Note: Descriptions are shown in the official language in which they were submitted.
t3~it~4rg-'
SOMATIC CELL GENE THERAPY
ACKNOWLEDGMENT
This invention was made with government support
under grants from the National Institutes of Health.
FIELD OF THE INVENTIOPT
The present invention relates generally to gene
therapy. More specifically, the present invention
relates to somatic cell gene therapy in humans and
animals.
BACKGROUND OF THE INVENTION
More than 1500 human genetic diseases are
caused by a single gene defect. It is possible that
many of these diseases can alleviated, at least in
part, if the deficient function can be supplied.
The concept of human gene therapy involves the
introduction of a functionally active "replacement"
gene into somatic cells of an affected subject to
correct the gene defect. Retroviral vectors, because
of their unique structure, mode of replication, and
ability to infect a wide variety of cells, including
stem cells, are ideally suited to transfer genetic
material into somatic cells (Verma, 1985).
To ensure a life long supply of the replacement
gene product, it is essential to introduce and express
the functionally active gene in cells that proliferate
during the entire adult life of the recipient. Because
pluripotent stem cells in bone marrow have both self
renewal capacity as well as the ability to give rise to
all hematopoietic lineages, they are a popular target
for the introduction of functionally active genes
(Miller, et al., 1984; Williams, et al., 1984; Keller,
et al., 1985: Dick, et al., 1985). Recently,
hepatocytes have been used as target cells for
introducing functionally active genes (Ledley, et al.,
1
1341246
1987; Wolfe, et al., 1987).
Although the number of stem cells in adult
marrow is low (0.01-0.1%), the use of high-titer
retrovirus has ensured infection and gene delivery into
these cells. The problem however has been that neither
the foreign genes nor the retroviral vector introduced
into these stem cells, the progenitor cells, or the
mature end cells are efficiently expressed (Williams,
et al., 1984; Joyner, et al., 1985).
To overcome this problem of inefficient
expression, the present invention discloses an
alternative strategy for somatic cell gene transfer.
The new strategy uses skin fibroblasts that are
infected with chimeric retrovirus containing a
functionally active endogenous or foreign
"replacement" gene. Once infected with the chimeric
retrovirus, the transduced fibroblasts are preferably
"fixed" in an extracellular collagen matrix, and then
implanted in the loose connective tissue of the skin.
Since this compartment of the dermis is highly
vascularized, the transduced fibroblasts, and thus
their "replacement" gene products, have direct access
to the circulatory system. As a result the needed
replacement gene products can easily and efficiently be
distributed to other parts of the body.
Recently two groups used mouse fibroblasts to
introduce and express foreign genes in mice (Selden, et
al., 1987; Carver, et al., 1987b). One group implanted
mice with a DNA transfected cell line and showed that
the recipient mice made the gene product (growth
hormone) but. maintained the graft only if the mice were
immunosuppressed (Selden, et al., 3987). The other
group, using a chimeric retroviral vector containing
the alphal-antitrypsin gene, produced a cell line from
2
1 341 246
a transduced cell and then transplanted cells from the
line into the peritoneal cavity of nude mice (Garver,
et al., L987b). In both cases, cell lines were
generated that would potentially be tumorigenic in
mice. Neither study addresses the issue of cell
maintenance in grafted mice without the use of harsh
immunosuppressive agents.
The method described herein obviates the need
for established cell. lines and uses instead, fibroblast
cells from recipient subjects. Use of a subject's own
cells minimizes the possibility of rejection. In
addition, culturing the cells in an extracellular
collagen matrix circumvents the problem of necrosis
that would ensue following subcutaneous injection
(Bell, et al., 1983). Finally, the high efficiency of
retroviral infection and expression in fibroblasts
(80%) essentially eliminates the need to identify
transduced cells by means of selectable markers, thus
greatly simplifying the overall endeavor of
introduction of foreign genes.
Clinical disease states that are candidates
for the gene therapy treatment method of the present
invention include hemophilia, endocrine deficiency,
alphal-antitrypsin, birth control, etc.
In addition to the work that has been done with
fibroblasts, at least one group has shown that
retroviral-mediated gene transfer can be used to
introduce a recombinant human growth hormone gene into
cultured human keratinocytes (Morgan, et al., 1987).
The transduced keratinocytes secreted biologically
active growth hormone into the culture medium. When
grafted as an epithelial sheet onto athymic mice, these
cultured keratinocytes reconstituted an epidermis that
was similar in appearance to that produced by normal
3
134 246 .
cells, but from which human growth hormone could be
extracted. Unfortunately, it was not possible to
determine rate of diffusion of human growth hormone
from the graft site to the bloodstream. This may have
been due to the fact that the surface skin graft does
not vascularize as efficiently and as quickly as the
embedded fibroblasts of the present invention.
BRIEF DESCRIPTION OF THE DRAWING
The following is a brief description of the
drawings. More detailed descriptions of the figures
are found in the Experimental Section of this
specification.
The drawings comprise five Figures.
Figure 1 (A, B & C) is composed of a schematic
drawing (A), and two photographs (B & C), all of which
relate to an analysis of recombinant human factor IX
retrovirus. Fig. 1A shows the structural arrangement
of the recombinant factor IX retrovirus pAFFIXSVNeo.
Figs. 1B and 1C are photographs of nitrocellulose
filters that illustrate proviral DNA and RNA
hybridizations, respectively.
Figure 2 is a graph that illustrates secretion
of human factor IX.
Figure 3 (A, B & C) is composed of a schematic
drawing (A), and two photographs (B & C), all of which
relate to embedding and implantation of transfected
fibroblasts. Fig. 3A is a schematic representation of
the protocol used to generate and graft the collagen
implants into the loose connective tissue of the skin
of the mouse model system. Figs. 3B & 3C are
photographs of mice that show the MEF (primary
fibroblast cells) and BL/6 (the tumor cell line)
collagen implants at day 14.
Figure 4 is a graph that compares the amount of
4
1341246
factor IX in human sera from both the MEF and BL/6
implants.
Figure 5 is a photograph of a nitrocellulose
filter that illustrates detection of mouse anti-human
factor IX antibodies in mice grafted with collagen
implants.
DEFINITIONS
In the present specification and claims,
reference will be made to phrases and terms of art
which are expressly defined for use herein as follows:
As used herein, LTR means long terminal repeat.
