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Neurturin antibody
United States Patent: 7,015,311
Issued: March 21, 2006
Inventors: Johnson; Eugene M. (St. Louis,
MO); Milbrandt; Jeffrey D. (St. Louis, MO); Kotzbauer; Paul T.
(Swarthmore, PA); Lampe; Patricia A. (St. Louis, MO)
Assignee: The National Institutes of
Health (Bethesda, MD)
Appl. No.: 476290
Filed: December 30, 1999
Executive MBA in Pharmaceutical Management, U. Colorado
A novel growth factor, neurturin, is
disclosed and the human and mouse amino acid sequences are identified.
Human and mouse neurturin genomic DNA sequences have been cloned and
sequenced and the respective cDNA sequences identified. The subcloning
into vectors and the preparation of cells stably transformed with the
vectors are also disclosed. In addition, methods for treating degenerative
conditions, tumor cells and obesity; methods for detecting gene
alterations and methods for detecting and monitoring patient levels of
neurturin are provided. Methods for identifying additional members of the
neurturin-GDNF family of growth factors are also provided.
The present invention is based upon the
identification, isolation and sequencing of a new growth factor, neurturin.
Surprisingly, this substance has been discovered to be able to promote
cell survival and, in particular, the survival of neurons. Prior to this
invention, neurturin was unknown and had not been identified as a discrete
biologically active substance nor had it been isolated in pure form.
The inventors herein have succeeded in discovering and isolating neurturin
from conditioned medium for CHO cells. The initial neuronal survival
promoting activity was identified by the inventors in a partially purified
preparation of this CHO-conditioned medium. Preparation of conditioned
medium for a given cell line is well known in the art (for example, see
Reid, in Methods in Enzymology Vol. LVIII, Cell Culture, Jakoby and
Pastan, Eds., Academic Press, San Diego, pp 161-164, 1979; Freshney,
Culture of Animal Cells in A Manual of Basic Technique, 2d Ed.,
Wiley-Liss, N.Y., p. 84, 1987 which are incorporated by reference). Thus,
although in the present work CHO cells were cultured and the conditioned
medium used to identify and to obtain neurturin in purified form, one
skilled in the art will readily appreciate that any cell that expresses
neurturin can be used as a source. Some of the cells that express
neurturin are identified below in Example 11 and the inventors herein
believe that any of the cells identified as expressing neurturin can be
used to obtain conditioned medium from which neurturin can be isolated.
In the isolation of neurturin from the CHO cell conditioned medium, an
initial crude conditioned medium can be obtained by centrifugation and/or
filtration to remove cellular debris. For further purification, one
skilled in the art will readily appreciate that any of a number of methods
known in the art can be used to isolate and purify neurturin from a
biological sample such as affinity chromatography, ion exchange
chromatography, preparative electrophoresis or the like wherein the
methods are used either individually or in combination.
The cell survival promoting effect of neurturin can be assessed in any
suitable system for assessing cell survival. The inventors herein believe
that neurturin can promote survival in a variety of different tissues
based upon what is known for other growth factors and upon the observation
that neurturin is expressed in a number of tissues in which it is believed
to have a survival promoting effect. In the work reported herein, neuronal
activity was assessed using a sympathetic neuronal survival assay
(sympathetic cervical ganglia, SCG) which has been extensively
characterized (Martin et al, J Cell Biol 106:829-844, 1989;
Deckwerth and Johnson, J Cell Biol 123:1207-1222, 1993 which are
incorporated by reference) (see FIG. 3). We also show the survival
promoting effects of neurturin on sensory neurons (See FIG. 10).
The SCG assay involved, in brief, the culturing of cells obtained from
superior cervical ganglia of rat embryo for 5 days at 37° C. in medium
containing nerve growth factor (NGF). The medium was then exchanged with a
medium containing no NGF and containing anti-NGF antiserum. Removal of NGF
results normally in death of the neurons in 24-72 hours. Neuronal survival
was visually assessed under a microscope on days 7-8. Maximum neuronal
survival criteria included lack of degeneration of both neuronal cell
bodies and neurites. Cell body degeneration was indicated when the
neuronal cell body was reduced in size, showed irregular membrane
swellings, contained vacuoles, or had lost refractility. A field of
neurites was scored as showing signs of disintegration when swellings and
blebs appeared along the neurite bundles. Survival was determined by
comparison with neurons grown in the presence of NGF (positive control) or
in the absence of NGF with NGF antisera (negative control).
Activity was quantitated by calculation of a "survival unit". The total
survival units in a sample were defined as the minimal volume of an
aliquot of the sample which produced maximal survival divided into the
total volume of that sample. For example, a volume of 600 ml was eluted
from the heparin agarose column and from this eluate, 12.5 μl was the
minimum volume that promoted maximal volume. Thus, the survival units in
the eluate from the heparin agarose column was 48,000. Specific activity
was calculated as the survival units divided by the mg total protein. The
intrinsic activity of neurturin is expressed herein in concentration units
of pg/ml or pM promoting maximal or half-maximal survival. As shown in
FIG. 5, a concentration-response curve of purified neurturin protein
indicates that the intrinsic activity of neurturin expressed as an EC50
is approximately 1.5 ng/ml or approximately 50 pM and an EC100
is approximately 3 ng/ml or approximately 100 pM.
Survival units were determined in an assay using approximately 1200
neurons in a 0.5 ml culture assay and a culture period of 48 hours
following addition of the fraction. Survival was assessed visually after
the 48 hours. Intrinsic activity as shown in FIG. 4 was determined in an
assay using approximately 2700 neurons and a culture period of 72 hours.
Survival was assessed by fixing the neurons and counting the number of
surviving neurons. Because the stability, as assessed by half-life of
activity, for neurturin decreases as the number of neurons increases, the
intrinsic activity measurement would be expected to be lower than that
predicted by Specific Activity determinations. The intrinsic activity
measurement would also be expected to be lower than that predicted by
specific activity because the survival was measured after 72 hours instead
of 48 hours.
The purification of neurturin is described in detail in Example 1 below.
The conditioned medium starting material was prepared from a derivative of
DG44 Chinese hamster ovary cells, DG44CHO-pHSP-NGFI-B (Day et al, J
Biol Chem 265:15253-15260, 1990 which is incorporated by reference).
The inventors herein have also isolated neurturin in partially purified
form from conditioned medium of other derivatives of DG44 Chinese hamster
ovary cells and these other cells could be used equally as well as the
DG44CHO-pHSP-NGFI-B cells as could the parent DG44 Chinese hamster ovary
Cells, ovary cells from other species and cells from other tissues such as
those known to express neurturin (See example 10). In preparing the
conditioned medium, cells were placed in serum free medium for 2 days at
which time conditioned medium is collected and the medium replenished.
This cycle was repeated to yield 5 harvests of conditioned medium from
each batch of CHO cells. The collected media was centrifuged to remove
cellular debris.
The first step in purification of neurturin from the CHO cell conditioned
medium involved the introduction of the conditioned medium onto a heparin
agarose column and the elution of partially purified neurturin therefrom.
This step resulted in an 111 fold increase in the specific activity and
purification of the protein. The buffer used to apply the medium to the
column contains 0.5 M NaCl. At this concentration of NaCl the neurturin
binds to the heparin agarose matrix. The inventors herein believe that
based upon their isoelectric points, LIF and CNTF would either not bind to
the heparin agarose matrix or be washed away from the matrix with buffer
containing 0.5 M NaCl. Thus, this step would be expected to isolate
neurturin from growth factors such as LIF and CNTF. After washing the
column, neurturin was eluted from the column using 1.0 M NaCl.
For further purification, the eluted material was then diluted and
introduced into a column containing SP SEPHAROSE® High Performance ion
exchange resin (Pharmacia, Piscataway, N.J.). Material eluted from this
column was further purified using fast protein liquid chromatography (FPLC)
on a Chelating Superose HR 10/2 column charged with Cu++
(Pharmacia, Piscataway, N.J.). Eluted fractions from the Cu++
superose column were introduced into a Mono S HR 5/5 cation exchange
column (Pharmacia, Piscataway, N.J.) for further FPLC purification. The
composition of the proteins in the Mono S fractions were analyzed using
non-reducing SDS-PAGE and silver staining.
Fractions collected from the columns at each stage of purification were
assayed for biological activity using the neuronal survival assay and for
protein content using the dye binding method of Bradford (Anal Blochem
72:248-254, 1976 which is incorporated by reference) with a Bio-Rad
protein assay dye reagent (Bio-Rad Laboratories, Inc., Hercules, Calif.).
The progressive purification using the above steps is shown in table 1.
The results of this analysis along with the results of the neuronal
survival assay of fractions revealed that a protein having an apparent
molecular weight of about 25 kD co-purified with the sympathetic neuron
survival activity.
The purified material isolated from CHO cell conditioned medium was used
to determine partial amino acid sequences of the protein in CHO cell
conditioned medium and subsequently as a basis for determining the
sequences in different species. The N-terminal amino acid sequence was
determined using an automated protein/peptide sequencer and the first 16
amino acids were considered to be, with uncertainty as to position 6, Ser-Gly-Ala-Arg-Pro-Xaa-Gly-Leu-Arg-Glu-Leu-Glu-Val-Ser-Val-Ser
where Xaa was an unknown amino acid (SEQ ID NO:3). Internal amino acid
fragments were obtained from the purified material following digestion
with protease enzymes and the sequences determined. Three internal
fragments thus obtained were (1) with uncertainty as to positions 1, 2 and
6, Xaa1-Cys-Ala-Gly-Ala-Xaa2-Glu-Ala-Ala-Val where
Xaa1 was unknown amino acid, Xaa2 was Ser or Cys (SEQ
ID NO:4); (2) with uncertainty as to positions 1, 2, 4, 10, 17 and 22, Xaa1-Xaa2-Val-Glu-Ala-Lys-Pro-Cys-Cys-Gly-Pro-Thr-
where Xaa1 and Xaa2 were unknown, Xaa3
was Gln or Glu (SEQ ID NO:5) and (3)
Tyr-His-Thr-Leu-Gln-Glu-Leu-Ser-Ala-Arg (SEQ ID NO:6). Based upon these
partial amino acid sequences, DNA probes and primers can be made and used
to obtain cDNA clones from different species based upon high sequence
conservation between mammalian species. The human cDNA and inferred amino
acid sequence is shown in FIG. 7 and the mouse cDNA and inferred amino
acid sequence is shown in FIG. 8.
