U.S. patent application number 12/034305 was filed with the patent office on 2008-06-19 for production of a biological factor and creation of an immunologically privileged environment using genetically altered sertoli cells.
This patent application is currently assigned to Sertoli Technologies, Inc.. Invention is credited to Paul Gores, Craig Halberstadt, Shaun A. Kirkpatrick.
Application Number | 20080145348 12/034305 |
Document ID | / |
Family ID | 23720111 |
Filed Date | 2008-06-19 |
United States Patent
Application |
20080145348 |
Kind Code |
A1 |
Kirkpatrick; Shaun A. ; et
al. |
June 19, 2008 |
PRODUCTION OF A BIOLOGICAL FACTOR AND CREATION OF AN
IMMUNOLOGICALLY PRIVILEGED ENVIRONMENT USING GENETICALLY ALTERED
SERTOLI CELLS
Abstract
The present invention provides a method of providing an
individual with a biological factor or intermediate thereof which
comprises introducing into the individual Sertoli cells genetically
altered to produce the biological factor or intermediate thereof.
The genetically altered Sertoli cells are administered in an amount
effective to produce the desired effect. Aside from producing the
biological factor or intermediate thereof, the engineered Sertoli
cells also create an immunologically privileged site. Vectors
comprising a promoter which functions in Sertoli cells operably
linked to coding sequence for a desired biological factor are also
provided as are Sertoli cells comprising such vectors. A
pharmaceutical composition comprising Sertoli cells genetically
altered to produce a biological factor is also provided.
Inventors: |
Kirkpatrick; Shaun A.;
(Tucson, AZ) ; Gores; Paul; (Charlotte, NC)
; Halberstadt; Craig; (Charlotte, NC) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Sertoli Technologies, Inc.
|
Family ID: |
23720111 |
Appl. No.: |
12/034305 |
Filed: |
February 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10219804 |
Aug 15, 2002 |
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12034305 |
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09433429 |
Nov 4, 1999 |
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10219804 |
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60107438 |
Nov 6, 1998 |
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Current U.S.
Class: |
424/93.21 ;
435/320.1; 435/366 |
Current CPC
Class: |
A61K 2035/122 20130101;
C12N 9/644 20130101; A61K 48/00 20130101; A61K 38/4846 20130101;
C12N 9/1051 20130101; A61K 38/28 20130101; C12Y 304/21022 20130101;
A61P 3/10 20180101; C12N 5/0683 20130101 |
Class at
Publication: |
424/93.21 ;
435/320.1; 435/366 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 15/63 20060101 C12N015/63; C12N 5/08 20060101
C12N005/08; A61P 3/10 20060101 A61P003/10 |
Claims
1. A method for providing an individual with a biological factor or
intermediate thereof which comprises introducing into said
individual a therapeutically effective amount of Sertoli cells,
said Sertoli cells genetically altered to produce said biological
factor or intermediate thereof and wherein said Sertoli cells
create an immunologically privileged site.
2. A method of treating a disease that results from a deficiency of
a biological factor which comprises introducing into a subject in
need of such treatment, a therapeutically effective amount of
Sertoli cells, said Sertoli cells genetically altered to produce
said biological factor or intermediate thereof and wherein said
Sertoli cells create an immunologically privileged site.
3. The method of claim 1 or 2 wherein said subject is a mammal.
4. The method of claim 3 wherein said mammal is human.
5. The method of claim 1 or 2 wherein said biological factor is a
hormone.
6. The method of claim 3 wherein said biological factor is a
hormone.
7. The method of claim 2 wherein said biological factor is insulin
and said disease is diabetes mellitus.
8. The method of claim 2 wherein said biological factor is Factor
IX and said disease is hemophilia B.
9. The method of claim 2 wherein said biological factor is B-UGT
and said disease is Crigler-Najjar (CN) disease.
10. The method of claim 1 or 2 wherein said introduction is by
transplantation.
11. The method of claim 1 or 2 wherein said Sertoli cells are
introduced into said individual in a dosage ranging from 10.sup.5
to 10.sup.10 cells.
12. The method of claim 10 wherein said transplantation is by
xenograft.
13. The method of claim 10 wherein said transplantation is by
allograft.
14. A pharmaceutical composition comprising Sertoli cells
genetically altered to produce a biological factor or intermediate
thereof admixed with a pharmaceutically acceptable carrier.
15. The pharmaceutical composition of claim 14 wherein said
biological factor is a hormone.
16. A compartmentalized kit comprising a first container adapted to
contain Sertoli cells genetically altered to produce a biological
factor or intermediate thereof.
17. The compartmentalized kit of claim 16 further comprising a
second container adapted to contain a pharmaceutically acceptable
carrier.
18. A vector comprising in the 5' to 3' direction a promoter which
functions in Sertoli cells operatively linked to a coding sequence
for a biological factor or intermediate.
19. The vector of claim 8 further comprising a 3' termination
sequence which functions in Sertoli cells.
20. The vector of claim 18 or 19 further comprising a signal
sequence coding for a signal peptide, said signal sequence located
downstream from said promoter and upstream to said coding sequence
for a biological factor or intermediate.
21. The vector of claim 18 or 19 wherein the coding sequence is the
coding sequence for human factor IX.
22. The vector of claim 20 wherein the coding sequence is the
coding sequence for human factor IX.
23. The vector of claim 18 or 19 wherein the coding sequence is the
coding sequence for B-UGT.
24. The vector of claim 20 wherein the coding sequence is the
coding sequence for B-UGT.
25. A Sertoli cell comprising the vector of claim 18 or 19.
26. A Sertoli cell comprising the vector of claim 20.
Description
FIELD OF THE INVENTION
[0001] Transplants of healthy organs or cells into a patient
suffering from a disease are often rejected by the body due to an
immune response initiated in response to the foreign tissue or
cells. Genetically altered cells administered during gene therapy
are often met with a similar immune response. The present invention
provides a method of cellular transplantation in which an
immunologically privileged site is created, thus alleviating the
rejection associated with conventional transplantation and gene
therapies.
[0002] Specifically, the present invention provides compositions
and methods for providing an individual with a biological factor or
intermediate thereof which comprises introducing into the
individual a therapeutically effective amount of Sertoli cells
genetically manipulated to produce the biological factor or
intermediate thereof and wherein the Sertoli cells create an
immunologically privileged site. A pharmaceutical composition
comprising genetically altered Sertoli cells which produce a
biological factor is also provided.
BACKGROUND OF THE INVENTION
[0003] Certain chronic diseases destroy the functional cells in
affected organs individuals with such diseases are often unable to
produce proteins or other biological products necessary to maintain
homeostasis and usually require numerous exogenous substances to
survive. Transplanting healthy organs or cells into an individual
suffering from such a disease may be necessary to save the
individual's life. This type of therapy is generally regarded as a
last alternative to curing an otherwise fatal condition. Such
transplants, however, are often rejected by the body due to an
immune response initiated in response to the foreign tissue or
cells. Presently, the only recourse to combat this immune response
is to administer chronic nonspecific immunosuppression agents. The
use of nonspecific immunosuppression agents however, is fraught
with unwanted side effects such as increased susceptibility to
infection, hypertension, renal failure and tumor growth.
