U.S. patent application number 11/880600 was filed with the patent office on 2007-11-15 for pigment epithelial cell of the eye, its production and use in therapy of an eye or cns disease.
Invention is credited to Stefan Kochanek, Ulrich Schraermeyer, Gabriele Thumann.
Application Number | 20070264244 11/880600 |
Document ID | / |
Family ID | 7675030 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264244 |
Kind Code |
A1 |
Kochanek; Stefan ; et
al. |
November 15, 2007 |
Pigment epithelial cell of the eye, its production and use in
therapy of an eye or CNS disease
Abstract
The present invention relates to a pigment epithelial cell of
the eye containing vector DNA of an adenoviral vector with large
DNA capacity, to the improved isolation and cultivation of these
cells and to methods for production and the use in the therapy of
an eye or nerve disease.
Inventors: |
Kochanek; Stefan; (Koeln,
DE) ; Schraermeyer; Ulrich; (Neuss, DE) ;
Thumann; Gabriele; (Koeln, DE) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
7675030 |
Appl. No.: |
11/880600 |
Filed: |
July 23, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10079609 |
Feb 21, 2002 |
7247479 |
|
|
11880600 |
Jul 23, 2007 |
|
|
|
60270746 |
Feb 22, 2001 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
435/371; 435/466 |
Current CPC
Class: |
A61P 43/00 20180101;
C12N 15/86 20130101; C12N 2503/02 20130101; A61K 35/30 20130101;
A61K 48/00 20130101; A61K 38/18 20130101; C12N 2710/10343 20130101;
A61K 38/57 20130101; C12N 5/0621 20130101; Y02A 50/30 20180101;
Y02A 50/473 20180101; C12N 2510/00 20130101; C12N 2830/008
20130101 |
Class at
Publication: |
424/093.21 ;
435/371; 435/466 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 43/00 20060101 A61P043/00; C12N 15/87 20060101
C12N015/87; C12N 5/06 20060101 C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 2001 |
DE |
10108412.9-41 |
Claims
1. A pigment epithelial cell of the eye, which comprises vector DNA
of an adenoviral vector with large DNA capacity comprising at least
one expressed nucleic acid operatively linked to a promoter.
2. The pigment epithelial cell of the eye as claimed in claim 1,
which is a retinal pigment epithelial cell or an iris pigment
epithelial cell.
3. The pigment epithelial cell of the eye as claimed in claim 1,
where the vector DNA comprises at least one therapeutic nucleic
acid, in particular a therapeutic gene, preferably for a
neurotrophic factor such as GDNF, PEDF, NGF, BDNF, CNTF, bFGF or
neurotrophin 3,4-5, an antiangiogenetic factor such as a soluble
VEGF receptor-1 (sflt-1), a dominant-negative VEGF receptor-2 (KDR)
or PEDF, an antioxidative factor such as superoxide dismutase,
catalase or various peroxydases, a lysosomal factor such as
alpha-mannosidase, beta-galactosidase,
N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, and
lipase, or a vasodilating factor such as NO synthase.
4. The pigment epithelial cell of the eye as claimed in claim 1,
where the vector DNA comprises any one or more of a constitutively
active, a regulatable or a tissue-specific promoter, or a
regulatable expression system.
5. The pigment epithelial cell of the eye as claimed in claim 1,
where the cell produces at least one therapeutic protein.
6. The pigment epithelial cell as claimed in claim 1, where the
cell is in a fixed assemblage of cells.
7. A pigment epithelial cell of the eye in the form of a fixed
assemblage of cells.
8. A cultivation system comprising at least one pigment epithelial
cell of the eye and a feeder layer.
9. A method for producing a pigment epithelial cell of the eye as
claimed in claim 1, which comprises genetically modifying the cell
with the aid of an adenoviral vector with large DNA capacity.
10. A method for producing a pigment epithelial cell of the eye as
claimed in claim 1, which comprises cultivating the cell in
serum-free medium or in the presence of a feeder layer.
11. A method for producing pigment epithelial cells of the eye in
the form of a fixed assemblage of cells as claimed in claim 7,
which comprises separating the assemblage of cells, in particular
enzymatically, from surrounding tissue.
12. A method for producing pigment epithelial cells, which
comprises cultivating the cells in a cultivation system as claimed
in claim 8.
13. A method of treating an eye disease, in particular of AMD, a
glaucoma, diabetic retinopathy or a genetic disease of the pigment
epithelium, which comprises using a pigment epithelial cell as
claimed in claim 1.
14. A method of treating an eye disease, in particular of AMD, a
glaucoma, diabetic retinopathy or a genetic disease of the pigment
epithelium, which comprises using a pigment epithelial cell as
claimed in claim 7.
15. A method of treating as claimed in claim 13 or 14, where the
pigment epithelial cell is transplanted into the eye, in particular
the choroid, into the papilla and/or into the vitreous.
16. A method of treating a nerve disease, in particular a disease
of the nervous system, preferably of the CNS, especially of
Parkinson's disease, which comprises using a pigment epithelial
cell.
17. The method of treating as claimed in claim 16, wherein the
pigment epithelial cell is a pigment epithelial cell as claimed in
claim 1.
18. The method of treating as claimed in claim 16, wherein the
pigment epithelial cell is transplanted into the nervous system, in
particular the CNS.
19. The method of treating as claimed in claim 13 or 14, wherein
the pigment epithelial cell is an autologous pigment epithelial
cell.
20. The method of treating as claimed in claim 16, wherein the
pigment epithelial cell is an autologous pigment epithelial
cell.
21. A medicament or diagnostic aid comprising a pigment epithelial
cell of the eye as claimed in claim 1 and other excipients or
additives.
22. The pigment epithelial cell of the eye as claimed in claim 1,
where the cell produces at least one therapeutic RNA.
23. The pigment epithelial cell as claimed in claim 1, where the
cell has been cultivated in the presence of a feeder layer.
24. The pigment epithelial cell as claimed in claim 1, where the
cell has been cultivated in serum-free medium.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Utility
application Ser. No. 10/079,609, filed Feb. 21, 2002, which claims
benefit of U.S. Provisional Application Ser. No. 60/270,746, filed
Feb. 22, 2001, and foreign patent application DE 10108412.9-41,
filed Feb. 21, 2001 in Germany.
[0002] The present invention relates to a pigment epithelial cell
of the eye comprising vector DNA of an adenoviral vector with large
DNA capacity, to the improved isolation and cultivation of these
cells and to methods for the production and the use in therapy of
an eye or nerve disease.
[0003] The five primary senses of touch, sight, hearing, taste and
smell serve to pick up information from the surroundings. About 75%
of our perceptions involve the sense of sight. This high percentage
makes it clear that sight is of predominant importance in our daily
life. Consequently, a weakening of our vision represents a great
intrusion into everyday life.
[0004] The eye consists of a compound lens system which produces an
image, which is inverted and reduced in size, of the surroundings
on the retina. The dioptic apparatus consists of the transparent
cornea, the iris forming the pupil, the lens and the vitreous body,
a gelatinous, transparent mass inside the eyeball between lens and
retina. FIG. 1 shows a schematic horizontal section through the
eye. The covering of the eyeball consists of 3 layers: the sclera,
the choroid and the retina. The retina in turn consists of an outer
layer, the retinal pigment epithelium (RPE), and an inner layer,
the neurosensory retina. collagen-rich extracellular matrix, the
iris stroma (it contains melanocytes, fibrocytes, nerves and blood
vessels) and the iris pigment epithelium (IPE).
[0005] The iris pigment epithelium is in two layers and consists of
an anterior and a posterior pigment epithelial cell layer (Freddo T
F (1996) Ultrastructure of the iris. Microsc Res Tech 33: 369-389).
The cells of the posterior iris pigment epithelium are connected by
tight junctions. The anterior pigment epithelium has in addition
smooth muscle cells (except in the region of the sphincter) which
contribute to dilatation of the iris (Freddo 1996, supra). The iris
pigment epithelium has the same embryological origin as the retinal
pigment epithelium. It is possible to obtain about
2.3.times.10.sup.5 IPE cells from a human iris, 90% of which
survive in cell culture (Hu D N, Ritch R, McCormick S A,
Pelton-Henrion K (1992) Isolation and cultivation of human iris
pigment epithelium. Invest Ophthalmol Vis Sci 33: 2443-2453). IPE
cells are highly pigmented and contain much eumelanin. Melanin has
the following protective functions. It is able to bind divalent
iron ions (Fe.sup.2+) and other toxic substances (e.g. Ca.sup.2+)
and thus remove them from the cytoplasm of the cell (Hill H Z
(1992) The function of melanin or six blind people examine an
elephant. Bioessays 14: 49-56). Melanin is additionally able to
convert Fe2+ into less toxic Fe.sup.3+ by redox reactions. On the
other hand, the melanin synthesis precursors dihydroxyindole (DHI)
and dihydroxyindolecarboxylic acid (DHIA) have a very strong
antioxidant effect which is stronger than that of alpha-tocopherol
(Memoli S, Napolitano A, d'Ischia M, Misuraca G, Palumbo A, Prota G
(1997) Diffusible melanin-related metabolites are potent inhibitors
of lipid peroxidation. Biochim Biophys Acta 1346: 61-68). Melanin
is able to eliminate toxic oxygen free radicals produced in the eye
by the high partial pressure of oxygen in combination with exposure
to light. Elements important for the normal function of the retina,
such as, for example, zinc, are moreover stored by melanin with
great efficiency. Zinc, as a cofactor for, for example,
antioxidative enzymes (superoxide dismutase) or connective
tissue-degrading enzymes (metalloproteinases), has several
important functions in the eye and in the central nervous system
(CNS).
[0006] The pigment epithelium plays an import part in metabolism
and in absorption of light in the eye. It is additionally
responsible for the outer blood-retina barrier and for disposing of
rejected photoreceptor cells. Consequently it forms an interesting
target for the gene therapeutic treatment of eye diseases.
[0007] To date, a few experiments on the genetic modification of
pigment epithelial cells have been described, but these provided
unsatisfactory results in terms of the duration and stability of
expression.
[0008] In a study with laboratory mice, a first generation
adenoviral vector which expressed the E. coli lacZ gene under the
control of the CMV promoter was used for gene transfer into the
retinal pigment epithelium by subretinal injection. First
generation adenoviral vectors (Gilardi et al., FEBS Letters 267,
60-62, 1990; Stratford-Perricaudet et al., Hum. Gene Ther. 1,
241-256, 1990) are characterized by deletions of the E1A and E1 B
genes. E1A and E1B have transforming and transactivating
properties. In some vectors there is also deletion of E3 in order
to increase the capacity to take up foreign DNA. Although the gene
transfer into the retinal pigment epithelium was efficient and very
good expression was observed shortly after injection in the retinal
pigment epithelium, the expression was transient. 6 weeks after the
injection, only a few lacZ-positive retinal pigment epithelial
cells were still observable (Li T, Adamian M, Roof D J, Berson E L,
Dryia T P, Roessler B J, Davidson B L (1994) In vivo transfer of a
reporter gene to the retina mediated by an adenoviral vector.
Invest Ophthalmol Vis Sci: 35, 2543-2549).
[0009] A further study carried out on laboratory rats with an
observation period of 14 days used a first generation adenovirus
which expressed the E.coli lacZ gene under the control of the Rous
Sarcoma Virus (RSV) promoter. Although the gene transfer into the
retinal pigment epithelium was efficient, and very good expression
was observed 7 days after the injection in the retinal pigment
epithelium, the expression was reduced one week later (Rakoczy P E,
Lai C M, Shen W Y, Daw N, Constable I J (1998) Recombinant
adenovirus-mediated gene delivery into the rat retinal pigment
epithelium in vivo. Australian and New Zealand Journal of
Ophthalmology 26 (Suppl.): S56-S58).
