Note: Descriptions are shown in the official language in which they were submitted.
W~93/14790 ~1 2 ~ O PC~/~S93/00494
-- 1 -
A METHOD FOR ~RANSPLANT}NG CELL~ I~T~ T~E B~IN
AND ~HE~APE~TIC U~ T~EREFOR
CROS5-R2FERENC~ ~O RE~ATED APP~ICA~ION
The present application is a continuation-in~part
of U~S application Serial No. 07/599,802, filed
Oc~ober 19, 1~90, the entire contents o~ which are
~ hereby incorporated by reference.
BAC~G~OUND OF T~E INYBNTION
Field of the Invention
The invention in the field of neuroscie~ce and
medicine~relates to methods for implantation or
transplantation o~ cells into the mammalian brain,
useful in treating neurological di~orders.
Descril~tio1_of the Backqround Art
: The clinical management of numerous neurological
disorders has been fru~trated by the progressive
nature of degenerative, traumatic or destructi~e
neurological
diseases and the~ limited efficacy and the serious
side-e~fects ~f available pha~macological agents.
: Because many such diseases involve destruction of
specific "neuronal clusters" or brain regions, it has
been:hoped ~ha~: grafting of neural cells or neuron-
3~ like cells directly into the affected brain region
might provi~e~therapeutic be~efit. Cell transplant
~ : approaches h~e taken on a major emphasis in current
: ~: Parkinson9s~disea~se~esearch1 and may prove useful in
promoting recovery from other debi.lita~ing diseases of
the nervous system incIuding Huntington's disease,
Alzheimer's disease, severe seizure disorders
~: :including epilepsy, familial dysautonomia, as well as
~12~
WO93/14790 PCT/US93/0~49~ ~.
-- 2
injury or trauma to the nervous system. In addition,
the characterization of factors which influence
neurotransmitter phenotypic expression in cells placed
into the brain may lead to a better understanding of
normal processes and indicate means by which birth
defects resulting from aberrant phenotypic expression
can be therapeutically prevented or corrected.
Neurons or neuronal~like cells can be grafted into the
central nervous system (CNS), in particular/ into the
brain, either as solid tissue blocks or as dispersed
cells. However, to date, a number of problems of both
a technical and ethical nature ha~e plagued the
development of clinically feasible grafting
procedures.
Parkinson's disease results from a selective loss
of dopaminergic nigrostriatal neurons, resulting in a
- loss ~f input from the~subs~antia nigra to the
striatum. Solid grafts of tissues potentially capable
of producing dopamine~ such as adult adrenal m~dulla
and embryonic substantia nigra (SN)~ have been used
extensively for experimental ~rafting in rats and
primates tr~ated with 6-hydroxydopamine (6-O~DA) to
destroy dopaminergic cells (Dunnett, S.B. et al.,
Brain Res. 215: 147-161 (19~ id. 229:457-470
: (1981); Morisha, J.M. et al., Exp. Neurol. 84:643-654
(1~84); Perlow, M.J. et al., Science 204:643-647
1979j). Grafts of embryonic SN have als~ been used
as therapy for primates lesisned with the neurotoxin
1-methyl~4-phenyl~1,2,3,4 tetrahydropyridine (MPTP),
which~p~oduces a Parkinson's~like disease (Redmond,
D.E. et a1 , L ncet 8490:1125-27 (1986)).
Stenevi et al. (Brain Res. 114:1-20 ~1976) found
that the best results were obtained with fetal CNS
3S neur~ns which were placed next to a rich vascular
supply. In fact, a review of the literature reveals
that tissue from almost every area of the fetal brain
~can be successfully transplanted if care is taken with
2 l) ~, ,3 ,!J
WO93/14790 ~ PCT/U~93/0~94
- 3 - .
procedural details (see, for example, Olson, L. A. et
al~, In: Neural Transplants: Development and Function, ::
Sladek, J.R. et al., eds, Plenum Press, New York,
1984, pp. 125-165).
Embryonic tissue provides an excellent source of
cells which will differentiate in a foreign
environment and become integrated with the host
tissue. For example, grafts of embryonic SN into 6- :
OHDA treated rats have been shown to produ~e dopamine,
to reduce apomorphine- or amphetamine-induced
rotation, to alleviate sensory deficits and to make
- synapses in the hosk strlatum (Dunnett et al., Morisha
et al., Perlow et al., su~ra). Grafted neurons are
also spontaneously active, thus mimicking normal adult
SN neuro~s (Wuer~hele, S.M. et al., In:
atecholamines, Part B, (E. Usdin et al., eds.~, A~Ro
Liss, Inc., ~ew York, pp. 333~341).
In contrast~to success~ul grafting of fetal
neural tissue, mature CNS neurons have never been
found ts survive in transplants ~Stenevi, U. ~_31~, ;
Brain~Res. 1~4:~-20 (lg7~)). The rPason fetal CNS
neurons survive grafting procedures while adult
neurons do not, while uncertain, is probably related
to several factors. First~ :fetal neurons are less
affected by low oxygen levels than mature n~urons
(Jilek, L., In: -Develo~mental Neurobi~l~y, Himwich, ..
W.A., ed., C.C. Th~oma~ Publisher~ Springfield, IL,
1970, pp. 331-369), and grafting procedures
: 30` necessarily involve periods of anoxia until an
adequatç blood supply to the transplant is
established. Secondly, fetal neurons seem to survive
best when they are taken during a rapid growth phase
and before connections are established with target
tissues ~Boer, G.J. et al., Neuroscience l5:lo87-llos~
(1985)). Also, fetal tissue may be especially
responsi~e to growth (or "survival") factors which are
known to be present in the milieu of the damaged host
2-i 2~30
W093/14790 PCT/VS93/0049
-- 4
brain (Nieto-Sampedro, M. et al., Science 217:860-861
(1982); Proc. Natl. Acad. Sci. USA 81:6250-6254
(1984)). .
However, d~spite the promise of ~etal tissue and
cell transplants, the art has turned to alte.rnate
sources of donor tissues for transplantation because
of the ethical, moral, and legal problems attendant to
utilizing fetal tissue in human medicine. These
sources include ~eural and par~neural cells from organ
donors and cultured cell lines. (See, for example:
Gash, D.M. et al., In: Meura Gr ~ in the
~ammalian CNS, Bjorklund, A. et al., eds, Elsevier,
Am~terdam, 1985, pp. 595-603; Gash, D.M. et al.,
$cience 233:1420-22 (1986~).
Although early clinical experiments using the
grafting approach did not result in long-las~ing
effects, an initial report of one study appeared more
promisinq (Madrazo et al., Soc Neurosci. Abstr~
; 20 12:563 (1986); for an overview, see: Lieberman, A. et
al., Adv._Tech. Stand.~Neurosurq. 17:65 76 (1990),
which is hereby incorpor~ted by reference). However,
the surgical procedure used required craniotomy or
full "open brain" surgery in which a portion of
2S healthy striatum:was removed and replaced with
"chunks" of fetal adrenal gland. The kherapeutic
results obtained were~somewhat controversial.
However, both the need:for serious neurosurgery in a~
already debilitated population and the need to use
fetal tissue makes~this approach undesirable.
Inl:fu~ther human studies (Lieber~an, supra;
Lindvall, 0., J._Neurol. Neurosurq. Psychiat., ~989,
: Special Supplement, pp. 39-54; Bakay, R.A~E.,
euro urq. Clin. N. A er. 1:881-~95
(1990~), autologous grafts have been attempted to
rep~ace the need for fetal material. In this
procedure the patients first underwent initial
abdominal surgery~for the removal of a healthy adrenal
~ W 0 93/14790 2~.3(. ~ PCT/~S93/00494
gland. The patient then was subjected to similar
neurosur~ery as that for the fetal adrenal transplant.
The ~urgical morbidity-mortality for the combined
adrenal~ctomy/neuros~rgery was expectedly high. The
ultimate therapeutic result was claimed to be as high
as 30% but may have been as low as one patient in the
series of six. There was no evidence khat the adrenal
material transplanted into these patients survi.ved. ...
Several additional observations suggest that
grafting adrenal cells should be a viable approach. :.
Adrenal medullary cells are derived from the neural
crest and, like sympathetic ~eurons, yrow processes in
vivo or in vitro in response to nerve growth ~ctor
(NGF) (Uns~ ker, K. et al., Proc. Natl. Acad. Sci. USA
75:3498-3 .~ ~1978)). Solid grafts of adrenal medulla ~.
from young rats can survive in the brain of 6 OHDA
treated rats for at least 5 months, produce dopamine
and reduce apomorphine induced rotation (Dunnett et
2~ al-, su~ra; Freed, W.~. t al l Ann._Neurol~ 8:510 519
(1980); Freed, W.J. et al. Science 222:937-~39
(1983)). These observations suggest that given the
appropriate environment, adren~l medullary cells have
the potential for growing catecholamine-synthesizing
~ 25 fibers into brain tissue.
