Note: Descriptions are shown in the official language in which they were submitted.
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NON-INVASIVE DELIVERY OF POLYPEPTIDES
THROUGH THE BLOOD-BRAIN BARRIER,
AND IN VIVO SELECTION OF ENDOCYTOTIC LIGANDS
FIELD OF INVENTION
This invention relates to methods for delivering polypeptides into brain and
spinal
tissue in humans and other mammals, and in other higher animals that have a
blood-brain
barrier (BBB). It also relates to methods for targeting delivery of
polypeptides to specific
populations or types of neurons entirely within the brain or spinal cord. It
further relates to
treatments for neurological disorders in humans, and to other forms of
treatment (such as to
regulate fertility, maturation, growth rates, etc.) in livestock and other
animals, using
polypeptides that normally cannot cross the BBB.
BACKGROUND OF THE INVENTION
The "blood brain barrier" (BBB) in higher animals helps ensure that neurons in
the
brain and spinal cord are not exposed to molecules in blood that would
interfere with
neuronal functioning if they penetrate the BBB. The BBB is not a single
membrane.
Instead, the capillaries in the brain and spinal cord are formed by
endothelial cells that form
"tight junctions". By contrast, capillary walls outside the brain and spinal
cord have "slit-
pores" between adjacent cells, which make those capillaries much more
permeable. The
BBB system is described in many textbooks and articles (e.g., Goldstein et al
1986;
Pardridge 1998; Rubin et al 1999; Banks 1999; Kniesel et al 2000; Kniesel et
al 2000).
Animal models for studying BBB permeation are described in Bonate 1995, and
early
efforts to develop cell culture models are described in de Boer et al 1999.
Although the BBB protects the brain and spinal cord, it also excludes many
therapeutic agents that could help treat diseases or injuries that affect the
nervous system. In
particular, with only a few exceptions, proteins and peptides cannot cross the
BBB in any
substantial quantities (e.g., Langer 1990). A small number of exceptions
involve limited
transport systems and certain types of neuronal receptors, such as transferrin
receptors
(e.g., Kastin et al 1999 and Granholm et al 1998). However, those limited
exceptions are
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not adequate for treating CNS disorders using various types of polypeptides.
The need for
better methods to transport therapeutic polypeptides (and other types of
useful drugs) across
the BBB is well-known, and many review articles have been published on the
subject,
include Zlokovic 1995, Cloughesy et al 1995, Davis et al 1995, Abbott et al
1996, Begley
1996, Kroll et al 1998, Pardridge et al 1998, Rochat et al 1999, and Rapoport
2000.
This invention relates to the delivery of polypeptides into brain or spinal
tissue that
is protected by the BBB. As used herein, polypeptide refers to any peptide
molecule
(formed by linking amino acids together) that has been formed, inside a living
cell, by gene
expression. Gene expression inside cells involves: (i) transcribing a DNA gene
sequence to
form a strand of messenger RNA (mRNA), followed by (ii) translating the mRNA
strand to
form a polypeptide chain made of amino acids bonded together in a precise
sequence. Any
such molecule formed by expression of a gene sequence is referred to herein as
a
polypeptide, regardless of whether the polypeptide is later processed by "post-
translation"
steps such as cleavage, cell secretion, glycosylation, cysteine cros slinking,
etc.
Some scientists use "protein" to describe polypeptides that are complete and
functional (as distinct from polypeptide fragments, or polypeptide precursors
that have not
yet been processed in ways necessary to render them active). In this
invention, a
polypeptide will not be of interest unless it is indeed capable of carrying
out an intended
function; therefore, the terms "polypeptide" and "protein" are used
interchangeably herein.
A polypeptide is of interest herein (and is covered by the claims) only if it
has three
traits. First: as discussed below, the polypeptide must be expressed by a
foreign gene (also
called an "exogenous" gene) that has been inserted into one or more classes of
neurons,
using an intervention method such as described herein. However, a foreign
(exogenous)
gene carried by a vector may have exactly the same sequence as a "native" or
"natural"
gene that is expressed normally inside the CNS. This can occur, for example,
in a
treatment that increases levels of a polypeptide that is no longer being
produced in adequate
amounts, in a person suffering from a disease.
Second: the polypeptide must be expressed in one or more classes of neurons
which
are then capable of releasing the polypeptide inside brain or spinal tissue
that normally is
protected by the blood-brain barrier. As used herein, phrases such as
"delivering
polypeptides through a blood-brain barrier" or "releasing polypeptides inside
(or into) brain
or spinal tissue" indicate and require that the foreign polypeptide(s) must
contact at least
one cell or class of cells that reside wholly within the BBB. This requirement
is not
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satisfied if a polypeptide is secreted by a BBB-straddling neuron only at
locations where the
polypeptide contacts other BBB-straddling neurons. Instead, in most
situations, this
requirement will be satisfied only if a foreign polypeptide is secreted by a
neuron into
cerebrospinal fluid (CSF) and/or synaptic fluid, at a location inside the BBB.
Third: a foreign polypeptide is of interest if and only if it is therapeutic
and/or
otherwise useful, and has a desired and useful (rather than pathogenic)
neuroactive
property. This excludes viral infections or other non-useful infections,
tests, and procedures
that involve attacks or challenges to brain or spinal tissue. Many tests have
been done on
lab animals, using viruses that infect neurons or glial cells, to test
antiviral drug candidates,
and to perform "tracer" studies that analyze neuronal networks and connections
by infecting
animals with a virus such as rabies, then sacrificing the animals after a
number of hours
and analyzing samples of brain tissue, to see which neurons were infected by
the viruses.