As used herein, factor IX refers to the blood
clotting factor gene or protein of the same name.
As used herein, pAFVXM refers to a retroviral
construct generated by Kriegler, et al., (1984).
pAFVXM is a progenitor construct for the recombinant
factor IX retrovirus, pAFFIXSVNeo. A replacement gene
of interest (or a cDNA for such a gene) can be linked
directly to the 5' LTR in the retrovirus by inserting a
BamHI/HindIII fragment from the gene or clone between
the BalII/HindIII sites of pAFVXM (Anson, et al.,
1984). pKoNeo is a neomycin phosphotransferase
expression plasmid.
As used herein, when reference is made to the
Greek letter psi, the name psi is sometimes substituted
for the symbol V.
As used herein, the letter g is sometimes used
to signify the symbol for the Greek letter gamma, Y.
As used herein, MEF means primary mouse embryo
fibroblasts.
As used herein, B1/6 refers to an immortalized
skin cell line derived from x-ray irradiated skin
fibroblasts obtained from C57BL/6J mice.
As used herein, psiFIXNeo means the cell line
5
1 3 ~ t 2 46
V~FIXNeo.
As used herein, moi means multiplicity-of-
infection.
As used herein, POLYBRENE is the trademark of
Sigma Chemical Company, St. Louis, MO for 1,5,-
Dimethyl-1,5-diazaundecamethylene polymethobromide;
Hexadimet.hrine bromide.
As used herein, DMEM means Dulbecco's modified
Eagle's medium.
As used herein, ELISA means enzyme linked
immunoabsorbant assay.
As used herein, FGF means fibroblast growth
factor. FGF is a angiogenic substance that can be used
in the present invention to stimulate vascularization
of the implanted fibroblasts.
As used herein, transduction refers to the
process of conveying or carrying over, especially the
carrying over of a gene from one cell to another by a
virus or retrovirus. A retrovirus that carries a gene
from one cell to another is referred to as a
transducing chimeric retrovirus. A eukaryotic cell
that has been transduced will contain new or foreign
genetic material (e.g., a replacement gene) in its
genome as a result of having been "infected" with the
chimeric transducing retrovirus.
As used herein, transfection of eukaryotic
cells is the acquisition of new genetic material by
incorporation of added DNA.
As used herein, the term skin technically means
to the body's largest organ. Skin consists of two
components, the epidermis and the dermis. The dermis
is a relatively inert structure which consists of
collagen and other matri~c materials. The epidermis
lies above the dermis and is separated from it by a
6
!,
1 3 ~ fi~2~8
basement membrane.
As used herein, the term fibroblasts refers to
flat, elongated connective tissue cells with
cytoplasmic processes at each end and an oval, flat
nucleus. Fibroblasts, which differentiate into
chondroblasts, collagenoblasts, and osteoblasts, form
the fibrous tissues in the body, e.g., tendons,
aponeuroses, plus supporting and binding tissues of all
sorts. Like other cells in the body, fibroblasts carry
an entire complement of genetic material. However,
only a small percentage of the genes contained in
fibroblasts are biologically functional; that is, most
of the genes in fibroblasts are not expressed at all or
are expressed at such low levels that the proteins they
encode are produced in undetectable amounts or at
concentrations which are not biologically functional or
significant. Using routine methods of molecular
biology it is now possible to introduce exogenous
genetic material (i.e., replacement genes) into
mammalian cells, thus enabling them to express genetic
materials not normally expressed. The transduced
fibroblasts of the present invention incorporate
exogenous genetic material, which they express, thereby
producing the gene products encoded by the incorporated
exogenous genetic material.
As used herein, a promoter is a specific
nucleotide sequence recognized by RNA polymerase, the
enzyme that initiates RNA synthesis. When exogenous
genes are introduced into fibroblasts using a
retroviral vector, the exogenous genes are subject to
retroviral control; in such a case, the exogenous
genes) is transcribed from an endogenous retroviral
promoter. It is possible to make retroviral vectors
that, in addition to their own endogenous promoters,
7
131246'
have exogenous promoter elements which are responsible
for the transcription of the exogenous gene(s). For
example, it is possible to make a construct in which
there is an additional promoter that is modulated by an
external factor or cue, and in turn to control the
level of exogenous protein being produced by the
fibroblasts by activating the external factor or cue.
As an illustration, the promoter for the gene which
encodes the metal-containing protein metallothionine is
responsive to Cd++ ions. Incorporation of this
promoter or another promoter influenced by external
cues makes it possible to regulate the production of
the proteins produced by the transduced fibroblasts of
the invention.
As used herein, subcutaneously means below the
basement membrane of the epidermis. Subcutaneous is
abbreviated as "s.c.".
As used herein, "i.p." means intraperitoneally.
As used herein, skin fibroblasts are fibroblast
cells that are normally found in the dermis portion of
the skin.
As used herein, syngeneic means isogenic, i.e.,
having the same genetic constitution.
As used herein, exogenous genetic material
means DNA or RNA, either natural or synthetic, that is
not naturally found in cells of a particular type: or
if it is naturally found in the cells, it is not
expressed in these cells in biologically significant
levels. For example, a synthetic or natural gene
coding for human insulin would be exogenous genetic
material to a yeast cell since yeast cells do not
naturally contain insulin genes: a human insulin gene
inserted into a skin fibroblast cell would also be an
exogenous gene to that cell since skin fibroblasts do
8
134r'~246
not express human insulin in biologically significant
levels.
As used herein, "exogenous" genetic material
and "foreign" genetic material mean the same thing, and
the terms "exogenous" and "foreign", when used to
describe genes or genetic material, are used
interchangeably.
A~; used herein, retroviral vectors are the
vehicles used to introduce replacement genes into the
skin fibroblasts. The following paragraphs contain
some general background information about retroviruses.
Retrovirus are RNA viruses; that is, the viral
genome is RNA. This genomic RNA is, however, reverse
transcribed into a DNA intermediate which is integrated
very efficiently into the chromosomal DNA of infected
cells. This integrated DNA intermediate is referred to
as a prom rus.
The retroviral genome and the proviral DNA have
three genes: c~aq, pol and env, which are flanked by two
long terminal repeat (LTR) sequences. The ,gag, gene
encodes the internal structural (nucleocapsid)
proteins; the pol gene encodes the RNA-directed DNA
polymerase (reverse transcriptase); and the env gene
encodes viral envelope gylcoproteins. The 5'and 3'
LTRs serve to promote transcription and polyadenylation
of virion RNAs.