: The potential for neuronal dif~erentiation of
chromaffin cells is even better elucida~ed by grafts `:
of dissociated adrenal chromaffin cells which grow
processes when injected into rat striatum. Cultured
adrenal medullary cells also dif~erentiate into
neuronal-likeicells with processes when cultured in
the presence of NGF ~Unsicker, supra). This
remarkable plasticity of adrenal cells is observed not
vnly in morphology~, but also in neurotransmitter
phenotypic expresslon. An array of neuropeptides
including vasoactive intestinal peptide, as well as
the monoamine, serotonin, are co-localized with
catecholamines in adrenal medullary cells
;
t.~ L n~ CJ U
W093/~4790 P~T/U~3/0049
-- 6
(Schultzberg, M. Neurosci. 3:1169-1186 (1978);
Lundberg, J.M, Proc. Natl. Acad. Sci. USA 76:~079-4082
(1979)). Expression of these various phenotypes can
be modulated by extrinsic signals. For example,
enkephalin and VIP expression are increased following
denervation of the adrenal in vivo, by treatment of
animals with ni~otinic blockers or after maintaining
adrenal cells in vitro (Tischler, A.S. Life Sci.
37:1881-1886 ~1985)). These observations taken
together with those of okher studie5 demonstratlng
that neurons derived from the neural crest can ~Witch
phenotype during normal development (Bohn, M.C. e5
al., De~el~ Biol. 82:1-10 (1981); Jonakait, G.M. et
~S al. Devel. Biol. 88:288-296 (19~1)) or following
experimental manipulation of the micromilieu
(LeDouarin, N.M. Science 231: 1515-1522 (19863) .~.
suggest that, in the future~ it may be possible to
control the neurotransmitter phenotype expressed by
grafted cells either before and/or after grafting.
An additional advantage of grafting dissociated
cells compared to blocks o~ tissue is that the cells
can be precultured with various ~ubstances such as
growth factors prior to grafting or they can be co-
grafted wi~h other cells or substances which promote~peci~ic parameters~of di~ferentiation. Furthermore,
glial ~ells may have~specific regional effects and
produ~e neuronal grow~h factors (Barbin, G. et al.,
: Devel. Neurosci. 7::296-307 ~1985); Schurch-Rathgeb, Y.
et al., Nature 273:308-309 (1978); Unsi~ker, K. et al.
Proc~ Na~l. Bcad. Sci _USA 81:2242-2245 (1~84);
Whitaker-Azmitia, P.M. et al~, Brain Res. 497:80-85
: tl989)). This suggests that co-~ransplanting cells
providing the desired neurotransmitters along with
3S specific types of glia which produce glial-derived
factors, may promote neuronal growth and the desired
differentiation o grafted cells.
. .
212~530 ;
WO93/147~ PCT/US93/00~94
- 7 - :
The lack of success in treatment of Parkinson's
Disease by transplantation of adult cells into the
brain may be due in large part to the failure, for
unknown reasons, of transplanted cells to thrive when
placed into the brain. It is generally known (and
also seen in unpublished studies by the present
inventor) that c~lls directly injected into the brain
die within about a two to four week period (see, for
1~ example, Itakura, T. et al., J. Neurosur~. 68:955-95
(1988~)~ Despite the potential promise of using
growth ~actors t as discussed above, actual attempts ko
use growth factors to prolong the transplanted cells'
survival have met with extremely limited success.
There is an additional, undesired, csmplication i.n the
use of neuronal growth factors with chromaffin cei}ls.
: Such factors often act to l'transform" the chromaffin
cells from a more ~ndocrine phenotype into a neuronal
phenokype, wherein ~otal secretion of dopamine i~ much
lower. Because of the extremely low probability of
these transplanted cells establishing proper synaptic
connec~ions in~the brain, the factor-induced neuronal
transformation will ul~imately result in cells
incapable of secreting sufficient quantities of
: 25 dopamine~ ~ ~
Thus, while the~feasibility of the transplant
approach has been established experiméntally, this
approach is severely;limited by the need for the use
of feta~ tissue, which ~is of limited availability and
,
of great political consèquence. In essence,
transplantation of human ~etal tissue from aborted
pregnancies:has been~prohibited in the United States.
It would thus be of great benefit if simple, routine
and safe meth~ods for the successful transplantation of
adult tissue into the brain were available for the
treatment of debilitating neurological disease~ :
One potential approach to this problem has been
attempted by Aeblscher and his colleagues, who have
2 ~ G ~3 0
W093/14790 PCT/US93fO049
-- 8
successfully implanted into the brain selectively
permeable biocompatible polymer capsules encapsulating
fragments of neural tissue which appeared to sur~ive
S in this environment (A~bischer, P. et al., Brain Res.
448:364-368 (1988~; Winn, S.RD et al., J. Biomed Mater
Res. 23:31-44 (lg8~). The polymer capsules, consisting
of a permselectlve polyvinyl chlo~ide acrylic
copolymer XM-50, completely prevented the invasion of
the encapsulated tissue by host cells. Based on the
permeability, antibodies and viruses would be expected
to be excluded as well. When dopamine-releasing
polymer rods were encapsulated into such a
permselective polymer and implanted into denervated
1~ striatum in rats, alleviation of experimentally- ;
induced Parkinson disease symptoms was achieved (Winn
: S.R. et al., ~æ~ ~el~ 105:244-50 (1989).
Furthermore, US Patent 4,892,538 (Aebischer et al.,
issued 1/9/90) discloses a cell culture device for
implantation in a subject for delivery of a
: neurotransmi~:ter comprisin~ secreting cells within a
: semipermeable membrane which permits diffusion of the
neuro~ransmitter while excludiny viruses, antibodies
and other detrimental ~gents present in the external
2~ environment. ~The semlpermeable membrane is of an ;~
acrylic copolymer~ polyvinylidene fluoride, :~
polyurethane, polyalginate, cellulose acetal,
polysulphone, polyvinyl alcohol, polyacrylonitrile, or
their derivatives or mixtures and permits diffusion of
solute of up to SO k~:molecular weight. This device
was said to be useful in treatment of
neurotransmitter-deficient conditions, such as
Parkinson's disease,~by sustained, local delivery of
` neurotransmit~ers,~precursors, a~onists, fra~ments,
35 etc., to a target area, especia~ly the brain. The :;
device may be made retrievable so that the contents
may be renewed or supplemented, and the cells are
~iZ~C3~
WO~3/14790 PCT/US~3/0~4~4
_ g _
protected against immunological response and viral
infection.
M~RY OF THE INV~TION
The in~entors have made the unexpected discovery
that ~y first culturing cells in vitro on a support
matrixr such as 90 ~m diameter glaS5 beadS9 such cells
including mature CNS neurons or cultured cells, can be
successfully transplanted into the mammalian brain.
The beads, to which the cells adhere, are
stereotaxical~y injected into ~he recipient's brain.
Cells 50 in3ected retain their vi~bility and are thus
effectlvely transplanted. They show prolonged
survival~ and viability in vivo, even when transplanted
acrQss ~pecies barriers.
Adult adrenal chromaffin cells which secrete
dopamine, when transplant~d in this manner, are able
to corre~t a deficit in dopamine in a rat model of
Parkinson's disease~ These results indicate that, not
only do these:ce~ survive for prolonged periods, but
: they continue to ~unc~ion and have therapeutic
efficacy.
~ 25 It i5 an objective of the present invention to
:~ overcome the aforementioned~deficiencies in the prior
k.
The present~ inYention provides a method for
: ~ grafting a~c~ll in ~the brain of a mammalian subject
: ` 30 comprising allowing ~he cell to attach to the surface
of a support`matrix in vitro, preferably by culturing
the cell:with the matrix, such that the cell is not
en~apsulate by~the~matrix, and implanting the support
:makrix wit~ the~attached cell into the brain.
~$he method includes support matrices made of
~: glass or ~ther~silicon oxides, polystyrene,
polypropylene,~polyethylene, polyacrylamide,
polycarbonate, polypentene, acrylonitrile polymer,
:
~: :
h ~ J ~j ~
WOg3/14790 P~T/US93/00~9
- 10 .. '
nylon, amylases, gelatin, collagen, natural or
modified polysaccharides, including dextrans and
celluloses (e.g. nitrocellulose), hyaluronic acid,
extracellular matr.ix, agar, or magnetite. Preferred
support matrices are beads, porous or nonporous, in
particular microbeads ha~ing a diameter from about 90
to about 150 ~m. .
The method of the present invention may employ -
cells of many different types, pre~erably 2ither cells
o~ neural or paraneural origin, such as adrenal
chroma~fin cells. Also useful are cell lines grown in
vit o. Cells not of neural or paraneural origin, such
g as fibroblasts, may also be used following
transfection with DNA encoding a neuropeptide or an
enzyme or set of enzymes which results in productlon
of neurotransmitter, or a neuronal growth factor.