[0004] In the fields of cell and organ transplantation, Selawry et
al., have demonstrated that Sertoli cells can be used to create an
artificial privileged environment when isolated from the testes and
then transplanted into heterologous sites. Privileged environments
have been created using both allogeneic and xenogeneic Sertoli
cells. Rajotte and Korbutt, 1997 Diabetes 46: 317-322. Similarly,
both rat Sertoli cell allografts and porcine Sertoli cell
xenografts have been shown to survive for at least two months in
the rat brain without cyclosporin A immunosuppression. Saporta, S.
et al., 1997 Exp. Neurology 146(2):299-304. Selawry, et al., have
also demonstrated the immunoprotection of islet cells placed in the
rat kidney capsule when cotransplanted with Sertoli cells. Long
term islet survival and functional recovery in a diabetic rat model
have been demonstrated. Selawry, H., et al., 1993 Cell Transplant
2:123-129. It has recently been suggested that Sertoli cells
express an immunosuppressant factor, Fas ligand (Fas-L),
responsible all or in part for providing the testis with its
immunoprivileged status. Sandberg, P. R., et al., 1997 Cell
Transplantation 6(2):191-193; Saporta, S., et al., supra.
[0005] Immune rejection of genetically altered cells during gene
therapy also remains a problem. In order to overcome the problem,
autologous cells, i.e., a patient's own cells, may be used. In
fact, most of the currently approved human gene therapy protocols
depend on genetic alteration of autologous cells. Such cells
however, may be difficult to obtain due to the diseased state of
the patient and are often destroyed during harvesting. Those cells
that survive harvesting are often difficult to grow in vitro and
require complicated culture conditions and purification
methodologies.
[0006] There is a need, therefore, for compositions and methods for
producing a biological factor in a subject wherein cells which are
genetically altered to produce the biological factor are readily
available and more easily manipulated in vitro. In addition, the
need exists for compositions and methods for producing a biological
factor in a subject who receives allogeneic or xenopeneic cells
which do not trigger an immune response requiring chronic
immunosuppression.
SUMMARY OF THE INVENTION
[0007] The present invention provides compositions and methods for
providing an individual with a biological factor or intermediate
thereof which comprises introducing into the individual a
therapeutically effective amount of Sertoli cells genetically
manipulated to produce the biological factor or intermediate
thereof and wherein the Sertoli cells create an immunologically
privileged site.
[0008] The present invention also provides a method of treating a
disease that results from a deficiency of a biological factor which
comprises administering to a subject in need of such treatment a
therapeutically effective amount of Sertoli cells genetically
transformed to produce the biological factor or an intermediate of
the biological factor and wherein the Sertoli cells create an
immunologically privileged site.
[0009] Preferably, the genetically altered Sertoli cells are
administered by transplantation. Transplantation may be by
xenograft or allograft.
[0010] The present invention also provides a pharmaceutical
composition comprising Sertoli cells genetically transformed to
produce a biological factor or intermediate thereof admixed with a
pharmaceutically acceptable carrier.
[0011] Also provided is a compartmentalized kit comprising a first
container adapted to contain Sertoli cells genetically altered to
produce a biological factor or intermediate thereof.
[0012] A vector comprising in the 5' to 3' direction a promoter
which functions in Sertoli cells operatively linked to a coding
sequence for a biological factor or intermediate is also provided
by the present invention. The vector may further comprise a 3'
termination sequence which functions in Sertoli cells and/or a
signal sequence coding for a signal peptide. The signal sequence is
located downstream from the promoter sequence and upstream to the
coding sequence for a biological factor or intermediate.
[0013] Sertoli cells comprising the subject vectors are also
provided.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides compositions and methods for
producing a biological factor in a subject. The compositions and
methods protect themselves from immune destruction by the subject,
thereby eliminating the need for chronic immunosuppression.
[0015] In accordance with the present invention, Sertoli cells, the
predominant cells of male testes, are used to produce a biological
factor such as a protein in vivo. The Sertoli cells used to produce
the biological factor are genetically altered and are generated by
either ex vivo gene transfer or else isolated from a transgenic
animal that expresses the biological factor in Sertoli cells. The
genetically altered Sertoli cells are then introduced into a
subject so that the biological factor is produced in that subject.
The resultant transplanted Sertoli cells, in addition to producing
a desired biological factor, also create an immunoprivileged
environment at the site of transplantation.
[0016] Preferably, the route of introduction is by subcutaneous
transplantation into such sites as the renal subcapsular space,
subcutaneous facie, liver subcapsular space, brain, hepatic portal
vein or omental pouch. The genetically altered Sertoli cells may
also be placed in a biocompatible device that allows for contact
with the vascular system, localizes the Sertoli cells, and enables
the Sertoli cells to survive long term. Such a device also provides
added protection from rejection by the immune system. An example of
such a biocompatible device is described in U.S. Pat. No.
5,182,111, issued to Aebischer et al. Immuno-isolation devices such
as alginate or thermoplastic capsules and hollow fibers may also
house the genetically altered cells in vivo. Such devices are
reviewed in Chang, P. L. 1997 IEEE Engineering in Medicine and
Biology, September/October, pp. 145-151.
[0017] In accordance with the present invention, a transgenic
animal that expresses a biological factor in Sertoli cells can be
made using any number of currently available systems. For example,
expression constructs having a promoter which functions in Sertoli
cells operably linked to coding sequence for a biological factor or
intermediate can used to transfect the testes of living mice by
microparticle bombardment and electroporation. Muramatsu T. et al.,
(1997) Biochem. and Biophys. Res. Commun. 233(1):45-49. Such
constructs may also be microinjected into pronuclei by means of
standard procedures as described in Hogan et al. (1986)
Manipulating the Mouse Embryo: a Laboratory Manual, Cold Spring
Harbor Laboratory Press, New York.
[0018] The present invention is useful in improving the general
health, well being, or appearance of an individual by supplying a
therapeutically effective amount of a biological factor or
intermediate thereof or else is useful in treating a disease that
results from a deficiency of a biological factor.
[0019] As defined by the present inventions a biological factor is
a protein or nonprotein compound that is necessary for cellular
metabolism and homeostasis. Further in accordance with the present
invention, a biological factor is a protein or nonprotein compound
that is absent, deficient or altered in a subject suffering from a
particular disease. As used herein, the term "protein" includes but
is not limited to peptides and polypeptides. The biological factor
may be a protein which enhances the immunosuppressive and growth
enhancing properties of Sertoli cells. When the biological factor
is a nonprotein compound, the Sertoli cells are genetically
engineered to produce a protein, such as an enzyme which protein is
necessary for one or more steps in the biosynthetic pathway of a
nonprotein compound. Thus, the Sertoli cells may be engineered to
produce a protein intermediate to a nonprotein biological
factor.