Adjacent to the 5' LTR are sequences necessary
for reverse transcription of the genome (the tRNA
binding site) and for efficient encapsidation of viral
RNA into particles (the ~ or psi site). (Mulligan,
1983; Mann et al., 1983; Verma, 1985.)
The various elements required for replication
of the retrovirus can be divided into cis- and trans-
acting factors. The trans-acting factors include
9
13~t 2~g
proteins encoded by the viral genome, which are
required for encapsidation of viral RNA, entry of
virions into cells, reverse transcription of the viral
genome, and integration of the DNA form of the virus
(i.e., the provirus) into host DNA. The cis-acing
factors include signals present in the viral RNA which
interact with the shove-described proteins and other
factors during virus replication.
If the sequences necessary for encapsidation
(i.e., packaging of retroviral RNA into infectious
virions) are missing from the viral genome, the result
is a cis defect which prevents encapsidation of genomic
RNA. The resulting mutant is however still capable of
directing synthesis of all virion proteins. When the
packaging signals are removed, viral RNA and proteins
are still synthesized, but no infectious particles are
made because viral RNA cannot be packaged into virions.
Mann, et al., (1983) used this strategy to create ~2
cell lines which supported the generation of infectious
transducing retroviruses without generating helper
murine leukemia viruses. Unfortunately, murine
leukemia virus env gene product is only able to infect
rodent cells, which limits the utility of ~2 cell
lines. On the other hand, amphotropic murine
retroviruses are able to infect a wide variety of cell
types, including human cells.
Using a strategy similar to the one described
by Mann, et al., for the production of the ~2 cell
lines, Verma and his colleagues generated a cell line
using the env gene product of the amphotropic viruses
(Verma, 1985; Miller, et al., 1985: Miller , et al.,
1986). As a result of this work, a wide-host-range,
packaging defective system was made available for the
generation of high-titer retroviruses containing
1341 2~6
exogenous genes (Verma, 1985; Miller, et al., 1985;
WO 86 00922. Such
retroviruses, or retroviral vectoxs, have general
utility for high-efficiency transduction of genes in
cultured cells, and specific utility for use in the
method of the present invention.
SUMMARY OF THE INVENTION
The present invention discloses a new gene
therapy method based on the use of transduced
fibroblasts that are implanted in the loose connective
tissue of tl-ie skin of the subject to be treated.
According to the invention, transduced fibroblasts are
preferably created by infecting fibroblast cells in
vitro with chimeric retroviruses that contain at least
one functionally active "replacement gene", i.e.,
foreign or exogenous genetic material that does not
normally occur in fibroblast cells, or if it does, is
not expressed by the fibroblast cells in biologically
significant concentrations. The transduced fibroblasts
are then preferably fixed by culturing them in vitro in
an extracellular matrix. Finally, the transduced
fibroblasts are implanted subcutaneously in the loose
connective tissue of the skin of the individual or
animal being treated. To insure rapid vascularization
of the implanted fibroblasts, an angiogenic substance
such as fibroblast growth factor is preferably placed
in the loose connective tissue along with the implant.
Because the fibrobiasts are implanted in a highly
vascularized compartment of the skin i.e., loose
connective tissue of the dermis, the transduced cells,
and thus their "replacement" gene products, have direct
access to the circulatory system. As a result, the
needed replacement gene products can easily and
efficiently be distributed to other parts of the body.
11
131245
When the gene therapy is no longer needed, the
implanted fibroblasts can be conveniently removed.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery
that transduced skin fibroblasts can be used for
somatic cell gene therapy when the transduced
fibroblasts are fixed in vitro in an extracellular
collagen matrix and implanted in the loose connective
tissue of the dermis of an subject to be treated. The
discovery makes it possible to overcome several
problems that have been encountered when prior art gene
therapy methods were used to treat animals or
individuals with genetic defects. Such problems
include: (1) inefficient expression of the foreign
"replacement" genes (Williams, et al., 1984; Joyner, et
al., 1985); (2) use of transduced cells that had the
potential to be tumorigenic to the animal or
individual being treated (Selden, et al., 1987; Garver,
et al., 1987b); (3) use of harsh immunosuppressive
agents to avoid rejection by the animal or individual
being treated (Selden, et al., 1987); (4) necrosis
following subcutaneous injection of cells (Bell, et
al., 1983); and (5) poor diffusion of the replacement
gene product (Morgan, et al., 1987). As will be
discussed more fully below, the present invention
preferably employs chimeric retroviruses to introduce
replacement genes into skin fibroblasts. Because of
the nigh efficiency of retroviral proviral infection
and expression in fibroblasts, the present invention
invention essentially eliminates the need to use marker
genes to identify transduced cells. This greatly
simplifies the overall problem of introducing
replacement genes into cells that will be used for gene
therapy. Since the invention preferably uses
12
~3~~ z~s
fibroblast cells from recipient individuals, it
obviates the need to use potentially tumorigenic cell
lines. Use of skin fibroblasts from the subject to be
treated minimizes the possibility of rejection, which
in turn lessons the need for harsh immunosuppressant
drugs. In addition, since the invention uses
transduced fibroblasts that preferably have been fixed
in vitro in an extracellular collagen matrix, the
problem of necrosis is also minimized. Finally, since
the invention implants the transduced fibroblasts into
the highly vascularized loose connective tissue of the
dermis, the replacement gene products are easily and
efficiently distributed to other parts of the body.
With regard to the fibroblasts employed herein,
preferably they will be skin fibroblasts from the
animal or individual to be treated with the gene
therapy. Fibroblasts from these subjects can easily be
obtained by skin biopsy, and then maintained in culture
until it is convenient to transduce them. General
methods for maintaining fibroblast cells in culture are
well known to those skilled in the art of tissue
culture. Such methods include culturing the cells in
Dulbecco-Vogt modified Eagle's medium with loo fetal
bovine serum. Such known methods can be used by the
skilled artisan, without undue experimentation, to
culture the fibroblast cells prior to transduction.
See generally, the Materials and Methods sections of
Palmer, at al., (1987).