The cDNA clone from mouse was 1.0 kb having an open reading frame of 585
nucleotides (SEQ ID NO:12) encoding the mouse pre-pro neurturin protein (SEQ
ID NO:8, FIG. 8). In addition, non-coding regions have been identified at
both the 5′ and 3′ ends of the coding region as shown in FIG. 9. (SEQ ID
NO:13, 5′ non-coding region, nucleic acids -348 through -1; SEQ ID NO:14,
3′ non-coding region, nucleic acids 589 through 675). The mouse neurturin
sequence can be used to obtain PCR primers for use in identifying homologs
from other species. A human 192 nucleotide fragment from human genomic DNA
was amplified by this method and further used to screen a human genomic
library to obtain clones containing the human neurturin genomic locus. The
human cDNA sequence was deduced from the sequencing of these clones. (FIG.
7, cDNA sequence of human pre-pro neurturin).
Reference to neurturin herein is intended to be construed to include
growth factors of any origin which are substantially homologous to and
which are biologically equivalent to the neurturin characterized and
described herein. Such substantially homologous growth factors may be
native to any tissue or species and, similarly, biological activity can be
characterized in any of a number of biological assay systems. Reference to
pre-pro neurturin herein is intended to be construed to include pre-pro
growth factors containing a pre- or leader or signal sequence region, a
pro-sequence region and neurturin as defined herein.
The terms "biologically equivalent" are intended to mean that the
compositions of the present invention are capable of demonstrating some or
all of the same growth properties in a similar fashion, not necessarily to
the same degree as the neurturin isolated from the CHO cell conditioned
medium herein or recombinantly produced human or mouse neurturin.
By "substantially homologous" it is meant that the degree of homology of
human and mouse neurturin to neurturin from any species is greater than
that between neurturin and any previously reported member of the TGF-β
superfamily or GDNF (For discussion of homology of TGF-β superfamily
members see Kingsley, Genes and Dev 8:133-46, 1994 which is
incorporated by reference).
Sequence identity or percent identity is intended to mean the percentage
of same residues between two sequences, referenced to human neurturin when
determining percent identity with non-human neurturin, referenced to
neurturin when determining percent identity with non-neurturin growth
factors and referenced to human GDNF when determining percent identity of
non-neurturin growth factors with GDNF, when the two sequences are aligned
using the Clustlal method (Higgins et al, Cabios 8:189-191, 1992)
of multiple sequence alignment in the Lasergene biocomputing software (DNASTAR,
INC, Madison, Wis.). In this method, multiple alignments are carried out
in a progressive manner, in which larger and larger alignment groups are
assembled using similarity scores calculated from a series of pairwise
alignments. Optimal sequence alignments are obtained by finding the
maximum alignment score, which is the average of all scores between the
separate residues in the alignment, determined from a residue weight table
representing the probability of a given amino acid change occurring in two
related proteins over a given evolutionary interval. Penalties for opening
and lengthening gaps in the alignment contribute to the score. The default
parameters used with this program are as follows: gap penalty for multiple
alignment=10; gap length penalty for multiple alignment=10; k-tuple value
in pairwise alignment=1; gap penalty in pairwise alignment=3; window value
in pairwise alignment=5; diagonals saved in pairwise alignment=5. The
residue weight table used for the alignment program is PAM250 (Dayhoff et
al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NBRF,
Washington, Vol. 5, suppl. 3, p. 345, 1978).
Percent conservation is calculated from the above alignment by adding the
percentage of identical residues to the percentage of positions at which
the two residues represent a conservative substitution (defined as having
a log odds value of greater than or equal to 0.3 in the PAM250 residue
weight table). Conservation is referenced to human neurturin when
determining percent conservation with non-human neurturin, referenced to
neurturin when determining percent conservation with non-neurturin growth
factors, and referenced to human GDNF when determining percent
conservation to non-neurturin growth factors with GDNF. Conservative amino
acid changes satisfying this requirement are: R-K; E-D, Y-F, L-M; V-I,
Q-H. The calculations of identity (I) and conservation (C) between mature
human and mature mouse neurturin (hNTN and mNTN, respectively) and between
each of these and mature human, rat and mouse GDNF (hGDNF, rGDNF and mGDNF,
respectively) are shown in table 2.
The degree of homology between the mature mouse and human neurturin
proteins is about 90% sequence identity and all neurturin homologs of
non-human mammalian species are believed to similarly have at least about
85% sequence identity with human neurturin. For non-mammalian species such
as avian species, it is believed that the degree of homology with
neurturin is at least about 65% identity. By way of comparison, the
variations between family members of the neurturin-GDNF family of growth
factors can be seen by comparing neurturin and GDNF. Human and mouse
neurturin have about 40% sequence identity and about 50% sequence
conservation with human, mouse and rat GDNF. It is believed that the
different family members similarly have a sequence identity of about 40%
of that of neurturin and about 40% of that of GDNF and within a range of
about 30% to about 85% identity with neurturin and within a range of about
30% to about 85% sequence identity with GDNF. Thus, a given non-neurturin
and non-GDNF family member from one species would be expected to show
lesser sequence identity with neurturin and with GDNF from the same
species than the sequence identity between human neurturin and neurturin
from a non-human mammalian species, but greater sequence identity than
that between human neurturin and any other known member of the TGF-β
superfamily member except GDNF (Kingsley, supra). In the case of pre-pro
neurturin, homologs of pre-pro neurturin in non-human mammalian species
can be identified by virtue of the neurturin portion of the amino acid
sequence having at least about 85% sequence identity with human neurturin
and homologs of pre-pro neurturin in non-mammalian species can be
identified by virtue of the neurturin portion of the amino acid sequence
having at least about 65% identity with human neurturin.
Neurturin can also include hybrid and modified forms of neurturin
including fusion proteins and neurturin fragments and hybrid and modified
forms in which certain amino acids have been deleted or replaced and
modifications such as where one or more amino acids have been changed to a
modified amino acid or unusual amino acid and modifications such as
glycosolations so long as the hybrid or modified form retains the
biological activity of neurturin. By retaining the biological activity, it
is meant that neuronal survival is promoted, although not necessarily at
the same level of potency as that of the neurturin isolated from CHO cell
conditioned medium or that of the recombinantly produced human or mouse
Also included within the meaning of substantially homologous is any
neurturin which may be isolated by virtue of cross-reactivity with
antibodies to the neurturin described herein or whose encoding nucleotide
sequences including genomic DNA, mRNA or cDNA may be isolated through
hybridization with the complementary sequence of genomic or subgenomic
nucleotide sequences or cDNA of the neurturin herein or fragments thereof.
It will also be appreciated by one skilled in the art that degenerate DNA
sequences can encode human neurturin and these are also intended to be
included within the present invention as are allelic variants of neurturin.
In the case of pre-pro neurturin, alternatively spliced protein products
resulting from an intron located in the coding sequence of the pro region
may exist. The intron is believed to exist in the genomic sequence at a
position corresponding to that between nucleic acids 169 and 170 of the
cDNA which, in turn, corresponds to a position within amino acid 57 in
both the mouse and human pre-pro neurturin sequences (see FIGS. 7 and 8).
Thus, alternative splicing at this position might produce a sequence that
differs from that identified herein for human and mouse pre-pro neurturin
(SEQ ID NO:11 and SEQ ID NO:12, respectively) at the identified amino acid
site by addition and/or deletion of one or more amino acids. Any and all
alternatively spliced pre-pro neurturin proteins are intended to be
included within the terms pre-pro neurturin as used herein.
Although it is not intended that the inventors herein be bound by any
theory, it is thought that the human and mouse proteins identified herein
as well as homologs from other tissues and species may exist as dimers in
their biologically active form in a manner consistent with what is known
for other factors of the TGF-β superfamily.
In addition to homodimers, the monomeric units of the dimers of neurturin
can be used to construct stable growth factor heterodimers or
heteromultimers comprising at least one monomer unit derived from
neurturin. This can be done by dissociating a homodimer of neurturin into
its component monomeric units and reassociating in the presence of a
monomeric unit of a second homodimeric growth factor. This second
homodimeric growth factor can be selected from a variety of growth factors
including GDNF or a member of the NGF family such as NGF, BDNF, NT-3 and
NT-4/5 or a member of the TGF-β superfamily, or a vascular endothelial
growth factor or a member of the CNTF/LIF family or the like.
Growth factors are thought to act at specific receptors. For example, the
receptors for TGF-β and activins have been identified and make up a family
of Ser/Thr kinase transmembrane proteins (Kingsley, Genes and Dev
8:133-146, 1994; Bexk et al Nature 373:339-341, 1995 which are
incorporated by reference). In the NGF family, NGF binds to the TrkA
receptor in peripheral sensory and sympathetic neurons and in basal
forebrain neurons; BDNF and NT-4/5 bind to trkB receptors; and NT-3 binds
primarily to trkC receptors that possess a distinct distribution within
the CNS (Tuszynski et al., Ann Neurol 35:S9-S12, 1994). The
inventors herein believe that GDNF, neurturin and as yet unknown members
of this family of growth factors act through specific receptors having
distinct distributions as has been shown for other growth factor families.
Thus, by forming heterodimers or heteromultimers of neurturin and one or
more other growth factors, the resultant growth factor would be expected
to be able to bind to at least two distinct receptor types preferentially
having a different tissue distribution. The resultant heterodimers or
heteromultimers would be expected to show an enlarged spectrum of cells
upon which it could act or provide greater potency. It is also possible
that the heterodimer or heteromultimer might provide synergistic effects
not seen with homodimers or homomultimers. For example, the combination of
factors from different classes has been shown to promote long-term
survival of oligodendrocytes whereas single factors or combinations of
factors within the same class promoted short-term survival (Barres et al.,
Development 118:283-295, 1993).
Heterodimers can be formed by a number of methods. For example, homodimers
can be mixed and subjected to conditions in which dissociation/unfolding
occurs, such as in the presence of a dissociation/unfolding agent,
followed by subjection to conditions which allow monomer reassociation and
formation of heterodimers. Dissociation/unfolding agents include any agent
known to promote the dissociation of proteins. Such agents include, but
are not limited to, guanidine hydrochloride, urea, potassium thiocyanate,
pH lowering agents such as buffered HCl solutions, and polar, water
miscible organic solvents such as acetonitrile or alcohols such as
propanol or isopropanol. In addition, for homodimers linked covalently by
disulfide bonds as is the case with TGF-β family members, reducing agents
such as dithiothreitol and β-mercaptoethanol can be used for
dissociation/unfolding and for reassociation/refolding.