[0020] Recombinantly produced proteins of the Sertoli cells may
comprise any number of structural, therapeutic or biologically
functional proteins i.e., a polypeptide or protein which affects
the cellular mechanism of the cell in which the biologically
functional protein is expressed or to which it is administered. For
example, the biologically functional protein may be a protein which
affects the cellular mechanism of the Sertoli cell in which the
biologically functional protein is expressed. The biologically
functional protein can also be a protein which improves the health
of a mammal by either supplying a missing protein, by providing
increased quantities of a protein which is underproduced in the
mammal, or by providing a protein which inhibits or counteracts an
undesired molecule which may be present in the mammal. The
biological factor may also be a factor which helps maintain or
improves the health and well being of the individual and includes
for example, peptides which stimulate or decrease appetite.
[0021] The biologically functional protein can be a protein which
is essential for normal growth or repair of cells. The biologically
functional protein may also be one which is useful in fighting
diseases such as cancer, atherosclerosis, sickle-cell anemia and
the thalassemias. Examples of such biologically functional proteins
are hemoglobin (.alpha., .beta. or .gamma.-globin), hematopoietic
growth factors such as granulocyte-macrophage colony stimulating
factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and
erythropoietin (EPO). Another example is tumor necrosis factor
(TNF), which is a molecule What can be used to treat cancer, and in
particular, tumors. The tumor suppressor p53 and retinoblastoma
(RB) are also contemplated. Various cytokines such as mast cell
growth factor (MGF) and interleukins 1-11 are also proteins which
are contemplated for production by the genetically altered Sertoli
cells of the present invention. Factor XIII (for treatment of
hemophilia A), Factor IX (for treatment of hemophilia B), IL-2 (for
enhancing the Sertoli cell's ability to provide immunoprotection),
insulin (for treatment of diabetes), and dopamine (for treatment of
Parkinson's disease) are also examples of proteins which can be
produced by the Sertoli cells in accordance with the present
invention.
[0022] B-UGT is yet another example of a protein which can be made
by the subject genetically altered Sertoli cells for the treatment
for Crigler-Najjar (CN) disease. Mammals normally have a liver
enzyme, bilirubin UDP-glucuronosyltransferase (B-UGT), which
conjugates bilirubin (an end product of the hemoglobin breakdown)
with glucuronic acid, rendering the molecule more water soluble and
excretable. Patients with Crigler-Najjar (CN) disease have a
deficiency in B-UGT and accumulate high serum levels of bilirubin.
Gunn rats are also incapable of bilirubin conjugation due to a
genetic defect in UDP-glucuronyl transferase. Gunn rat fibroblasts
expressing recombinant B-UGT have been transplanted
intraperitonealy into Gunn rats with resultant correction of the
genetic defect.
[0023] Thus, any protein which can be produced through recombinant
DNA means is contemplated for production by the subject genetically
altered Sertoli cells and for use in the methods of the present
invention. Many well known procedures exist for the preparation of
DNA sequences which code for a desired protein. For example,
oligonucleotides of various lengths can be synthesized by known
procedures. Several such oligonucleotides can be assembled into
longer, double stranded molecules. Alternatively, DNA molecules
having the desired coding sequences can be synthesized by use of
the enzyme reverse transcriptase using messenger RNA related to the
desired polypeptide as a template for the action of reverse
transcriptase (cDNA cloning). Another possibility for preparing DNA
sequences coding for the desired protein is the cloning of genomic
DNA fragments obtained from a gene bank or library. In many
instances, the DNA encoding the protein of interest is commercially
available.
[0024] In some instances, the recombinantly produced protein is
beneficial in exerting its effect on the Sertoli cell in which it
is produced. IL2 is an example of such a protein. In most other
instances however, the recombinantly produced protein of the
genetically altered Sertoli cell benefits the subject only when
such protein reaches other non-Sertoli cells. Thus, in a preferred
embodiment of the invention, the recombinantly produced protein is
exported across the Sertoli cell membrane and outside the Sertoli
cell, i.e., secreted. In this aspect of the invention, the
recombinantly produced protein will have on its N-terminal end a
signal peptide (N-terminal leader sequence). The signal peptide
enables the transport of the recombinantly produced protein out of
the Sertoli cell where it is produced and into the intercellular
spaces and capillaries of the surrounding cells. Once transported
out of the Sertoli cell, the recombinantly produced protein enters
the circulatory system of the subject.
[0025] The signal peptide may be encoded by the same gene from
which the promoter is derived, e.g., FSHR. In this embodiment of
the invention, the recombinantly produced protein is most often a
chimeric protein. The native signal peptide of the recombinantly
produced protein may also used. In yet another embodiment, the
signal peptide may be from a protein other than the recombinantly
produced protein or protein whose corresponding gene provides the
promoter for the expression vector. Many different proteins have
signal peptides at their N-terminal ends. The skilled artisan is
aware of many different sequences known as signal sequences, which
code for signal peptides. Such signal sequences are useful for
incorporation into expression vectors of the present invention in
order to ensure secretion of the recombinantly produced protein out
of the Sertoli cell.
[0026] Sertoli cells which are to be genetically altered to produce
a desired biological factor are first isolated using methods known
in the art such as those described in Cheng et al., 1987 J. Biol.
Chem. 26:12768-12779. Sertoli cells can be separated from other
testicular cells such as Leydig cells, peritubular cells and germ
cells, using conventional techniques. For example, the testes of a
male mammal, such as a human, mouse, rat, pig, boar or ram, are
first collected by castration. The testes are then chopped into
several pieces and subsequently washed by centrifugation.
[0027] Testicular Leydig cells can be removed from the tissue
suspension using digestion agents such as trypsin and DNase. The
remaining cell suspension is then washed by centrifugation several
times. The pellet is resuspended in collagenase, incubated and
washed by centrifugation to eliminate peritubular cells within the
testes. Testicular germ cells can be removed by incubating the
pellet with hyaluronidase and DNase. After several washings by
centrifugation, the Sertoli cells can be collected for genetic
transformation. Similar methods of isolating Sertoli cells are
described in Selawry, H. P. and Cameron, D. F., (1993) Cell
Transplantation 2:123-129; and Korbutt, G. S., et al, (1997)
Diabetes 46:317-322.
[0028] Established Sertoli cell lines may also be used to produce a
biological factor in accordance with the present invention. For
example, an immortalized rat Sertoli cell line (SerW3) has been
established. Pognan, F., et al., 1997 Cell Biology and Toxicology
13:453-463. Fresh primary rat Sertoli cells were immortalized with
the T antigens of the Simian virus (SV40) which are known to
immortalize heterologous cells but not transform them. The SerW3
Sertoli cell line has structural and biochemical properties similar
to those of primary cells in culture or Sertoli cells in testicular
sections. Pognan F., supra. A prepubertal rat Sertoli cell line
known as 93RS2 has also recently been developed. Jiang, C., et al.,
1997 J. Andrology 18(4):393-399. Conditionally immortalized Sertoli
cells lines have also been established from H-2K.sup.b-tsA58
transgenic mice. Walther N., et al., 1996 Exp. Cell Res.
225:411-421
[0029] In accordance with the present invention, either primary or
cultured Sertoli cells may be used to produce a biological factor.
Whether primary cells or cultured cells, the Sertoli cells of the
present invention may be obtained from species such as rat, human,
porcine, murine, bovine or other species. These distantly related
Sertoli cells function as "universal" biological factor producing
cells since upon administration, such cells will not trigger an
immune rejection, even in a distantly related or non related
recipient subject.