Exogenous genetic material or genes especially
useful in the invention are those genes that encode
secretory proteins. Such useful genes include, but are
not limited to, genes that encode blood clotting
factors such as human factors VIII and IX: hormone
genes such as the genes encoding for insulin,
13
134 2~6
parathyroid hormone, lutenizing hormone releasing
factor (LHRH), alpha and beta seminal inhibins, and
human growth hormone; enzyme genes: genes encoding
inhibitor substances such as alphal-antitrypsin, and
genes encoding substances that function as drugs, e.g.,
genes encoding the diphtheria and cholera toxins.
(Genes that encode useful "gene therapy" proteins,
e.g., many enzyme proteins, that are not normally
secreted can be used in the invention if they are
"functionally appended" to a signal protein sequence
that will "transport" them across the fibroblasts'
limiting membranes and into the extracellular space. A
variety of such signal sequences are known and can be
used by those skilled in the art without undue
experimentation.)
It is possible to use vehicles other than
retroviruses to genetically engineer the fibroblasts of
the present invention. However, chimeric retroviruses
are the preferred agents used to incorporate new
genetic material into the skin fibroblasts.
Retroviruses and helper-free replication-defective
viral vectors are well known and can be adapted for use
in the present invention without undue experimentation.
Examples of such retroviruses are disclosed in the
Experimental Section of present specification:
additional examples are disclosed and discussed in
Palmer, et al., (1987): Miller, et al., (1986); St.
Louis and Verma (1987): Miller et al., (1985): and in
WO 86 00922 which has
been assigned to the Salk Institute for Biological
Studies, San Diego, CA. Helper-free replication-
defective viral vectors that have a dominant selectable
marker such as the neomycin resistance gene or the
mutant DHFR gene (Miller, et al., 1985) can be used in
14
13 ~ ~ 2 ~i ~
the present invention. However, since the efficiency of
retroviral infection is about 80%, use of a dominant
selectable marker to identify transfected cells is
usually not necessary.
It is possible, through the use of a
recombinant retrovirus to introduce new genetic
material into fibroblasts without altering the
functional characteristics of the recipient
fibroblasts. Therefore, retroviral vectors useful in
the method of the present invention will have a cloning
site. The presence of such a site makes it possible to
introduce exogenous genetic material into the vector
and have it expressed by fibroblasts co-cultivated with
the recombinant virus. Methods for introducing
exogenous genetic material into the retroviral vectors
are known and can be used by the skilled artisan
without undue experimentation. For example, useful
methods are disclosed in the Experimental Section of
this specification and in Palmer, et al, (1987). (In
Palmer, et al., 1987, see especially the Materials and
Methods section of the publication.) Additional
methods and helpful details are disclosed in Verma,
(1985); Miller, et al., (1985); Miller, et al., (1986);
St. Louis and Verma, (1987), and in
WO 86 00922 which has been assigned
to the Salk Institute for Biological Studies, San
Diego, CA.
A cell line that produces recombinant
amphotropic chimeric retrovirus is used in co-
cultivation with the fibroblasts to be "transduced".
The dam cell line, which can be modified using standard
techniques to include chimeric retrovirus, is available
from the American Type Culture Collection, Rockville,
Maryland. For example, a dam line which produces a
B
13~1~24s
chimeric retrovirus can be constructed as follows: the
exogenous gene or cDNA of interest is ligated into a
cloning site in the retroviral vector. (Such a vector
could also carry a selectable marker such as the Neo
gene.) Chimeric retroviruses that carry the exogenous
gene or DNA of interest are isolated and transfected
into ~Uam, cells. ~Uam cells that produce the chimeric
virus construct are isolated, e.g., as 6418 resistant
colonies if the chimeric retrov:irus carried the Neo
gene as a selectable marker.
Co-cultivation methods are well known to those
skilled in the art and can be used in the present
invention without undue experimentation. Generally,
the methods can be summarized as follows: On day one,
fibroblast cells to be "infected" with chimeric
retrovirus are seeded in conventional culture medium at
approx. 5 x 106 cells per 60-mm culture dish. On day
two, the culture medium is replaced with medium from
cells that produce chimeric retrovirus. On day three,
the infected fibroblasts are suspended with an enzyme
such as trypsin. (Although it would not usually be
necessary due to the high efficiency of bulk infection,
if the chimeric retrovirus carried a selectable marker,
the fibroblast cells would be grown in culture dishes
containing selective media. Resistant colonies (i.e.,
those formed from cells that have been transduced by
the chimeric retrovirus) would then be scored after an
appropriate amount of time (10-12 days). Fibroblasts
from the resistant colonies would contain the new
genetic material carried by the transducing chimeric
retroviruses.)
According to the invention, once the skin
fibroblasts have been transduced with chimeric
retrovirus, they are preferably "fixed" in vitro in an
16
~34~ 2~g
extracellular matrix. See generally, Elsdale, et al.,
(1972) and Bell, et al., (1979). A preferred method
for "'fixing" the transduced fibroblasts in vitro in an
extracellular matrix is discussed in the Experimental
Section of this specification. In summary, the
fibroblasts are preferably fixed by culturing them in
an extracellular matrix composed of collagen (either
natural or synthetic) and culture medium. The cells
are cultured at about 37° C. for about 3 days, during
which time the collagen contracts to a tissue-like
structure. Once contracted, the "artificial"
fibroblast tissue grafts can be implanted into the
loose connective tissue in the dermis of the recipient
subject. While the extracellular collagen matrix is
preferred (since it is easy, inexpensive and
effective), those skilled in the art will realize that
other collagen-like materials, both natural and
synthetic, could be used to generate the extracellular
matrix into which the transduced fibroblasts become
fixed.
To ensure rapid vascularization of the grafted
implant, it is preferable to insert basic fibroblast
growth factor along with each graft. The growth factor
can be conveniently supplied by first applying it to a
piece sterile sponge, e.g., as gelfoam (Upjohn), which
is then implanted in the connective tissue along with
each graft.
The present invention makes it possible to
genetically engineer skin fibroblasts that can secrete
a variety of useful gene products (e. g., clotting
factors, immunoregulatable factors, hormones and
drugs). when these transduced fibroblasts are
implanted into the dermis of an individual or animal,
the secreted gene products diffuse into the
17
131246
bloodstream, and thus are carried to various parts of
the body.