Heterodimers can also be made by transfecting a cell with two or more
factors such that the transformed cell produces heterodimers as has been
done with neurotrophin. (Heymach and Schooter, J Biol Chem
270:12297-12304, 1995).
Another method of forming heterodimers is by combining neurturin
homodimers and a homodimer from a second growth factor and incubating the
mixture at 37° C.
When heterodimers are produced from homodimers, the heterodimers may then
be separated from homodimers using methods available to those skilled in
the art such as, for example, by elution from preparative, non-denaturing
polyacrylamide gels. Alternatively, heterodimers may be purified using
high pressure cation exchange chromatography such as with a Mono S cation
exchange column or by sequential immunoaffinity columns.
It is well known in the art that many proteins are synthesized within a
cell with a signal sequence at the N-terminus of the mature protein
sequence and the protein carrying such a leader sequence is referred to as
a preprotein. The pre-portion of the protein is cleaved during cellular
processing of the protein. In addition to a pre-leader sequence, many
proteins contain a distinct pro sequence that describes a region on a
protein that is a stable precursor of the mature protein. Proteins
synthesized with both pre- and pro-regions are referred to as
preproproteins. In view of the processing events known to occur with other
TGF-β family members as well as the sequences determined herein, the
inventors believe that the form of neurturin protein as synthesized within
a cell is the pre-pro neurturin. The pre-pro neurturin is believed to
contain an N-terminal 19 amino acid signal sequence (human pre-signal
sequence, SEQ ID NO:15, FIG. 7, amino acids 1 through 19 encoded by SEQ ID
NO:17, FIG. 7, nucleic acids 1 through 57; mouse pre-signal sequence, SEQ
ID NO:16, FIG. 8, amino acids 1 through 19, encoded by SEQ ID NO:18, FIG.
8, nucleic acids 1 through 57). It is known that the full length of a
leader sequence is not necessarily required for the sequence to act as a
signal sequence and, therefore, within the definition of pre-region of
neurturin is included fragments thereof, usually N-terminal fragments,
that retain the property of being able to act as a signal sequence, that
is to facilitate co-translational insertion into the membranes of one or
more cellular organelles such as endoplasmic reticulum, mitochondria,
golgi, plasma membrane and the like.
The signal sequence is followed by a pro-domain which contains an RXXR
proteolytic processing site immediately before the N-terminal amino acid
sequence for the mature neurturin. (human pro-region sequence, SEQ ID
NO:19, FIG. 7, amino acids 20 through 95 encoded by the nucleic acid
sequence SEQ ID NO:20, FIG. 7 nucleic acids 58 through 285; mouse
pro-region sequence, SEQ ID NO:22, FIG. 8, amino acids 19 through 95
encoded by nucleic acid sequence SEQ ID NO:21, FIG. 8, nucleic acids 58
through 285).
The pre- and pro-regions together comprise a pre-pro sequence identified
as the human pre-pro sequence (SEQ ID NO:23, FIG. 7, amino acids 1 through
95 encoded by SEQ ID NO:25, nucleic acids 1 through 285) and the mouse
pre-pro sequence (SEQ ID NO:24, FIG. 8, amino acids 1 through 95 encoded
by SEQ ID NO:26, nucleic acids 1 through 285). The pre-region sequences
and pro-region sequences as well as the pre-pro region sequences can be
identified and obtained for non-human mammalian species and for
non-mammalian species by virtue of the sequences being contained within
the pre-pro neurturin as defined herein.
Using the above landmarks, the mature, secreted neurturin molecule is
predicted to be approximately 11.5 kD which is likely to form a disulfide
linked homodimer of approximately 23 kD by analogy to other members of the
TGF-β family. The predicted approximately 23 kD protein is consistent with
the 25 kD protein purified from CHO cell conditioned media being a
homodimer. The inventors herein have detected an approximately 11.5 kD
protein from conditioned medium of Chinese hamster ovary cells transfected
with the neurturin expression vector (pCMV-NTN-3-1) using SDS-PAGE under
reducing conditions and this protein is thought to be the monomer.
The nucleotide sequences of pre- and/or pro-regions can also be used to
construct chimeric genes with the coding sequences of other growth factors
or proteins and, similarly, chimeric genes can be constructed from the
coding sequence of neurturin coupled to sequences encoding pre- and/or
pro-regions from genes for other growth factors or proteins. (Booth et
al., Gene 146:303-8, 1994; Ibanez, Gene 146:303-8; 1994;
Storici et al., FEBS Letters 337:303-7, 1994; Sha et al J Cell
Biol 114:827-839, 1991 which are incorporated by reference). Such
chimeric proteins can exhibit altered production or expression of the
active protein species.
A preferred neurturin of the present invention has been identified and
isolated in purified form from medium conditioned by CHO cells. Also
preferred is neurturin prepared by recombinant DNA technology. By "pure
form" or "purified form" or "substantially purified form" it is meant that
a neurturin composition is substantially free of other proteins which are
not neurturin.
Recombinant human neurturin may be made by expressing the DNA sequences
encoding neurturin in a suitable transformed host cell. Using methods well
known in the art, the DNA encoding neurturin may be linked to an
expression vector, transformed into a host cell and conditions established
that are suitable for expression of neurturin by the transformed cell.
Any suitable expression vector may be employed to produce recombinant
human neurturin such as, for example, the mammalian expression vector pCB6
(Brewer, Meth Cell Biol 43:233-245, 1994) or the E. coli pET
expression vectors, specifically, pET-30a (Studier et al., Methods
Enzymol 185:60-89, 1990 which is incorporated by reference) both of
which were used herein. Other suitable expression vectors for expression
in mammalian and bacterial cells are known in the art as are expression
vectors for use in yeast or insect cells. Baculovirus expression systems
can also be employed.
Neurturin may be expressed in the monomeric units or such monomeric form
may be produced by preparation under reducing conditions. In such
instances refolding and renaturation can be accomplished using one of the
agents noted above that is known to promote dissociation/association of
proteins. For example, the monomeric form can be incubated with
dithiothreitol followed by incubation with oxidized glutathione disodium
salt followed by incubation with a buffer containing a refolding agent
such as urea.
By analogy with the N-terminal sequence and internal fragments of the
neurturin purified from CHO cell conditioned medium, the mature mouse
sequence was deduced and from this the mature human form was predicted
using the sequence from the human gene. The amino acid sequence of the
mature human form is as shown in FIG. 5 (hNTN, SEQ ID NO:1). The material
purified from CHO cell conditioned medium is considered to be mature
neurturin and may exist as a dimer or other multimer and may be
glycosylated or chemically modified in other ways. As noted above, the
mouse and human nucleic acid sequences suggest that neurturin is initially
translated as a pre-pro polypeptide and that proteolytic processing of the
signal sequence and the "pro" portion of this molecule results in the
mature sequence, referenced herein as "mature neurturin", as obtained from
medium condition by CHO cells and as exists in human and in non-human
species in homologous form. The present invention, therefore, includes any
and all "mature neurturin" sequences from human and non-human species and
any and all pre-pro neurturin polypeptides that may be translated from the
neurturin gene.
It is believed that the coding sequence for the pre-pro-neurturin
polypeptide begins at the first ATG codon encoding methionine at the 5′
end of the clone (position 1 in FIG. 9) which is positioned in the same
reading frame as the sequence encoding the amino acid sequences obtained
from the purified neurturin. Downstream from the first codon is the
largest open reading frame containing the coding sequence for the pre- and
pro-regions followed by the coding sequence for the mature mouse neurturin.
Sequence analysis of the murine neurturin genomic clones identified a 0.5
kb intron located between nucleotide 169 and 170 of the pre-pro neurturin
from the cDNA clones. This intron is located in the coding sequence of the
pro-region of the pre-pro-neurturin protein. Thus, it is believed that the
mouse neurturin gene contains at least two exons, one of which contains
the coding sequences upstream from the splice site and the other contains
the coding sequence downstream (FIG. 8, SEQ ID NO:29, SEQ ID NO:30). It is
known that the gene for GDNF contains an intron located at an analogous
position and an alternately spliced form of GDNF has been detected by
RT-PCR experiments (Suter-Crazzolara and Unsicker, Neuroreport 5:
2486-2488, 1994 which is incorporated by reference). This alternate form
results from the use of a splice site in the second coding exon located 78
bp 3′ to the original splice site reported. The alternately spliced form
encodes a GDNF protein with a deletion of 26 amino acids relative to the
originally reported form. The two forms are expressed in different ratios
in different tissues. We have not detected alternately spliced forms of
neurturin in RT-PCR and RACE experiments using mouse P1 brain and P1 liver
cDNAs. The possibility exists, however, that alternate splice sites in the
neurturin gene may be utilized in different tissues.
The coding sequence of the human neurturin CDNA has been deduced from the
sequence of the human neurturin genomic clones. The coding sequence of the
human cDNA, like that of the mouse cDNA, is interrupted by an intron
between nucleotides 169 and 170 of the coding sequence. Thus, the human
neurturin gene is believed to contain at least two exons, one of which
contains the coding sequence upstream from the splice site and the other
contains the coding sequence downstream (FIG. 7, SEQ ID NO:27, SEQ ID
NO:28). The splice sites at the intron-exon junctions of the human and
mouse genes have been conserved.
From the deduced amino acid sequence of human neurturin, the earlier
predicted N-terminal sequence lies between positions 286 and 339 and the
predicted internal sequences lie between positions 385 and 417, positions
474 and 533, and positions 547 and 576. The TGA stop codon at positions
592-594 terminate the open reading frame.
The predicted length of the purified pre-pro neurturin is 197 amino acid
residues for the human pre-pro neurturin (SEQ ID NO:7) and 195 amino acid
residues for the mouse pre-pro neurturin (SEQ ID NO:8). The predicted
molecular weight of this polypeptide is 22.2 kD for mouse and 22.4 kd for
human. The predicted length of the purified neurturin is 100 amino acid
residues and its predicted monomeric molecular weight is 11.5 kD. There
are no N-linked glycosolation sites, however, potential O-linked
glycosolation sites occur at amino acid residues in positions 18, 26, 80,
86 and 95 in human neurturin. Glycosylation at any one or combination of
these sites would increase the molecular weight of the molecule.