[0030] In accordance with the present invention, the isolated
Sertoli cells may be genetically altered to produce a desired
biological factor using any number of well known methods. For
example, a DNA or RNA sequence coding for a particular protein is
first placed within a vector which can replicate within a Sertoli
cell. Thus, the present invention also provides vectors for
expression of proteins in the Sertoli cell. Within the vector,
coding sequence for a particular protein is operably linked at its
5' end to a promoter which functions in human, mouse, rat, pig,
boar or ram cells, including Sertoli cells. In a preferred
embodiment, the vector also contains a 3' termination sequence
operably linked to the 3' end of the coding sequence.
[0031] The vectors of the present invention can be constructed by
standard techniques known to one of ordinary skill in the art and
found, for example, in Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y., or
any of a myriad of laboratory manuals on recombinant DNA technology
that are widely available. A variety of strategies are available
for ligating fragments of DNA, the choice of which depends on the
nature of the termini of the DNA fragments and can be readily
determined by the skilled artisan.
[0032] The vectors of the present invention may also contain other
sequence elements to facilitate vector propagation, isolation and
subcloning; for example, selectable marker genes and origins of
replication that allow for propagation and selection in bacteria
and host cells. Selectable marker genes can include ampicillin and
tetracycline resistance genes for propagation in bacteria or
neomycin, hygromycin or zeocin resistance for selection in
mammalian cells. Sequences for heterologous genes coding for
structural, therapeutic or biologically functional proteins as well
as sequences for selectable markers and reporter genes are well
known to the skilled artisan. Examples of reporter genes include
GFP, luciferase, CAT, and .beta.-galactosidase. By "heterologous
genes" is meant coding sequences or parts thereof which are
artificially introduced into Sertoli cells.
[0033] Inducible expression systems are presently available where
exogenously administered factors function to induce gene
expression. Such systems are especially useful in selection of
Sertoli cells which have been transformed with an expression vector
of the present invention. The REV TET-ON.TM. and REV TET-OFF.TM.
systems available from Clonetech Laboratories, Palo Alto, Calif.,
and the Ecdysone-Inducible Mammalian Expression System, available
from Invitrogen, Carlsbad, Calif., are especially helpful in this
regard.
[0034] Promoters which function in Sertoli cells are known to those
of skill in the art. For example, the promoter for transferrin or
clusterin may be used. In order to limit the expression of the
desired biological factor or intermediate to Sertoli cells only,
the follicle stimulating hormone receptor (FSHR) promoter may be
employed. Other promoters, upstream 5 regulatory sequences and 3'
regulatory sequences including termination sequences which may be
used in the vectors of the present invention include sequences from
Cytomegalo virus (CMV), Simian Virus 40 (SV40), the Moloney Murine
Leukemia (MOMLV) virus, Herpes-virus, pox-virus, and
Adeno-associated virus (AAV), Epstein-Barr virus (EBV).
[0035] The clusterin gene has been studied in mouse, rat, and
human. Herault et al., 1992 Nucleic Acids Research 20:6377-6383;
Wong et al., 1993 J. Biol. Chem. 268:5021-5031, Wong et al., 1994
Eur. J. Biochem. 221:917-925; Jordan-Starck et al., 1994, J. Lipid
Res. 35: 194-210; Rosemblit and Chen, 1994 J. No. Endocrinology 13:
69-76. The gene is expressed in the male reproductive tissues at
different levels and is most highly expressed in the Sertoli cells.
Rosemblit et al., 1996, J. Mol. Endocrinology. 16:287-296. It has
been found in rat and mouse that androgens and cAMP negatively
regulate clusterin gene expression. Pignataro et al., 1992
Endocrinology 130(5):2745-2750. Clusterin regulatory sequences such
as the promoter and 5' flanking sequences from rat have been
identified as located between nucleotides -266 and +54. Potential
negative regulatory DNA elements may be present at upstream
locations such as -266 to -714 and from -714 to -1298. Rosemblit et
al., 1996, J. Endocrinology 16(3):287-296. The human clusterin gene
promoter region has been published. Michel et al., 1997 Biochem. J.
328:45-50; Wong et al. 1994 Eur. J. Biochem 221:917-925. Between
mammalian clusterin promoters, a relatively modest conservation
exists; most of the homology is confined within a very proximal
domain with upstream regions being completely divergent. Michel,
supra.
[0036] The promoter from the FSH receptor (FSHR) gene may also be
used in the expression vectors of the present invention. FSHR gene
expression is limited to Sertoli cells of the testis and granulosa
cells of the ovary. Gromoll et al., 1996 Genomics 35:308-311. Thus,
this tissue specific promoter is highly useful for the compositions
and methods of the present invention. The FSHR gene has been
isolated and characterized in mouse, rat and human. Goetz et al.,
1996J. Biol. Chem. 271(52):33317-33324. In rat, primer extension
and S1 nuclease experiments have located two major transcriptional
start sites; one at -80 and one at -98 relative to the
translational start site. Transient expression studies utilizing a
chimeric gene constituting 830 bp of DNA 5' to the translational
start site operably linked to the chloramphenicol acetyltransferase
(CAT) gene have demonstrated that this portion of the gene acts as
transcriptional promoter in rat Sertoli cells. Heckert, L. L, et
al., 1992 Mol. Endocrin. 6(1):70-80.
[0037] The human FSHR gene is 54 kb, consisting of 10 exons and 9
introns. The gene encodes 695 amino acids including a signal
peptide of 18 amino acids. The core promoter region is located
between the translational start codon and 225 bp upstream. As in
rat and mouse, in the human gene, a consensus CAAT or TATA box is
not located within this promoter region. Gromoll et al., supra. The
promoter region of the human FSHR gene has been finely mapped and
been shown to consist of a conserved consensus E box sequence and
an initiator-like region (InR) sequence. A 114-base pair region
spanning -143 to -30 which encompasses the E box and InR has been
demonstrated as sufficient for conferring greater than 75% promoter
function. Mutations in the InR, however, result in significant
reduction of FSHR promoter activity. Goetz, supra. The sequence of
the 5' flanking region of human FSHR gene has also been published.
When transiently transfected with gene constructs containing 1486
bp of the 5' flanking region the FSHR gene (including deletions
thereof), operably linked to the CAT gene, Chinese hamster ovary
(CHO), primary rat Sertoli cells, and human granulosa-lutein cells
directed significant expression of CAT. The promoter proximal
region has been allocated to the region from -225 to -1 bp. Gromoll
et al., 1994 Molecular and Cellular Endocrinology 102:93-102.
[0038] Another regulatory sequence useful for the compositions and
methods of the present invention includes the transferrin gene
promoter. Transferrin, an iron-transport protein, is expressed at a
high level in the liver but at lower levels in other organs such as
the brain, Sertoli cells of the testis, mammary glands and fetal
muscle. Sertoli cells synthesize and secrete testicular
transferrin. P Schaeffer et al., 1993 J. Biol. Chem.
268(31):23399-23408; Guillou et al., 1991 J. Biol. Chem.