[0010] Another study carried out on 6-week-old RCS rats used a
first generation adenoviral vector which expressed the green
fluorescence protein (GFP) gene under the control of the CMV
promoter (Anglade E, Csaky K G (1998) Recombinant
adenovirus-mediated gene transfer into the adult rat retina. Curr
Eye Res 17: 316-321). Although the gene transfer into the retinal
pigment epithelium after subretinal injection was efficient, and 30
to 90% of the retinal pigment epithelium were GFP-positive in the
region of the injection site 3 days after the injection, GFP
expression was no longer detectable 6 days later (that is 9 days
after the injection).
[0011] Whereas first generation adenoviral vectors were used in the
abovementioned examples of gene transfer in the region of the eye,
in a further publication there was use of an adenoviral vector
which is referred to as adenovirus minichromosome (EAM) for
subretinal injection of mice (Kumar-Singh R, Farber D B (1998)
Encapsidated adenovirus mini-chromosome-mediated delivery of genes
to the retina: application to the rescue of photoreceptor
degeneration. Hum Mol Genet 7: 1893-1900). This comprises a vector
which does not express any viral proteins. The vector expressed the
beta unit of cyclic GMP phosphodiesterase (PDE) under the control
of the natural PDE promoter. The vector also expressed the E.coli
lacZ gene under the control of the CMV promoter. In addition, the
vector contained various E.coli plasmid elements (Plasmid backbone,
ampicillin resistance gene, E.coli origin of replication). After
production, the vector was characterized by pronounced variability
of its genome. Monomeric and dimeric structures were observed, the
latter in head-to-head, head-to-tail and tail-to-tail orientation.
Because of this variability and the presence of plasmid sequences
including antibiotic resistance, this vector is unsuitable for
therapeutic use. The gene transfer experiments were carried out on
rd mice which represent an animal model of retinal degeneration and
are characterized by a mutation, which causes the degeneration, in
the beta unit of the PDE gene. In this study, expression was
detected exclusively in the neuronal portion of the retina but not
in the retinal pigment epithelium. Although the neuronal cells are
post-mitotic and thus no longer able to divide, expression of the
PDE gene was only transient. Various methods (RT-PCR, Western blot
analysis and determination of the PDE activity) were used to
demonstrate that expression was no longer detectable 4 months after
the injection.
[0012] To date, only the nonviral transfection reagent
Lipofectamine has been used for transfecting IPE cells. In this
study, the plasmid pXCN2-bFGF which expresses the rat bFGF cDNA was
produced. The plasmid additionally contains a neomycin resistance
gene. Cultivated rat IPE cells were transfected with this plasmid.
The cells expressed the bFGF cDNA in vitro, and the authors write
that degeneration of photoreceptors was delayed by up to 4 weeks in
the RCS rat after subretinal transplantation as cell suspension
(Tamai M, Yamada K, Takeda N, Tomita H, Abe T, Kojima S, Ishiguro I
(1997) bFGF transfected iris pigment epithelial cells rescue
photoreceptor cell degeneration in RCS rats. In: La Vail M, eds.
Degenerative retinal diseases. 323-328). However, since, as shown
in the work mentioned, the same effect, namely delayed degeneration
of photoreceptors, was observed also in rats which had received IPE
cells after transfection with a control plasmid by subretinal
injection, and was not improved or extended by the transfection,
this effect was not one which could be attributed to a targeted
gene transfer but was explicable solely by the transplantation of
the IPE cells. In addition, bFGF expression after transplantation
was not demonstrated.
[0013] The publications mentioned therefore do not disclose an
expression system for pigment epithelial cells of the eye with
which long-term stable expression of an introduced gene can be
observed. Long-term stable expression of such a gene is, however,
necessary for the therapy of a large number of hereditary and
acquired eye diseases. For many applications it is precisely the
long-term production of therapeutic proteins which is the crucial
factor for achieving a therapeutic effect.
[0014] It is therefore an object of the present invention to
provide pigment epithelial cells of the eye which can be employed
in therapy.
[0015] The object is achieved by providing a pigment epithelial
cell of the eye.
[0016] It has now been found, surprisingly, that a pigment
epithelial cell of the eye which comprises vector DNA of an
adenoviral vector with large DNA capacity shows long-term stable
expression of at least one introduced gene and thus can be employed
in therapy.
[0017] One aspect of the present invention is a pigment epithelial
cell of the eye which comprises a vector DNA of an adenoviral
vector with large DNA capacity.
[0018] A pigment epithelial cell of the eye means an epithelial
cell of the eye in which pigment, for example melanin, is
incorporated. An example of a pigment epithelial cell of the eye is
a retinal pigment epithelial cell (RPE) or an iris pigment
epithelial cell (IPE).
[0019] An adenoviral vector of large DNA capacity is understood by
the skilled worker to be adenoviruses which comprise no viral
coding DNA sequences (Kochanek S, Clemens P R, Mitani K, Chen H H,
Chan S, Caskey C T (1996) A new adenoviral vector: Replacement of
all viral coding sequences with 28 kb of DNA independently
expressing both full-length dystrophin and beta-galactosidase. Proc
Natl Acad Sci U.S.A. 93: 5731-5736; Fisher K J, Choi H, Burda J,
Chen S J, Wilson J M. (1996); Recombinant adenovirus deleted of all
viral genes for gene therapy of cystic fibrosis. Virology 217:
11-22; Kumar-Singh R, Chamberlain J S (1996) Encapsidated
adenovirus minichromosomes allow delivery and expression of a 14 kb
dystrophin cDNA to muscle cells. Hum Mol Genet 5: 913-921). These
adenoviruses contain only the viral ends with inclusion of the
inverted terminal repeats (ITRs) and the packaging signal. The
capacity to take up foreign DNA is, for example, up to about 37 kb,
because the predominant part of the adenoviral genome has been
deleted.
[0020] Adenoviruses are particularly important as expression
vectors, especially in the framework of gene therapy. One advantage
of adenoviral vectors is the fact that these vectors are able to
transduce replicating and nonreplicating cells efficiently in vitro
and in vivo.
[0021] Various systems for producing adenoviral vectors of large
DNA capacity have been described (Kochanek S (1999) High-capacity
adenoviral vectors for gene transfer and somatic gene therapy. Hum
Gene Ther 10: 2451-2459). The advantage of these adenoviral vectors
with large DNA capacity compared with first and second generation
adenoviral vectors is in particular the larger capacity. This makes
it possible to introduce one or more genes or expression cassettes
into the pigment epithelial cells.
[0022] After uptake of the adenoviral vector into the cell, the
coat of the vector is normally broken down in endosomes. The
remaining vector DNA is then transported into the cell nucleus and
usually does not integrate into the cellular genome.
[0023] One example of an adenoviral vector with large DNA capacity
is a vector which expresses the human alphal-antitrypsin gene
(Schiedner G, Morral N, Parks R J, Wu Y, Koopmans S C, Langston C,
Graham F L, Beaudet A L, Kochanek S (1998) Genomic DNA transfer
with a high capacity adenovirus vector results in improved in vivo
gene expression and decreased toxicity. Nature Genetics 18:
180-183). Another example is a vector which expresses the
dystrophin gene and E.coli lacZ genes (Kochanek S, Clemens P R,
Mitani K, Chen H H, Chan S, Caskey C T (1996) A new adenoviral
vector: Replacement of all viral coding sequences with 28 kb of DNA
independently expressing both full-length dystrophin and
beta-galactosidase. Proc Natl Acad Sci U.S.A. 93: 5731-5736). In a
preferred embodiment, HC-AdFK7 or HC-AdhCMV.PEDF is used as
adenoviral vector with large DNA capacity. HC-AdFK7 is an
adenoviral vector with large DNA capacity which expresses the
enhanced green fluorescence protein (EGFP) under the control of the
human cytomegalievirus promoter. HC-AdhCMV.PEDF is an adenoviral
vector with large DNA capacity which expresses the human pigment
epithalial cell-derived factor (PEDF) gene under the control of the
cytomegalovirus promoter. In this vector, the PEDF protein is
tagged by attachment (expression as fusion protein) of a
polyhistidine epitope, so that the protein can easily be detected
by use of an anti-polyhistidine antibody.
[0024] As shown in the examples, pigment epithelial cells can be
transduced very efficiently in vitro with an adenoviral vector of
large DNA capacity. As likewise shown in the examples,
transplantation of these genetically modified cells is followed by
a long-term gene expression which can be detected continuously for
at least 4 months. It is evident that the transplantation site is
not critical in this connection. After transplantation of the
genetically modified pigment epithelial cells both into the eye in
the subretinal space and, particularly surprisingly, into the CNS
in the Corpus striatum led to long-term gene expression detectable
for at least 4 months (eye) and at least 2 months (CNS).
[0025] Most experiments with these vectors have to date been
carried out in the liver and in the skeletal muscle. Although liver
gene transfer with a vector expressing human alpha 1-antitrypsin
into baboons (Morral N, O'Neal W, Rice K, Leland M, Kaplan J,
Piedra P A, Zhou H, Parks R J, Velji R, Aguilar-Cordova E,
Wadsworth S, Graham FL, Kochanek S, Carey K D, Beaudet A L (1999)
Administration of helper-dependent adenoviral vectors and
sequential delivery of different vector serotype for long-term
liver-directed gene transfer in baboons. Proc Natl Acad Sci USA 96:
12816-12821) was followed by longer-term expression (longer than
one year) in two of three animals, in contrast to the present
invention there was observed to be a continuous decrease in
expression, which was still 19% of the initial levels in one of the
animals after 16 months, and still 8% in the second animal after 24
months. In a third animal there was complete loss of expression
within 10 weeks. There was speculation about the reason for the
slow decrease in expression in the two animals with prolonged
expression. Both growth of the animals and slow cell division of
the hepatocytes were discussed. In the final analysis, the cause of
the slow loss of expression is not explained. In the animal in
which rapid loss of expression was observed, production of
antibodies directed against human alphal-antitrypsin was
observed.
[0026] A distinct decrease in expression of a LacZ reporter gene
after gene transfer into the liver with an adenoviral vector of
large DNA capacity within 30 days after the injection was observed
in a further study (Parks R J, Bramson J L, Wan Y, Addison C L,
Graham F L (1999) Effects of stuffer DNA on transgene expression
from helper-dependent adenovirus vectors. J Virol 73:
8027-8034).
[0027] Skeletal muscle is a tissue which, in terms of the natural
turnover of the cells, resembles the pigment epithelial cells of
the eye. Skeletal muscle cells are postmitotic cells. This means
that they are similar to pigment epithelial cells in no longer
dividing. There has been particular experience in gene transfer
into the skeletal muscle of laboratory animals using adenoviral
vectors of large DNA capacity. The gene transfer in these
experiments was effected by direct injection into the tissue,
similar to the present invention. Although expression over a
prolonged period was observed after gene transfer using an
adenoviral vectors of large DNA capacity which expressed both the
dystrophin cDNA and the E.coli LacZ gene, it was observed that
expression once again decreased within 84 days (Chen H H, Mack L M,
Kelly R, Ontell M, Kochanek S, Clemens P R (1997) Persistence in
muscle of an adenoviral vector that lacks all viral genes. Proc
Natl Acad Sci USA 94: 1645-1650). There was complete loss of
expression after 84 days in immunocompetent animals displaying no
tolerance to E.coli beta-galactosidase.
[0028] In contrast to the experiments described in the literature,
the stability of expression of genes introduced by gene transfer
with an adenoviral vector of large DNA capacity into the pigment
epithelial cell was surprisingly high. The advantages of adenoviral
vectors of large DNA capacity in the transfection according to the
invention of pigment epithelial cells of the eye compared with
known transfection systems are accordingly [0029] the ensuring of
stable gene expression; [0030] the possibility of achieving
regulated gene expression by use of constitutive, tissue-specific,
regulatable promoters and regulatable expression systems; [0031]
the lack of immunogenicity and toxicity of the vector; [0032] the
high transduction efficiency on use of pigment epithelial
cells.