Different possible cleavage sites may be present in the pre-pro-neurturin
sequence. The amino acid sequence of the mature mouse neurturin (FIG. 5,
SEQ ID NO:2) is predicted from alignment with the N-terminal amino acid
sequence of the purified chinese hamster neurturin. A four residue RRAR
cleavage site (amino acids 92-95) is found immediately before the
predicted N-terminal amino acid of mature mouse neurturin. This RRAR
sequence fits the RXXR consensus sequence at which members of the TGF-β
superfamily are usually cleaved. This putative RRAR cleavage sequence is
conserved in human neurturin. However, the mature human neurturin is
predicted to have a two amino acid N-terminal extension relative to mature
mouse neurturin when cleaved at this sequence. Since neurturin contains
other sequences which fit the RXXR consensus (for example the sequence
RRRR at amino acids 90-93) and the specificities of proteases involved in
this cleavage are not completely understood, the possibility exists that
in some situations, neurturin is cleaved at sites other than the above
RRAR sequence, and the mature neurturin protein may have a variable number
of amino acids preceding the cysteine residue at position 101 in the mouse
sequence (pre-pro protein) and position 103 in the human sequence. Such
alternate cleavage sites could be utilized differently among different
organisms and among different tissues of the same organism. The N-terminal
amino acids preceding the first of the seven conserved cysteines in the
mature forms of members of the TGF-β family vary greatly in both length
and sequence. Furthermore, insertion of a ten amino acid sequence two
residues upstream of the first conserved cysteine does not affect the
known biological activities of one family member, dorsalin (Basler, K.,
Edlund, T., Jessell, T. M., and Yamada, T., (1993) Cell 73:687-702). Thus
neurturin proteins which contain sequences of different lengths preceding
the cysteine 101 in mouse and cysteine 103 in human would be likely to
retain their biological activity.
The inventors herein believe that at a minimum the sequence of neurturin
that will show biological activity will contain the sequence beginning at
cysteine 103 and ending at cysteine 196 for human neurturin (FIG. 7, SEQ
ID NO:31) and beginning at cysteine 101 and ending at cysteine 194 for
mouse neurturin (FIG. 7, SEQ ID NO:32). Thus, within the scope of the
present invention are amino acid sequences containing SEQ ID NO:31 and
amino acid sequences containing SEQ ID NO:32 and nucleic acid sequences
encoding these amino acid sequences.
The present invention includes nucleic acid sequences including sequences
that encode human and mouse neurturin (FIG. 5). Also included within the
scope of this invention are sequences that are substantially the same as
the nucleic acid sequences encoding neurturin. Such substantially the same
sequences may, for example, be substituted with codons more readily
expressed in a given host cell such as E. coli according to well
known and standard procedures. Such modified nucleic acid sequences would
be included within the scope of this invention.
Specific nucleic acid sequences can be modified by those skilled in the
art and, thus, all nucleic acid sequences which encode for the amino acid
sequences of pre-pro neurturin or the pre-region or the pro-region or
neurturin can likewise be so modified. The present invention thus also
includes nucleic acid sequence which will hybridize with all such nucleic
acid sequences—or complements of the nucleic acid sequences where
appropriate—and encode for a polypeptide having cell survival promoting
activity. The present invention also includes nucleic acid sequences which
encode for polypeptides that have neuronal survival promoting activity and
that are recognized by antibodies that bind to neurturin.
The present invention also encompasses vectors comprising expression
regulatory elements operably linked to any of the nucleic acid sequences
included within the scope of the invention. This invention also includes
host cells—of any variety—that have been transformed with vectors
comprising expression regulatory elements operably linked to any of the
nucleic acid sequences included within the scope of the present invention.
Methods are also provided herein for producing neurturin. Preparation can
be by isolation from conditioned medium from a variety of cell types so
long as the cell type produces neurturin. A second and preferred method
involves utilization of recombinant methods by isolating a nucleic acid
sequence encoding neurturin, cloning the sequence along with appropriate
regulatory sequences into suitable vectors and cell types, and expressing
the sequence to produce neurturin.
A mammalian gene family comprised of four neurotrophic factors has been
identified including nerve growth factor (NGF), brain derived neurotrophic
factor (BDGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). These
factors share approximately 60 percent nucleic acid sequence homology (Tuszynski
and Gage, Ann Neurol 35:S9-S12, 1994 which is incorporated by
reference). The neurturin protein displays no significant homology to the
NGF family of neurotrophic factors. Neurturin shares less than about 20%
homology with the TGF-β superfamily of growth factors. However, neurturin
shows approximately 40% sequence identity with GDNF. In particular, the
positions of the seven cysteine residues present in both neurturin and
GDNF are exactly conserved. The inventors herein believe that other
unidentified genes may exist that encode proteins that have substantial
amino acid sequence homology to neurturin and GDNF and which function as
growth factors selective for the same or different tissues and the same or
different biological activities. A different spectrum of activity with
respect to tissues affected and/or response elicited could result from
preferential activation of different receptors by different family members
as is known to occur with members of the NGF family of neurotrophic
factors (Tuszynski and Gage, 1994, supra).
As a consequence of members of a particular gene family showing
substantial conservation of amino acid sequence among the protein products
of the family members, there is considerable conservation of sequences at
the DNA level. This forms the basis for a new approach for identifying
other members of the gene family to which GDNF and neurturin belong. The
method used for such identification is cross-hybridization using nucleic
acid probes derived from one family member to form a stable hybrid duplex
molecule with nucleic acid sequence from different members of the gene
family or to amplify nucleic acid sequences from different family members.
(see for example, Kaisho et al. FEBS Letters 266:187-191, 1990
which is incorporated by reference). The sequence from the different
family member may not be identical to the probe, but will, nevertheless be
sufficiently related to the probe sequence to hybridize with the probe.
Alternatively, PCR using primers from one family member can be used to
identify additional family members. The above approaches have not
heretofore been successful in identifying other gene family members
because only one family member, GDNF was known. With the identification of
neurturin herein, however, unique new probes and primers can be made that
contain sequences from the conserved regions of this gene family. In
particular, three conserved regions have been identified herein which can
be used as a basis for constructing new probes and primers. The new probes
and primers made available from the present work make possible this
powerful new approach which can now successfully identify other gene
family members. Using this new approach, one may screen for genes related
to GDNF and neurturin in sequence homology by preparing DNA or RNA probes
based upon the conserved regions in the GDNF and neurturin molecules.
Therefore, one embodiment of the present invention comprises probes and
primers that are unique to or derived from a nucleotide sequence encoding
such conserved regions and a method for identifying further members of the
GDNF-neurturin gene family. Conserved region amino acid sequences include
Val-Xaa1-Xaa2-Leu-Gly-Leu-Gly-Tyr in which Xaa1
is Ser or Thr and Xaa2 is Glu or Asp (SEQ ID NO:33); Glu-Xaa1-Xaa2-Xaa3-Phe-Arg-Tyr-Cys-Xaa4-Gly-Xaa5-Cys-Xaa6-Xaa7-Ala
in which Xaa1 is Thr or Glu, Xaa2 is Val or Leu, Xaa3
is Leu or Ile, Xaa4 is Ala or Ser, Xaa5 is Ala
or Ser, Xaa6 is Glu or Asp and Xaa7 is Ala or Ser (SEQ
ID NO:34); and Cys-Cys-Arg-Pro-Xaa1-Ala-Xaa2-Xaa3-Asp-Xaa4-Xaa5-Ser-Phe-Leu-Asp
in which Xaa1 is Thr or Val or Ile, Xaa2 is Tyr or Phe, Xaa3
is Glu or Asp, Xaa4 is Glu or Asp and Xaa5 is
Val or Leu (SEQ ID NO:35). Nucleotide sequences containing a coding
sequence for the above conserved sequences or fragments of the above
conserved sequences can be used as probes. Exemplary probe and primer
sequences include nucleic acid sequences encoding amino acid sequences,
ID NO:40 and SEQ ID NO:41 and, in particular, nucleic acid sequences, SEQ
ID NOS:47, and SEQ ID NOS:48.
Hybridization using the new probes from conserved regions of the nucleic
acid sequences would be performed under reduced stringency conditions.
Factors involved in determining stringency conditions are well known in
the art (for example, see Sambrook et al., Molecular Cloning, 2nd
Ed., 1989 which is incorporated by reference). Sources of nucleic acid for
screening would include genomic DNA libraries from mammalian species or
cDNA libraries constructed using RNA obtained from mammalian cells cloned
into any suitable vector.
PCR primers would be utilized under PCR conditions of reduced annealing
temperature which would allow amplification of sequences from gene family
members other than GDNF and neurturin. Sources of nucleic acid for
screening would include genomic DNA libraries from mammalian species
cloned into any suitable vector, cDNA transcribed from RNA obtained from
mammalian cells, and genomic DNA from mammalian species.
DNA sequences identified on the basis of hybridization or PCR assays would
be sequenced and compared to GDNF and neurturin. The DNA sequences
encoding the entire sequence of the novel factor would then be obtained in
the same manner as described herein. Genomic DNA or libraries of genomic
clones can also be used as templates because the intron/exon structures of
GDNF and neurturin are conserved and coding sequences of the mature
proteins are not interrupted by introns.
Although neurturin has been purified on the basis of its ability to
promote the survival of a particular neuronal type, this factor will act
on other neuronal cell types as well. For example, neurturin is shown
herein to promote the survival of nodose sensory ganglia neurons (see
Example 3). Neurturin is also likely to promote the survival of
non-neuronal cells. Indeed, all the growth factors isolated to date have
been shown to act on many different cell types (for example see Scully and
Otten, Cell Biol Int 19:459-469, 1005; Hefti, Neurotrophic
Factor Therapy 25:1418-1435, 1994 which are incorporated by
reference). It is known that NGF acts on sympathetic neurons, several
types of sensory neurons and certain populations of CNS neurons. GDNF,
which is more closely related to neurturin, has been shown to act on
dopaminergic, sympathetic, motor and several sensory neurons (Henderson et
al. supra, 1994; Miles et al, J Cell Biol 130:137-148, 1995; Yan et
al, Nature 373:341-344, 1995; Lin et al, Science
260:1130-1132, 1993; Trupp et al, J Cell Biol 130:137-148, 1995;
Martin et al Brain Res 683:172-178, 1995; Bowenkamp st al J Comp
Neurol 355:479-489, 1995 which are incorporated by reference). Thus,
it is likely that in addition to peripheral sympathetic and sensory
neurons, neurturin can act on a wide variety of central and peripheral
neuronal cell types.
