polypeptides include
as a primary example, those polypeptides that can compensate for defective
or deficient species in an animal, or those that act through toxic effects
to limit or remove harmful cells from the body.
Therapeutic polynucleotides provided by the invention can also code for
immunity-conferring polypeptides, which can act as endogenous immunogens
to provoke a humoral or cellular response, or both. The polynucleotides
employed according to the present invention can also code for an antibody.
In this regard, the term "antibody" encompasses whole immunoglobulin of
any class, chimeric antibodies and hybrid antibodies with dual or multiple
antigen or epitope specificities, and fragments, such as F(ab).sub.2,
Fab', Fab and the like, including hybrid fragments. Also included within
the meaning of "antibody" are conjugates of such fragments, and so-called
antigen binding proteins (single chain antibodies) as described, for
example, in U.S. Pat. No. 4,704,692, the contents of which are hereby
incorporated by reference.
Thus, an isolated polynucleotide coding for variable regions of an antibody
can be introduced, in accordance with the present invention, to enable the
treated subject to produce antibody in situ. For illustrative methodology
relating to obtaining antibody--encoding polynucleotides, wee Ward et al.
Nature, 341:544-546 (1989); Gillies et al., Biotechnol. 7:799-804 (1989);
and Nakatani et al., loc. cit., 805-810 (1989). The antibody in turn would
exert a therapeutic effect, for example, by binding a surface antigen
associated with a pathogen. Alternatively, the encoded antibodies can be
anti-idiotypic antibodies (antibodies that bind other antibodies) as
described, for example, in U.S. Pat. No. 4,699,880. Such anti-idiotypic
antibodies could bind endogenous or foreign anti | | |