T~e present invention includes a method for
treating a neurologi-al disease in a subject which
comprises yra~ting an effective number of cells
capable of t~eating the disease according to the ab~ve
` methods. Diseases which can be treated according to
the present in~ention include t but are not li~ited to, .
~:Parkinson's disease, Alzheimer's disease, Huntington's
disease, epilepsy, familial dysautonomia, and
traumatic brain injury.
~.:
B~IE DESaRIPTI~ O~ THE DRAWTN~S
Figure 1 is a photomicrograph of a section of the :~
brain of a rat implan~ed with adult adrenomedullary
cells on a glass microbead. Thè section was fixed an
stained histochemically for ty~osine hydroxylase (TH)
using horse radish peroxidase. The darkly stained
areas indicate cells containing TH. The pattern of
s~aining shows a~needle tract entering the section on ~.
the right side. The large circular stained area in
the center represents the bulk of the implanted cells.
The injected cells form a "gland-like'l pattern, with
.
`:
2~
` W093/14790 PC~/VS93/~04~4
11 -- -
some extending back in-to the needle tract. (Thickness: :
20 microns; magnification: 100x)
Figure 2 is a graph showing effects of
kransplanting cells attached to glass beads on
apomorphine-induced turning behavior in rats. Rats
received either control beads, or beads to which
adrenal chromaffin cells or retina.l pi~ment epithelial ::
(RPE) cells had been attached.
l~ Figure 3 is a graph showing the ~urvi.~al of
cells, either transplanted alone or transplanted a~ter
lncubation with glass beads, transplanted into rat
brains.
Figure 4 is a photomicrograph showing a glass
bead surrounded by associated viable pigmented adrenal
chromaffin cells six months after transplantation into
a rat brain ~Enlargement: 400x).
: .,
DE8CRIPTION OF ~E PRBF~RRED_EMBODIMENTS
~0 ~he present invention is based on the unexpected
discovery by the inventor~ hat adl~lt cells or
cultured cells which cannc norma `y be tr~nsplanted
into a mammalian brain and survive, can be made to
survive by first attaching them to a support matrix.
A number of different cell types are useful for
the present in~sntion. Typically, a cell will be
s~lected based on~its ability to provide a missing
s~bstance to ~he reciplent brain. Missing substances
can be neurotransmitters or other neurally-active
molecules, the absence of which results in
neurologicaI disease:or dysfunction. It is important
- that the transplante~d cell not grow as a tumor once it
: is inserted into the recîpient.
The referen~es cited below are all incorporated
: 35 by reference ~erein,~whether specifically incorporated
or not~
: By the term:"neural or paraneural originl' is
in~ended a cell which is derived from the embryonic
: :
2~2~ 630
3/1479~ PCT/US93/0~94 .:.
- 12 -
neural crest. A preferred example of a cell of
paraneural origin is a adrenal medullary chromaffin
cell. The precursor cells to the ma~malian adrenal
medulla are o~ neural crest origin and possess the
potential to develop along either neuronal or
~ndocrine lines of differentiation tBohn, M~C. et al.,
1981l suPra, Devel. Biol. ~9:299-308 (1982); Unsicker,
K., DeveloP. Biol. 10~:259 268 (1985)). Chromaffin
cells from ~he rat, monkey, and human adrenal medulla,
when removed from adrenal cortical influences and
exposed to nerve growth factor (NGF~, chanye fro~ an ;`
endocrine to a neuronal phenotype ~Notter, M.E. et
al., Cell__Tiss~ Res. 244:69-70 (1986); Stromberg~
et al., Exp. Brain Res. 60:335-349 (1985); Unsicker,
K. et al^, ~978, suPra). When co grafted with
cerebral cortical or hippocampal tiSsue into the
anterior chamber of the rat eye, adrenal chromaffin
cells form nerve fibers which innervate the adjacent
co grafted brain tissue (Olson, Lo A. et al., Exp.
Neurol. 70414-426 (1980)). Another paraneural cell
type is a retinal pigment epithelium cell (Sony, M-K
et al., J ell. Phy_iol. 148:1~6-203 (1990)).
One source of donor cells are eskablished neural
cell lines. Many neuronal clones exist which have
been used extensively as model systems of development
since they are electrically active with appropriate
surface receptors,:specific neurotransmitters, synapse
;~ forming properties and the ability to diff~rentiate
3~ morphologica1Iy and biochemically into normal neurons.
; I Neural llin~s may express a tremendous amount of
genetic information which corresponds to the genetic
expression seen in CNS neurons. Such cells are
described in the following references: Kimhi, Y. et
al., Proc. Natl. Acad. Sci. USA 73:462-466 (1976); In:
Excitable Cells in Tissue Culture, ~elson, P~Go et
al., eds., Plenum Press, New Yo~k, 1977, pp. 173-2~5);
Prasad, K.M. et al., In: Con~rol of Proliferation_of
:
..
, 3 ~
WO93/14790 PCT/US93/004g4
- 13 -
Animal Cells, Clarkson, B. et al., eds., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1974,
pp. 58l-594); Puro, D.G. et al., Proc. Natl. Acad.
Sci. USA 73:3544-3548 (l976~; Notter, M.F. et al.,
Devel. Brain Res. 26:59-68 tl986); Schubert, D. et
al., Proc. Natl~ Acad. Sci. USA 67:247-254 (l970);
Kaplan, B.B. et al., In: Bas.ic and Clinical Aspects of
Molecular Neurobioloqy, Guffrida-Stella, A.M. et al.,
eds., Milano Fondozione International Manari~i,
1982)).
A major advantage of these cultured cells has .
been the potential to manipulate the environment, as
well as the cells themselves, in controlling the
phen~type and genotype.
For example, human neuroblastoma cells ~rom the
IMR 32 cell line can survive and express cholinergi~
markers in primate brain nine months after
transplantation (Gash, D.M. et al., Science 233:1420-
2 ~lg86)). These cells are preferably treated torender them morphologically and biochemically
differentiated in vitro and must be rendered
permanently amitotic before implantation, which
further ~id$ in their survival (Gash et al., supra;
Gupta, M. et al.,~ n ~ rg~ l9:2l-29 (l985~
Whereas untreated pheochromocytoma/neuroblastoma
ce:lls ~PCl2 cells) which grafted into the adult rat
striatum showed~no survival after two weeks in vivo
(Hefti, F. et al.j Brain Res.~ 348:283-288 (1985)),
treatment in vitro with NGF to stimulate neural cell
differe~tiation followed by exposure to antimi~otic
agents to inhibit cell proliferation, does not
deleteriously affect catecholamine neurotransmitter
expression nor Yiability and is of value for
: 35 stimu~ting performance following transplant.
Therefore, in ~ne embodiment of the present invention,
cell line cells are mvdulated in vitr~ with the
appropriate growth or differentiation factor and with
~ 1 h ~ ~ 3 ~
WO93/14790 PCT/US93/0049
an ~mitotic agent before transplantation in order to ::~
promote c~ll survival and prevent expres~ion of the
malignant potential. ~.:
It will be apparent to one of ordinary skill in
the art that examination of the neural cell surface of
clonal cells in vitro using con~entional methods will
permit the selection of appropriate cells for use
according to the present invention. During normal
development and dif~erentiation, the neuronal cell
sur~ace undergoes significant changes which account
fvr the migration, recognition and integration of
neurons in the CNS. For example, when mouse C1300
clonal~ neural cell lines are treat~d with antimitotic
15 agents or growth modulators such as prostaglandin E~ ;
and cyclic AMP~ new l~w molecular weight cell surface
glycopr~teins are produced (Bottenstein, J.E~, In:
Functionall~Di~ferentiated Cell Lines, Sato, G.H.,
ed., Alan R. Liss, New York, 1981). CNS surface
gangli~sides are induced which bind tetanus toxin
(Notter, M.F., 1986, upra~ while specific lectin ~:
binding glycoproteins appear which are similar to
normal central nervous system glycopxoteins.
Additionally, receptors for neurotransmitters and
: 25 neuropeptides~ on neuroblastoma cells can be modified
in a manner ~imilar to:cells found in the CNS (Costa, :~
L.G.:et_al., Biochem. Pharmacol. 9I:3407-3413 (1982).
~nother important source of potential graft
~ 30 ma~erial are cells engineered by somatic cell
: ' hy~ridization, a process which can immor~alize single
~ ~ neurons. Somatic~cell hybridizatiQn is a powerful
: ~ cell biologic tool used not only to generate cell
: lines with a variety of genotypes but to analyze the .;
mechanisms regulatlng the expression of vario~s
phenotypes of dif~erentiation. Fusing cells which
differ:in the expxession of specific genes allows for
the exploration of the mechanisms controlling gene
5 ~ !~
W~93/147gO - PCT/US93/~0~94
- 15 -
expression while chromosome alterations occur at rates
to generate genetically different cell lines. Hybrid
cells can be formed which retain the properties of
differentiated cell5. Hybrids derived from fusion of
sympathetic ganglia and neuroblastoma cells can
syn~hesize dopamine (Greene~ ~.A. et al., Proc Natl.