It is also likely that neurturin will act on non-neuronal cells to promote
their survival, growth or function. This expectation is based upon the
activity of known growth factors. Although NGF is the prototypical
neurotrophic factor, this growth factor also acts upon mast cells to
increase the number of mast cells when injected into newborn rats (Aloe,
J Neuroimmunol 18:1-12, 1988). In addition, mast cells express the
trk receptor and respond to NGF such that NGF is a mast cell secretogogue
and survival promoting factor (Horigome et al., J Biol Chem
269:2695-2707, 1994 which is incorporated by reference). Moreover, members
of the TGF-β superfamily act on many cell types of different function and
embryologic origin.
The inventors herein have identified several non-neuronal tissues in which
neurturin is expressed including blood, bone marrow, neonatal liver and
mast cells. This suggests a role for neurturin in hematopoiesis,
inflammation and allergy.
Neurotrophic factors of the NGF family are thought to act through
factor-specific high affinity receptors (Tuszynski and Gage, 1994, supra).
Only particular portions of the protein acting at a receptor site are
required for binding to the receptor. Such particular portions or discrete
fragments can serve as agonists where the substances activate the receptor
to elicit the promoting action on cell survival and growth and antagonists
to neurturin where they bind to, but do not activate, the receptor or
promote survival and growth. Such portions or fragments that are agonists
and those that are antagonists are also within the scope of the present
Synthetic, pan-growth factors can also be constructed by combining the
active domains of neurturin with the active domains of one or more other
growth factors. (For example, see Ilag et al., Proc Nat'l Acad Sci
92:607-611, 1995 which is incorporated by reference). These pan-growth
factors would be expected to have the combined activities of neurturin and
the one or more other growth factors. As such they are believed to be
potent and multispecific growth factors that are useful in the treatment
of a wide spectrum of degenerative diseases and conditions including
conditions that can be treated by any and all of the parent factors from
which the active domains were obtained. Such pan-growth factors might also
provide synergistic effects beyond the activities of the parent factors (Barres
et al., supra).
Pan-growth factors within the scope of the present invention can also
include chimeric or hybrid polypeptides that are constructed from portions
of fragments of at least two growth factors. Growth factors of the TGF-β
superfamily are structurally related having highly conserved sequence
landmarks whereby family members are identified. In particular, seven
canonical framework cysteine residues are nearly invariant in members of
the superfamily (Kingsley, Genes & Dev 8:133-146, 1994 which
is incorporated by reference)(see FIG. 17). Chimeric polypeptide molecules
can, therefore, be constructed from a sequence that is substantially
identical to a portion of the neurturin molecule up to a crossover point
and a sequence that is substantially with a portion of another TGF-β
superfamily member extending on the other side of the corresponding
crossover point in the other TGF-β superfamily member. Such portions of
neurturin are preferably from about 10 to about 90, more preferably from
about 20 to about 80 and most preferably from about 30 to about 70
contiguous amino acids and such portions of another, non-neurturin TGF-β
superfamily member are preferably from about 10 to about 90, more
preferably from about 20 to about 80 and most preferably from about 30 to
about 70 contiguous amino acids. For example, a particular crossover point
might be between the third and fourth canonical framework cysteine
residues. One such exemplary construct would contain at the 5′ end a
sequence comprised of the human neurturin sequence from residue 1 through
the third canonical framework cysteine residue 39 and up to residue 68 but
not including the fourth canonical framework cysteine residue 69. The 3′
end of the hybrid construct would constitute a sequence derived another
TGF-β superfamily member such as, for example, GDNF which is another TGF-β
superfamily member that is closely related to neurturin. Using GDNF as the
other TGF-β family member, the hybrid construct from the crossover point
would be comprised of a sequence beginning at the fourth canonical
framework cysteine residue 101 of human GDNF and continuing through
residue 134 at the 3′ end of human GDNF. A second exemplary hybrid
construct would be comprised of residues 1 through 100 of human GDNF
beginning at the 5′ end of the construct, contiguously linked with
residues 69 through 102 of human neurturin. The above constructs with
neurturin and GDNF are intended as examples only with the particular TGF-β
family member being selected from family members including but not limited
to transforming growth factor-β1 (TGFβ1), transforming growth factor-β2
(TGFβ2), transforming growth factor-β3 (TGFβ3), inhibin β A (INHβA),
inhibin β B (INHβB), the nodal gene (NODAL), bone morphogenetic proteins 2
and 4 (BMP2 and BMP4), the Drosophila decapentaplegic gene (dpp),
bone morphogenetic proteins 5-8 (BMP5, BMP6, BMP7 and BMP8), the
Drosophila 60A gene family (60A), bone morphogenetic protein 3 (BMP3),
the Vg1 gene, growth differentiation factors 1 and 3 (GDF1 and GDF3),
dorsalin (drsln), inhibin a (INHα), the MIS gene (MIS), growth factor 9
(GDF-9), glial-derived neurotropic growth factor (GDNF) and neurturin
(NTN) (see FIG. 18). In addition, the crossover point can be any residue
between the first and seventh canonical framework cysteines molecules of
neurturin and the particular other family member.
In constructing a particular chimeric molecule, the portions of neurturin
and portions of the other, non-neurturin growth factor are amplified using
PCR, mixed and used as template for a PCR reaction using the forward
primer from one and the reverse primer from the other of the two component
portions of the chimeric molecule. Thus, for example a forward and reverse
primers are selected to amplify the portion of neurturin from the
beginning to the selected crossover point between the third and fourth
canonical cysteine residues using a neurturin plasmid as template. A
forward primer with a short overlapping portion of the neurturin sequence
and a reverse primer are then used to amplify the portion of the other,
non-neurturin growth factor member of the TGF-β superfamily from the
corresponding crossover point through the 3′ end using a plasmid template
containing the coding sequence for the non-neurturin TGF-β family member.
The products of the two PCR reactions are gel purified and mixed together
and a PCR reaction performed. Using an aliquot of this reaction as
template a PCR reaction is performed using the neurturin forward primer
and the reverse primer for the non-neurturin growth factor. The product is
then cloned into an expression vector for production of the chimeric
Chimeric growth factors would be expected to be effective in promoting the
growth and development of cells and for use in preventing the atrophy,
degeneration or death of cells, particular in neurons. The chimeric
polypeptides may also act as a receptor antagonists of one or both of the
full length growth factors from which the chimeric polypeptide was
constructed or as an antagonist of any other growth factor that acts at
the same receptor or receptors. Such polypeptides can also be used as
foodstuffs, combustible energy sources, and viscosity-enhancing solutes.
The present invention also includes therapeutic or pharmaceutical
compositions comprising neurturin in an effective amount for treating
patients with cellular degeneration and a method comprising administering
a therapeutically effective amount of neurturin. These compositions and
methods are useful for treating a number of degenerative diseases. Where
the cellular degeneration involves neuronal degeneration, the diseases
include, but are not limited to peripheral neuropathy, amyotrophic lateral
sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease,
ischemic stroke, acute brain injury, acute spinal chord injury, nervous
system tumors, multiple sclerosis, peripheral nerve trauma or injury,
exposure to neurotoxins, metabolic diseases such as diabetes or renal
dysfunctions and damage caused by infectious agents. Where the cellular
degeneration involves bone marrow cell degeneration, the diseases include,
but are not limited to disorders of insufficient blood cells such as, for
example, leukopenias including eosinopenia and/or basopenia, lymphopenia,
monocytopenia, neutropenia, anemias, thrombocytopenia as well as an
insufficiency of stem cells for any of the above. The above cells and
tissues can also be treated for depressed function.
The compositions and methods herein can also be useful to prevent
degeneration and/or promote survival in other non-neuronal tissues as
well. One skilled in the art can readily determine using a variety of
assays known in the art for identifying whether neurturin would be useful
in promoting survival or functioning in a particular cell type.
In certain circumstances, it may be desirable to modulate or decrease the
amount of neurturin expressed. For example, the inventors herein have
discovered that overexpression of neurturin in trangenic mice results in
obesity with the accumulation of large amounts of fat subcutaneously and
in the liver. It is believed that such overproduction of neurturin in
humans can alter metabolism such that additional adipose tissue is
produced. In such a disease condition, it would be desirable to modulate
or decrease the amount of neurturin present and treatments to modulate or
decrease neurturin can involve administration of neurturin antibodies,
either polyclonal or monoclonal, the use of antisense polynucleotides to
modulate neurturin expression, or hybrid or chimeric polypeptides with
antagonist properties.
Thus, in another aspect of the present invention, isolated and purified
neurturin antisense oligonucleotides can be made and a method utilized for
diminishing the level of expression of neurturin by a cell comprising
administering one or more neurturin antisense oligonucleotides. By
neurturin antisense oligonucleotides reference is made to oligonucleotides
that have a nucleotide sequence that interacts through base pairing with a
specific complementary nucleic acid sequence involved in the expression of
neurturin such that the expression of neurturin is reduced. Preferably,
the specific nucleic acid sequence involved in the expression of neurturin
is a genomic DNA molecule or mRNA molecule that encodes neurturin. This
genomic DNA molecule can comprise regulatory regions of the neurturin
gene, the pre- or pro-portions of the neurturin gene or the coding
sequence for mature neurturin protein. The term complementary to a
nucleotide sequence in the context of neurturin antisense oligonucleotides
and methods therefor means sufficiently complementary to such a sequence
as to allow hybridization to that sequence in a cell, i.e., under
physiological conditions. The neurturin antisense oligonucleotides
preferably comprise a sequence containing from about 8 to about 100
nucleotides and more preferably the neurturin antisense oligonucleotides
comprise from about 15 to about 30 nucleotides. The neurturin antisense
oligonucleotides can also include derivatives which contain a variety of
modifications that confer resistance to nucleolytic degradation such as,
for example, modified internucleoside linkages modified nucleic acid bases
and/or sugars and the like (Uhlmann and Peyman, Chemical Reviews
90:543-584, 1990; Schneider and Banner, Tetrahedron Lett 31:335,
1990; Milligan et al., J Med Chem 36:1923-1937, 1993; Tseng et al.,
Cancer Gene Therap 1:65-71, 1994; Miller et al., Parasitology
10:92-97, 1994 which are incorporated by reference). Such derivatives
include but are not limited to backbone modifications such as
phosphotriester, phosphorothioate, methylphosphonate, phosphoramidate,
phosphorodithioate and formacetal as well as morpholino, peptide nucleic
acid analogue and dithioate repeating units. The neurturin antisense
polynucleotides of the present invention can be used in treating
overexpression of neurturin or inappropriate expression of neurturin such
as in treating obesity or in modulating neoplasia. Such treatment can also
include the ex vivo treatment of cells.