266(15):9876-9884; Skinner et al. 1980 J. Biol. Chem.
255:9523-9525. The human gene has been isolated and the regulatory
sequences identified. Adrian et al., 1986 Gene 49:167-175.
5'-deletion analysis in transient expression systems have indicated
the region of -125/+39 as the promoter proximal region. Schaeffer
et al., 1989 J. Biol. Chem. 264:7153-7160. The region spanning -52
to +30 TATA box is sufficient to activate a basal level of
transcription. Guillou et al., 1991 J. Biol. Chem. 266:9876-9884.
Transcriptional activation in Sertoli cells involves the
interaction of DNA-binding proteins with the PRI-TATA box couple or
the PRI-PRII couple. Guillou et al., 1991 J. Biol. Chem.
266:9876-9884. The transferrin promoter has also been found to
contain two critical response elements designated Sertoli element 1
(SE1) and Sertoli Element 2 (SE2). Through sequencing analysis, SE2
has been found to contain an E-box element. E-box response elements
have been demonstrated to respond to basic-helix-loop-helix (bHLH)
transcription factors. bHLH transcription proteins are a class of
transcription factors which are involved in the induction and
progression of cell differentiation. Chaudhary, J., et al., 1997
Endocrinology 138(2):667-675.
[0039] Transgenic mice transformed with genetic constructs in which
a 670-bp segment of the regulatory sequence from the human
transferrin gene operably linked to the CAT gene or three different
apolipoprotein E (apoE) alleles have exhibited expression of such
genes in different portions of their brain. Bowman, B. H., et al.,
1995 Proc. Nat., Acad. Sci. USA 92(26):12115-12119. The 5' flanking
region of the mouse transferrin gene has also been isolated and
used to drive expression of human growth hormone (hGH) in
transgenic mice.
[0040] Those skilled in the art are familiar with the methodologies
required to subclone all or a portion of the clusterin, FSHR, and
transferrin genes, and to isolate the respective regulatory regions
such as the promoter and 5' and 3' regulatory sequences. For
example, a clusterin, FSHR, or transferrin gene promoter and
upstream regulatory sequences may be generated from a mouse, rat,
bovine, porcine, or human genomic clone having either or both
excess 5' flanking sequence or downstream coding sequence via
exonuclease III-mediated deletion. This is accomplished by
digesting appropriately prepared DNA with exonuclease III (exoIII)
and removing aliquots at increasing intervals of time during the
digestion. The resulting successively smaller fragments of DNA may
be sequenced to determine the exact endpoint of the deletions.
There are several commercially available systems which use
exonuclease III (exoIII) to create such a deletion series, e.g.,
Promega Biotech, "Erase-A-Base" system. Alternatively, PCR primers
can be defined to allow direct amplification of the desired
promoters and 5' regulatory regions.
[0041] A clusterin, FSHR, or transferrin 3' regulatory sequence,
including 3' termination sequence, may be isolated from a mouse,
rat, bovine, porcine, or human genomic clone having either or both
excess 3' flanking sequence or upstream coding sequence via the
same exoIII mediated deletion methodologies described above.
[0042] The 5, and 3' regulatory sequences and the DNA sequences
which code for a desired protein and signal peptide can be modified
for preparation of an expression vector by a variety of procedures.
For example, the ends of the DNA prepared as described above can be
ligated with the enzyme DNA ligase to short double-stranded DNA
molecules which contain the nucleotide sequence recognized by
specific restriction endonucleases, so called linker molecules.
Digestion of these molecules with a specific restriction
endonuclease following the ligation will generate termini
corresponding to the specified restriction endonuclease recognition
site at the ends of the prepared DNA sequence.
[0043] Thus, the present invention also provides a recombinant
expression vector comprising in the 5' to 3' direction: a promoter
which functions in a Sertoli cell and coding sequence for a
biological factor or intermediate. In another embodiment, a subject
vector comprises in the 5' to 3' direction: a promoter which
functions in a Sertoli cell, coding sequence for a biological
factor or intermediate and a 3' regulatory sequence including a
termination sequence. In yet another embodiment, a vector is
provided comprising in the 5' to 3' direction: a promoter which
functions in a Sertoli cell, a signal sequence, and coding sequence
for a biological factor or intermediate. In still another
embodiment, there is provided a vector which comprises in the 5' to
3' direction: a promoter which functions in a Sertoli cell, a
signal sequence, coding sequence for a biological factor or
intermediate, and a 3' regulatory sequence including a termination
sequence. Other embodiments include any of the above delineated
vectors with additional 5' regulatory sequence in addition to the
promoter.
[0044] The subject vectors may then be introduced into the Sertoli
cell. The introduction of DNA into Sertoli cells can be
accomplished through various well known procedures. For example,
the subject vectors may comprise viral sequences required for
packaging, reverse transcription, and integration into a host
genome in addition to a promoter which functions in Sertoli cells
operably linked to coding sequence for a biological factor or
intermediate as described supra. vectors having such sequences are
useful for transfecting into helper cells from which recombinant
virions can be produced. Virions containing the subject vectors are
then used to transfect Sertoli cells.
[0045] Physical/chemical techniques such as calcium phosphate
transfection, complex formation with polycations or lipids,
electroporation, particle bombardment and microinjection into
nuclei may also be used to transfect Sertoli cells. Preferably, the
introduction of the subject vectors into Sertoli cells is
accomplished using liposomes.
[0046] Lipofectin reagents are commercially available, e.g., the
LipofectAMINE.RTM. system available from Gibco/BRL. In this
methodology, positively charged and neutral lipids form liposomes
that complex with the subject negatively charged DNA vector
constructs. The DNA-liposome complexes are applied to the primary
or cultured Sertoli cells and are taken up by endocytosis. The
endosomes undergo breakage of membranes and the DNA constructs are
released within the Sertoli cells. The DNA enters the nucleus of
the Sertoli cell through nuclear pores and facilitates integration
or homologous recombination into the chromosomes of the Sertoli
cell.
[0047] After transfer of the genetic construct into Sertoli cells,
cells are selected using the appropriate antibiotic. Detection of
the expression of the heterologous gene is then performed using an
appropriate assay. The assay used to detect expression depends of
the nature of the heterologous sequence. For example, reporter
genes exemplified by chloramphenicol acetyl transferase (CAT)
.beta.-galactosidase and luciferase, are commonly used to assess
transcriptional and translational competence of chimeric
constructions. Standard assays are available to sensitively detect
the reporter enzyme in a transgenic organism. For example, a CAT
assay may be employed which detects mono and diacetylated
chloramphenicol derivatives by thin layer chromatography. The
chloramphenicol derivatives may be quantitated by phosphor
imaging.
[0048] Sertoli cells transformed with vectors comprising the
reporter gene, .beta.-galactosidase, can be identified by detecting
the cleaved galactosidase moiety by light emission. This method is
best performed using a luminometer or scintillation counter.
[0049] The introduction of Sertoli cells genetically altered to
produce a desired biological factor or intermediate into a subject
such as a mammal is accomplished by conventional techniques. In a
preferred embodiment, introduction is by subcutaneous
transplantation. Examples of preferred locations for
transplantation include the renal subcapsular space, liver
subcapsular space, omental pouch, subcutaneous facie, the brain and
the hepatic portal vein. Immunoprivileged environments may be
created at the site of transplantation using either allogeneic or
xenopeneic Sertoli cells.