Acad. Sci USA 82:4923-4927 (1975) while brain cell
hybrids express choline acetyltr~nsferase ~Minna, J.D.
1~ et al., Genetics 79:373-383 (197$)). Therefore,
embryo~ic precursors to dopaminergic neurons from the
CNS can be fused with mîtotic cells to incorporate
both genomes into a sinyle one that loses extra
chromosomes over time and results in a new hybrid
line. It is within the skill of the art to produce
suzh hybrid neural or paraneural cells without undue
experimentation, screen them for the desired traits,
such as dvpamine secretion, and select those having
the best potential for tr~nsplankation.
Another source of cells for transplantatio~
according to the present invention is the adrenal
medulla. This neural crest-derived tissue has been
involved in clinical trials (see ~ackground) to treat
Parkinson's disease. Adult monkey adrenal medulla can ~-
be cultured in vitro ~or at least about threP weeks as
single cells (Notter, M.F. et al., Cell Tiss. Res.
: 244:69-76 (lg86)~. These cells respond to NGF by
: ~ p~enotypic alteration from the epithelioid, glandular
: morphology, to~a neuronal morphology in which cells
show extensive neuritic arborizations containing
microtu~ular arraysO~ This neuronal phenotype appears
: : to:be critical for long term survival of rat medullary
:
cells in host CNS~as well as their integration with
host tissue (Stromberg,:L. et al., su~ra).
Transplanted adrenal medulla tissue can correct
~unctional deficits resulting from nigrostriatal
dopamine depletion in rats (see, for example, Freed et
al., 1981, ~PE~)- Thisl however, is thought to ~e
W093/147~0 2 ~ PCT/US93/0049~ ;
- 16 - :
brought about by diffusion of dopamine from the ::~
transplant, a phenomenon that decreases three to six
months after transplantation. N~F treatment of the ::
transplanted cells induces fiber outgrowth from the
transplant into the host and induces a longer lasting
behavioral recovery (at least a year). Indeed,
without NGF treatment, few chromaffin cells
transplanted either to rat (Freed et al.., supra) or
rhesus monkey caudate nucleus (Morihisia, J.M. et al.,
EX~_NeUrQ1. 84:643-653 (1984) using prior art
echniques survive.
~`Similarly, retinal pigment epithelial cells
secrete dopamine and other factcrs and may be used for
brain implants according to the present invention (Li,
L. et al., Exp. E~e Res. 47:771-785 (1988); Lui, G.M.
~_31 , Proc. Int'l. 50c. EYe ResO 6:172 (1990); Li,
L. t al., Inv. Ophthal. Vis._Sci. 31(Suppl):59$
(1~90, abstr. 2915-13); Sheedlo, H.J. et al., ibid.,
~C abs~rO 2916-14; Fektorovich, E.G~ et al., ibid.
(abstr. 2~17-15); Song, M-K _t al., supra~O
: Cells txansplanted into the ~amm~lian brain
~: according to the pre~ent invention ha~e shown survival
in the absence of added growth factors. Howe~er, an
additional embodiment of the present invention is
directed to transplantatlon of cells attached to a
support matrix combined wi.th the treatment, either in
..
vitro prior to tr~nsplant, in vivo after transplant,
~r both, with~the appropriate growth/differentiation
factor.
Human adrenal medullary cells can be maintained
n vitro having the neuronal phenotype for at least
nine weeks (Notter, M.F. et al., Schmitt Neurol Sci.
~: ~Ym~, June 30, 1987, abstr.). NGF induces this
3~ conversion from the:glandular state. Adrenal
medullary cells co-cultured with C6 glioma cells
exhibit extensive ne~ritic ar~orization and intimate
contact with glloma cells. These astrocytic cells,
:
2 i ~ J lJ
W093/14790 PCT/US93/OV494
- 17 -
treated with antimitotic agents to inhibit mitosis,
are known to produce a growth factor similar to NGF
which sustains sympathetic neurons in vitro (Barde,
5 Y~ YE~ 274:818 (197B))~ Grafted glial cells may
play an important role in ~unctional recvvery of
neurons ~nd may be an important source of trophic
factors (Doering, L.C. et al~, J. Neurolog. Sc1
63:183-196 (1984~; Gumple, J. et al., Neurosci. Lett.
?0 37: 307-311 (19843). Therefore, a~other embodiment of
the present invention involves ~o-culture of neural or
paraneural cells with:glial cells, their co-incubation
witX a support matrix, followed by implan~ation of ~he
support matrix carrying both cell types.
In additional embodiments of the present
inventio~J, cells which are not of neural or paraneural
origin, but which have been altered to produce a
substance of neurological interest, ~re used.
preferred cell type is a human for~skin ~i~roblast
which is easily obtained ~nd cultured, and survives
: upon transplantation into the rat brain using the
: : methods o~ the present in~ention (see Example III~.
~or use in the present invention, the cells are
ge~etically altered,~using methods known in the art,
to express neuronal growth factors, n~urotransmit~ers,
neuropeptides,~or~enzymes invGlved in bxain
metabolism. (See, for exampIe, Ga~e, F~H. et al.,
Neur~scien~e _ :795-807 (1987); Rosenberg, ~.B. Pt
al., Science 242~:~1575-1578 (1988); Shimohama, S. ek
3~ al., ol. Brain_Res. 5:271-278 (1989); which are
lhereby incorporated~by re~erence).
: Standard reference works setting forth the
general principles~:o~ recombinant DNA technology and ~;
~ ~ cell biologyO which are hereby incorporated by
:~ ~: 35 reference, include Watson, J.D., et al., Molecular
Biologv of the Gene, Volumes I and II,
Benjamin/CUmmings Publishing Co., Inc~, Menlo Park, CA
(1987); Darnell, J.E. et al., Molecular Cell Bioloq~,
~'
2 ~ S 3 ~
WO 93/14790 PCr/US93/0049
-- 18 ~
Scientif ic American Books , Inc ., New York , NY ( 198 6 ~;
Lewin, B.M., Genes II , John Wiley & Sons, New York, NY
(1985); Old, R.W. et al., PrinciPles of Gene
Manipulati~n: An Introduction to Genetic En~ineerinq,
2nd Ed., University of Cali~ornia Press, Berke~ey, CA
(1981); Maniatis T. et al. (Molecular Cloninq: A
LaboratorY Manual, Cold Spring Harbor Press, Cold
Spring Harbor, NY ~1982)); Sambrook, J. et al.
10 ( olecular Cloninq: A Laboratory Manual, 2nd Edition, :~
Cold Spring Harbor Press, Cold Spring Harb~r, NY
.(1989) and Albers, ~ et al., Molecular Bî~loqY of the
Cell, 2nd Ed., Garland Publishing, Inc., New York, NY
89)-
The recombinant DNA molecules useful for the
methods~of the present invention can be produced
through any of a Yariety of means, such as, for
example, DNA or RNA synthesis, or more preferably, by
application of recombinant DNA techniques. Techniques
zO ~or synthesizing such moIecules are disclosed by, for
example, Wu, R., et al. (Proq. Nucl~ Acid. Res. Molec.
Biol. 21:101-141 (1978)). Procedures for constructing
recombin~nt molecules are disclosed in detail by
Sambrook et al., supra).
Typicallyj the gene or genes of interest are
cloned from a library of expression vectors which has
been prepared by r-loning DNA or, more preferably, cDNA
(from a cell capable of expressing the gene) into an
expression vector. The library is then screened for
members capa~le of expressing the gene product of
~ I interest, such as a neurotransmitter-synthesizing
enzyme, using antibody binding with an antibody
specific for the~ene product. DNA, or more
preferably cDNA, is extracted and purified from a cell
which is capable of expressing the gene product of
interest~ The purified cDNA is fragmentized (by
shearing, endonuclease digestion, etc.) to produce a
pool of DNA or cDNA fragments. DNA or cDNA fragments
; 3 3
W093/14790 PCT/US93/00~94
-- 19 --
from this pool are then cloned into an expression
vector in order to produce a library of expression
vectors whose members each contain a unique cloned DNA
or cDNA ~ragment.
- An l'expression vector" is a vector which (due to
the presence o~ appropriate transcriptional and/or
translational control sequences) is capable of
expressing a DNA (or- cDNA) molecule which has been
cloned into the vector and of thereby producing a
polypeptide or prot~in. Expression of the cloned
sequences occurs when the expre~sion vestor is
introduced into an appropriate host cell~ An
appropriate mammalian host cell would be any mammalian
cell capable of expressing the cloned sequences.