The therapeutic or pharmaceutical compositions of the present invention
can be administered by any suitable route known in the art including for
example intravenous, subcutaneous, intramuscular, transdermal, intrathecal
or intracerebral or administration to cells in ex vivo treatment
protocols. Administration can be either rapid as by injection or over a
period of time as by slow infusion or administration of slow release
formulation. For treating tissues in the central nervous system,
administration can be by injection or infusion into the cerebrospinal
fluid (CSF). When it is intended that neurturin be administered to cells
in the central nervous system, administration can be with one or more
agents capable of promoting penetration of neurturin across the
blood-brain barrier.
Neurturin can also be linked or conjugated with agents that provide
desirable pharmaceutical or pharmacodynamic properties. For example,
neurturin can be coupled to any substance known in the art to promote
penetration or transport across the blood-brain barrier such as an
antibody to the transferrin receptor, and administered by intravenous
injection. (See for example, Friden et al., Science 259:373-377,
1993 which is incorporated by reference). Furthermore, neurturin can be
stably linked to a polymer such as polyethylene glycol to obtain desirable
properties of solubility, stability, half-life and other pharmaceutically
advantageous properties. (See for example Davis et al. Enzyme Eng
4:169-73, 1978; Burnham, Am J Hosp Pharm 51:210-218, 1994 which are
incorporated by reference).
The compositions are usually employed in the form of pharmaceutical
preparations. Such preparations are made in a manner well known in the
pharmaceutical art. One preferred preparation utilizes a vehicle of
physiological saline solution, but it is contemplated that other
pharmaceutically acceptable carriers such as physiological concentrations
of other non-toxic salts, five percent aqueous glucose solution, sterile
water or the like may also be used. It may also be desirable that a
suitable buffer be present in the composition. Such solutions can, if
desired, be lyophilized and stored in a sterile ampoule ready for
reconstitution by the addition of sterile water for ready injection. The
primary solvent can be aqueous or alternatively non-aqueous. Neurturin can
also be incorporated into a solid or semi-solid biologically compatible
matrix which can be implanted into tissues requiring treatment.
The carrier can also contain other pharmaceutically-acceptable excipients
for modifying or maintaining the pH, osmolarity, viscosity, clarity,
color, sterility, stability, rate of dissolution, or odor of the
formulation. Similarly, the carrier may contain still other
pharmaceutically-acceptable excipients for modifying or maintaining
release or absorption or penetration across the blood-brain barrier. Such
excipients are those substances usually and customarily employed to
formulate dosages for parenteral administration in either unit dosage or
multi-dose form or for direct infusion into the cerebrospinal fluid by
continuous or periodic infusion.
Dose administration can be repeated depending upon the pharmacokinetic
parameters of the dosage formulation and the route of administration used.
It is also contemplated that certain formulations containing neurturin are
to be administered orally. Such formulations are preferably encapsulated
and formulated with suitable carriers in solid dosage forms. Some examples
of suitable carriers, excipients, and diluents include lactose, dextrose,
sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,
alginates, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl-
and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil,
and the like. The formulations can additionally include lubricating
agents, wetting agents, emulsifying and suspending agents, preserving
agents, sweetening agents or flavoring agents. The compositions may be
formulated so as to provide rapid, sustained, or delayed release of the
active ingredients after administration to the patient by employing
procedures well known in the art. The formulations can also contain
substances that diminish proteolytic degradation and promote absorption
such as, for example, surface active agents.
The specific dose is calculated according to the approximate body weight
or body surface area of the patient or the volume of body space to be
occupied. The dose will also be calculated dependent upon the particular
route of administration selected. Further refinement of the calculations
necessary to determine the appropriate dosage for treatment is routinely
made by those of ordinary skill in the art. Such calculations can be made
without undue experimentation by one skilled in the art in light of the
activity disclosed herein in assay preparations of target cells. Exact
dosages are determined in conjunction with standard dose-response studies.
It will be understood that the amount of the composition actually
administered will be determined by a practitioner, in the light of the
relevant circumstances including the condition or conditions to be
treated, the choice of composition to be administered, the age, weight,
and response of the individual patient, the severity of the patient's
symptoms, and the chosen route of administration.
In one embodiment of this invention, neurturin may be therapeutically
administered by implanting into patients vectors or cells capable of
producing a biologically-active form of neurturin or a precursor of
neurturin, i.e. a molecule that can be readily converted to a
biological-active form of neurturin by the body. In one approach cells
that secrete neurturin may be encapsulated into semipermeable membranes
for implantation into a patient. The cells can be cells that normally
express neurturin or a precursor thereof or the cells can be transformed
to express neurturin or a precursor thereof. It is preferred that the cell
be of human origin and that the neurturin be human neurturin when the
patient is human. However, the formulations and methods herein can be used
for veterinary as well as human applications and the term "patient" as
used herein is intended to include human and veterinary patients.
Cells can be grown ex vivo, for example, for use in transplantation or
engraftment into patients (Muench et al., Leuk & Lymph
16:1-11, 1994 which is incorporated by reference). Neurturin can be
administered to such cells to elicit growth and differentiation. Thus, in
another embodiment of the present invention, neurturin is used to promote
the ex vivo expansion of cells for transplantation or engraftment. Current
methods have used bioreactor culture systems containing factors such as
erythropoietin, colony stimulating factors, stem cell factor, and
interleukins to expand hematopoietic progenitor cells for erythrocytes,
monocytes, neutrophils, and lymphocytes (Verfaillie, Stem Cells
12:466-476, 1994 which is incorporated by reference). These stem cells can
be isolated from the marrow of human donors, from human peripheral blood,
or from umbilical cord blood cells. The expanded blood cells are used to
treat patients who lack these cells as a result of specific disease
conditions or as a result of high, dose chemotherapy for treatment of
malignancy (George, Stem Cells 12(Suppl 1):249-255, 1994
which is incorporated by reference). In the case of cell transplant after
chemotherapy, autologous transplants can be performed by removing bone
marrow cells before chemotherapy, expanding the cells ex vivo using
methods that also function to purge malignant cells, and transplanting the
expanded cells back into the patient following chemotherapy (for review
see Rummel and Van Zant, J Hematotherapy 3:213-218, 1994 which is
incorporated by reference). Since neurturin is expressed in the developing
animal in blood, bone marrow and liver, tissues where proliferation and
differentiation of progenitor cells occur, it is believed that neurturin
can function to regulate the proliferation of hematopoietic stem cells and
the differentiation of mature hematopoietic cells. Thus, the addition of
neurturin to culture systems used for ex vivo expansion of cells could
stimulate the rate at which certain populations of cells multiply or
differentiate, and improve the effectiveness of these expansion systems in
generating cells needed for transplant.
It is also believed that neurturin can be used for the ex vivo expansion
of precursor cells in the nervous system. Transplant or engraftment of
cells is currently being explored as a therapy for diseases in which
certain populations of neurons are lost due to degeneration such as, for
example, in parkinson's disease (Bjorklund, Curr Opin Neurobiol
2:683-689, 1992 which is incorporated by reference). Neuronal precursor
cells can be obtained from animal or human donors or from human fetal
tissue and then expanded in culture using neurturin or other growth
factors. These cells can then be engrafted into patients where they would
function to replace some of the cells lost due to degeneration. Because
neurotrophins have been shown to be capable of stimulating the survival
and proliferation of neuronal precursors cells such as, for example, NT-3
stimulation of sympathetic neuroblast cells (Birren et al., Develop
119:597-610, 1993 which is incorporated by reference), neurturin could
also function in similar ways during the development of the nervous system
and could be useful in the ex vivo expansion of neuronal cells.
In a number of circumstances it would be desirable to determine the levels
of neurturin in a patient. The identification of neurturin along with the
present report showing that neurturin is expressed by a number of tissues
provides the basis for the conclusion that the presence of neurturin
serves a normal physiologic function related to cell growth and survival.
Indeed, other neurotrophic factors are known to play a role in the
function of neuronal and non-neuronal tissues. (For review see Scully and
Otten, Cell Biol Int 19:459-469, 1995; Otten and Gadient, Int J
Devl Neurosciences 13:147-151, 1995 which are incorporated by
reference). Endogenously produced neurturin may also play a role in
certain disease conditions, particularly where there is cellular
degeneration such as in neurodegenerative conditions or diseases. Other
neurotrophic factors are known to change during disease conditions. For
example, in multiple sclerosis, levels of NGF protein in the cerebrospinal
fluid are increased during acute phases of the disease (Bracci-Laudiero et
al., Neuroscience Lett 147:9-12, 1992 which is incorporated by
reference) and in systemic lupus erythematosus there is a correlation
between inflammatory episodes and NGF levels in sera (Bracci-Laudiero et
al. NeuroReport 4:563-565, 1993 which is incorporated by
Given that neurturin is expressed in blood cells, bone marrow and mast
cells, it is likely that the level of neurturin may be altered in a
variety of conditions and that quantification of neurturin levels would
provide clinically useful information. Furthermore, in the treatment of
degenerative conditions, compositions containing neurturin can be
administered and it would likely be desirable to achieve certain target
levels of neurturin in sera, in cerebrospinal fluid or in any desired
tissue compartment. It would, therefore, be advantageous to be able to
monitor the levels of neurturin in a patient. Accordingly, the present
invention also provides methods for detecting the presence of neurturin in
a sample from a patient.
The term "detection" as used herein in the context of detecting the
presence of neurturin in a patient is intended to include the determining
of the amount of neurturin or the ability to express an amount of
neurturin in a patient, the distinguishing of neurturin from other growth
factors, the estimation of prognosis in terms of probable outcome of a
degenerative disease and prospect for recovery, the monitoring of the
neurturin levels over a period of time as a measure of status of the
condition, and the monitoring of neurturin levels for determining a
preferred therapeutic regimen for the patient.
To detect the presence of neurturin in a patient, a sample is obtained
from the patient. The sample can be a tissue biopsy sample or a sample of
blood, plasma, serum, CSF or the like. Neurturin is expressed in a wide
variety of tissues as shown in example 10. Samples for detecting neurturin
can be taken from any of these tissues. When assessing peripheral levels
of neurturin, it is preferred that the sample be a sample of blood, plasma
or serum. When assessing the levels of neurturin in the central nervous
system a preferred sample is a sample obtained from cerebrospinal fluid.
In some instances it is desirable to determine whether the neurturin gene
is intact in the patient or in a tissue or cell line within the patient.