[0033] In a preferred embodiment, therefore, the adenoviral vector
comprises a therapeutic nucleic acid, in particular a therapeutic
DNA, which does not originate from the adenoviral vector. This
might be, for example, a therapeutic gene. A therapeutic gene is
understood by the skilled worker to be a gene whose expression
product can be used for the therapy or diagnosis of a disease.
[0034] A nucleic acid means a polymer which is cleaved on
hydrolysis into sugars, in particular pentoses, especially ribose
and deoxyribose, heterocyclic organic bases, in particular adenine,
cytosine, guanine, thymine and uracil, and phosphoric acid. The
nucleic acid may be, for example, a DNA or RNA. A therapeutic
nucleic acid is a nucleic acid which itself or the product thereof
has a therapeutic effect.
[0035] A gene means a linear DNA section which codes for a protein
or an RNA. The therapeutic gene which is introduced by gene
transfer into the pigment epithelial cell may vary in nature. The
choice is determined by the therapeutic aim. For example, a gene
which codes for a neurotrophic factors can be used. Examples of
neurotrophic factors are the glial cell-derived neurotrophic factor
(GDNF) and the pigment epithelial cell-derived factor (PEDF). It is
also possible, for example, to use genes which prevent
neoangiogenesis. One example is the soluble receptor for the
vascular endothelial cell growth factor (VEGF) which is called
soluble vascular endothelial cell growth factor receptor-1 (sflt1)
(Roeckl W, Hecht D, Sztajer H, Waltenberger J, Yayon A, Weich H A
(1998) Differential binding characteristics and cellular inhibition
by soluble VEFG receptors 1 and 2. Experimental cell research
241:161-1709. Another example is a dominant-negative VEGF receptor
2 (KDR) (Machein M R, Risau W, Plate K H (1999) Antiangiogenic gene
therapy in a rat glioma model using a dominant-negative vascular
endothelial growth factor receptor 2. Hum Gene Ther 10: 1117-1128).
Further therapeutic genes might be, for example, NGF, BDNF, CNTF,
bFGF or neurotrophin 3,4-5.
[0036] PEDF has a very strong neurotrophic and neuroprotective
effect (King G L, Suzuma K (2000) Pigment-epithelium-derived
factor--a key coordinator of retinal neuronal and vascular
functions. N Engl J Med 342: 349-351). This factor is produced by
RPE under normoxic conditions. Production is stopped during
hypoxia. This greatly promotes neovascularization. In age-related
macular degeneration (AMD) the damaged RPE cells produce too little
PEDF. This produces uncontrolled neoangiogenesis. The central
effect of PEDF in the eye is to prevent neogenesis of vessels.
[0037] It is therefore possible according to the present invention
for a genetically modified pigment epithelial cell to be a pigment
epithelial cell which, after genetic modification with a
PEDF-expressing adenoviral vector of large DNA capacity, secretes
PEDF. This cell can then, for example, be transplanted into the
subretinal space near the macula of patients following surgical
removal of neovascularization membranes. The pigment epithelial
cell is thus able on the one hand to replace the removed retinal
pigment epithelium, and on the other hand to produce the PEDF
factor essential for preventing neovascularization. Vision is
stabilized in this way. PEDF is additionally able to protect from
glutamate-mediated neurotoxicity.
[0038] It is additionally possible, depending on the cause of the
disease, for various therapeutic genes to be expressed, singly or
in combination, by the adenoviral vectors of large DNA
capacity.
[0039] In another preferred embodiment, the gene is under the
control of a viral or nonviral promoter which has constitutive,
tissue-specific and/or regulatable activity.
[0040] A promoter which has constitutive activity means a promoter
which mediates transcription of the downstream gene in virtually
all tissues and virtually independently of the physiological state
of the cell. An example of a promoter which has constitutive
activity is the SV40 or the cytomegalovirus promoter.
[0041] A tissue-specific promoter means a promoter which mediates
transcription of the downstream gene only in a particular tissue.
Use of the tissue-specific promoter allows a protein or a
functional RNA to be expressed tissue-specifically in IPE or in RPE
cells. An example of such a tissue-specific promoter is the
transthyretin promoter, which has good activity in RPE and in IPE
cells.
[0042] A regulatable promoter means a promoter which mediates the
transcription of a gene for example depending on the metabolic
situation in the cell, the concentration of a molecule or the
temperature. Gene expression can be controlled quantitatively and
qualitatively by use of a regulatable promoter. An example of a
regulatable promoter is a promoter which is activated in the event
of hypoxia through inclusion of a hypoxia-sensitive element (Boast
K, Binley K, Iqball S, Price T, Spearman H, Kingsman S, Kingsman A,
Naylor S (1999) Characterization of physiologically regulated
vectors for the treatment of ischemic disease. Hum Gene Ther 10:
2197-2208).
[0043] However, it is also possible to use a regulatable expression
system which, for example, is induced or inactivated on
administration of a medicament. An example of such a system is a
tetracycline-dependent gene expression system (Freundlieb S,
Schirra-Muller C, Bujard H (1999) A tetracycline controlled
activation/repression system with increased potential for gene
transfer into mammalian cells. J Gene Med 1:4-12).
[0044] After transduction of the pigment epithelial cell with an
adenoviral vector with large DNA capacity, the cell is able to
produce therapeutic proteins or RNAs. The therapeutic protein is a
protein which causes a therapeutic effect. An analogous statement
applies to a therapeutic RNA, for example an antisense RNA or a
ribosyme. Examples of therapeutic proteins are the neurotrophic
factors PEDF, GDNF, NGF, BDNF, CNTF, bFGF or neurotrophin 3,4-5
(Friedman W J, Black I B, Kaplan D R (1998) Distribution of the
neurotrophins brain-derived neurotrophic factor, neurotrophin-3,
and neurotrophin-4/5 in the postnatal rat brain: an
immunocytochemical study. Neuroscience 84: 101-114) and factors
with antiangiogenetic activity, such as, for example, the soluble
VEGF receptor-1 (sflt-1), the dominant-negative VEGFR-2 (KDR), and
once again PEDF, which also has an antiangiogenetic activity in
addition to its neurotrophic function (Dawson D W, Volpert O V,
Gillis P, Crawford S E, Xu H, Benedict W, Bouck N P (1999) Pigment
epithelium-derived factor: a potent inhibitor of angiogenesis.
Science 285: 245-248).
[0045] Further examples of therapeutic genes are lysosomal enzymes
(Cingle K A, Kalski R S, Bruner W E, O'Brien C M, Erhard P,
Wyszynski R E (1996) Age-related changes of glycosidases in human
retinal pigment epithelium. Curr Eye Res 115: 433-438)
alpha-mannosidase, beta-galactosidase,
N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase,
and lipase. These enzymes play an important part in the breakdown
of visual cell membranes and may be reduced in AMD.
[0046] Some more examples are genes which code for anti-oxidative
enzymes (superoxide dismutase, catalase, peroxidases) because they
may likewise be involved in the pathogenesis of AMD (Frank R N,
Amin R H, Puklin J E (1999) Antioxidant enzymes in the macular
retinal pigment epithelium of eyes with neovascular age-related
macular degeneration, J Ophthalmol 127: 694-709).
[0047] Further examples are genes for gene products which are able
to increase choroidal blood flow, for example NO synthases, because
reduced choroidal blood flow may be involved in the pathogenesis of
AMD (Luksch A, Polak K, Beier C, Polska E, Wolzt M, Domer G T,
Eichler H G, Schmetterer L (2000) Effects of systemic NO synthase
inhibition on choroidal and optic nerve head blood flow in healthy
subjects. Invest Ophthalmol Vis Sci 41: 3080-3084).
[0048] A further aspect of the present invention is a pigment
epithelial cell in a fixed assemblage of cells, called a cell
sheet. To date, only single cell suspensions of autologous IPE
cells have always been transplanted during experimental therapy of
AMD. The advantage of such cell sheets is that the cells can be
sited distinctly better by the transplantation technique, and that
migration of cells away from the site of transplantation is
prevented. Pigment epithelial cells in a fixed assemblage of cells
are characterized by the assemblage of cells consisting of at least
about 100, preferably about 1000, particularly preferably about
10000, pigment epithelial cells, and the latter not being separable
from one another by moderate shear forces, in particular by
repeated, for example ten-fold, movement up and down in a solution
using a pipette.
[0049] A further aspect of the present invention is a cultivation
system comprising at least one pigment epithelial cell of the eye
and a feeder layer. The growing of IPE and RPE cells after
isolation thereof is usually very time-consuming. The cultivation
system of the invention allows large numbers of IPE and RPE cells
to be produced in a very short time.
[0050] A feeder layer is understood by the skilled worker to mean
cells which are cocultivated with other cells (target cells) and
have a beneficial effect on the growth of the target cells. A
beneficial effect may mean, for example, a faster growth of the
cells or prevention of differentiation or dedifferentiation. This
takes place, for example, by the cells of the feeder layer
secreting molecules into the medium which then have a beneficial
effect on growth of the target cells.
[0051] Inactivated fibroblasts are normally used as feeder layer
for cultivating embryonic stem cells. Inactivation of the
fibroblasts can be achieved, for example, by treatment with
mitomycin C or by exposure to y rays. It is possible, for example,
to use fibroblasts from a mammal, in particular mouse or human. In
one implement, the fibroblasts and pigment epithelial cells of the
same species, in particular of the same individual, are used.
However, the fibroblasts may also be a permanent cell line, for
example STO fibroblasts or 3T3 fibroblasts, or primary embryonic
fibroblasts. The production of fibroblasts is known to the skilled
worker (e.g. Abbondanzo S, Gadi I, Stewart C (1993) Derivation of
embryonic stem cell lines. Methods in Enzymology 225: 803-823).
[0052] The cultivation system might, for example, comprise a
culture vessel in which the pigment epithelial cells of the eye and
the feeder layer are cultivated directly adjacent to one another,
in particular one on top of the other, in a suitable medium. The
different cells [lacuna], however, also be cultivated in a culture
vessel spatially separate from one another so that the exchange of,
for example, factors to stimulate growth takes place solely through
the medium.
[0053] A further aspect of the present invention is a method for
the production of a pigment epithelial cell of the eye, where the
cell is genetically modified with the aid of an adenoviral vector
of large DNA capacity.
[0054] A genetic modification is understood by the skilled worker
to be any alteration of the genetic information of the cells. This
can be achieved, for example, by addition, insertion, substitution
and/or deletion of one or more nucleotides. In a particular
embodiment, the genetic modification is brought about by gene
transfer, it being possible for the gene to be, for example,
present extrachromosomally in the cell.
[0055] Gene transfer means the introduction of one or more genes
into, for example, a cell. In the present invention it is possible,
for example, for at least one gene to be introduced with the aid of
an adenoviral vector with large DNA capacity into a pigment
epithelial cell. cDNAs are normally used. However, it is also
possible to use the genes themselves (including their introns and
exons). In another embodiment, however, it is also possible to
introduce a genetically modified, naturally occurring gene or
synthetic nucleic acids into the pigment epithelial cell.
[0056] A further aspect of the present invention is a method for
the production of a pigment epithelial cell of the eye comprising
an adenoviral vector of large DNA capacity, where the cell is
cultivated in serum-free medium, in the presence of a feeder layer
and/or in a fixed assemblage of cells.
[0057] To isolate pigment epithelial cells in a fixed assemblage of
cells, the iris or a part of the iris, or the retina, in particular
in the peripheral retinal region, is separated, for example
mechanically or enzymatically, in particular with Accutase,
chondroitinase and/or heparinase, in particular from the stroma and
the basal membrane. The cell sheet can then be cultivated further
in cell culture. If required, the cell sheets can be broken down
into single cells by renewed incubation with Accutase.
[0058] It has been possible to show within the scope of the present
invention that a cell culture medium which contains no serum, for
example fetal calf serum, has a beneficial effect on the growth of
pigment epithelial cells of the eye.