Pro edures for p~eparing cDNA and for producing a
genomic library are disclosed by Sambroo~. et al.
( u~ra).
A DNA sequence encoding a product or products the
~xpression of which is desired for the present
invention may be recombined with Yector DNA in
accordance with conventional techniques, including
blunt-ended or~staggered-ended termini for ligation,
restriction enzyme digestion to provide ~ppropriate
: 25 termini, filling in of cohesive ends as appropriate,
: alkaline phosphatase treatment to a~oid undesirable
joining,~and ligation~with appropriate ligases.
Techni~ues for such manipulations are disclosed by
Sambrook et al~, supra, and are well known in the art.
3~ A nucleic:acid molecule, such as ~N~, is said to
be "capable of expressing" a polypeptide if it
contains nucleotide sequences which con~ain
transcriptional and translational regulatory
information and such sequences are 'l~perably linked"
~o nucleotlde sequences which encode the polypeptide.
An operable linkage is a linkage in which the
regulatory DNA sequences and the DNA sequence sought
to:be expressed are connected in such a way as to
,:
2i~G.3~ ~
WO93/14790 PCT~US93/0049a
- 20 -
permit gene expression. The precise nature of the
regulatory regions needed for gene expression may vary
from organism to organism, but shall in general
include a promoter region which, in prokaryotes,
contains both the promoter (which directs the
initiation of RNA transcription~ as well as the DNA
sequences which, when transcribed into ~NA, will
signal the initiation of protein synthesis. Such .
regions will normally include those 5'-non-coding
sequences involved with initiation of transcription .;
and translation, such as the TATA box, capping
seq-uence, CAAT sequence, and the like. :~
If desired, the non-coding region 3' to the gene
sequence coding for the protein may be obt~ined by the
above-described methods. This region may be retained
: for its transcriptional termination regulatory
sequences, such as termination and poly~denylakion.
Thus, by retaining the 3'-region naturally contiguous
: 20 to the DNA ~equence coding for the protein, the
: transcriptional termination signals may be provided.
Where the transcriptional termination signals are not
satisfactorily functional in the expression host cell,
~hen a 3' reglon functional in the host cell may be
: ~5- substituted.
Two DNA s quenoes (such as a promoter region
s~quence and a coding sequence) are said to be
~perably link d if the nature of the linkage between
;~ the~two DNA s:equences does not (l) result in the
intr~duction of a frame-shift mutation, (2) interfere
with th,e ability of the promoter region sequence'to` ` .
direct the transcription of:the coding sequence, or
(3j interfere with:~the:ability:of the coding se~uence
: to~e ~ranscribed~by the promoter region sequence. A
promoter regiQn would ~e operably linked to a DNA
.:
- sequence if the promoter were capable of effecting ~.
transcriptlon of:that DNA sequence. Thus, to express
:
:~ W093/~479~ ' 3 PCT/US93/00494
- 21 -
the pro~ein, transcriptional and translatlonal signals
recognized by the host cell are necessary.
A promoter is a double-stranded DNA or RNA
molecule which is capable of binding RNA polymerase
and promoting the transcription of an "operably
linked" nucleic acid se~uence. As used herein, a
"promoter sequence" is ~he sequence of the promoter
which is found on that stran~ of the DMA or RNA which
1~ is transcribed by the RNA polymerase. A "promoter
sequence complement" is a nucleic acid molecule whose
sequence is the complement of a "promoter sequence."
Hence, upon extension of a primer DN~ or ~NA adjacent
ko a single-stranded "promoter sequence complement"
or, of a "promoter sequence," a double-stranded
molecule is created which will contain a functional
promoter, iP tha~ extens}on proceeds towards the
"promoter sequence" or the "promoter se~uence
complement.l' This functional pro~oter will direct the
transcription of a nucleic acid molecule which is
operably linked to that strand of the double-stranded
molecule which contains ~he "promoter sequence" ~and
not that strand ~f the molecule which contains the :~
I'promoter sequence complement'~
:: 25 The promoter sequences useful for producing cells
for the present invention may be either eukaryotic or
viral. Suitable promoters are repressible, or, more
: ~ preferably, constitutive. ~seful eukaryotic promoters
include the p~omoter of the mouse metallothio~ein I
gene (~amer, D~., et al., J.~Mol. API~L~D~ 1:273_288
(1982~); the TK promoter of Herpes virus (McKnight,
S.l 5~1~ 31:355-3G5~(1982));~the SV40 early promoter
~ (Benoist, C.~ et al. r Nature (London~ 290:304-310
: (19Sl)). A preferrP.d promoter, in particular for
human fibroblasts, i5 the collagen promoter (Prockop,
D.~. et al., N. Enq. J. Med 30i:13-23l 77 85 (1979);
Eyre, D.R., Science 207:1315-1322 (1980); Martin, G.R.
:
7 ~
WOg3/14790 PCT/U~93/0049
- 22 - :
et al., Trends Bioch. Sci. 10:285-2~7 ~19~5); which
references are hereby incorporated by reference).
Also intended within the scope of the present -~:
in~ention are cells attached to, or m.ixed with~ a
support matrix, according to the invention, which are
frozen and stored in a frozen state using methods well
known in the art. Following thawing, the matrix~bound :~
~ells are implanted into a recipient brain.
The cPlls useful in the methods of the present
in~ention may be xenogeneic (- heterologous, i.e., ;~
derived from a species different from the recipient),
al~ogeneic (= homologous, iOeO, derived from a
genetically different member o* the same species as
the reci~ient) or autologous, wherein the recipient
also serves as the donor.
The number of cells needed to achieve the
purposes of the present invention is ~ariable .:
depending on the SiZP, age, weight of the subject, the
~O nature of the underlying disease, other concurrent
: therapies, ~nd the like~ This can be determine~ by
one of skill in the art without undue experimentation.
~: In an adult human, an effective number of cells
a~tached to a support matrix are in the range of about
1 x 103 to about 1 x 107 cells, more preferably about 5
x ~n3 to about 1 x 106 cells. Alternatively, the
effective amount of transplanted cells can be :~
~: determined in~terms o~f mass of cells added to a volume
of beads, for example 40 mg of c~ll mass per ml of
bead5. : :
I Materials of which the support matrix can be
:~ comprised include th~ose materials to which cells
adhere ~ollowing in vitro incubation, and on which
: cells can grow,~and which can ~e implanted into a
mammalian brain without producing a toxic reaction, or
an in~lammatory or gliosis reaction which would
destroy the implanted cells or otherwise interfere
with their biological or therapeutic activity. Such
:
2i2~
W093/t~7~0 ~ PCT/USg3/0~494
- 23 -
materials may be synthetic or natural chemical
substances or substances having a biological origin.
The matrix materials include, but are not limited to,
glass and o~her silicon oxides, polystyrene,
polypropylene, pol~ethylene, polyvinylidene fluoride,
polyurethane, polyalginate, polysulphone, polyvinyl
alcohol, acrylonitrile polymers, polyacrylamide,
polycarbonate, polypentene, nylon, amylases, gelatin,
collagen, natural and modified polysaccharidest
including dextrans and celluloses (e~g.
nitrocellulose), agar, and magnetite. Either
.
resorbable or non-resorbable materials may be used.
Also intended are extracellular matrix materials,
which are well-known in the art (see below~.
Extracellular matrix materials may be obt~ined
commercially or prepared by growing cells which
secrete such a matrix, removing the ~ecreting cells, :~
and allowing the cells which are to be transplanted to
interact with and adhere to the matrix.
The support matrix of the present invention is
distinguished~from that described by Aebischer and his
colleagues (see above~ in that, according to the
present invention, the ce.lls are attached to or
: 25 coating the surface of the support; they are not
encapsulated within a closed compartment, where their
survival would be questionable given the exclusion
capacity of the~disclosed encapsulatinq supports.
Furthermore, the support matrix of the present
invention presents no requirement that the material
have p~rticular permeability~properties, such aslthe
selective permea~ility of the Aebischer deviGes which
~: allow low molecular weight substances ~o cross but
:~ ex~lude larger molecules (~50 kD~.
: ~ 3S When a~tached to the support according to the
~: present invention,:the cells used for transplantation
are generally on the "outer surface" of the support.
.
The support may be solid or~porous. However, even in
Zi~33D
WO93/14790 PCT/US93~004~ .
- 24 - .:
a porous support, the cells are in direct contact with
the external m.ilieu without an intervening membrane or
other barrier. Thus, according to the present
invention, the cells are considered to be on the
"outer surface" of the support even though the surface
to which they adhere may be in ~he form of internal
folds or convolutions of the porous support materi~l
which are not at the exterior o~ the particle or bead
its~lf.