By an intact neurturin gene it is meant that there are no alterations in
the gene such as point mutations, deletions, insertions, chromosomal
breakage, chromosomal rearrangements and the like wherein such alteration
might alter production of neurturin or alter its biological activity,
stability or the like to lead to disease processes or susceptibility to
cellular degenerative conditions. Thus, in one embodiment of the present
invention a method is provided for detecting and characterizing any
alterations in the neurturin gene. The method comprises providing an
oligonucleotide that contains the neurturin cDNA, genomic DNA or a
fragment thereof or a derivative thereof. By a derivative of an
oligonucleotide, it is meant that the derived oligonucleotide is
substantially the same as the sequence from which it is derived in that
the derived sequence has sufficient sequence complementarily to the
sequence from which it is derived to hybridize to the neurturin gene. The
derived nucleotide sequence is not necessarily physically derived from the
nucleotide sequence, but may be generated in any manner including for
example, chemical synthesis or DNA replication or reverse transcription or
Typically, patient genomic DNA is isolated from a cell sample from the
patient and digested with one or more restriction endonucleases such as,
for example, TaqI and AluI. Using the Southern blot protocol, which is
well known in the art, this assay determines whether a patient or a
particular tissue in a patient has an intact neurturin gene or a neurturin
gene abnormality.
Hybridization to the neurturin gene would involve denaturing the
chromosomal DNA to obtain a single-stranded DNA; contacting the
single-stranded DNA with a gene probe associated with the neurturin gene
sequence; and identifying the hybridized DNA-probe to detect chromosomal
DNA containing at least a portion of the human neurturin gene.
The term "probe" as used herein refers to a structure comprised of a
polynucleotide which forms a hybrid structure with a target sequence, due
to complementarity of probe sequence with a sequence in the target region.
Oligomers suitable for use as probes may contain a minimum of about 8-12
contiguous nucleotides which are complementary to the targeted sequence
and preferably a minimum of about 20.
The neurturin gene probes of the present invention can be DNA or RNA
oligonucleotides and can be made by any method known in the art such as,
for example, excision, transcription or chemical synthesis. Probes may be
labelled with any detectable label known in the art such as, for example,
radioactive or fluorescent labels or enzymatic marker. Labeling of the
probe can be accomplished by any method known in the art such as by PCR,
random priming, end labelling, nick translation or the like. One skilled
in the art will also recognize that other methods not employing a labelled
probe can be used to determine the hybridization. Examples of methods that
can be used for detecting hybridization include Southern blotting,
fluorescence in situ hybridization, and single-strand conformation
polymorphism with PCR amplification.
Hybridization is typically carried out at 25-45° C., more preferably at
32-40° C. and more preferably at 37-38° C. The time required for
hybridization is from about 0.25 to about 96 hours, more preferably from
about one to about 72 hours, and most preferably from about 4 to about 24
Neurturin gene abnormalities can also be detected by using the PCR method
and primers that flank or lie within the neurturin gene. The PCR method is
well known in the art. Briefly, this method is performed using two
oligonucleotide primers which are capable of hybridizing to the nucleic
acid sequences flanking a target sequence that lies within a neurturin
gene and amplifying the target sequence. The terms "oligonucleotide
primer" as used herein refers to a short strand of DNA or RNA ranging in
length from about 8 to about 30 bases. The upstream and downstream primers
are typically from about 20 to about 30 base pairs in length and hybridize
to the flanking regions for replication of the nucleotide sequence. The
polymerization is catalyzed by a DNA-polymerase in the presence of
deoxynucleotide triphosphates or nucleotide analogs to produce
double-stranded DNA molecules. The double strands are then separated by
any denaturing method including physical, chemical or enzymatic. Commonly,
the method of physical denaturation is used involving heating the nucleic
acid, typically to temperatures from about 80° C. to 105° C. for times
ranging from about 1 to about 10 minutes. The process is repeated for the
desired number of cycles.
The primers are selected to be substantially complementary to the strand
of DNA being amplified. Therefore, the primers need not reflect the exact
sequence of the template, but must be sufficiently complementary to
selectively hybridize with the strand being amplified.
After PCR amplification, the DNA sequence comprising neurturin or pre-pro
neurturin or a fragment thereof is then directly sequenced and analyzed by
comparison of the sequence with the sequences disclosed herein to identify
alterations which might change activity or expression levels or the like.
In another embodiment a method for detecting neurturin is provided based
upon an analysis of tissue expressing the neurturin gene. Certain tissues
such as those identified below in example 10 have been found to express
the neurturin gene. The method comprises hybridizing a polynucleotide to
mRNA from a sample of tissues that normally express the neurturin gene.
The sample is obtained from a patient suspected of having an abnormality
in the neurturin gene or in the neurturin gene of particular cells. The
polynucleotide comprises SEQ ID NO:11 or a derivative thereof or a
fragment thereof.
To detect the presence of mRNA encoding neurturin protein, a sample is
obtained from a patient. The sample can be from blood or from a tissue
biopsy sample. The sample may be treated to extract the nucleic acids
contained therein. The resulting nucleic acid from the sample is subjected
to gel electrophoresis or other size separation techniques.
The MRNA of the sample is contacted with a DNA sequence serving as a probe
to form hybrid duplexes. The use of a labeled probes as discussed above
allows detection of the resulting duplex.
When using the CDNA encoding neurturin protein or a derivative of the cDNA
as a probe, high stringency conditions can be used in order to prevent
false positives, that is the hybridization and apparent detection of
neurturin nucleotide sequences when in fact an intact and functioning
neurturin gene is not present. When using sequences derived from the
neurturin cDNA, less stringent conditions could be used, however, this
would be a less preferred approach because of the likelihood of false
positives. The stringency of hybridization is determined by a number of
factors during hybridization and during the washing procedure, including
temperature, ionic strength, length of time and concentration of formamide.
These factors are outlined in, for example, Sambrook et al. (Sambrook, et
al., 1989, supra).
In order to increase the sensitivity of the detection in a sample of mRNA
encoding the neurturin protein, the technique of reverse
transcription/polymerization chain reaction (RT/PCR) can be used to
amplify cDNA transcribed from mRNA encoding the neurturin protein. The
method of RT/PCR is well known in the art (see example 10 and FIG. 6
The RT/PCR method can be performed as follows. Total cellular RNA is
isolated by, for example, the standard guanidium isothiocyanate method and
the total RNA is reverse transcribed. The reverse transcription method
involves synthesis of DNA on a template of RNA using a reverse
transcriptase enzyme and a 3′ end primer. Typically, the primer contains
an oligo(dT) sequence. The cDNA thus produced is then amplified using the
PCR method and neurturin specific primers. (Belyavsky et al, Nucl Acid
Res 17:2919-2932, 1989; Krug and Berger, Methods in Enzymology,
Academic Press, N.Y., Vol. 152, pp. 316-325, 1987 which are incorporated
by reference).
The polymerase chain reaction method is performed as described above using
two oligonucleotide primers that are substantially complementary to the
two flanking regions of the DNA segment to be amplified.
Following amplification, the PCR product is then electrophoresed and
detected by ethidium bromide staining or by phosphoimaging.
The present invention further provides for methods to detect the presence
of the neurturin protein in a sample obtained from a patient. Any method
known in the art for detecting proteins can be used. Such methods include,
but are not limited to immunodiffusion, immunoelectrophoresis,
immunochemical methods, binder-ligand assays, immunohistochemical
techniques, agglutination and complement assays. (for example see Basic
and Clinical Immunology, Sites and Terr, eds., Appleton & Lange,
Norwalk, Conn. pp 217-262, 1991 which is incorporated by reference).
Preferred are binder-ligand immunoassay methods including reacting
antibodies with an epitope or epitopes of the neurturin protein and
competitively displacing a labeled neurturin protein or derivative
As used herein, a derivative of the neurturin protein is intended to
include a polypeptide in which certain amino acids have been deleted or
replaced or changed to modified or unusual amino acids wherein the
neurturin derivative is biologically equivalent to neurturin and wherein
the polypeptide derivative cross-reacts with antibodies raised against the
neurturin protein. By cross-reaction it is meant that an antibody reacts
with an antigen other than the one that induced its formation.
Numerous competitive and non-competitive protein binding immunoassays are
well known in the art. Antibodies employed in such assays may be
unlabeled, for example as used in agglutination tests, or labeled for use
in a wide variety of assay methods. Labels that can be used include
radionuclides, enzymes, fluorescers, chemiluminescers, enzyme substrates
or co-factors, enzyme inhibitors, particles, dyes and the like for use in
radioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme-linked
immunosorbent assay (ELISA), fluorescent immunoassays and the like.
Polyclonal or monoclonal antibodies to the neurturin protein or an epitope
thereof can be made for use in immunoassays by any of a number of methods
known in the art. By epitope reference is made to an antigenic determinant
of a polypeptide. An epitope could comprise 3 amino acids in a spacial
conformation which is unique to the epitope. Generally an epitope consists
of at least 5 such amino acids. Methods of determining the spatial
conformation of amino acids are known in the art, and include, for
example, x-ray crystallography and 2 dimensional nuclear magnetic
One approach for preparing antibodies to a protein is the selection and
preparation of an amino acid sequence of all or part of the protein,
chemically synthesizing the sequence and injecting it into an appropriate
animal, usually a rabbit or a mouse (See Example 12).
Oligopeptides can be selected as candidates for the production of an
antibody to the neurturin protein based upon the oligopeptides lying in
hydrophilic regions, which are thus likely to be exposed in the mature
Antibodies to neurturin can also be raised against oligopeptides that
include one or more of the conserved regions identified herein such that
the antibody can cross-react with other family members. Such antibodies
can be used to identify and isolate the other family members.
Methods for preparation of the neurturin protein or an epitope thereof
include, but are not limited to chemical synthesis, recombinant DNA
techniques or isolation from biological samples. Chemical synthesis of a
peptide can be performed, for example, by the classical Merrifeld method
of solid phase peptide synthesis (Merrifeld, J Am Chem Soc 85:2149,
1963 which is incorporated by reference) or the FMOC strategy on a Rapid
Automated Multiple Peptide Synthesis system (DuPont Company, Wilmington,
Del.) (Caprino and Han, J Org Chem 37:3404, 1972 which is
incorporated by reference).