[0059] A further aspect of the present invention is the use of
genetically modified pigment epithelial cells for the therapy of
eye diseases, possibilities being both a hereditary and an acquired
eye disease. Examples of acquired or hereditary eye diseases are
age-related macular degeneration, glaucoma and diabetic
retinopathy.
[0060] Age-related macular degeneration (AMD) is the commonest
reason for legal blindness in western countries. Atrophy of the
submacular retinal pigment epithelium and the development of
choroidal neovascularizations (CNV) results secondarily in loss of
central visual acuity. For the majority of patients with subfoveal
CNV and geographic atrophy there is at present no treatment
available to prevent loss of central visual acuity. Early signs of
AMD are deposits (druses) between retinal pigment epithelium and
Bruch's membrane. During the disease there is sprouting of choroid
vessels into the subretinal space of the macula. This leads to loss
of central vision and reading ability. An example of a therapeutic
gene which can be employed for therapy of AMD is the PEDF gene.
[0061] Glaucoma is the name given to a group of diseases in which
the pressure in the eye increases abnormally. This leads to
restrictions of the visual field and to the general diminution in
the ability to see. The commonest form is primary glaucoma; two
forms of this are distinguished: chronic obtuse-angle glaucoma and
acute angle closure. Secondary glaucoma may be caused by
infections, tumors or injuries. A third type, hereditary glaucoma,
is usually derived from developmental disturbances during
pregnancy. The aqueous humor in the eyeball is under a certain
pressure which is necessary for the optical properties of the eye.
This intraocular pressure is normally 15 to 20 millimeters of
mercury and is controlled by the equilibrium between aqueous
production and aqueous outflow. In glaucoma, the outflow of the
aqueous humor in the angle of the anterior chamber is blocked so
that the pressure inside the eye rises. Glaucoma usually develops
in middle or advanced age, but hereditary forms and diseases are
not uncommon in children and adolescents. Although the intraocular
pressure is only slightly raised and there are moreover no evident
symptoms, gradual damage occurs, especially restriction of the
visual field. Acute angle closure by contrast causes pain, redness,
dilation of the pupils and severe disturbances of vision. The
cornea becomes cloudy, and the intraocular pressure is greatly
increased. As the disease progresses, the visual field becomes
increasingly narrower, which can easily be detected using a
perimeter, an ophthalmological instrument. Chronic glaucoma
generally responds well to locally administered medicaments which
enhance aqueous outflow. Systemic active substances are sometimes
given to reduce aqueous production. However, medicinal treatment is
not always successful. If medicinal therapy fails, laser therapy or
conventional operations are used in order to create a new outflow
for the aqueous humor. Acute glaucoma is a medical emergency. If
the intraocular pressure is not reduced within 24 hours, permanent
damage occurs.
[0062] A numbers of growth or neurotrophic factors are able to
withstand the survival of glaucomatous neurons. These include NGF,
BDNF, CNTF, bFGF and neurotrophin 3,4-5. It would be possible in a
preferred embodiment to use genetically modified pigment epithelial
cells which contain as therapeutic gene the gene for NGF, BDNF,
CNTF, bFGF and/or neurotrophin 3,4-5 for the therapy of glaucoma.
These factors would then be able to regulate the survival by
activating specific metabolic pathways. Many of these factors have
a short half-life. Stable expression of these factors is
accordingly of considerable therapeutic importance.
[0063] Diabetic retinopathy arises in cases of diabetes mellitus
[lacuna] thickening of the basal membrane of the vascular
endothelial cells as a result of glycosilation of proteins. It is
the cause of early vascular sclerosis and the formation of
capillary aneurysms. These vascular changes lead over the course of
years to diabetic retinopathy. The vascular changes cause
hypoperfusion of capillary regions. This leads to lipoid deposits
(hard exudates) and to vasoproliferation. The clinical course is
variable in patients with diabetes mellitus. In age-related
diabetes (type II diabetes), capillary aneurysms appear first.
Thereafter, because of the impaired capillary perfusion, hard and
soft exudates and dot-like hemorrhages in the retinal parenchyma
appear. In later stages of diabetic retinopathy, the fatty deposits
are arranged like a corona around the macula (retinitis circinata).
These changes are frequently accompanied by edema at the posterior
pole of the eye. If the edema involves the macula there is an acute
serious deterioration in vision. The main problem in type I
diabetes is the vascular proliferation in the region of the fundus
of the eye. The standard therapy is laser coagulation of the
affected regions of the fundus of the eye. The laser coagulation is
initially performed focally in the affected areas of the retina. If
the exudates persist, the area of laser coagulation is extended.
The center of the retina with the site of sharpest vision, that is
to say the macula and the papillomacular bundle, cannot be
coagulated because the procedure would result in destruction of the
parts of the retina which are most important for vision. If
proliferation has already occurred, it is often necessary for the
foci to be very densely pressed on the basis of the proliferation.
This entails destruction of areas of the retina. The result is a
corresponding loss of visual field. In type I diabetes, laser
coagulation in good time is often the only chance of saving
patients from blindness.
[0064] One example of a genetically related disease of the pigment
epithelium is autosomal recessive severe retinal dystrophy which
starts in childhood and is caused by mutation in the RPE65 gene (Gu
S M, Thompson D A, Srikumari C R, Lorenz B, Finckh U, Nicoletti A,
Murthy K R, Rathmann M, Kumaramanickavel G, Denton M J, Gal A
(1997) Mutations in RPE65 cause autosomal recessive childhood-onset
severe retinal dystrophy. Nat Genet 17: 194-197. Correction of the
pathological phenotype is to be expected from introduction of the
RPE65 gene with the aid of an adenoviral vector with large DNA
capacity.
[0065] It was additionally completely surprising that pigment
epithelial cells of the eye can also be transplanted into the CNS.
It was possible to show within the framework of the present
invention that pigment epithelial cells survived the observation
period of 5 weeks. Histological examination revealed no evidence of
induction of damage to neural cells. Instead it was possible to
observe that the pigment epithelial cells formed intensive contacts
with neurons.
[0066] To date, a number of different cell types have been employed
in animal experiments or in patients with Parkinson's disease in
clinical studies:
[0067] Examples are fetal cells obtained from brains of human
fetuses. Fetal cells from the ventral midbrain or dopaminergic
neurons have already been transplanted in clinical studies on more
than 300 patients with Parkinson's disease (for review, see Alexi
T, Borlongan C V, Faull R L, Williams C E, Clark R G, Gluckman P D,
Hughes P E (2000) (Neuroprotective strategies for basal ganglia
degeneration: Parkinson's and Huntington's diseases. Prog Neurobiol
60: 409-470). A number of different cell types, including
non-neuronal cells, e.g. cells from the adrenal cortex, Sertoli
cells on the gonads or glomus cells from the carotid bodies,
fibroblasts or astrocytes, have been used in patients with
Parkinson's disease or in animal models with the aim of replacing
dopamine spontaneously or after gene transfer (Alexi et al. 2000,
supra). The survival rate of transplanted fetal dopaminergic
neurons is 5-8%, which was enough to cause a slight improvement in
the signs and symptoms (Alexi et al. 2000, supra).
[0068] In recent years, neuronal stem cells from brains of adult
vertebrates have been isolated, expanded in vitro and reimplanted
into the CNS, after which they differentiated into pure neurons.
Their function in the CNS remains uncertain, however. Neuronal
precursor cells have also been used for gene transfer (Raymon H K,
Thode S, Zhou J, Friedman G C, Pardinas J R, Barrere C, Johnson R
M, Sah D W (1999) Immortalized human dorsal root ganglion cells
differentiate into neurons with nociceptive properties. J Neurosci
19: 5420-5428). Schwann cells which overexpressed NGF and GDNF had
neuroprotective effects in models of Parkinsonism (Wilby M J,
Sinclair S R, Muir E M, Zietlow R, Adcock K H, Horellou P, Rogers J
H, Dunnett S B, Fawcett J W (1999) A glial cell line-derived
neurotrophic factor-secreting clone of the Schwann cell line SCTM41
enhances survival and fiber outgrowth from embryonic nigral neurons
grafted to the striatum and to the lesioned substantia nigra. J
Neurosci 19: 2301-2312).
[0069] The advantage of pigment epithelial cells, compared with
cells used to date, especially on use of endogenous (autologous)
cells, is that they are not rejected by the immune system and thus,
as expected, have a very high survival rate. In addition, they
replace natural melanin pigment which is lost in the substantia
nigra of Parkinsonian patients. This melanin is able to detoxify
free Fe.sup.++ and thus has a beneficial effect on the progress of
the disease.
[0070] Another aspect of the present invention is therefore the use
of pigment epithelial cells for the therapy of nerve diseases, in
particular a disease of the nervous system, preferably of the CNS,
especially of Parkinson's disease.
[0071] An example of a common disease of the CNS is Parkinson's
disease which is a chronic degenerative disease of the brain. The
disease is caused by degeneration of specialized neuronal cells in
the region of the basal ganglia. The death of dopaminergic neurons
results in reduced synthesis of dopamine, an important
neurotransmitter, in patients with Parkinson's disease. The
standard therapy is medical therapy with L-dopa. L-Dopa is
metabolized in the basal ganglia to dopamine and there takes over
the function of the missing endogenous neurotransmitter. However,
L-dopa therapy loses its activity after some years.
[0072] Pigment epithelial cells spontaneously produce some factors
which have a neuroprotective effect. Examples of such factors are,
which are produced, for example, by IPE cells, are nerve growth
factor (NGF), ciliary neurotrophic factor (CNTF), basic fibroblast
growth factor (bFGF) or factors with an angiogenic activity such
as, for example, vascular endothelial growth factor (VEGF) or
platelet-derived growth factors A and B (PDGF A+B). An example of a
neurotrophic factor which can be produced in the genetically
modified IPE cells after gene transfer is glial cell-derived
neurotrophic factor (GDNF).
[0073] It is additionally possible to utilize the natural
protective function of pigment epithelial cells. In Parkinson's
disease, transplanted IPE cells may display a neuroprotective
effect due to the antioxidant effect of their melanin granules.
This could be caused by the ability of melanin and its precursors
to bind Fe.sup.2+ and other toxic substances (e.g. Ca.sup.2+) and
thus remove them from the cell cytoplasm (Hill H Z (1992) The
function of melanin or six blind people examine an elephant.
Bioessays 14: 49-56) or to have a very strong antioxidant effect.
IPE cells have a high melanin content and also continue to form
melanin when they are located in the retina, the subretinal space
or the CNS. Unambiguous proof thereof is the presence of numerous
early stages of melanogenesis (premelanosomes), detectable by
electron microscopic studies. Melanin has antioxidant properties,
protects from lipid peroxidation, is able directly to bind oxygen
free radicals (Hill H Z (1992) The function of melanin or six blind
people examine an elephant. Bioessays 14: 49-56) and can prevent
the formation of new oxygen free radicals by binding metal cations
(Memoli S, Napolitano A, d'lschia M, Misuraca G, Palumbo A, Prota G
(1997) Diffusible melanin-related metabolites are potent inhibitors
of lipid peroxidation. Biochim Biophys Acta 1346: 61-68). If highly
pigmented iris pigment epithelial cells are introduced into tissue
with high oxidative stress, for example in the substantia nigra of
patients with Parkinson's disease, or into the papilla of glaucoma
patients, or into the vicinity of the macula of AMD patients, then
a neuroprotective effect occurs simply through the presence of the
melanin in the IPE cells.