The implanted transduced fibroblasts of the
present invention can be used in a variety of
applications. For example, the implanted fibroblasts
can serve as a continuous drug delivery system to
replace present regimes that require periodic
administration (by ingestion, injection, etc.) of a
needed substance. In another example, the transduced
and implanted fibroblasts could be used to provide
continuous delivery of insulin. This would be very
useful since it would eliminate the need for daily
injections of insulin. Genetically engineered
fibroblasts can also be used for the production of
clotting factors. Hemophiliacs lack a protein called
Factor VIII, which is involved in blood clotting.
Factor VIII is now administered by injection. Like
insulin, it could be made by transduced fibroblasts.
Transduced and implanted fibroblasts could also be used
to deliver growth hormone.
Another application for transduced fibroblasts
produced by the present invention is in fertility
control. Several hormones, including lutenizing
hormone releasing factor (LHRH) and the seminal and
ovarian inhibins, are being studied for their ability
to regulate fertility. Continuous administration of
LHRH results in a sterile individual: yet when
administration of the hormone is stopped, fertility
returns. Rather than taking LHRH injections or oral
medication, one could implant collagen fixed
fibroblasts carrying the LHRH gene, and thus provide a
continuous supply of the hormone.
Transduced fibroblasts having foreign or
exogenous genetic material introduced according to the
18
present invention can also be used for drug delivery,
especially in animals. In this way certain drugs can
be continuously delivered to the animals, thus
eliminating the need to incorporate the drugs into the
animals' food or water.
In each of the cited applications for the
transduced fibroblasts of the present invention, the
amount of replacement gene product delivered to the
subject can be controlled by controlling such factors
as: (1) the type of promoter used to regulate the
replacement gene (e.g., use of a strong promoter or an
weak one); (2) the nature of the promoter, i.e.,
whether the promoter is constitutive or inducible; (3)
the number of transduced fibroblasts that are present
in the implant; (4) the size of the implant; (5) the
number of implants, (6) the length of time the implant
is left in place, etc.
Without further elaboration, it is believed
that one of ordinary skill in the art can, using the
preceding description, and the following Experimental
Section, utilize the present invention to its fullest
extent. The material disclosed in the Experimental
Section is disclosed for illustrative purposes and
therefore should not be construed as limiting the
appended claims in any way.
EXPERIMENTAL SECTION
ABSTRACT
Mouse primary skin fibroblasts were infected
with a recombin~3nt retrovirus containing human factor
IX cDNA. Bulk infected cells capable of synthesizing
and secreting biologically active human factor IX
protein were embedded in collagen and the implant
grafted under the epidermis. Sera from the
transplanted mice contain human factor IX protein for
19
1343 246
at least 10-12 days. Loss of immunoreactive human
factor IX protein in the mouse sera is not due to graft
rejection. Instead, the mouse serum contains anti-
human factor IX antibodies, which react with the
protein.
EXPERIMENTAL METHODS
Construction and infection
by recombinant factor IX retroviruses
The recombinant pAFFIXSVNeo is based on a
retroviral construct pAFVXM generated by Kriegler, et
al. (Kriegler, et al., 1984). A human factor IX cDNA
was linked directly to the 5' long terminal repeat
(LTR) by inserting a 1.6 kilobase kb BamHI/ HindIII
fragment from the clone CVI between the BctlII and
HindIII sites of pAFVXM (Anson, et al., 1984). The
entire expression unit from the neomycin
phosphotransferase expression plasmid (pKoNeo) was
excised by partial HindIII digestion and inserted into
the HindIII site of the above factor IX viral construct
(Fig. lA). "Helper free" recombinant ecotropic virus
in ~2 cells was generated as described (Miller, et
al., 1986; Mann, et al., 1983). The titres of
recombinant retrovirus expressed from drug resistant
clones were done essentially as described (Miller, et
al., 1986).
Primary mouse embryo fibroblasts (MEF) were
obtained from day 17 embryos of C578L/6J mice (Todaro,
et al., 1963). The BL/6 line is an immortalized skin
cell line derived from x-ray irradiated skin
fibroblasts obtained from C57BL/6J mice. The skin
fibroblast cell line BL/6, and NIH3T3 TK- cells were
infected with recombinant retroviruses from the cell
line, ~UFIXNeo 4, at a multiplicity-of-infection (moi)
of 1-2 in the presence of POLYBRENE at 8 ug/ml: MEF
~~~~ ~~s
cells were infected at a moi of 5.
Implantation of infected mouse fibroblasts in mouse
Infected BL/6 and FIEF cells were cultured in
vitro in an extracellular matrix composed of rat tail
type I collagen (1 mg/ml: Sigma) and Dulbecco's
modified Eagle's medium (DIEM) supplemented with 10%
fetal bovine serum in a 5-cm dish (Elsdale, et al.,
1972; Bell, et al., 1979). The cells were cultured at
37°C for 3 days during which the collagen lattice
contracted to a tissue-like structure (1/25th the area
of the original gel). Once contracted, two artificial
tissues containing approximately 4 x 106 infected
fibroblasts were grafted into the loose connective
tissue of the d'rmis in the midback of a recipient
C57BL/6 mouse. To ensure rapid vascularization of the
grafted tissue, a 2-mm2 piece of gelfoam (Upjohn)
containing 2 ug of basic fibroblast growth factor was
inserted into the loose connective tissue along with
each graft. Serum samples were drawn at two day
intervals and analyzed for the presence of human factor
IX by ELISA.
Analysis of secreted factor IX
Levels of antigenic factor IX were assayed by
ELISA a~ described by Anson, et al., (1987).
Biologically active human factor IX was immunoaffinity
purified using A7 antibody (Anson, et al., 1987; Smith,
et al., 1987). The amount of biologically active
protein was determined by a one step clotting assay
using canine fa~~tor IX deficient plasma (Goldsmith, et
al., 1978). This assay is based on the ability of the
sample to decrease the prolonged activated partial
thromboplastin time of congenital factor IX-deficient
plasma. Purified human factor IX was used as a
control.
21
1341 ~~46
RESULTS
Transduction of Neomycin Resistance
and Expression of Human Factor IX
The titres of helper-free ~ FIXNeo virus
produced in the various cell lines ranged from 3 x 105
to 7 x 105 6418 resistant colony forming units per ml
when assayed by NIH3T3 TK- cells. As measured by
ELISA, all of the virus producing cell lines secreted
essentially the same levels of factor IX into the
culture media (approx. 200 ng/ml). All infected and
drug resistant cell lines were also found to secrete
factor IX into the culture media, albeit at different
levels (see Fig. 2).