Polyclonal antibodies can be prepared by immunizing rabbits or other
animals by injecting antigen followed by subsequent boosts at appropriate
intervals. The animals are bled and sera assayed against purified
neurturin protein usually by ELISA or by bioassay based upon the ability
to block the action of neurturin on neurons or other cells. When using
avian species, e.g. chicken, turkey and the like, the antibody can be
isolated from the yolk of the egg. Monoclonal antibodies can be prepared
after the method of Milstein and Kohler by fusing splenocytes from
immunized mice with continuously replicating tumor cells such as myeloma
or lymphoma cells. (Milstein and Kohler Nature 256:495-497, 1975;
Gulfre and Milstein, Methods in Enzymology: Immunochemical Techniques
73:1-46, Langone and Banatis eds., Academic Press, 1981 which are
incorporated by reference). The hybridoma cells so formed are then cloned
by limiting dilution methods and supernates assayed for antibody
production by ELISA, RIA or bioassay.
The unique ability of antibodies to recognize and specifically bind to
target proteins provides an approach for treating an over expression of
the protein. Thus, another aspect of the present invention provides for a
method for preventing or treating diseases involving over expression of
the neurturin protein by treatment of a patient with specific antibodies
to the neurturin protein.
Specific antibodies, either polyclonal or monoclonal, to the neurturin
protein can be produced by any suitable method known in the art as
discussed above. For example, murine or human monoclonal antibodies can be
produced by hybridoma technology or, alternatively, the neurturin protein,
or an immunologically active fragment thereof, or an anti-idiotypic
antibody, or fragment thereof can be administered to an animal to elicit
the production of antibodies capable of recognizing and binding to the
neurturin protein. Such antibodies can be from any class of antibodies
including, but not limited to IgG, IgA, IgM, IgD, and IgE or in the case
of avian species, IgY and from any subclass of antibodies.
Claim 1 of 3 Claims
1. Isolated or purified
antibodies which specifically bind with a neurturin polypeptide, wherein
said polypeptide is identified by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7 or
SEQ ID NO:8, and wherein said polypeptide promotes survival of superior
cervical ganglion cells or nodose ganglion cells.
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full


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Once upon a time there was only one firm on the lips of any business' IT team when it came to company phones – BlackBerry.
A lot has changed since then and BlackBerry's fortunes have taken a turn for the worse, leaving the door wide open for rivals to court the enterprise business.
Now the rest of the pack is hot on its heels, if not already overtaking it, with the likes of Apple, Samsung, Nokia and HTC all vying for a piece of the business pie.
This is great for any potential business buyer, as you now have a wide choice of handsets to choose from, allowing you to select the best phones for your company's requirements.
In no particular order, here are the top 10 smartphones for business use and business users:
HTC One M8
It's been heralded as the "best mobile phone in the world", and rightly so, as the HTC One M8 fuses superb design with excellent functionality.
If you're looking to make a statement the next time you enter a meeting the One M8's full metal body puts it up there with the iPhone 5S in terms of premium style and boardroom presence.
The full HD, 5-inch display is beautiful to look at and the punchy quad-core processor and 2GB of RAM ensures you'll never be short of power.
There are plentiful amounts of bells and whistles on the HTC One M8 and the Android KitKat operating system allows for a wealth of customisation - although HTC has heavily skinned it with its Sense 6 overlay.
You may find that overlay gets in the way of core business functions, so it's worth checking out the HTC One M8 before hand, but if the interface is manageable you'll be more than happy getting this out in meetings.
The One M8 is also HTCpro certified, which adds additional encryption and mobile device management tools to the handset especially for enterprise customers.
- Read: HTC One M8 review
Sony Xperia Z2
If you're looking for something a little more understated, yet still premium and powerful the Sony Xperia Z2 is just the ticket.
A sleek, industrial design coupled with powerful specs including a quad-core processor, 3GB of RAM, 5.2-inch full HD display and an impressive 20.7MP camera means the Xperia Z2 is a high performer all round.
Built in noise cancelling technology means calls will be clearer, and your music tracks crisper when you find yourself in noisy environments.
The Xperia suite of business apps including Email, Calendar and Contacts are also present on the Z2, allowing you to keep working even when you're away from your desk.
Sony has also built in a host of security features for businesses into the Xperia Z2 including device, network and storage security.
- Read: Sony Xperia Z2 review
iPhone 5S
More and more businesses are turning to Apple's smartphone offerings, and while it produces great looking, powerful devices, they certainly come at a price.
The iPhone 5S is the latest in Apple's line up, and while it only builds fractionally on the iPhone 5 it continues to turn heads in every circle.
Apple offers a wide range of enterprise resources on the iPhone 5S with support for SSL VPN and data protection APIs, enabling third party app makers to produce fully secure solutions.
There's even a fingerprint scanner hidden beneath the home key on the iPhone 5S, adding another layer of security to the handset.
On the topic of apps, the iPhone family is best placed when it comes to business focussed software, with thousands of applications available on the App Store aimed at making work easier - not to mention Apple's own fleet of business software which is now available for free.
You also get Siri, your own personal digital assistant who listens to your voice and brings you whatever you need, from meeting reminders to the latest footy scores.
The iPhone 5S runs Apple's latest software, iOS 7, which features a fresh new look allowing it to keep pace with the rapidly evolving Android platform.
If your company has already upgraded to the iPhone 5 there really is no need to make the switch to the 5S, you're better off waiting for the iPhone 6 at least.
- Read: iPhone 5S review
Sony Xperia Z1 Compact
If your budget can't stretch to a fleet of the latest iPhone and the last thing you want is a load of 5-inch+ smartphones stretching jacket pockets and employee's palms then the pint-sized Z1 Compact is the phone for you.
The Xperia Z1 Compact packs in the same power as its bigger brother, the Xperia Z1, but it comes in a smaller package with a 4.3-inch display.
It may not be a full HD display, but the 720p resolution is fine at this size and it means the Z1 Compact is infinitely more pocketable than its high-end Android rivals.
As well as impressive quad-core power and 2GB of RAM the Z1 Compact also features Android KitKat, a 20.7MP rear camera, plus a dust and waterproof chassis.
Not bad for a smartphone which is just a touch larger than the iPhone 5S.
Samsung Galaxy Note 3
Working on your smartphone is usually problematic due to the restrictive screen, but manufacturers are looking to ease the issues with supersized handsets.
The Samsung Galaxy Note 3 arrives with a huge 5.7-inch, full HD display and its very own S-Pen stylus, merging the line between smartphone and tablet.
If your business is creatively minded then you may find the accuracy of the S-Pen and the ability to draw a real advantage.
You can also annotate documents, emails and anything else on screen using the stylus, allowing you to provide instant feedback to colleagues.
Multitasking has always been tricky on smartphones, partly due to the screen size and the fact that it's not really possible to have two apps on screen at once.
Samsung has overcome this problem on the Galaxy Note 3 by allowing you to run two apps side by side on the screen, so you could be looking at the latest financials while tapping out a quick email to the shareholders.
Of course, the size of the handset may hamper some as it isn't the most pocket friendly of devices, nor is it the lightest.
Also, some of the applications, especially those designed for use with the S-Pen, are overly complicated in places so intensive training may be required for some staff.


text:
For my last wedding of the fall season I had the opportunity to travel to Milwaukee Wisconsin for Rachel and Bryan's wedding shindig! Milwaukee has a similar vibe to the Fort Point District of Boston, with several converted buildings, except for one main difference, most of the brick you'll find in Milwaukee is cream, hence the cities nickname "Cream City."
Rachel and Bryan are also my seasons winners (if I was running a contest) for the best photo bomb, see below! Thanks again for having me back to your fabulous city Rachel and Bryan, I had a blast!
Salon: Actaea Works
Getting Ready: Hotel Metro
Ceremony + Reception: The Hamilton
Cupcakes: Classy Girl Cupcakes


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This hotel is conveniently located and caters well for both business and leisure guests. It is close to Leeds yet has a quiet spot near to the Yorkshire Dales. The lovely gardens lead on to wildlife w...More about Mercure Leeds Parkway Hotel
Originally the residence of a local chapel minister, this modern, well-established hotel provides well-equipped, comfortable bedrooms. It also boasts a cosy bar and pleasant restaurant, serving an ima...More about Dalesgate Hotel
With stunning views of the Wharfedale countryside this beautiful hotel, owned by the Duke and Duchess of Devonshire, dates back to the 17th century. Bedrooms are elegantly furnished; those in the old ...More about The Devonshire Arms Country House Hotel & Spa


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21 March 2012
Selected students and staff from Cardiff are celebrating today as it was announced that a trio of Torchbearers have been chosen to represent the University as part of Samsung's nomination programme for the London 2012 Olympic Torch Relay.
In addition to its global search for the world's unsung heroes, Samsung, a presenting partner of the London 2012 Olympic Torch Relay teamed up with 31 university partners across the UK last year to create this unique Torchbearer nomination programme.
Selecting from staff, students and the community around the university, the programme aimed to find individuals who always go the extra mile and was designed to reflect the core value of the Olympic movement and the rich heritage of each institution – helping Samsung achieve its ambition of making London 2012 Everyone's Olympic Games.
The three lucky Torchbearers, Ruby Miller, a student in the School of Healthcare Studies, Guylee Simmonds, a student in the Welsh School of Architecture, and Stuart Vanstone, Head of Sport at the University will each carry the Olympic Flame through Cardiff as it travels on its 70 day journey before the start of the London 2012 Olympic Games.
Stuart Vanstone comments: "The announcement that I am one of the London 2012 Torchbearers is fantastic and very exciting. Furthermore the opportunity for Ruby and Guylee to run with the torch in Cardiff is a once in a lifetime opportunity. We have been working extensively with Samsung, one of the torch relay presenting partners and are grateful for the opportunity they have given to us.
"Alongside this, the numerous projects that Cardiff is running ahead of the Games will have a lasting legacy for the University and the city and places us at the forefront of Olympic activity in Wales."
The University representatives make up part of the 8,000 Torchbearers who will carry the Olympic Flame across the UK. Starting in Land's End on 19th May 2012, the Olympic Flame's journey will reach more than 1000 cities, towns and villages across the UK, with 95% of the UK population being within one hour's distance of this historic event. The Olympic Flame will be travelling through the streets of Cardiff from 25-26 May 2012, with local street celebrations taking place along the route. The Olympic Flame will finish its epic route and arrive at the Olympic Stadium in London on 27th July 2012 to mark the start of the Olympic Games. For the full Olympic Torch Relay route, click here and to learn more about some of Samsung's other successful Torchbearers visit www.samsung.com/uk/london2012
Cardiff University Sport
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