[0074] An example of a protein with a good therapeutic potential
for the therapy of patients with Parkinson's disease is glial
cell-derived neurotrophic factor (GDNF), a survival factor for
dopaminergic neurons. GDNF has effects even in picomolar
concentrations on the survival rate and growth of dopaminergic
neurons from embryonic brain. Animal experimental studies have
shown that direct gene transfer into the substantia nigra of a GDNF
expression cassette using various vectors (first generation
adenoviral vectors, AAV vectors or lentiviral vectors) was able to
protect dopaminergic neurons in the 6-OHDA rat model (Mandel R J,
Spratt S K, Snyder R O, Leff S E (1997) Midbrain injection of
recombinant adeno-associated virus encoding rat glial cell
line-derived neurotrophic factor protects nigral neurons in a
progressive 6-hydroxydopamine-induced degeneration model of
Parkinson's disease in rats. Proc Natl Acad Sci U.S.A. 94:
14083-14088). In a preferred embodiment, GDNF is expressed in
autologous IPE cells after transduction with an adenoviral vector
of large DNA capacity. The cells genetically modified in this way
are then implanted stereotactically at the site of action, for
example into the striatum. In a further embodiment, the gene coding
for the therapeutic protein undergoes regulated expression, for
example through use of a cell-specific promoter. In a preferred
embodiment, the therapeutic gene is expressed regulatably, for
example by use of a system which can be regulated by
tetracycline.
[0075] In a preferred embodiment of this invention, pigment
epithelial cells are implanted at the desired site in the CNS. The
cells can be put at the desired site of action for example by
injection, for example during a stereotactic operation. This makes
it possible to produce specific therapeutic molecules in situ.
[0076] In a particularly preferred embodiment there is the use of
autologous pigment epithelial cells. The use of autologous pigment
epithelial cells has the following advantages compared with other
cell types: no rejection reactions occur because the pigment
epithelial cells used are derived from the patients themselves; in
terms of developmental history, these are neuroepithelial cells
histogenetically related to brain cells.
[0077] In a preferred embodiment of this invention, pigment
epithelial cells, in particular autologous IPE cells, which are
genetically modified are used for the therapy of CNS diseases, such
as, for example, Parkinson's disease.
[0078] The pigment epithelial cells of the invention can
additionally be used in transplantation. In one embodiment of the
invention, the pigment epithelial-cells of the invention can
display their therapeutic effect through transplantation into the
eye.
[0079] The pigment epithelial cells can be transplanted, for
example, into the choroid and exert there a therapeutic effect
through production of pigment epithelial-endogenous factors or
through production of therapeutic molecules after genetic
modification with an adenoviral vector of large DNA capacity.
[0080] However, the pigment epithelial cells can also be employed
in the region of the papilla. It has been possible to show within
the scope of the present invention that, after injection into the
posterior part of the vitreous, the cells migrate into the optic
disk and there integrate into the tissue assemblage. This opens up
the possibility of therapy of diseases manifested, for example, in
the optic disk.
[0081] The pigment epithelial cells can also be injected into the
vitreous, in particular through a transscleral access into the
posterior part of the vitreous. It was possible to show, by
fundoscopic inspection of RCS rats, that the IPE cells are found
entirely on the papilla throughout an observation period of 2
months. It was histologically evident that the IPE cells had
migrated into the optic disk and there formed intensive contact
with the blood vessels and axons. Electronic microscopy shows no
damage to neuronal cells or proliferation of IPE cells in the
vitreous. This embodiment of the invention makes it possible, for
example, to have direct access to the optic disk and to release or
activate, with or without genetic modification, neuroprotective
mediators in or in the direct vicinity of the papilla.
[0082] A further embodiment of the present invention relates to the
use of adenoviral vectors with large DNA capacity for the genetic
modification of pigment epithelial cells in vivo. As shown in the
examples, in vivo transduction of RPE cells leads, through
subretinal injection with an adenoviral vector of large DNA
capacity, to a surprisingly stable expression for at least 6
months.
[0083] A further aspect of the present invention relates to a
medicament or diagnostic aid comprising a pigment epithelial cell
of the invention and suitable excipients and/or additives. Suitable
excipients and additives, which serve, for example, to stabilize or
preserve the medicament or diagnostic aid, are generally known to
the skilled worker (see, for example, Sucker H et al. (1991)
Pharmazeutische Technologie, 2.sup.nd edition, Georg Thieme Verlag,
Stuttgart). Examples of such excipients and/or additives are
physiological saline solutions, Ringer dextrose, dextrose, Ringer
lactate, demineralized water, stabilizers, antioxidants, complexing
agents, antimicrobial compounds, proteinase inhibitors and/or inert
gases.
[0084] The following figure and the examples are intended to
explain the invention further without restricting it thereto.
DESCRIPTION OF THE FIGURE
[0085] FIG. 1 Schematic cross section through the right eye.
[0086] The iris pigment epithelium is located on the side of the
iris facing the lens. The macula is the region (about 6 mm in
diameter) directly surrounding the fovea.
[0087] FIG. 2 Genomic structure of HC-Ad.PEDF.
[0088] Features from left to right: left terminus of adenovirus
type 5 (nt 1-440) including the packaging signal .psi.; 20 kb of
stuffer DNA fragment derived from the human HPRT locus (gene map
positions 1777-21729, locus: HUMHPRTB); hCMV promoter and PEDF cDNA
with a C-terminal 6His tag; SV40 poly A; 6.5 kb human fragment of
C346 (cosmid map positions 10205-16750, locus: HUMDXS455A); right
terminus of adenovirus type 5 (nt 35818-35935).
EXAMPLES
[0089] 1. Isolation of Cell Sheets
[0090] To isolate IPE cells in assemblages of cells (cell sheets),
iridectomies were collected fresh from the operating theater after
a trabeculectomy or a basal iridectomy, brought in F12 medium
((HAM) with L-glutamine, Gibco, Life Technologies, Paisely,
Scotland) and directly processed further. Basal iridectomies of
glaucoma patients or pieces of iris from rats or pigs were treated
with Accutase (Cat. No. L11-007, PAA Laboratories) in Dulbecco's
PBS with 0.5 mmol/l EDTA.times.Na for 15-20 min. The tissue which
can be obtained by an iridectomy has an area of about 3.5 mm.sup.2
and contains about 20000 IPE cells. The cell layers were pipetted
up and down very carefully with F12 medium and pipetted out on
polystyrene. It was possible to detach the IPE cells completely as
double cell layer with intact basal membrane from the stroma under
the stereomicroscope as was demonstrable by examination under the
electron microscope. It was possible to remove this basal membrane
completely by incubation with 0.1 U/ml chondroitinase ABC (Sigma)
and 2.4 U/ml heparinase (Sigma) in PBS at pH 7.4 and at 37 degrees
Celsius for 2 hours. It was then possible to break the cell sheets
down into single cells by renewed Accutase incubation for 5
minutes.
[0091] To isolate RPE cells in assemblages of cells (cell sheets),
autologous RPE cell sheets and single cells were mechanically
detached in the periphery of human eyes after local retinotomy, and
aspirated with a canula. It was possible to remove 50,000
peripheral RPE cells locally without the patients later complaining
of unpleasant serious losses of visual field. The removal of 50,000
RPE cells in each case was possible at several peripheral sites on
the eye.
[0092] 2. Growing of IPE and RPE Cells by Cultivation on
Fibroblasts
[0093] IPE or RPE cells obtained from iridectomies or eyes of organ
donors were cultivated on fibroblasts (mouse 3T3 fibroblast cell
line), which served as feeder layer, in F12 medium. The cells
become adherent to the fibroblasts within one day and start to
proliferate. The number of cells tripled or quadrupled on the
fibroblasts within 3 days. The fibroblasts had previously been
treated with 40 .mu.g/ml mitomycin C so that they die after no more
than 10 days. A pure culture of pigment epithelial cells is
obtained after this time.
[0094] 3. Injection of IPE Cells
[0095] IPE cells were isolated as single cell suspension as
described under 1. An animal model of age-dependent macula
degeneration and of retinal degeneration caused by a specific
phagocytosis defect and degeneration of the RPE is the Royal
College of Surgeons Rat (RCS rat). Under Ketanest/Nembutal
anesthesia, the upper conjunctiva of dystrophic RCS rats (18 days
old) was opened by an incision 4 mm long and 4 mm posterior of the
limbus. Kolibri forceps were used to hold the conjunctiva near the
limbus firmly.
[0096] 3.1. Subretinal Injection
[0097] In the subretinal injection, a 26 gage canula was used to
pierce the sclera, choroid and retinal pigment epithelium at the
level of the equator as far as the vitreous. A Hamilton syringe
with a blunt 32 gage canula was introduced anteriorally 2-3 mm
tangentially between retina and RPE. 60000 IPE cells were injected
in 0.5 .mu.l of cell culture medium (F 12 (HAM) with L-glutamine,
Gibco, Life Technologies, Paisely Scotland). The RCS rats were
sacrificed after completion of the observation period of 6 and 8
months. The eyes were enucleated. The corneas were removed and the
remaining parts of the eyes were fixed in 3% glutaraldehyde. Areas
with transplanted iris cells were easily identifiable from the
pigmentation and were excised and embedded for electron microscopic
investigations in accordance with a routine protocol. Under the
electron microscope, surviving, i.e. morphologically intact, IPE
cells were detectable in the subretinal space for up to 8 months
after transplantation. Surviving photoreceptors with inner segments
but without outer segments were present for up to 6 months after
subretinal transplantation.
[0098] 3.2. Injection into the Vitreous
[0099] Injection into the vitreous took place at the same site of
the subretinal injection in 6 eyes. However, the canula was
introduced like a secant of a circular arc 1-2 mm deep into the
vitreous. 60000 IPE cells were injected close to the papilla. The
vitreous and the lens remained clear in the observation period of 2
months. The IPE cells in all 6 eyes formed a macroscopically or
funduscopically visible collection on the papilla. The histology
showed that the IPE cells migrated into the optic disk. The cells
were highly pigmented and there was no evidence of cell damage or
proliferation.
[0100] 3.3. Injection into the Choroid
[0101] The site chosen for injection into the choroid was the same
as for subretinal injection. The sclera was cut with a pointed
scalpel through an incision 1 mm long until the choroid was
visible. The canula was placed perpendicular to the eyeball on the
incision site and 60000 IPE cells in 0.5 .mu.l of F12 medium were
injected into the choroid. IPE cells transplanted into the choroid
in 15 eyes, compared with 6 untreated eyes, led to a survival of
photoreceptors for up to 6 months. Both the number of surviving
photoreceptors/mm of retina (p=0.020) and the maximum nucleus
height (p=0.019) were significantly different in the Mann-Whitney
test from the untreated eyes (Table 1). TABLE-US-00001 TABLE 1
Median, 25.sup.th and 75.sup.th percentiles of the number of
photoreceptor cell nuclei still present, of the maximum thickness
of the photoreceptor layer in semithin sections 6 months after
transplantation of IPE cells into the choroid is indicated. Maximum
height of Photoreceptor the photoreceptor Number nuclei cell of
eyes [mm.sup.-1] nuclei IPE transplantation 15 Median 12.3 1.0
25.sup.th Percentile 0.0 0.0 75.sup.th Percentile 45.5 3.0 Control
without treatment 6 Median 0.0 0.0 25.sup.th Percentile 0.0 0.0
75.sup.th Percentile 0.0 0.0 Mann-Whitney test P value 0.020
0.019
[0102] 4. Injection of Rat IPE Cells into the CNS
[0103] For the stereotactic implantation method, Wistar rats were
anesthetized by intraperitoneal injection of 1 ml of avertin (2 g
of tribromoethanol 3,3,3 (dry), 1 g of pentanol (liquid), 8 ml of
100% ethanol and 90 ml of 0.9% Nacl) per 100 g of body weight. The
cranium was fixed in a precisely reproducible manner at three bone
points, the external auditory canals and the maxilla so that the
calvaria was horizontal at the level of the bregma. After the
medial front-occipital skin incision, 1.5 cm long, the periostium
was pushed aside in order to have a clear view of the cranium
sutures which served as reference point for the stereotactic
coordinates. The coordinates were determined on the basis of the
atlas of Praxinos and Watson (Praxinos G. Watson C. The rat brain
in stereotactic coordinates. 1986;2 end Dr., Academic Press,
Sydney):
[0104] The puncture site was 1.5 mm in the frontal direction and 2
mm to the right parietally from the bregma. The upper portion of
the striatum is at a depth of 4.5 mm. The hole with a diameter of
about 0.5 mm was drilled at the appropriate position using a
precision shaft drill (Proxxon, Minimot 40IE) avoiding damage to
the dura. 5-10 .mu.l of the cell suspension were introduced through
this drilled hole with a 25 .mu.l N-702-N Hamilton syringe with
fixed needle, injecting at a depth of 5 mm measured from the
surface of the dura. 60000 IPE cells from Long Evans rats were
injected into the striata of each of 4 Wistar rats. Before the
needle was withdrawn, 2 min were allowed to elapse so that the cell
suspension was able to diffuse into the tissue and the resulting
local pressure could diminish. Otherwise there was a risk that
cells could have followed the withdrawn needle into the puncture
channel or into the overlying tissue sections. For the same reason,
a further 30 sec were allowed to elapse after the needle had been
withdrawn 4 mm, before it was completely removed. A skin suture was
then applied.