The organization of the integrated recombinant
retrovirus in the virus producing cell line was
determined by Southern blot analysis of SstI digested
(SstI cleaves once in each LTR to generate a 5.1-kb DNA
fragment (Fig. 1B) genomic DNA. All infected cells
displayed a single band of the expected size of
approximately 5.1 kb which hybridizes to both the
factor IX cDNA and the Neo probe, therefore ruling out
any detectable rearrangements. Furthermore, the size
of this band in infected NIH3T3 TK , BL/6, and MEF
cells is identical to that found in the virus producing
cell line ~FIXNeo 4 (compare lanes 5 and 9 to other
lanes).
The RNA blot analysis of the RNA isolated from
~FIXNeo 4, infected NIH3T3 TK , BL/6 and MEF is shown
in Figure 1C. When hybridized to factor IX probe, only
one major transcript of the expected size of 5.1 kb,
corresponding to full length viral RNA could be
detected in the infected cells. Hybridization with Neo
probe reveals an additional 2.2 kb transcript that is
the predicted size of the mRNA species, the synthesis
22
1341 24~
of which is initiated from the simian virus 40 early
promoter and is terminated in the 3' LTR (Fig. 1C).
Ratios of the steady state levels of the 5.1 kb and the
2.2 kb transcripts varied within the different infected
cell types. From results shown in Figure 1, it was
concluded that the ~FIXNeo recombinant retrovirus is
properly integrated and expressed in the infected
cells.
Secretion of Factor IX Protein
Because human factor IX is a secretory protein
it was important to verify if it is secreted into the
medium of the infected cells. Figure 2 shows that both
rate and extent of antigenic factor IX released into
the medium is dependent on the cell type rather than on
the relative amounts of the factor IX transcripts. For
instance, steady state levels of factor IX transcript
in infected NIH3T3 TK- cells is much higher than in
BL/6 cells (Fig. 1C); yet the rate and amount of factor
IX secreted in the latter cell type is much higher.
Both the virus producing cell line y~F'IXNeo 4 and
infected skin fibroblast cell line BL/6, secreted
antigenic factor IX at similar rates, approximately 5.7
ng per ml/hr for 3 x 106 cells and 5.0 ng per ml/hr for
3 x 106 cells, respectively. This rate was almost 3
fold higher than the rate of factor IX secretion seen
for infected MEF (1.75 ng per ml/hr for 3 x 106 cells)
and infected NIH3T3 cells (1.65 ng per ml/hr for 3 x
106 cells). These results indicate that the rate of
synthesis and/or secretion may be a property of the
cell type, rather than the levels of expression.
Secreted Human Factor IX Protein
Is Biologically Active
The primary translation product of factor IX
gene undergoes extensive post-translational
23
1341 246
modification which include addition of sialic
carbohydrates (Chavin, et al., 1984; Fournel, et al.,
1985), vitamin K-dependent conversion of glutamic acid
residues to Ycarboxy/glutamic acid (Suttie, 1980) and
~-hydroxylation of aspartic acid residue 64 (Ferlund,
et al., 2983). The Ycarboxylation of factor IX is
essential for clotting activity and this modification
generally occurs in the liver, the primary source of
factor IX synthesis in the body. Two different
approaches were taken to assess biological activity of
human factor IX secreted from cells in culture: (i) The
infected mouse embryo fibroblasts were cultured in
factor IX deficient canine serum obtained from
hemophiliac dogs, supplemented with epidermal growth
factor (10 ng/ml) and vitamin K (25 ng/ml). Media
harvested after 48 hr incubation was monitored for
activity by a one step assay (Goldsmith, 1978).
Conditioned media from MEF cells contained biologically
active human factor IX at 210 ng/ml which is similar to
the levels seen with ELISA assays (Fig. 4). (ii)
Because BL/6 cells did not attach to the tissue culture
dish in canine sera, a different approach had to be
resorted to. Infected BL/6 cells were grown in 10%
total calf serum supplemented with vitamin K (25
ng/ml), and the media harvested after 48 hr incubation
was applied to an immunoaffinity column containing
human factor IX monoclonal antibody A-7 (Anson, et al.,
1987; Smith, et al., 1987). This monoclonal antibody
recognizes the calcium binding domain of human factor
IX protein, thus discriminating between carboxyl-
lacking factor IX and biologically active carboxyl
human factor IX. One-hundred and sixty ml of the media
obtained from BL/6 cells containing 32 ~,g of antigenic
human factor IX (determined by ELISA) was passed
24
~3~~ 2~s
through the column. Nearly 3.5 ug of the biologically
active material was recovered from the column. This
represents over 10% of the total antigenic factor IX in
the starting sample. No biologically active factor IX
could be identified from uninfected MEF or BL/6 cells.
Despite lack of information on the extent of
carboxylation or other post-translational
modifications, it is concluded that the infected cells
used for subsequent implantation studies synthesize
biologically active human factor IX.
Detection of Human Factor IX In Mice
Grafted With Infected Fibroblasts
Infected MEF cells and BL/6 cells were cultured
in an extracellular matrix, composed of collagen,
before grafting. A tumor cell line, BL/6, was chosen
in addition to MEF because it has an advantage in
growth and vascularization and thus would increase our
chances of detecting secreted factor IX in the sera.
Attachment of the cells to the collagen resulted in a 3
dimensional array of cells stacked on top of one
another. After the primary fibroblast cells (MEF) or
the tumor cell line BL/6 contracted in the collagen
gel, the cells were grafted into the loose connective
tissue of the mid-back dermis of a recipient syngeneic
C57BL/6 mouse (Fig. 3A). Figure 3B shows that the
inserted implants were extensively vascularized by day
14. A similar extent of vascularization was also
detected in 2~ day implants (data not shown).
The serum levels of the human clotting factor
were measured in engrafted mice by ELISA over a 34 day
period. Figure 4 shows that the average levels of
human factor IX in 3 mice progressively increased from
20 ng/ml at day 2 to a peak of 97 ng/ml 7 days after
grafting the BL/6 cells into the mice. The 4 mice
~3~~ Z~s
grafted with the infected MEF fibroblasts showed a
similar pattern of increase in which an average peak of
25 ng/ml of factor IX was detected at day 9. This rise
was followed by a rapid decline to near non detectable
levels of serum human factor IX at day 16 in both the
BL/6 and MEF grafts. A minor peak of factor IX was
seen at day 20 in mice with either graft, which was
followed by loss of any detectable factor IX antigen.