[0050] In accordance with the present invention, an exogenous
biological factor may be administered following the transplantation
of the genetically altered Sertoli cells until the transplanted
Sertoli cells produce a therapeutically effective amount of the
biological factor. For example, in the treatment of diabetes,
insulin may be administered following the transplantation of
insulin-producing Sertoli cells until the transplanted Sertoli
cells produce a therapeutically effective amount of insulin.
[0051] The Sertoli cells which produce the biological factor or
intermediate can be transplanted using any technique capable of
introducing the cells into a subject such as parenteral
administration or subcutaneous administration following surgical
exposure to a desired site. Prior to transplantation, the recipient
mammal is anesthetized using local or general anesthesia according
to conventional techniques. For example, after the mammal is
anesthetized, the Sertoli cells can be injected into a tissue mass,
thereby creating an immunoprivileged site. In a preferred
embodiment, the subject to be treated is a mammal. In an even more
preferred embodiment, the mammal is a human.
[0052] In accordance with the present invention, introduction of
Sertoli cells genetically altered to produce a biological factor or
intermediate also results in the creation of an immunologically
privileged site in the treated subject. An immunologically
privileged site as defined by the present invention is a site in
the subject where the immune response produced in response to the
transplanted cells is suppressed due to immuno-suppressive agents
produced by the transplanted Sertoli cells.
[0053] As used herein, the term "allogeneic" refers to tissues or
cells of two genetically dissimilar subjects of the same species.
The term "xenogeneic" refers to tissues or cells of two genetically
dissimilar subjects of different species. The term "allograft" as
used in the present invention describes the transfer of tissues or
cells between two genetically dissimilar subjects of the same
species. The term "xenograft" in the present invention describes
the transfer of tissues or cells between two subjects of different
species.
[0054] Sertoli cells are introduced in an amount effective to
provide an immunologically privileged site. Such an effective
amount is defined as that which prevents immune rejection of the
transplanted, genetically altered Sertoli cells. Immune rejection
can be determined for example histologically, or by functional
assessment of the biological factor produced by the cells.
[0055] The genetically altered Sertoli cells producing the
biological factor are introduced in a therapeutically effective
amount. By "therapeutically effective amount" is meant an amount
effective to produce the desired effect. For example, in the case
of a peptide which stimulates the appetite, a therapeutically
effective amount is an amount which stimulates the appetite. In the
case of a peptide which depresses the appetite, a therapeutically
effective amount is an amount which depresses the appetite. In the
case of a particular disease, "a therapeutically effective amount"
is that amount effective to treat the disease. For purposes of the
present invention, the terms "treat" or "treatment" include
preventing, inhibiting, reducing the occurrence of and/or
ameliorating the physiological effects of the disease condition
treated.
[0056] The ordinary skilled artisan can determine the appropriate
amount of cells producing the biological factor or intermediate by
methods known in the art. The amount of Sertoli cells is dependent
upon the amount of factor being produced by the cells and the known
therapeutically effective amount of the factor necessary to produce
the desired effect or treat the disease. The precise
therapeutically effective amount of genetically altered Sertoli
cells can be determined by a physician with consideration of
individual differences in age, weight, the particular disease to be
treated, stage of the disease, and condition of the patient. It can
generally be stated that a therapeutic composition comprising the
subject genetically altered Sertoli cells should be preferably
administered in an amount of at least about 1.times.10.sup.1 to
about 1.times.10.sup.10 cells per dose.
[0057] After transplantation of the genetically altered Sertoli
cells producing a biological factor or intermediate, an
immunosuppressive agent may be administered for such a time during
which the Sertoli cells become established and create the
immunoprivileged site. Such immunosuppressive agents include for
example, cyclosporine, tacrolimus, despergualin and monoclonal
antibodies to, e.g., T cells. In a preferred embodiment the
immunosuppressive agent is cyclosporine. In another preferred
embodiment cyclosporine is administered at a dosage of from 0.5 mg
to 200 mg/kg body weight. In a most preferred embodiment
cyclosporine is administered at a dosage of from 5 mg to 40 mg/kg
body weight.
[0058] More generally, the immunosuppressive agent can be
administered for a time sufficient to permit the transplanted
Sertoli cells become functional. This period extends from the point
prior to or immediately following the transplantation of the
Sertoli cells to the point at which the cells are capable of
producing therapeutically effective amounts of the biological
factor or intermediate. In a preferred embodiment, the sufficient
period of time to administer an immunosuppressive agent is about 40
to about 100 days following transplantation of the Sertoli cells.
In a more preferred embodiment, the sufficient period of time is
about 50-60 days.
[0059] One embodiment of this invention is directed to a method of
treating Type I and Type II diabetes mellitus by transplanting into
the renal subcapsular space, Sertoli cells which have been
genetically altered to produce insulin. In another embodiment of
the invention, there is provided a method of treating hemophilia B
by transplanting into the renal subcapsular space, Sertoli cells
which have been genetically altered to produce Factor IX. In still
another embodiment of the invention, there is provided a method of
treating Crigler-Najjar (CN) disease by transplanting into the
renal subcapsular space, Sertoli cells which have been genetically
altered to produce bilirubin UDP-glucuronosyltransferase
(B-UGT).
[0060] Another aspect of the present invention provides a
pharmaceutical composition comprising Sertoli cells genetically
altered to produce a biological factor or intermediate and a
pharmaceutically acceptable carrier. In a preferred embodiment, the
Sertoli cells are rat, murine, porcine, bovine or human Sertoli
cells. As used herein, a pharmaceutically acceptable carrier
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic agents and the like.
The use of such media and agents is well-known in the art.
[0061] The present invention is also directed to a kit for
treatment of a disease. In one embodiment, the kit is
compartmentalized to receive a first container adapted to contain
Sertoli cells genetically altered to produce a biological factor or
intermediate thereof that is absent, produced at low levels, or
defective in the individual to be treated in an amount effective to
produce the desired effect or treat the disease. In a preferred
embodiment, the Sertoli cells are rat, murine, bovine, porcine or
human and are provided in an amount of from 10.sup.1 to 10.sup.10
cells. In a more preferred embodiment, Sertoli cells are provided
in an amount of from 10.sup.5 to 10.sup.10 cells. In another
preferred embodiment the Sertoli cells that produce a biological
factor or intermediate are cells that have been transformed with
DNA encoding the factor or intermediate.
[0062] The invention is further illustrated by the following
specific examples which are not intended in any way to limit the
scope of the invention.
EXAMPLE 1
[0063] Sertoli cells are isolated from Lewis rats and cultured for
two to seven days using standard techniques (Selawry, H. P. and
Cameron, D. F., 1993 Cell Transplantation 2:123-129; Korbutt, G.