[0105] After 5 weeks, the brains were perfusion-fixed with 3%
glutaraldehyde in cacodylate buffer. Pigmented areas were excised
from the striatum and embedded for electron microscopy.
[0106] The transplanted areas were easily identifiable
macroscopically by the pigmentation. Under the electron microscope,
the IPE cells had intact mitochondria and plasma membranes. They
were highly pigmented, contained melanogenesis stages and formed
contact zones with neurons. The IPE cells were always found singly
without contacts with other IPE cells. They were also found 3-4 mm
away from the puncture channel, which suggests active migration of
the cells. The neurons adjacent to the IPE cells were
morphologically intact. Immunocompetent cells (macrophages,
lymphocytes) were not observed.
[0107] 5. Genetic Modification
[0108] Isolated and adherent rat and human IPE cells were
transduced in vitro with 20, 50 and 100 MOI (multiplicity of
infection) of the adenoviral vector with large DNA capacity HC
adenovirus "HC-AdFK7" which harbors the EGFP (enhanced green
fluorescent protein) under the control of the human CMV
(cytomegalovirus) promoter as transgene. For this purpose, 80%
confluent cell cultures in F12 complete medium were incubated with
the appropriately diluted virus stock solution at 37.degree. C. for
24 hours. The medium was changed the next morning. Expression of
the transgene was checked as green fluorescence inside the cells
under a fluorescence microscope with FITC filter [lacuna] 24, 48
and 72 hours and then each subsequent week.
[0109] As soon as 24 hours after the transduction a slight
fluorescence was visible and became distinctly stronger in
subsequent days. The human IPE cells were 100% transduced with 100
MOI, and the rat cells were 80% transduced with 20 MOI and 100%
transduced with 50 and 100 MOI. Expression was detectable in vitro
for a period of up to 8 weeks or longer.
[0110] In a further experiment, the adenoviral vector of large DNA
capacity AdhCMV.PEDF was constructed. This vector expresses the
human PEDF cDNA under the control of the human CMV promoter. The
PEDF protein is additionally tagged by a poly-histidine epitope
expressed as fusion protein with the PEDF. This vector was produced
by a standard method (Schiedner G, Morral N, Parks R J, Wu Y,
Koopmans S C, Langston C, Graham F L, Beaudet A L, Kochanek S
(1998) Genomic DNA transfer with a high capacity adenovirus vector
results in improved in vivo gene expression and decreased toxicity.
Nature Genetics 18: 180-183) in Cre-recombinase-expressing 293
cells and purified by CsCl density gradient centrifugation.
[0111] Cytokeratin-positive human IPE cells from the 2.sup.nd
passage were transduced with the HC-Ad.CMV.PEDF vector. For this
purpose, 80% confluent cell cultures in F12 complete medium were
incubated with the appropriately diluted virus stock solution at 37
C. for 24 hours. The medium was changed next morning. Expression of
the transgene and secretion of the PEDF into the culture
supernatant were checked in an ELISA using specific
anti-polyhistidine antibodies after 72 hours. The culture
supernatants contained 150 ng PEDF/ml. This corresponds to a
production of 60 pg of PEDF per 1000 cells in 72 hours. It was
possible to detect in another ELISA using another antibody which
specifically recognizes the human PEDF protein that human IPE cells
do not, in contrast to human RPE cells, spontaneously produce
PEDF.
[0112] IPE Sheet Transfection
[0113] It was possible to transfect not only single cells but also
cell sheets. IPE cell sheets could be transfected after enzymatic
removal of the basal membranes. The cell sheets with intact basal
membrane could not be transfected, as was demonstrated by PCR.
[0114] For transfection of cell sheets, pig eyes were brought to
the laboratory, and processed further, from the slaughterhouse
immediately after the animals were slaughtered. The anterior
segment was removed by a circular cut about 2 mm behind the limbus.
The iris was then removed by a blunt dissection from the posterior
direction and incubated in 1 ml Accutase at 37.degree. C. for 15
min. The IPE is then detached from the stroma of the iris using a
glass pipette bent in a flame. Estimation of the area of the
individual IPE cell sheets obtained resulted in between 40,000 and
70,000 IPE cells per cell sheet.
[0115] The IPE sheets were incubated with 200 MOI of the
EGFP-expressing adenoviral vector HC-AdFK7 for 24 h. The medium was
changed after 24 hours. The IPE sheets were cultivated in F12
complete medium for 6 days and investigated for EGFP fluorescence,
but it was not possible to demonstrate this with certainty because
of the morphology of the cells with very densely concentrated
melanin granules. DNA was then obtained from the cells using the
QIAmp DNA mini kit (Qiagen). The manufacturer's instructions were
followed. The transgene was detected by PCR using the primers
prod1, which binds in the region of the CMV promoter, and prod2,
which binds in the region of the EGFP sequence, to the DNA of
HC-AdFK7. With successfully transduced cells, prod1 and prod2
produced a PCR product with a length of about 700 base pairs. A
plasmid pFK7 with the same insert as is to be found in HCAdFK7
served as positive control.
[0116] 6. Subretinal Transplantation of Genetically Modified
Cells
[0117] 8 Wistar rats received subretinal transplantation, in 8
eyes, of IPE cells, transduced with HC-AdFK7 vector, from Long
Evans rats (albinotic) by the same method as described for
untransfected IPE cells.
[0118] Four eyes into which IPE cells had been transplanted were
enucleated after 2 months, embedded in tissue freezing medium
(Jung, Heidelberg, Germany) and frozen at -80 C. Cryostat sections
(7 .mu.m) were, after thawing, embedded in Kaiser's glycerol
gelantin (Merck, Darmstadt, Germany) and examined under a Zeiss
Axiophot light microscope with an excitation wavelength of 400-400
nm and an emission wavelength of 470 nm. The subretinally
transplanted IPE cells showed distinct expression of green
fluorescent proteins.
[0119] For evaluation using the scanning laser ophthalmoscope (SL),
the other 4 transfected rats were examined 14 days and 4 months
after the transfection. The animals were anesthetized with Ketanest
and evaluated using the scanning laser ophthalmoscope (Rodenstock,
Munich). This entailed scanning of the retina of the rats with
mydriasis in fluo mode with the infrared laser (780 nm), the argon
green laser (514 nm) and argon blue laser (488 nm). In this mode,
the instrument uses a fluorescein cutoff filter suitable for
observing the EGFP fluorescence. The images were recorded by S-VHS
video. The analog video images were copied digitally onto DV, and
bitmaps were produced from representative sections and evaluated
for area and intensity using the software Optimas 6.1.
[0120] During the observation period of 4 months, the intensity of
fluorescence caused by the transfected IPE cells, and the extent of
the transplanted areas in the fundus remained constant in all 4
eyes, i.e. expression of the transfected genes remained unchanged
at the protein level.
[0121] 7. Subretinal Injection of Free Vector for Genetic
Modification of RPE Cells of the Host (in vivo Gene Therapy)
[0122] For subretinal injection of free vector in vivo, various
concentrations of an HC adenovirus "HC-AdFK7" which harbors the
EGFP (enhanced green fluorescent protein) gene under the control of
a CMV (cytomegalovirus) promoter as transgene were injected
subretinally into Wistar rats. Expression of the transgene were
evaluated using the scanning laser ophthalmoscope (Rodenstock,
Munich). This entailed scanning of the retina of the rats with
mydriasis in fluo mode with an infrared laser (780 nm), the argon
green laser (514 nm) and argon blue laser (488 nm). In this mode,
the instrument uses a fluorescein cutoff filter suitable for
observing the EGFP fluorescence. The images were recorded by S-VHS
video. The analog video images were copied digitally onto DV, and
bitmaps were produced from representative sections and evaluated
for area and intensity using the software Optimas 6.1.
[0123] During the observation period of 6 months, the intensity of
the fluorescence caused by the transfected IPE cells, and the
extent of the transplanted areas in the fundus remained constant in
all 4 eyes, i.e. expression of the transfected genes remained
unchanged at the protein level. After 6 months, the animals were
sacrificed, and the eyes were fixed in 3% glutaraldehyde. The
anterior segments of the eyes were removed and the remaining
posterior optic cups were divided into four. After the retinas had
been removed, the sclera, choroid with pigment epithelium were
examined under a fluorescence microscope (Axiovert Zeiss,
Oberkochen, Germany) using a 450-490 nm excitation filter and a 520
nm emission filter (AF Analysentechnik, Tubingen, Germany). This
revealed the typically hexagonal shape of transduced and
EGFP-positive pigment epithelial cells.
[0124] 8. Transplantation of Genetically Modified Cells into the
CNS
[0125] For transplantation of genetically modified cells into the
CNS, IPE cells (60000) which had been transduced with HC-AdFK7
vector and expressed EGFP from Long Evans rats were
stereotactically injected as described above into the striatum of
each of 4 Wistar rats.
[0126] After 8 weeks, the animals were sacrificed by cervical
dislocation under CO.sub.2 anesthesia. The brains were dissected
out. Pigmented areas with transplanted cells were excised from the
striatum and frozen in tissue freezing medium (Jung, Heidelber,
Germany). The fluorescence caused by the expression of EGFP by the
IPE cells was detectable in frozen sections 8 weeks after
transplantation in pigmented cells.
[0127] 9. Prevention of Choroidal Neovascularization [lacuna]
Genetically Modified and PEDF-Expressing IPE Cells in vivo
[0128] IPE cells were cotransfected with the adenoviral vector of
large DNA capacity HC-AdFK7, which harbors the EGFP (enhanced green
fluorescent protein) gene under the control of a CMV
(cytomegalovirus) promoter as reporter gene, and simultaneously
with the PEDF-expressing adenoviral vector of large DNA capacity
HC-AdCMV.PEDF in vitro and, after 6 days, transplanted into the
subretinal space of Long Evans rats (60,000 cells/eye) (1.sup.st
experimental group). The PEDF expression cassette in the adenoviral
vector of large DNA capacity contained a poly-HIS epitope for
detecting the protein using an anti-HIS antibody in addition to the
PEDF-encoding sequence. One week after injection, the rats were
anesthetized, the pupils were dilated and the rats received 3-4
laser burns around the optic nerve with a blue-green argon laser
(Coherent, Inc., Santa Clara, Calif., USA). The energy of the laser
was 90 mW for 100 ms, and the diameter of the beam was 100 .mu.m. A
second group of rats received only laser burns without genetic
modification by cell transplantation or free vectors. After 16
days, the rats were anesthetized and received 0.5 ml Liquemin i.p.