In parallel experiments, 10~ infected BL/6 or MEF cells
were injected directly into the peritoneal cavity of
the recipient C57BL/6 mice. Serum levels of human
factor IX in th_= injected animals followed a similar
profile as that seen with the grafts (data not shown).
EX~lanted Grafts Make Factor IX
The decline in serum levels of antigenic human
factor IX in animals that were either grafted or
injected i.p. was not associated with the necrosis of
cells in the grafts. BL/6 cells in the collagen matrix
grew as an aggressive tumor at the site of the graft.
The tumor continued to grow until the animals were
sacrificed at day 32. Mice with grafts containing
infected MEF were visibly vascularized upon gross
inspection until day 28, however, by day 120 the extent
of vascularization was reduced but the implant was
still viable (data not shown). Additionally, cells
explanted at various times during the course of the
experiment praduced factor IX when grown in culture
(Table I). The explanted BL/6 cells grew well in
culture and secrete antigenic factor IX at levels
similar to that before grafting. The MEF cells
explanted from the grafts at days 14 and 21 grew well
in culture, but produced slightly lower levels of
factor IX. Ce?~.s explanted at day 28 did not grow
well, and the low level of factor IX secreted from
26
1341 246
these cells is perhaps a consequence of this poor
growth.
Detection of Serum Anti-Factor IX Antibodies
To further investigate the decline of serum
levels of human factor IX it was reasoned that the
recipient animal mounted an immunological response
against the highly immunogenic human factor IX protein.
To test whether mice bearing grafts with infected BL/6
or MEF cells are generating anti-factor IX IgG
antibodies, pooled serum samples were used to probe
immunologic blots containing purified human factor IX
protein (Figure 5). The levels of anti-human factor IX
IgG antibodies were not detectable in mice with MEF
grafts at day 7 to day 21. Slightly higher levels of
serum antibodies were detected in mice with BL/6 grafts
during this period, presumably because they are
releasing more factor IX. Maximum levels of anti-human
factor I~; antibodies were detected at day 28 in mice
with either graft. The mice with BL/6 grafts exhibited
the highest level of xeno-antibodies. Pooled serum
drawn from mice 28 days after i.p. injection with
infected MEF also showed anti-factor IX IgG antibodies
albeit at much lower levels. Naive animals which have
not been exposed to infected BL/6 or MEF cells do not
make anti-human factor IX antibodies. These
observations world suggest that human factor IX is
continuously produced in grafted mice but is not
detectable due to a large pool of mouse anti-human
factor IX antibodies.
DISCUSSION
This example presents the development and
characterization of a different approach of gene
product delivery into an animal model system. The BL/6
cells and MEF cells infected with a helper free
27
1347 24~
recombinant retroviral vector containing the human
clotting factor IX cDNA secrete partially biologically
active clotting factor at a rate 10 fold higher than
seen with another retroviral vector containing the
human clotting factor cDNA (Anson, et al., 1987). In
addition, the example demonstrates that those
genetically modified cells can be reintroduced into the
loose connective tissue of the dermis of a syngeneic
mouse. Grafts are quickly vascularized in the presence
of angiogenic factor, fibroblast growth factor, and
remain vascularized for at least 28 days. Grafts
containing the BL/6 cells grow as aggressive tumors
over this period while the size of the grafts
containing the MEF cells does not increase over the
same period. The clotting factor secreted from the
infected cells in the graft is accessible to the
circulatory compartment and can easily be detected in
serum of the recipient. Functional status of the
infected cells in the grafts can be measured by
monitoring serum levels of human factor IX or by the
ability of explanted cells to continue secreting the
human protein. However, C57BL/6 mice recognize the
human blood clotting factor as foreign and thus mount a
strong humoral immune response against it. Although a
humoral response against factor IX clearly exists,
there does not appear to be a major cell mediated
response against the cells in the grafts. The cells in
the graft are still viable after 28 days of
implantation and continue to synthesize factor IX
protein.
Even though the data presented here was
obtained from mouse embryo fibroblasts, it should be
noted that the observations have been extended by
infecting adult hemophiliac dog fibroblasts with factor
28
1 3 4 ~ 2 46
IX retrovirus.
It should also be noted that in normal
individuals, levels of factor IX protein are
approximately 5 ug/ml of plasma. Although the levels
reported here are lower by several orders of magnitude,
it should be remembered that individuals containing 0.5
~.g of biologically active factor IX per ml in plasma do
not show the symptoms of hemophilia. The low levels of
factor IX can be increased either by making improved
vectors capable of generating large amounts of factor
IX proteins or, alternatively, by grafting more cells.
According to the data presented here, up to 25 ng of
factor IX per hr can be generated from an implant
containing 4 x 106 cells (Fig. 4). In larger animals
multiple grafts of up to 108 cells can be easily
implanted, increasing the levels of factor IX protein
to that required to alleviate the deficiency.
Culturing infected cells in a defined medium
(without fetal bovine serum) and improved technology
for reconstitution of living skin would also increase
the efficiency of the system (Bell, et al., 1983).
Moreover, impro~,red surgical skills may ensure that the
implant would lay flat in the dermal compartment of the
mouse skin to allow more uniform vascular development
and hence impro-~e cell viability during the brief
period required for vascularization. Although the
extent of cell viability has not yet been determined in
grafts containing MEF cells, experiments in rats have
shown that transplanted fibroblasts persist for at
least 13 months (Bell, et al., 1983).
In conclusion, this example has shown that skin
fibroblasts can be used as a viable mode of
introduction and expression of foreign genes in
mammals. The process of manipulation of genetically
29
1341 246
engineered fibroblasts appears to be both less complex
and cumbersome than the widely accepted use of bone
marrow transplantation far somatic cell gene therapy.
DETAILED DESCRIPTION OF THE FIGURE LEGENDS
Figure 1. Analysis of recombinant human factor
IX re~rovirus. (a) pAFFIXSVNeo. Arrows indicate
transcripts that initiate at either the promoter
located in the 5' LTR, or the simian virus 40 early
promoter located between the two LTRs, and terminate at
the polyadenylation signal in the 3' LTR. Bars
indicate the putative initiation site of transcription.