The configuration of the support is preferably
spherical, as in a bead, but may be cylindrical,
elliptical, a flat sheet or strip, a needle or pin
shape, and the like. A pre~erred form of support
matrix is a glass bead. Another preferred bead is a
polystyrene bead. Bead sizes may range from aboùt 10
~m to 1 cm in diameter, pre~erably from about 90 to
about 150 ~m. For a description of various
microcarrier beads, see, for Pxample, Fisher Bîotech
Sour e 87-88, Fisher Scienti~i¢ Co., 1987, pp. 72 75;
Si~a Cell Culture Cataloq, Siyma Chemical Co~, St.
Louis, 1991, pp. 162-163; Ventrex PrQduct Catalo~,
Ventrex Laboratories, 1989; these references are
hereby incorporated by reference. The upper limit on
: 25 the bead size is dicta~ed by the bead's stimulation of ~`
undesired host reactions such as gliosis, which may :
interfere with the function of the transplan~ed cells
or cause da~age to the surrounding brain tissue. Su h
limitations are readily determinable by one of skill
in the art.
To improve cell adhesion, survival and function,
the solid matrix may optionally be coated on its
external surface with factors known in the art to
: promote cell adhesion, growth or survival~ Such
~actors include cell adhesion molecules, extracellular
matrix, such as, for example, fibronectin, laminin,
~ ~ collagen, elastin, gycosaminoglycans, or proteoglycans
: (see: Albers,~ B. ~ , pp. 8~2-834) or growth
W~93/14790 PCT/US93/00494
- ~5 -
factors, such as, for example, NGF. Alternatively, if
the solid matrix to which the implanted cells are
attached is constructed of porous material, the growth-
or survival-promoting factor or factors may be
incorporated into the matrix material, from which they
would be slowly released after implantation in vivo.
In an alternate embodiment of the present
invention, cells growing on, or mi~ed with, resorbable
matrices, such as, for example, collagen, can be
lmplanted in sites of neurological interest other than ~:
the brai~i, in order to promote neuronal regrowth or ~:~
recovery. For example, cells attac~ed to the matrix
D of the invention may be implanted lnto the spinal
cord, or placed in, or adjacent to, the optic nerve.
Th~ matrix material on which the cells to be ::
implanted grow, or with which the cells are mixed, may
be an endogenous product of the implanted cells
themselves. Thus, for example, the matrix material ::.
may ~e extracellular matrix or basement membrane
material which is produced and secreted by the ~rery ~`
cells to be implanted.
: : The meth~ds of the present invention are useful
for treating a number of human neurological dis~ase. :~
25 Parkinson's Disease can be treated according to the
: present invention by implanting dopamine-producing
:~ cells in the~recipient's striatum. Alzheimer's
~: disea5e involves a deficit in cholinergic cells in the
nucleus ba~alis. Thus, according to the invention, a ~.
3~ subject having Alzheimer's disease or at ri~k therefor
may be implanted wlth cells pro!ducing acetylcholine. ~;
Huntington's disease involves a gross wasting of s
: the head of the caudate nucleus and putamen, usually ;;
accompanied:by moderate disease of the gyrus. A
subject suffering from Huntington's disease can be
~ t~eated:by implanting cells producing the
: : neurotransmit~ rs gamma amino butyrlc acid (GABA),
,
acetylcholine, or a mixture thereof. According to the ~.
~;'
,.,
~ ~L r ~ ; s3 0
WO93/14790 PCT/U~g3tOO~
- 26
present invention, the support matrix material to
which such cells are attached is preferably implanted
into the caudate and putamen.
Epilep~y is not truly a single disease but rather
is a symptom produced by an underlying abnormality.
One skilled in the art will appreciate that each .
epileptic subject will have damage or epileptic foci
which are unique for the individual. Such foci can be
localized using a combination of diagnostic methods
well-known in the art, including
electroencephalography, computerized axial tomography
~nd magnetic resonance imaging. A patient
suffering from epilepsy can be treated according to
the present invention by implanting the support matrix
material to which GABA-producing cells are attached
: into the affected site. Since blockers of glutamate
receptors and NMDA receptors in the brain have been
used to ~ontrol experimen~al epilepsy, cells producing
molecules which block excitatory amino acid pathways
may he used according to the invention. Thus
implantation of cells which have been modified as
described herein to produce polyamines t such as
spermidine~ in larger than normal quantities may be
useful for treating epilepsy,
The methods of the present invention are intended
for use with any~ mammal which may experience the
beneficial effects of the methods of the in~ention.
Foremost among such mammals are humans, although the
invent:ion is not intended to be so limited, and is
~also applicable~to veterinary uses.
Havlng now~generally described the in~ention, the
same will be more readily understood through reference
to the following examples which are provided by way of
illustration, and are not intended to be limiting of
the present invention, unless specified.
:: :
2i~.)6 1ù
WO g3/14790 Pcr/uss3/004s4
- 27 -
. .
EX~MPLE I
Adrenal chromaffin cells from adult rats were
prepared according to known methods (see Example IV,
below; see also, Inoue, M. et al., A. J. PhYsiol. 257
(Cell Physiol.):C906-912 (~989))~ After sacrifice by
nembutal anesthesia followed by decapitation adrenal
glands were removed. The medulla was freed of
capsular and cortical material by care~ul
microdissectîon. The tissue was cut into 2 or 3
pieces and incubated for 30 minutes with 0.2% ..
collage~ase in calcium-free balanced salt solution ~t,
co~taining l~O~mM NaCl, 5 mM KCl, 5.2 mM MgCl2, 5 mM
HEPES, lO mM D-glucose, at pH 7.4. During the enzy~e
treatment, the preparation was shaken gently by
bubbling through 99-9% 2- After digestion, the tissue
was washed three or four times with the above salt
solution and then triturated gently in a pasteur
pipet, ~
The dissociated~chromaffin cells were transferred
to:cell culture flasks~ containing serum free medium
(Ventrex PC 1) and;we:re cultured overnight at 37C in
a 5~ C2 atmosphere. Under these conditions t the cells
re~ain ~heir viability for no more than a few days.
Sterilized glass~bead:s (Ventrex, 90 micron) were added
to the culture fl~ask. Tha cells attached to the beads
and essentially formed a monolayer on the beads.
~ .
2~ 630
WOg3/14790 PCr~US~3/00~94
- 28 -
Within Z4 hrs. of preparation in vitro, l ~o 5 ~l
aliquots of the cell-carrying bead suspension were
injected into the brains of anesthetized adult rats.
Later a~alysis, at times when the cells would not have
survived in culture, the cells were found to be viable
in vivo in the site surrounding the injection, (see
Figure l) as determined by immunocytochemical staining
for tyrosine hydroxy~ase according to the Weiner
procedure (see: Kilpatrick, D.L. et al., J. Neurochem.
35:679 (1980); HoXfelt, R. et al., Handbook of
S _Hemical_Neuroanatomy, Elsevier Science Publisheris,
Amsterdam, lg85). The gla~s beads produced no
necrotic damaqe above that typical of the injection
itself.
EXAM?~E II
:
:Adrenal medullae were removed from adult guine~
: : 25
pigs~according to me~hods ~escribed in Examples I and
IV and subjec~ed~to:the procedure described in Example
The cells~on glass microbeads were injected into
::
rat brain. The~cells survived for a peri~d of at
least 21 days in a completely healthy state. No signs
of lmmunologica~l rejection were evident.
EXAMPLE III
~; :
.
:
WO93/14790 2~ )3~, Pcr/Us93/01)494
~ 29 ~
Cultured human foreskin fibroblasts were attached
to glass microbeads as described above and injected
into the rat brain. The cells were localized by
immunof-uorescence using human IgG which selectively
bound to the fibroblasts, ~ollowed by a fluorescein-
conjugated goat anti-human Ig~ anti~ody. The
implanted human cells survived ~or a period of at
least 21 days in a completely healthy state. No signs
of~immunological rejection were evident.
; 15 E~AMPLE IV
Adrenal ~hromaffin Cells Transplanted on Microcarriers
Correct a Lesion of Niarostriat~l Dopaminer~ic Cells
:,
Studies were conducted to test the ~bility of
: 2~ adrenal chroma~fin cells attached ko ;nicrocarriers,
shown above to su~vive for prolonged periods in the
:
brain, to carry out a physiological function, i r e,
dopamine secretion, and corrert a lesion in the
dopamine ~ystem.
25~ A standard animal model for Parkinsonls disease
was used. Nigrostrlatal dopaminergic neurons are
~ :: destr~yed locally and selecti~ely by stereotaxic
: ~ in~ection:of 6-OHDA. Subsequent injection of
- apomorphine,~a dopaminérgic agonist, induces
rota~ional or ~urning bPhavior in lesione~ but not in
normal rats. Therapy of the Parkinson-like symptoms
; is:assessed by~a~decrease in this turning behavior.