(Roche, Grenzach-Wyhlen, Germany). The ascending aorta was
canulated and, after the right atrium was opened, the blood was
washed out with 50 ml of Ringer lactate solution (Stereofundin,
Braun, Melsungen, Germany). This was followed by perfusion with 20
ml of Ringer solution with 5 mg/ml FITC dextran (Sigma Deisenhofen,
Germany). The eyes were enucleated, pierced with a scalpel at the
level of the limbus and fixed in 4% paraformaldehyde overnight. The
next day, the anterior segment of the eyes was removed up to a
short distance behind the ora serrata by a circular incision. The
remaining optic cup was divided into quadrants by 4 radial
incisions, and the retinas were removed. Quadrants consisting of
pigment epithelium, choroid and sclera containing laser scars were
incubated in tris buffer (TBS) for 4.times.10 min and then in 0.5 M
NH.sub.4CL (Sigma, Deisenhofen, Germany) and 0.25% Triton (Serva,
Heidelberg, Germany) for 10 min. After two further washes, the
specimens were incubated with 5% BSA (Albumin, Bovine Fraction
Sigma, Deisenhofen, Germany). Some of the specimens were incubated
with antibodies against histidine (anti-His antibody, Qiagen,
Hilden, Germany) in order to detect the histidine residues in the
PEDF. The primary antibodies were visualized using anti-mouse IgG
coupled to the fluorescent dye Cy3 (Rockland, Gilbertsville, Pa.,
USA).
[0129] Other specimens were treated with rat anti-mouse CD 31
(PECAM-1, Pharmingen, San Jose, Calif., USA) in order to visualize
the endothelial cells. This was followed by a 2.sup.nd incubation
with anti-rat IgG-biotin (Amersham, Pharmacia Biotech Europe GmbH,
Freiburg, Germany) with subsequent localization of the biotin by
Fluorolink Cy3 (Amersham Life Sciences, Braunschweig, Germany).
Some choroid specimens underwent double labeling of PEDF and
endothelial cells. In these cases, PEDF expression was visualized
with Cy3 as described, and the PECAM-biotin complex was visualized
with streptavidin-Alexa Fluor 350 (MoBiTec, Gottingen,
Germany).
[0130] The flatmount specimens were evaluated under the
fluorescence microscope (Axiophot, Zeiss, Oberkochem, Germany).
[0131] In group 1 there was no observable escape of FITC-dextran
nor an increased occurrence of CD 31-positive cells in 16 of 19
laser scars when PEDF-expressing IPE cells were present at a
distance of 100-1000 .mu.m from the scar. The expression of PEDF by
the transplanted IPE cells was demonstrated with anti-His
antibodies.
[0132] In the control group 2 (only laser burn), neovascularization
was present in 9 of 12 laser scars. This was evident from the
escape of dextran-FITC in and around the scar region and from the
presence of flattened CD 31-positive endothelial cells in and
around the scar region.
[0133] Neovascularization was thus detected in the same eye with a
functional (dextran leakage) and an immunological method (direct
detection of the newly produced endothelial cells with antibodies).
These flatmount specimens allow the entire choroid to be assessed.
These results show that neovascularization is inhibited by
transplantation of IPE cells which express PEDF of an adenoviral
vector of large DNA capacity.
[0134] 10. Endogenous and HC-Ad Vector Mediated Production of PEDF
in IPE and RPE Cells
[0135] Endogenous PEDF production in nontransduced RPE and IPE
cells was determined. RPE cells secreted 96+9.5 ng/ml PEDF within
72 h (n=4), while endogenous PEDF was not detected in supernatants
from IPE cells (detection limit 1.56 ng/ml). Thus, IPE cells, in
contrast to RPE cells, were found to lack the expression of PEDF, a
protein that is believed to play a key role in the homeostasis of
the retina.
[0136] The vector HC-Ad.PEDF was constructed to express the human
PEDF carrying a C-terminal 6-His tag (FIG. 2). IPE (Hu, D. N.,
Ritch, R., McCormick, S. A. & Pelton-Henrion, K. (1992) Invest
Ophthalmol Vis Sci 33, 2443-2453) and RPE (Chang, C. W., Defoe, D.
M. & Caldwell, R. B. (1997) Invest Ophthalmol. Vis. Sci. 38 ,
188-195) cell cultures were prepared from Long Evans rats. Early
passage cultures of choroidal endothelial cells (hCEC) (positive
for v.Willebrand factor) were obtained from donor eyes (Hoffmann,
S., Spee, C., Murata, T., Cui, J. Z., Ryan, S. J. & Hinton, D.
R. (1998) Graefes Arch Clin Exp Ophthalmol 236, 779-784).
1.times.10.sup.6 IPE cells were transduced with 50 MOI of
HC-Ad.PEDF for 24 h. 48 h and 72 h after medium change supernatants
were collected. PEDF released into the medium was detected by ELISA
with mouse penta-his (5 .quadrature.g/ml; Qiagen) and anti-PEDF
antibodies (1 .mu.g/ml, Chemicon). Endogenous PEDF production was
determined from nontransduced IPE and RPE cells. Standard curves
were generated from known amounts of recombinant PEDF.
[0137] Following transduction of IPE cells with HC-Ad.PEDF vector
IPE cells (1.times.10.sup.6 cells/dish, n=4) secreted 250+38 ng/ml
PEDF into the supernatant within 72 h. IPE cells secreted
functionally active PEDF at high levels following HC-Ad mediated
gene transfer.
[0138] To examine effects of IPE-produced PEDF on hCEC function IPE
cells were transduced with 50 MOI of HC-Ad.PEDF or 50 MOI of
HC-Ad.FK7. Undiluted, 1:10 or 1:100 diluted supernatants
(conditioned media=CM) collected 72 hours after medium change were
added to hCECs.
[0139] To analyze proliferation, 1.times.10.sup.3 hCECs/well seeded
in 96-well plates were exposed to CM with or without 50 ng/ml VEGF
(Sigma) and with or without 1 .mu.g/ml anti-PEDF antibody. Five
days later, cellular proliferation was determined using the WST-1
proliferation assay (Roche). Nondiluted and 1:10 diluted CM from
HC-Ad.PEDF transduced IPE cells reduced VEGF-stimulated
proliferation of hCECs without having any effect on unstimulated
proliferation. Anti-PEDF antibody (1 .mu.g/ml), present in the CM,
abolished the inhibitory effect. CM from HC-Ad.FK7 transduced and
nontransduced IPE cells did not influence both stimulated and
nonstimulated proliferation of the cells.
[0140] To analyze migration, 5.times.10.sup.3 hCECs/insert were
incubate in modified Boyden chambers (Tang, S., Gao, Y. & Ware,
J. A. (1999) J. Cell Biol. 147, 1073-1084) (FluoroBlock inserts,
Becton Dickinson) in CM with or without VEGF (50 ng/ml) for 8 h at
37.degree. C. Migrated cells were visualized by the nuclear
fluorescence dye (DAPI, Alexis) and counted in three random
fields/membrane. CM from HC-Ad.PEDF transduced IPE cells reduced
the migration of hCECs towards angiogenic VEGF from 47.5+5.9 to
14.3+5.7 cells/membrane (P<0.001).Exposure to CM from HC-Ad.FK7
transduced and nontransduced IPE cells did not influence hCECs
migration.
[0141] To determine the formation of capillary-like tubes in
response to PEDF, 1.times.10.sup.4 hCECs/well were seeded in
96-well plates coated with VEGF-containing ECM gel (Chemicon) for
24 h in CM. CM from HC-Ad.PEDF infected IPE cells suppressed
neovascular tube formation while supernatants from HC-Ad.FK7
transduced and nontransduced IPE cells had no effect.
[0142] 11. Long-Term EGFP Expression Following Subretinal
Transplantation of HC-Ad.FK7 Transduced IPE Cells
[0143] IPE cells were transduced with 50 MOI of HC-Ad.FK7 or were
co-transduced with 50 MOI of HC-Ad.PEDF and HC-Ad.FK7. Before
transplantation the medium was changed, the cells were washed twice
with PBS, and suspensions of 5.times.10.sup.4 cells/.mu.l were
transplanted (1 .mu.l/eye). Transplantation was performed as
aforementioned.
[0144] EGFP fluorescence in Wistar rats was monitored 7 days, 1, 2,
3 and 4 months after subretinal injection of HC-Ad.FK7 transduced
IPE cells by Scanning Laser Ophthalmoscopy (SLO, Rodenstock,
Germany), and on RPE-choroidal flatmounts (McMenamin, P. G. (2000)
Invest Ophthalmol Vis Sci 41, 3043-3048) by fluorescence microscopy
(Axioplan, Zeiss, Germany). Four months after injection, the areas
of HC-Ad.FK7 transduced IPE transplants were subjected to electron
microscopy. PEDF expression in IPE transplants was visualized in 4%
paraformaldehyde fixed RPE-choroidal flatmounts and in paraffin
sections using penta-his antibody (5 .mu.g/ml) and Cy3 (Amersham)
or peroxidase (Amersham) conjugated secondary antibodies.
[0145] Seven days after transplantation of HC-Ad.FK7 transduced IPE
cells areas of patchy and/or continuous fluorescence was observed
by SLO at the site of injection. Three months later, areas of
bright EGFP fluorescence were still present in the same eyes with
comparable extension and intensity as at 1 and 2 month. Four months
after surgery, EGFP expressing IPE cells were found integrated into
the host RPE layer as determined in RPE-choroidal flatmounts. By
electron microscopy, pigmented IPE cells formed a second layer on
the RPE of the Wistar hosts. The rod outer segments facing the
transplants appeared morphologically intact. Thus, genetically
modified IPE cells formed a monolayer following transplantation and
stably expressed an EGFP reporter for at least 4 months without
adverse effects.
[0146] 12. Effects of HC-Ad Vector Mediated PEDF Expression from
Transplanted IPE Cells in a Model of Oxygen-Induced Retinal
Neovascularization
[0147] A previously described model of ischemia-induced retinopathy
(Smith, L. E., Wesolowski, E., McLellan, A., Kostyk, S. K.,
D'Amato, R., Sullivan, R. & D'Amore, P. A. (194) Invest
Ophthalmol Vis Sci 35, 101-111) was set up by using Wistar rats.
Animals in the normoxia group were maintained in room air
throughout the experiment. Rats in the hyperoxia groups were
exposed to 75% oxygen from postnatal day 7 (P7) to P12, then
removed to room air and immediately transplanted subretinaly with
1) nontransduced IPE cells; 2) IPE cells transduced with HC-Ad.FK7;
3) IPE cells co-transduced with HC-Ad.PEDF and HC-Ad.FK7. At P 22,
the animals were anesthetized and perfused with 50 mg/ml
fluorescein isothiocyanate-dextran (Sigma) as described (Smith et
al., supra). The neovascularization was investigated on
retinal-RPE-choroidal flatmounts using a fluorescence microscope.
To quantify neovascularization the length of newly formed tortuous
blood vessels with diameters larger than 25 .mu.m on the inner
surface of peripheral retina was determined by computer assisted
morphometry (Openlab software; ImproVision, Inc., Lexington, USA).
For quantification the angiographic images were digitalized and
subsequently processed. Vessels were classified according to their
diameters. Measurements of the vessel length were taken in areas of
800 000 .mu.m.sup.2 per eye located up to 200 .mu.m from the
transplanted IPE cells or in corresponding peripheral regions of
hyperoxia controls. Additionally to the vessel length, a second
independent quantification of neovascularization was performed by
evaluation of the total vascularized area on the same peripheral
regions. The fluorescence images were evaluated by setting of
threshold level of fluorescence, above which the superficial
retinal vessels but not the vessels of retinal deep plexus were
captured. Vascularized areas were normalized to the total evaluated
peripheral retinal area. Furthermore, to confirm epiretinal
localization of the superficial pathological vessels 10 .mu.m
serial frozen sections were prepared cut through half of the eye.
Immunofluorescence microscopy was performed as aforementioned.