S., et. al., 1997 Diabetes 46:317-322). For example, testicles from
15 to 21-day-old Lewis rates are collected in Hanks' balanced salt
solution (HBSS), chopped into 1-mm pieces with scissors, and
digested for 10 minutes at 37 C with collagenase (2.5 mg/ml; Sigma
Type V, St. Louis, Mo.) in HBSS. The digest is washed three times
with calcium- and magnesium-free HBSS containing 1 mmol/EDTA and
0.5% bovine serum albumin (Sigma) (HBSS/EDTA), digested for 10
minutes at 37.degree. C. with trypsin (25 .mu.g; Boehringer) in
HBSS/EDTA, and washed four times in HBSS; the final cell pellet is
resuspended in HAM's F10 media supplemented with 10 mmol/l
D-glucose, 2 mmol/l L-glutamine, 50 .mu.mol/l
isobutylmethylxanthine, 0.5% bovine serum albumin, 10 mmol/l
nicotinamide, 100 U/ml penicillin, 100 ng/ml streptomycin, and 5%
Lewis rat serum (not heat inactivated). The tissue is passed
through a 500-.mu.m mesh, placed in nontissue culture-coated Petri
dishes and incubated for 48 hours at 37.degree. C. (5% CO.sub.2)
before transplation.
[0064] An expression vector containing the CMV promoter and the
gene for .beta.-galactosidase is used to transfect the Sertoli
cells. For example, pCMVbeta (available from Clontech, Palo Alto,
Calif.) contains the human cytomegalovirus early gene promoter, an
intron (splice donor/splice acceptor) and polyadenylation signal
from SV40 along with the full-length E. coli beta-galactosidase
gene with eukaryotic translation signals. Alternatively, an
expression vector constituting the chloramphenicol
acetyltransferase gene (CAT) under control of the SV40 promoter
(pCAT3, available from Promega, Madison, Wis.) is used to transfect
the Sertoli cells.
[0065] After about two to seven days in culture, the Sertoli cells
are transfected using the lipofectin method of Gibco/BRL
(Rockville, Md.) and following the instructions provided by the
manufacturer. The calcium phosphate method coupled with
hyperosmotic shock (10% glycerol) as described in Whaley et al.
(1995) Endocrinology 136:3046-3053, may also be used.
[0066] Following transfection, in order to select those Sertoli
cells transformed by an expression vector, an appropriate assay is
employed. For example, Sertoli cells transfected with the CAT gene
undergo a CAT assay which detects mono and diacetylated
chloramphenicol derivatives such as the assay described in Gorman
et al., (1982) Mol. Cell. Biol. 2(9):1044-1051. The chloramphenicol
derivatives may be quantitated by phosphor imaging.
[0067] Sertoli cells transfected with the lacZ gene are identified
by detecting the cleaved galactosidase moiety using light emission.
A luminometer or scintillation counter is best suited for this
purpose. Transfected Sertoli cells are plated at 5.times.10.sup.5
cells per plate. Cells are plated in triplicate and expression is
assayed at 0, 2, 4, 8, 12, 18, 28 and 42 days. If expression is
lower than expected, the expression vector may be altered by
incorporating the pCAT enhancer.
[0068] After selection of cells with stable integration of the
genetic construct, the selected cells are allowed to expand in
culture (about 15-21 days) After expansion, the cells are collected
for transplantation.
EXAMPLE 2
[0069] The collected Sertoli cells from Example 1 are then pooled
and transplanted in a normal rat under the kidney (renal)
sub-capsular space. The transplantation protocol used to transplant
the stably transfected cells is the same as that used for
transplantation of islet cells and Sertoli cells described in
Rajotte et al. (1997) Diabetes 46:317-322. For example, each
recipient receives between about 5.5.+-.0.3 to
11.+-.0.4.times.10.sup.6 transgenic Sertoli cells. Cells are
aspirated into polyethylene tubing such as PE-50, pelleted by
centrifugation, and gently placed under the left renal subcapsular
space of anesthetized (e.g., halothane-anesthetized) animals. The
number of transgenic Sertoli cells grafted in each animal is
assessed by measuring the DNA content of triplicate representative
aliquots of each cell preparation before transplantation, and
calculations are based on the finding that freshly isolated
testicular cells contain about 20 pg DNA per cell. Therefore, the
total DNA content transplanted per 20 pg DNA per testicular cells
equals the testicular cell number.
[0070] A predetermined number of rats undergo transplantation with
the genetically altered Sertoli cells. Both isografts (Lewis rat
transformed Sertoli cells transplanted into Lewis rats) and
allogafts (Lewis rat transformed Sertoli cells transplanted into
Wistar-Furth rats) are performed. As controls, Lewis rats receive
both isografts and allografts of untransformed Sertoli cells
transplanted under the kidney capsule. Also as a control, islets
isolated from Lewis rat pancreatic tissue is transplanted into
Wistar-Furth recipients.
[0071] A portion of the rats with transplanted Sertoli cells are
sacrificed for histological examination at various time points
post-transplantation. The time points post-transplantation are
approximately 5, 10, 15, 20, 30, 50 and 90 days. The histological
examination is performed in triplicate at each time point.
[0072] Kidney tissue containing the different grafts is examined
histologically using immunohistochemical staining e.g. for
clusterin, to ensure that the Sertoli cells are still viable, i.e.,
have not been rejected, and that .beta.-galactosidase continues to
be produced.
EXAMPLE 3
[0073] Lewis rat Sertoli cells are transfected with a retroviral
vector constituting the alkaline phosphatase gene. The
RetroExpress.RTM. system, available from Clontech is especially
useful. After selection and expansion as described in Example 2,
transfected Sertoli cells are transplanted into Wistar-Furth rats
as described in Example 2.
EXAMPLE 4
[0074] Porcine Sertoli cells are isolated similarly to the methods
described in Korbutt et al. (1997) Diabetes 46:317-322 and Selawry
(1993) Cell Transplantation 2:123-129. Testes are removed from
recently expired pigs with scissors and forceps. The testes are
placed in a beaker containing approximately 100 ml Hank's Balanced
Salt Solution plus CaCl.sub.2 (HBSS, Life Technologies),
supplemented with an antibiotic such as gentamycin.
[0075] A piece of testicular tissue is removed from the testes. The
tunica albugeia is removed from the tissue leaving a smaller
aliquot of about 1.45 to 1.63 g. The parenchyma is minced with
scissors and between about 1.45 and 1.63 g of testis is placed in a
50 ml centrifuge tube and the sample is washed by centrifugation
twice with Hank's+CaCl.sub.2. The total volume for each wash is
between 35 to 40 ml and the sample is centrifuged at 1000 rpm for
about three minutes.
[0076] 30 ml of Hank's plus CaCl2 is added to the pellet followed
by 33 mg of collagenase. The sample and media are transferred to a
flask, capped with sterile gauze and foil, and placed in a
shaker/water bath at 37.degree. C. for 15 minutes (shaker setting
approximately 6.5). Cells are transferred to a 50 ml centrifuge
tube and washed two times with Hank's plus CaCl.sub.2. To the cells
are added 400 .mu.l trypsin(stock solution of 2 mg/ml), 200 .mu.l
DNase (stock solution of 1 mg/ml), 40 ml of Hank's (WITHOUT Calcium
or Magnesium, Life Technologies). The cells and media are
transferred to a flask and placed in a shaking water bath for ten
minutes at 37 C. Cells and media are then transferred to a 50 ml
centrifuge tube and washed three times with Hank's plus CaCl2.