~:~ 35 ~ Male Sprague-Dawley rats, obtained from Tacona at
a weight of 120-150 grams, were maintained in the Bery
~: Institute at NYU Medical Center~, an approved animal
:: : : : :
:: :
2~2.^~b~
WO 93/14790 PCI/US~3/004194
- 30 -
~..
facility, and were provided with food and water ad
libitum. Animals were lesioned when they had attained
a weight of 180 to 20~ grams.
Destruction of 5ubstantia Ni~ra ~SN)
A lesion was induced in the SN by stereotaxic
application of the selective neurotoxic agent, 6~0HDA~
Rats were anesthetized using sodium pentobarbital, 45
mgJkg, i.p. Animals were placed in a Kop~-type
stereotaxic instrument. For lesioniny the right side,
~he following coordinates were used:
Rostral Caudal: ~4.~mm; Dorsal Ventral: -8 r lmm;
Lateral Medial: -1., -2.0mm; Jaw Bar: -3.3mm.
After t~e induction of Nembutal anesthesia, the
rat's h~ad was shaved using an Oster clipper with
surgical blade. The rat was then placed into the
stereotaxic apparatus and its head approximately
positioned. A 3/4 inch rostral-caudal incision was
made midline in the cranium. The ~kull was gently~
: scraped using a #10 scalpel blade to remove the
pericranial membranes. The bregma was located and the ~:
injection needle was plac~d directly o~er this
landmark. Stereotaxic coordinates were then correc~ed
for the individual animal using the position directly
: over the bregma as the zero-value coordinates. For a
: right-sidP unilateral lesion of the SN, the rostral- .
: ~ caudal coordinate wa~ subtracted from the zeroed value
and the latera~l-medial coordin~te was subtracted from
the zeroed value.
~! These correGtion; pl~aced the needle directly above the
SN. ~ :
: :: The:needle:wa~ then lowered to touch the skull
~: ~ and the point of contact was marked with a pencil.
The neadle was then raîsed out of the way and a hole
was dril;ed through the cranium usîng a Vremel Flex-
~: shaft drill equipped wîth a #253 handpiece and a #6
: ~ dental bit. The needle was repositioned above the
:~ :
h ~ h i~
WO 93/1~790 P~T/US93/004g4
- 31 - :~
opening in the s~ull and then lowered into the hole so
that it sat just inside the entrance. The stereotaxic
coordinate of this position was recorded as the
dorsal-ventral z~ro value with the needle at the dura
mater. l~he dorsal-ventral value was then subtracted
from this value to give the desired final coordinate.
The needle was then lowered to the corrected dorsal
ventral coordinate for the injection.
The 6 OHDA hydxobromide in a vehicle of isotonic
~0.9%) saline con~aining 0.2 mg/ml ascorbic acid was
used at a concentra~ion of 8 ~g/4 ~l. The ayent was
prepared immediately prior to injection to minimize
oxidakive deyradation of this drug and was kept on ice
until it was used. The drug was injected using a
perfusion pump attached to a 23 gau~e needle at a rate
of l ~l/min until a total volume of 4 ~1 had been ..
injected~ Prior to removal, the needle was allowed to
remain in place for an additi~nal 5 minutes to allow
~0 for i~filtration of the drug into the desired area.
The surgical site was closed usiny Clay-~dams 9mm
wound clips, and the rats were allowed to recover.
After the surgery, the animal was placed into a warm
container and allowed to recover from the anesthesia
25 prior to its return;to its normal housing in Berg ..
` Institute. By the next day, the rats had regai~ed
normal beha~ior~
: BQhavioral Testinq
To assess the effects of the SN lesion, animals
! were the apomorphine-induced rotational movement
paradigm was used. Rats were injected suhcutaneQusly
with apomorphine; Q.25 mg/kg, and were observed for
signs of dopamine-induced stereotypical behavior:
licking, ptosis of the eyelids r gnawing, etc. The
time of onset was recorded. The animal was observed
for the initiation of rotational movement, and the
time was recorded. Rats lesioned in their SN with 6-
3 ~ ~
W~93/147gO PCT/US93/0049
- 32 -
OHDA lesioned rat are known to exhibit tight
contr~lateral rotations when injected with
apomorphine.
Five minutes after apomorphine administration,
~uantitation of the rotational movement wa5 initiated.
Only complete 360 turns were recorded. Quantitation
of movement continued for 25 minutes. Rats were
scored on the basis of turns per minute. Rats
1~ exhibiting less than 7 apomorphine-induced turns per
minute were rejected ~rom the study. Rats were tested
at weekly intervals until the rotational beha~ior
s~abilized, usually at three to four weeks post-
injection, and remained stable for an additional two
testing periods. In general, rats exhibited 9 to 10
apomorp~ine-induced turns per minute at this time, and
were ready f~r transplant studies. ::
Pre~aration Qf Chromaf~in_Cells
Donor rats were anesthetized using sodium
pentobarbital, 45 m~kg, i.p.. A~ter the induction of
: anesthesia, ~he rat was placed on its back in a
sterile surgical field and the adrenal gland was
removed under sterile conditions. An ~T~-shaped
2 5 incision was mads for the length of the abdominal
cavity and the skin~flaps were pulled back. The
peritoneum was then opened and the kidneys were
exposed. The kidney and its associated adrenal gland
: was freed from thP peri~enaI fat pad by blunt
dissection. The kidney/adrenal gland was removed from
i the animal and placed into a cul~ure dish containing
~C-l medium (Ventrex). The capsule was opened and the
entire adrenal gland was carefully dissected free of
: the ~idney.
The free adrenal was transferred to a 60mm
: culture dish containing Ventrex PC-1 supplemented
medium. The adrenal was minced as finely as possible
using a scalpel. The medium was carefully decanted
:
~i2~ ,3a
W093/14790 PCT/U$93/~0494
- 33 - ~;
"'"
and replaced with 10 ml sterile complete PC-1 medium ~:
containing 0.1~ Trypsin/0.2% collagenase. The adrenal
was incubated in this solution for 30 mins. in a 37C
5 incubator in an atmosphere of 5% carbon dioxide in -~
air.
Followîng this incubation, the cell mixture was
made hom~geneous by repeatedly drawing it up and down
through a sterile lO ml plastic pipett~ and was then
passed through a lO0 ~m cell sieve. The clean
filtrate was placed in a sterile tube and centrifuged
at 200 x g for 5 min. The supernatant was decanted
and the cell-containing pellet was resuspended in :~
complete PC-1 medium, which included the "~erum-like"
lS factors, (3-4 ml if cells were to be cultured on .:
;microca~riers). The mixture was shaken gently and
placed in a 37C incubatvr.
;,
Culturinq the_~ells on Microcarriers
~ 20 Ventrex glass microcarriers (Ventreglas, 90-125
: ~m) were sterilized by p}acing the beads in 10 ml of
sterile distilled water per 1 gram of b ads and :~
heating to 121~C;for ~5 min. The solution was allowed
to cool to room temperature and the water was
2~ discarded. The microcarriers were then suspended into
a small volume of cultur.e medium and allowed to stand
for 30 mins. The medium was removed and the beads
~ ~ .
~ pproximately 0.21 g3 were added to the previously
; ;~descri:bed cell pr~epara~ion. The resulting mixture was
shaken for 2 hours and an additlonal 4 ml of complete
:PC-l was a~ded. The culture flask was then incubated
: overnight at; 37GC in a 5% ~C2 atmosphere to allow the ~.
cells to adhere to the microcarriers. ~
:~ Additional cell types which have been testing by ~.
: 35 the present i~ventors using this methodology include
: human retinal pigment epithelial cells and human
foreskin fibrobl~sts. These cell types have heen
tested only for survival in the recipient animal's
~123~3,~
W093/~4790 ~T/US93/00494
- 34 -
brain, and have given positive results in experiments
lasting up to four months. (see Example I).
Transplantation of Cells
The cells were injected into caudate/putamen
region of the brain using a stereotaxic injection.
The Atlas coordinates used for the injection were:
Rostral Caudal: -3.14; Dor~al Ventral: ~7.4;
1~ Lateral Medial: -5.0; Jaw Bar: -3.3. Corrected values
were derived as pre~iously described.
. In initial experimenks, a 23 gauge needle was
used for th~ injection. There was a tendency for the
microcarriers to sediment during the injection and
clog the needle. This problem was partially
allevia~ed using a larger needle. However, the use o
~: a straigh~ bore vr beveled needle resulted also in
brain tissue enterin~ the bore and plugging the
needle. This was been corrected using a needle with
: ~ 20 the tip bent over to protect the opening. Using this
:~ ~ : : type of needle, it has bee~ pos~ible to eYenly inject
the cells into the brain.
As a result of this modification, the stereotaxic
coordinates required further correction for the actual
opening of the~needle rather than it~ tip. This was
: :: ` ~ acco~plished by measurement of the tip-opening
: dis~ance (typically 1 - 2 mm~ and appropriate :~
: rsduction of~the~:values gi~en above. Once khe needle
: : was properly:placed, the suspension containing
~icrocarrier-adhering cells was i~jected a~ a rate of
4 ~l/min until a final volume of 20 ~1 had been
:: injected. The total number of ~eads injected varied
somewha~ due to sedimentation during the injection,
but was calculated to be about 170-200 beads.