[0148] At P22 on flatmounts from hyperoxia-exposed nontransplanted
animals, large peripheral areas with dilated radial vessels,
microaneurysms and hemorrhages, typical for retinal
neovascularization were observed. These were not present in retinas
from normoxia controls. Typical signs of peripheral retinal
neovascularization characterized by vascular tufts, dilated vessels
with abnormal tortuous courses on the inner surface of the
peripheral retina were evident. The epiretinal position of the
newly formed pathological vessels was evident in cryosections. IPE
cell transplants were localized in the peripheral areas of
flatmounts between the RPE layer and the neuronal retina. IPE cells
were identified by EGFP expression and their preserved hexagonal
shape. HC-Ad.PEDF transduced IPE implants prevented the formation
of pathological tortuous vessels in the areas of transplantation.
Immunofluorescence microscopy documented the expression of PEDF
from genetically modified IPE transplants. The overall effects of
the IPE transplants on the formation of pathological vessels are
summarized in. IPE transplants were located in the periphery where
typical pathological vessels are usually formed after exposure to
hyperoxia in this model (Smith et al., supra). To quantify the
neovascularization the length of dilated tortuous vessels on the
inner surface of the retina with diameters larger than 25 .mu.m
were measured on retinal areas of 800 000 .mu.m per eye. Evaluated
areas were localized up to 200 .mu.m from IPE transplants or in
corresponding peripheral regions of hyperoxia controls. Similar as
in normoxia controls, in the vicinity of PEDF expressing IPE cells
pathological epiretinal vessels were absent. Nontransduced IPE cell
transplants did not influence pathological vascularization compared
to hyperoxia controls. Additionally to the vessel length, the sizes
of vascularized areas (superficial vessels) were determined as an
independent paramenter of neovascularization. Close to PEDF
expressing IPE cells the sizes of vascularized areas were reduced
to 0.0153.+-.0.0081 .mu.m.sup.2 blood vessels per .mu.m.sup.2
tissue in comparison to 0.0546.+-.0.014 .mu.m.sup.2 blood
vessels/.mu.m.sup.2 tissue in retinal regions with transplanted
non-transduced IPE cells. Thus, subretinal transplantation of
PEDF-expressing IPE cells prevented neoangiogenesis in a model of
oxygen-induced neovascularization.
[0149] 13. Effects of HC-Ad Vector Mediated PEDF Expression from
Transplanted IPE Cells in a Model of Laser Induced Choroidal
Neovascularization (CNV)
[0150] IPE cells were subretinally transplantated into 4-5 months
old Long Evans rats. Six days later laser photocoagulation (100
.mu.m spot size, 0.1 sec duration, 150 mW) was performed using
blue-green setting of a coherent Novus 2000 argonlaser (Coherent
Inc., USA) close to the transplanted IPE cells (3 burns/eye). Ten
days after transplantation choroidal neovascularization was
evaluated on RPE-choroidal flatmounts by labeling endothelial cells
with a rat anti-mouse CD31 (PECAM-1) monoclonal antibody (1
.mu.g/ml; Becton Dickinson). The sizes of the areas occupied with
endothelial cells were determined by computer assisted morphometry.
The burns were classified: type I--100% occupied; type
II--completely free from endothelial cells; type III--both occupied
and endothelial cell free areas present.
[0151] Laser scars were identified on bright field images by
pigment clumping. Fluorescence micrography of a laser burn from
nontransplanted laser controls after labeling of the endothelial
cells with the anti-PECAM-1 antibody revealed that the burn (type
I) is completely (100%) occupied with newly formed endothelial
cells. IPE transplants were localized surrounding the laser
burnsig. The existence of EGFP expressing IPE cells (co-transduced
with HC.AdFK7 and HC-Ad.PEDF) close to 3 laser burns could be
prooven. These IPE transplants strongly expressed PEDF as detected
by staining with the penta-his antibody. The presence of PEDF
expressing IPE transplants close to the site of damage prevented
formation of new vessels within the laser burns. These laser burns
were classified as type II burns, completely free from endothelial
cells. Furthermore, PEDF expressing IPE cells (about 100)
surrounding the burn reduced the area of neovascularisation
compared to nontransplanted laser controls. In this case, newly
formed endothelial cells occupied 67% (47,400 .mu.m.sup.2) of the
whole area within the burn (70,500 .mu.m.sup.2). Endothelial cells
remained at the margin of the scar and did not proliferate or
migrate into the center of the burn. The areas free of endothelial
cells within the burn were localized close to transplanted IPE
cells. Table 2 summarizes the classification of laser burns based
on the size of CNV areas in the different experimental groups. Both
EGFP expressing IPE and nontransduced IPE transplants did not
influence laser induced CNV formation. Thus, subretinal
transplantation of PEDF-expressing IPE cells prevented
neoangiogenesis in a model of laser-induced neobascularization.
TABLE-US-00002 TABLE 2 Classification of laser burns based on the
sizes of CNV areas. Experimental Groups Close to Close to PEDF EGFP
Close to Non Laser expressig IPE expressig transduced Evaluated
controls cells IPE cells IPE cells laser burns n = 31 (%) n = 18
(%) n = 6 (%) n = 5 (%) I. Burns Completely 28 90.3 0 0 6 100 5 100
vascularized II. Burns Completely 0 0 6 33.3 0 0 0 0 free from new
vessels III. Burns with vascularized 3 9.7 12 66.6 0 0 0 0 and
non-vascularized areas
[0152] 14. Effects of HC-Ad Vector Mediated PEDF Expression from
Transplanted IPE Cells on Photoreceptor Rescue in a Model of
Retinal Degeneration
[0153] Two months after transplantation of IPE cells into 20 days
old RCS rats (Schraermeyer, U., Kociok, N. & Heimann, K. (1999)
Invest Ophthalmol Vis Sci 40, 1545-1556) the animals were
sacrificed and 5 .mu.m thick paraffin sections were prepared cut
through half of the eye. After H&E staining, rescue effects
were quantitated by determining the number of rows and size of
areas with preserved photoreceptor nuclei in all sections.
Rhodopsin expression was detected with rhodopsin antibody (5
.mu.g/ml, Leinco Technol.) and peroxidase-labeled secondary
antibody. The sizes of areas with preserved rhodopsin-containing
outer segments were determined using computer assisted
morphometry.
[0154] Only few photoreceptor nuclei were present close to IPE
cells which had been transduced with the EGFP-expressing HC-Ad.FK7
vector alone. However, several rows of photoreceptor nuclei were
preserved adjacent to the HC-Ad.PEDF vector transduced IPE
transplants. In these areas by immunofluorescence microscopy PEDF
was found to be expressed from HC-Ad.PEDF transduced IPE
transplants. In these sections the number of photoreceptor rows was
significantly higher (4.4+0.68, P<0.05) compared to HC-Ad.FK7
transduced (2.18+0.29) and nontransduced IPE transplants
(2.2+0.55). In addition, in sections with PEDF expressing cells
(n=9), the areas with preserved photoreceptor nuclei (more than 5
rows) had a length of 2.6+1.0 mm. Preserved rhodopsin was present
in rod outer segments of the survived photoreceptors in the
vicinity of PEDF expressing cells. In the same sections a rhodopsin
positive area of 53 947+24 656 .mu.m.sup.2 (n=5) measured. In
sections with HC-Ad.FK7 transduced or in nontransduced IPE
transplants rhodopsin staining was not detectable. Thus, subretinal
transplantation of PEDF-expressing IPE cells prevented
photoreceptor degeneration in a model of retinal degeneration.
[0155] 15. Effects of HC-Ad Vector Mediated PEDF Expression in RPE
Cells in a Model of Laser Induced Choroidal Neovascularization
[0156] 5.times.10.sup.6 infectious particles of HC-Ad.PEDF and
HC-Ad.FK7 in a final volume of 0.5 82 l were injected into the
subretinal space of 4-5 months old Long Evans rats. Thereafter,
laser photocoagulation (100 pm spot size, 0.1 sec duration, 150 mW)
was performed using blue-green setting of a coherent Novus 2000
argonlaser (Coherent Inc., USA) close to the site of injection (3
burns/eye). Ten days after transplantation choroidal
neovascularization was evaluated on RPE-choroidal flatmounts by
labeling endothelial cells with a rat anti-mouse CD31 (PECAM-1)
monoclonal antibody (1 .mu.g/ml; Becton Dickinson). The sizes of
the areas occupied with endothelial cells were determined by
computer assisted morphometry. The burns were classified as
indicated in table 2 (type I to III).
[0157] In such lesions areas free of endothelial cells were always
present. Expression of PEDF by the transplants was analyzed using
specific antibodies. In the controls (only laser-burns) only 10% of
the laser-burns lacked neovascularization, whereas in the other 90%
the areas of these scars were completely filled with endothelial
cells as detected by staining using a CD 31 antibody. All (100%)
scars were completely free of newly formed endothelial cells if
PEDF expressing RPE cells were surrounding the lesions at a
distance of 100 .mu.m or closer. In this model choroidal
neovascularization was detected by an immunological method in the
same laser lesion. Thus, injection of the PEDF-expressing HC-Ad
vector into the subretinal space prevented neoangiogenesis in a
model of laser-induced neovascularization.
[0158] 16. Effects of HC-Ad Vector Mediated PEDF Expression in RPE
Cells in a Model of Oxygen-Induced Retinal Neovascularization
[0159] In these experiments Wistar rats were used. Animals in the
normoxia group were maintained in room air throughout the
experiment. Rats in the hyperoxia groups were exposed to 75% oxygen
from postnatal day 7 (P7) to P12, then removed to room air and
immediately injected subretinaly with 5.times.10.sup.6 infectious
particles of HC-Ad.PEDF and HC-Ad.FK7 in a final volume of 0.5
.mu.l. At P 22, the animals were anesthetized and perfused with 50
mg/ml fluorescein isothiocyanate-dextran (Sigma, Deisenhofen,
Germany). The neovascularization was investigated on
retinal-RPE-choroidal flatmounts using a fluorescence microscope.
To quantify neovascularization the length of newly formed tortuous
blood vessels with diameters larger than 25 .mu.m on the inner
surface of peripheral retina was determined by computer assisted
morphometry (Openlab software; ImproVision, Inc., Lexington, USA).
Measurements of the vessel length were taken in areas of 800 000
.mu.m.sup.2 per eye located up to 200 .mu.m from the transfected
RPE cells or in corresponding peripheral regions of hyperoxia
controls. Furthermore, to confirm epiretinal localization of the
superficial pathological vessels 10 .mu.m serial frozen sections
were prepared cut through half of the eye.
[0160] At P22, on flatmounts from hyperoxia-exposed nontransplanted
animals, large peripheral areas with dilated radial vessels,
microaneurysms and hemorrhages, typical for retinal
neovascularization were observed. These were not present in retinas
from normoxia controls. Typical signs of peripheral retinal
neovascularization characterized by vascular tufts, dilated vessels
with abnormal tortuous courses on the inner surface of the
peripheral retina were evident. The epiretinal position of the
newly formed pathological vessels was evident in cryosections.
Transfected RPE cells were localized in the peripheral areas of
flatmounts. Transfected RPE cells were identified by EGFP
expression. HC-Ad.PEDF transduced RPE cells prevented the formation
of pathological tortuous vessels in the areas of transfection.
Immunofluorescence microscopy documented the expression of PEDF
from genetically modified RPE cells. Transduced RPE cells were
located in the periphery where typical pathological vessels are
usually formed after exposure to hyperoxia in this model. At a
distance of 200 .mu.m or larger from transfected RPE cells dilated
tortuous newly formed vessels on the inner surface of the retina
with diameters larger than 25 .mu.m were regularly present. Similar
as in normoxia controls, in the vicinity of PEDF expressing RPE
cells pathological epiretinal vessels were absent. Thus, oxygen
induced retinal neovascularization was locally inhibited by HC-Ad
vector mediated expression of PEDF in RPE cells.
[0161] 17. Statistics
[0162] All data are means.+-.SEM from at least three experiments
with 3 to 6 determinations. For multiple comparisons one-way
analysis of variance (ANOVA) was used with subsequent post hoc
analysis (Duncan test).
* * * * *