The restriction endonuclease cleavage sites SstI,
HindIII, BamHI, BalII and Clal are diagnostic sites
used during the construction of the vector or
subsequent characterization of the provirus in the
genome of infected cell lines. (b) Proviral DNA.
Genomic DNA isolated from either uninfected or infected
~FIXNeo ~, NIH3T3 TK , BL/6, MEF cells, and from the
virus producing cell line ~FIXNeo 4 was digested with
Sstl fractionation by agarose gel electrophoresis,
transferred onto a nitrocellulose membrane and
hybridized to either a nicktranslated 1.6 kb factor IX
cDNA probe (lanes 1-8) or 1.4 kb HindIII to BamHI Neo
DNA probe (lanes 9-12). Under these conditions of
hybridization, human factor IX cDNA does not hybridize
to mouse DNA. (c) RNA transcripts. Total RNA (10 ug)
isolated from uninfected and infected cells and cell
lines was subjected to RNA blot analysis.
Figure 2. Secretion of Human Factor IX. Rate
of secretion of human factor IX by the virus producing
cell line ~FIXNeo 4 (shown by open squares) and by
infected NIH3T3 TK- cells (shown by solid squares),
BL/6 cells (shorn by solid diamonds), and MEF cells
(shown by open diamonds). Cells were seeded at 3 x 106
13 4 '~ ~ ~i !~
cells per 5 cm dish in 4 ml of medium. At each
indicated time point 100 ul of medium was removed and
assayed for human factor IX by enzyme linked
immunoabsorbant assay (ELISA) (Anson, et al., 1987).
The mouse anti-human monoclonal antibody, FXC008,
generated by Bajaj, et al. (Bajaj, et al., 1985) was
used as the primary antibody, whereas pooled normal
human sera were used as a standard. Each time point
was done in triplicate and thus represE:nts an average
amount of factor IX secreted over a 48 hr period.
Curves were corrected for the slight increase in cell
number over this period.
Figure 3. Embedding and Implantation. (A)
Schematic representation of the protocol used to
generate and graft the collagen implants into the loose
connective tissue of the skin of the mouse model
system. (B and C) View of both MEF (B) and BL/6 (C)
collagen implants at day 14. The grafts (white area)
and the high degree of vascularization are clearly
visible. The implant is 0.75 cm in diameter. FGF,
fibroblast growth factor.
Figure .~. Factor IX in Human Sera. Average
(four mice for IvEF; three mice for BL/6) amount of
human factor IX detected in the sera of mice that
received two collagen implants containing approximately
4 x 106 cells each. Sera was drawn from each animal at
the indicated times. Levels of circulating human
factor IX were determined by ELISA: levels of factor IX
fluctuated 2- to 3-fold between experiments.
Figure 5. Detection of mouse anti-human factor
IX antibodies in mice grafted with collagen implants.
Purified human factor IX was subjected to PAGE under
denaturing conditions and then transferred onto
nitrocellulose as described (Towbin, et al., 1979).
31
1341 246
The nitrocellulose strips were treated with blocking
solution for 2 hr followed by 1:100 dilution of naive
normal mouse serum; 1/100 dilution mouse monoclonal
anti-factor IX antibody FXC008 (lane 2); 1:100 dilution
of serum from mouse harboring grafts containing
infected MEF cells drawn at day 7, day 14, day 20, and
day 28 (lanes 3-6); 1:100 dilution of serum from mouse
harboring grafts containing infected BL/6 cells drawn
at day 7, day 15, day 21, and day 29 (lanes 7-10).
After overnight incubation at 37°C the strips were
washed, incubated with 1251-labeled goat anti-mouse IgG
antibody, and then subjected to autoradiography as
described (Glenney, 1986).
32
1341 2~6
Table I. Amount of antigenic factor IX secreted from
cells explanted from grafts. Tissue explanted from the
grafts at times indicated after implantation were
cultured in vitro. When cells were confluent medium
was replaced; after 48 hr, levEls of secreted factor IX
secreted into the culture were assayed by ELISA.
Collagen
Days after explants,, n
Implantation BL/6 MEF
14 180 40
23 210 27
28 150 11
33
1341 24g _
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The following patent and journal publications
are referred to in the specification.
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~3412'~8~::
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23. Miller, A.D., Law, M-F., & Verma, I.M.
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24. Mor~an, J.R., Barrandon, Y., Green, H., &
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25. Mulligan, R.C. (1983), "Construction of
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26. Palmer, T.D., Hock, R.A., Osborne, W.R.A.,
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PATENT PUBLICATIONS
1. WO 86 00922, Applicant: The Salk Institute
For Biological Studies; Inventors: I.M.
Verma, A.G. Miller, and R.M. Evans; Title:
Retroviral Gene Transfer Vectors.
2. United States Patent 4,624,944, issued
November 25, 1986 to the Regents of the
University of California for "Human Seminal
Alpha Inhibins".
SPECIFICATION SUMMARY
From the foregoing description, one of
ordinary skill in the art can understand that the
present invention is a new somatic cell gene therapy
method. According to the invention, transduced
fibroblasts are preferably created by infecting
fibroblast cells in vitro with chimeric retroviruses
that contain at least one functionally active
"replacement gene". Such replacement genes can be
37
1341 24~~
either foreign genetic material that is not found in
fibroblast cells, or native genetic material that is
found in fibroblast cells but not normally expressed in
biologically significant concentrations in these cells.
Since the invention uses transduced fibroblasts from
the individual or animal to be treated, the possibility
of rejection is minimized. In addition, since the
invention implants the transduced fibroblasts in the
highly vascularized loose connective tissue of the
dermis, the transduced cells, and thus their
"replacement" gene products, have direct access to the
circulatory system. As a result the needed replacement
gene products can easily and efficiently be distributed
to other parts of the body. When the gene therapy is
no longer needed, the implanted fibroblasts can be
conveniently removed.
Since the fibroblasts can be transduced to
express a variety of replacement genes, the method of
the invention has many important applications for both
humans and animals. For example, the method can be
used to treat diseases caused by genetic defects, to
deliver drugs to individuals and animals, and to ad-
minister birth control hormones.
without departing from the spirit and scope
of this invention, one of ordinary skill can make
various changes and modifications to the invention to
adapt it to various usages and conditions. As such,
these changes and modifications are properly,
equitably, and intended to be, within the full range of
equivalence of the following claims.
38