~: :
Current es~imates are that approximately 175 beads
(ea~h holding 2-5 cells) need to be injected to obtain
an excellent therapeutic response. This number
'
.
6 3 Q
WO93/14790 P~T/US93/~04~4 :
,,.~,
extrapolates to 50,000 to 75,000 beads in the human,
occupying a volume of about 0.5 ml.
5 ~ecovery and Testing of Animals Receiving Cell -~
Implants _ _ _ __ :
After injection, the surgical site was closed ~:~
using Clay-Adams 9 mm. wound clips, and the rat was
allowed to recover. For the ~irst two hours after the
surgery, the animal was kept in a warmed container to
recover from the anesthesia. The animal was then :;:
returned to its normal housing in Berg Institute. By
~ t~e~next day, the ra~s had regained normal behavior.
N~ morbidity or mortality was associated ~ith the cell ','!i
implantation procedure. `
Tes.ting of anima~s ~or rotational behavior began
kwo days after the surgery and continued at weekly
intervals for one month and at monthly intervals
thereafter. Rats have been tested for up to 4.5 ~
months. Successful function of the implant~d cells in
2~ ~:
secreting dopa~ine was measured as a reduction in the .~
apomorphine-induced turning behavior described above ~.
: in the unilaterall~y 6-OHDA-lesioned rats.
~ .:
Histoloaic Anal~is~ j
~ ,
: Rats were anesthetized with sodium pentobarbital,
- .,
60 mg/mg i.p~. the~chest cavity was opened to expose
the heart. A 22~gauge butterfly needle was inserted
into the bottom~f the left ventricle, and the right
atrium was cut. The butter~.~y needle was attach~d to
a peris~altic pump~and isotonic saline was pumped
through the heart until the solution leavin~ the cut
atrium appeared:clear. In general, 200 - 400 ml of
5aline we~e required.~ The perfusion solution was then ~
changed to 1~ glutaraldehyde/ 1% paraformaldehyde/ 0.1 :~.
M sodium phosphate, pH 7.2~ The perfusion was
continued with ~his fixative until the liver achieved
,
a whitish color. This required approximately 400 ml
~;
2 1 f~ 3 (~ ;
WO g3/14790 ' Pcr/vss3/004s4
- 3~
of perfusion fluid. After successful perfusion, the
rats were stiff. The rat was removed from the
perfusion apparatus and decapitated. The head was
placed into a solution of 2~ glutaraldehyde and
allowed ko soak fo~ 2 hours. At the end of this time,
the brain was removed and frozen-sectioned in a
microtome at a thickness o~ 26 to 28 ~m. Sections
were transferred to nu~b red test-tubes containing
phosphate buffered saline, pH 7.2. Sections were
examined at low power under a dis~ec~ing microscope to
select those ~or ~urther study.
The sections were stained as appropriate and
m~unted on gelatin-coated slides. To gelatin-coat the
microscope slides, a filtered solution containing 3 g
gelatin, 0.3 g chromium potassium sulfate in 600 ml
distilled w~ter was freshly prepar~d and the slides
were individually immersed once in this solution. The
slides were allowed to dry in open air overnight. The
~0 section was then mounted on the slide and dehydrated
by immersing for 5 minutes each in 70% alcohol, 90%
alcohol, 95% alcohol, twice in 100~ alcohol and twice
in ~ylene. Co~erslips were then mounted using
Permount solution.
RESULTS
Two groups of rats served as lesion controls.
One group was not treated. A second group was
injected with glass~beads in the putamen-caudate
3~ region. Both these control groups exhibited
rotational behavior in the rahge of 9~10 turns/min
~see Figure 2~. ;
: When beads containin~ adrenal chromaffin cells
were injected into the same area of the brain, an
immediate reduction of the rotational beha~ior
occurred, to a level of about 40~50% of the control `~
values. This change represents a substantial
therapeutic effect. It is noteworthy that in short- :
WO 93/14790 2 i 2 ,~ PCI/I S93/00494
-- 37 --
term studies using fetal cel~s, such as those
discussed in the Background section above, similar
decrea~es in rotation were observed. The apomorphine :.
challenge using the present dose represents an extreme
test~ With lower doses of apomorphine, turning can be
reduced to 0, but false positives bec~me Inore
frequent.
The experimental findings discussed above
xepresent r~sults from 8 control and 10 treated rats,
exceeding the number re~uired for statistical
signific~nce at a p value of 0.001.
Similar experiments were conducted with retinal
pigment epithelial which had been cultured with beads
and then injected. Similar or somewhat improved
results were obtained, which may be rela~ed to the
fact that these cells produce high levels of dopamine
(See Figure 2).
A econd effect observed in these studies relates
: ~ 20 to the fact that:6-OHDA lesioned rats do not maintain
a normal growth curve. The rats which h~ve been
implanted with a~renal chroma~fin cells or retinal
pigment epithelium cells on beads regain a normal
~rowth curve.
: 2S Histological analysis of the brains of rats
: exhibiting no or only limited ef~ec~s of the .
transplanted cells indicated that only a very small
number of cells had been in~ected. In such
nonresponders, injection coordinates were found to
have been inaccurate. When the results of these
: particular animals were removed from the analysis, the
a~eraqe rotation af~er cell transplant was reduced
from g ~ 0.5 turns/min for non transplanted controls
to 5.1 + 1.2 turns/min ~or transplanted rats.
Statistical analysis using Student's t test yielded a ..
p-value between 0~05 and 0.001, indicating a high
2 i ~ ,J
W093/14790 PCT~US93/0~4~4
- 38 -
level of statistical significance of this difference.
Histological examination of brains of the
experimental animals showed distinct clusters of well-
pigmented cells with no sign of v~scular infiltrate or
inf lammatory reaction. The slides were ~urther
examined by Dr. Victor Sapirstein of the Nathan Kline
Institute of Psychiatric Research (Orangeburg, New
York) who concurred in these ~indings.
EXRMPLE V
Long Term Sur~ival o~ Cells Tr~nsplanted
in~o the Braln with Beads
~5
Adrenal chromaffin cells (prepared as described
in Example IV~ in numbers ranging from 103 to 107 were
injected into the caudate-putamen area of rak brains
in v~lumes of 5-~0 ~l. At ~arious times thereafter
ranging from 1 ~o 180 days (see Figure 3), animals
: : were sacrificed, and t~eir brains were sectioned into
20-50 ~m sections. The sections were examined at 400x
and t~e densities of cells were calculated from the :.
cells/micrcscopic ~ield. Results were expressed as
th~ ~ of cells sur~iving (viable cells as a percent of
injected cells). Each experimental group contained 6 :
rats.
The results shown in Figures 3 and 4 demonstrate
that cells injected alone (in th absence of beadsj
died rapidly, with only 30% survival at 24 hours.
~ i Viability further decreased at a slower rate over the
:: next~period of days t~ weeks. At some time between 30
and 180 day all ~he implanted cells died. This
: observa~ion is related with the many literature
: reports showing that therapeutic effects of cells
: 35
: transplanted into the brain greatly diminish over a :~
few month period (see, for example, Lieberman, supra;
Lindvall, ~Er~; Bakay, ~.A.E., supra).
:
W093/14790 2 ~ 2 3 6 3 ~ PCT/US93J004~4
39
The upper cur~e in Figure 3 shows survival of
cells injected after being grown on beads as described
in Example IV. There was no rapid cell loss during
the initial 24 hours. Furthermore, close to 100% of
the injected bead-adherent cells remained viable in
vivo for the entire test period of l80 days. Figure 4
is a photomicrograph of a section of a rat ~rain six
mont~s a~ter transplantation of adrenal chromaffin
cells showi~g deeply pigmented viable cells attached
to the bead and in the adja~ent area.
Similar res~lts have been obtained using retinal
pigment epithelial cells (:in 6 rats).in a ~0 day
r st~dy. In addition, similar results w~re also
obtained in a 30 day study with human fibroblasts on
beads transplant~d into two ra~s.
Having now fully described this invention, it
will be appre~.iated by those skilled in the art that :.
the same can be performed within a wide rznge o~
equivalent parameters, concentrations, and conditions
without departing from the spirit and scope of the
invention ~nd without undue experimentation.
While this invention has been described in
connecti~n with specific embodiments thereof, it will
be understood that it is capable of further
modifications. This application is intended to cover
any variations, uses, or adaptations of the inventions
following, in general, the principles of the invention
and including such departures from the present
disclosure as come within known or customary practice
: within the art to which the invention pertains and as
may be applied to the essential features hereinbefore
set forth as follows in the scope of the appended
claims.