After the final wash, 20 ml of Media 199 (Sigma BioSciences, St.
Louis, Mo.) plus 5% Horse Serum plus antibiotics [herein referred
to as "Media 199"] is added to the tube, the pellet resuspended and
the suspension poured through a 500 micron cell collector into a
beaker. An additional 10 ml of Media 199 is poured over the cells.
Passing of the cells is facilitated by gently pushing with the
plunger of a 10 ml syringe. An additional 10 ml of Media 199 is
poured over the cells and the process repeated. The cells are
diluted further with another 10 ml of Media 199 (total volume 50
ml). At this point, a sample may be placed under a phase/contrast
microscope to confirm the predominance and integrity of Sertoli
cells in the preparation.
[0077] In order to estimate cell number based on correlation to DNA
content (see Example 2), 2 ml of cell suspension is removed to a
small plastic centrifuge tube, centrifuged at 2000 rpm for ten
minutes, the media removed, and the resulting pellet, resuspended
in the media remaining in the tube. The sample may be placed in a
-50.degree. C. freezer prior to DNA analysis.
[0078] To either non-stick Falcon 1001 plates or stick plates
(Falcon 3003) is added 3 ml cell suspension and 12 ml Media 199. In
addition, 0.2 ml of antibiotic/antimycotic was added to each plate.
Plates are placed in an incubator at 37 C and 2% C0.sub.2. Cells
are cultured for two to seven days.
[0079] Alternatively, the above-described method is altered as
follows. After the step of adding 30 ml of Hank's+CaCl.sub.2 to the
minced testes parenchyma, instead of adding 33 mg of collagenase,
30 mg of trypsin (from 2 mg/ml stock) and 0.6 mg of DNase (from 1
mg/ml stock) is added and the suspension is transferred to a flask
such as an Erlenmeier flask. The flask is placed in a shaking water
bath for 30 minutes at 37.degree. C. Following this step, the
suspension is transferred to a 50 ml centrifuge tube and the cells
are washed three times with Hank's+CaCl.sub.2. Cells are
resuspended in 40 ml of 1M glycine+2 mM EDTA containing 0.01% soy
trypsin inhibitor+0.8 ml DNase. The suspension is incubated for ten
minutes at room temperature. Following the incubation step, cells
are washed three times by centrifugation with Hank's+CaCl.sub.2.
After the last centrifugation, cells are suspended in 40 ml of
Hank's+CaCl.sub.2+0.2 mg of DNase (1 mg/ml stock) and 40 mg
collagenase. The suspension is transferred to a flask such as an
Erlenmeir flask and shaken in a 37.degree. C. waterbath for ten
minutes. After transferring cells to a 50 ml centrifuge tube, cells
are washed three times with Hank's. Thirty ml of Media 199 is added
to the pellet and the cells are resuspended and poured through a
500 micron cell collector into a beaker. Passing of the cells is
facilitated by gently pushing with the plunger of a 10 ml syringe.
An additional 20 ml of Media 199 is poured over the cells and the
process repeated. Cells are resuspended in a final volume of 50 ml
and may be examined under a phase/contrast microscope for the
presence and integrity of Sertoli cells.
[0080] For DNA content analysis, (see Example 2), 2 ml of cell
suspension is removed to a small plastic centrifuge tube,
centrifuged at 2000 rpm for ten minutes, the media removed and the
resulting pellet, resuspended in the media remaining in the tube.
The cells are stored in a -50.degree. C. freezer.
[0081] Non-stick plates receive 5 ml of cells plus an additional 10
ml of Media 199. Stick plates receive 3 ml of cells and 12 ml of
Media 199. All plates receive 0.2 ml of antibiotic/antimycotic and
plates are incubated at 37 C and 2% CO.sub.2.
[0082] The porcine Sertoli cells are transfected via the Lipofectin
method with the expression vectors as detailed in Example 1 or via
the retroviral system described in Example 3. The transfected
porcine Sertoli cells are transplanted to the renal subcapsular
space of Wistar-Furth rats as described in Example 2. The grafts
are assayed as described in Example 2.
EXAMPLE 5
[0083] Mouse, rat, and porcine Sertoli cells are genetically
altered to express the human Factor IX gene. The constructs used to
transfect the Sertoli cells contain the human Factor IX gene under
the control of a promoter such as the CMV promoter, rat, mouse or
porcine transferrin gene promoter, human transferrin gene promoter,
rat, mouse or porcine FSHR gene promoter, and human FSHR gene
promoter.
[0084] An expression vector comprising a promoter which functions
in rat Sertoli cells is operably linked to the cDNA for human
factor IX cDNA. Human factor IX cDNA is described in Palmer et al.,
1989 Blood 73:438-445. Initially, the CMV promoter is employed.
Transfected rat Sertoli cells are used for transplantation into
normal rats (controls) and in factor IX deficient rats. A factor
IX-deficient mouse model for hemophilia B gene therapy has been
developed where the clotting factor IX (FIX) gene has been
disrupted by homologous recombination. Wang, L. et al. 1997 Proc.
Natl. Acad. Sci. U.S.A. 94(21):11563-11566. The same experiment is
repeated using pig Sertoli cells rather than rat Sertoli cells
followed by transplantation into rats. In a third experiment,
Sertoli cell-specific promoters such as those of the rat
transferrin, clusterin, or PSH receptor genes operably linked to
the cDNA for human factor IX are used to transfect rat Sertoli
cells followed by transplantation into rats. In a fourth
experiment, Sertoli cell-specific promoters such as those of the
rat transferrin, clusterin, or FSH receptor genes operably linked
to the cDNA for human factor IX are used to transfect porcine
Sertoli cells followed by transplantation into rats.
[0085] After transplantation, the graft is examined at time
intervals of 5, 10, 15, 20, 30, 50, and 90 days and assayed for
factor IX production by immunohistochemistry as described in Wang,
L., 1997 supra.
EXAMPLE 6
[0086] Gunn rats are incapable of bilirubin conjugation due to a
genetic defect in UDP-glucuronyl transferase. As a result, Gunn
rats may be used as a model of congenital enzymatic deficiency to
test the metabolic activity of transplanted cells. Seppen J., et
al., 1997 Hum. Gene. Ther. 8(1):27-36.
[0087] In this experiment, mouse, rat and porcine Sertoli cells are
transfected with a gene construct having the bilirubin
UDP-glucuronosyltransferase (B-UGT) gene (Seppen, J. 1997 supra)
under the control of a promoter which functions in rats such as the
rat transferrin, clusterin or FSH receptor genes and transplanted
into Gunn rats.
[0088] After Sertoli cells expressing B-UGT are found to
glucuronidate bilirubin present in cell culture media, the cells
are transplanted under the kidney capsule and/or liver capsule of
Gunn rats. Animals exhibiting a B-UGT deficiency corrected by the
Sertoli cell transplantation are detected by the presence of normal
serum bilirubin concentrations and bilirubin glucuronides in the
bile.
* * * * *