Although such tests have used viruses to introduce foreign polypeptides into
BBB-protected
brain or spinal tissue, it should be clear that inflicting those types of
infections on research
animals (most of which were killed, as part of the experiment) were
pathogenic, rather than
therapeutic. Accordingly, this invention is substintially different from such
tracer and other
pathogen tests, since this invention is designed to provide a method for
introducing
therapeutic or otherwise useful and beneficial polypeptides into BBB-protected
tissue, in
fields such as human medical therapy, and in breeding of livestock or
endangered species.
The reference to "neuroactive" is also a necessary part of the third factor
discussed
above. As used herein, "neuroactive" polypeptides are polypeptides that can
exert a
therapeutic or otherwise useful and/or desirable result or effect, if properly
delivered to a
desired and intended region of brain or spinal tissue that is protected by the
BBB. Various
examples are discussed below and listed in the tables herein.
It should be noted that useful neuroactive polypeptides may include
polypeptides that
will exert direct effects only on glial cells, without requiring direct
effects on neurons, if
the treated glial cells will generate a response that leads to a therapeutic
or other beneficial
result among neurons that are in fluid communication with the treated glial
cells.
Also, the term "polypeptide" include variants, derivatives, and fragments of
naturally occurring or genetically engineered polypeptides. Examples include:
(i) chimeric
or fusion polypeptides, which have a neuroactive portion derived from one
gene, and a
"leader" sequence (to increase stability, transport, secretion, or some other
useful trait or
activity) from some other gene; and, (ii) fragments or portions of
polypeptides (such as a
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single-chain binding fragment which has been isolated from the variable domain
of a
monoclonal antibody) that have a desirable form of neuroactivity.
The following section offers an overview of four categories of neuroactive
polypeptides that can offer therapeutic benefits in people or animals
suffering from
neurological disorders.
Neurotrophic Factors
The root word "-troph" comes from the Greek work for "nourishment" or "food".
"Trophic" implies that a certain molecule is involved in the stimulation,
growth,
nourishment, sustenance, or similar support of a certain system. "Neurotrophic
factors" are
molecules that promote neuronal growth, cause the formation of new synaptic
connections
between neurons, or carry out other stimulating or supporting neuronal
activities. However,
this term excludes: (i) nutrients (such as oxygen, glucose, amino acids, and
nucleotides)
that are required by all cells, and (ii) neurotransmitters which directly
modulate nerve
impulses between neurons (such as glutamate, acetylcholine, dopamine,
serotonin, etc.).
Most neurotrophic factors function in a manner similar to hormones; they are
secreted by one type of cell, and subsequently interact with other cells.
However, they
differ from hormones in that, after secretion, they typically interact only
with neighboring
cells. In some respects, they act in a manner comparable to "paracrine"
factors; this term
describes hormone-like molecules that act only locally, while "endocrine"
factors act on the
entire system and can affect cells distant from the secreting cell.
Neurotrophic factors are discussed in articles such as Lindsay 1994, Snider et
at
1994, Bothwell et at 1995, Lewin et at 1996, and Skaper et at 1998, and in
numerous US
patents. The first neurotrophic factor that was discovered to stimulate nerve
cell growth was
called "nerve growth factor" (NGF). Later, other neurotrophic factors were
discovered, and
they required more elaborate names, such as brain-derived neurotrophic factor
(BDNF),
glial-cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor,
and names with
numbers in series, such as neurotrophin-3, neurotrophin-4, etc.
Many neurotrophic factors are polypeptides, and these offer potential
therapies for
many CNS disorders, including: (1) treatments for brain damage caused by
physical
injuries, such as automobile accidents, concussions, etc.; (2) brain damage
caused by a
stroke, cardiac arrest, near-drowning or suffocation, loss of blood, or other
problems
involving ischemia (inadequate blood flow) or hypwda (inadequate oxygen
supply); and, (3)
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neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease,
Huntington's
disease, amyotrophic lateral sclerosis, etc. These potentials are discussed in
articles such as
Hefti 1994, Thoenen et al 1994, Ibanez et al 1995, McMahon and Priestley 1995,
and Yuen
and Mobley 1996.
For convenience, terms herein such as disorder, damage, and injury refer to
any
CNS disorder (whether from trauma, disease, etc.) that might be prevented or
treated by
one or more therapeutic polypeptides, if such polypeptides can be delivered to
the proper
regions of the brain or spinal cord.
Hypothalamic Releasing Factors
The hypothalamic releasing factors are short polypeptides that are made and
secreted
by nerve cells in the hypothalamus. These polypeptides in turn stimulate the
pituitary gland
to secrete various hormones that control and affect numerous important body
functions
involved in growth, metabolism, water and salt balance of the blood,
reproduction, etc. are
potent, and are the subject of considerable study. Articles that describe
hypothalamic
releasing factors include Turnbull et al 1999, Phelps et al 1999, and
Sawchenko et al 2000.
Peptide Neurotransmitters
A number of peptide neurotransmitters have been described, including Substance
P,
enkephalins, endorphins, vasointestinal peptide (VIP), calcitonin gene related
peptide
(CGRP), galinin, somatostatin, etc. In many cases, peptide neurotransmitters
act in a
manner similar to classical neurotransmitters, such as glutamate or
acetylcholine; they are
released by a neuron at a synapse, where a binding reaction to a specific
receptor on the
other neuron at the synapse stimulates or inhibits the transmission of a nerve
signal (also
called a nerve impulse, firing, or depolarization). In other cases, binding of
a peptide
neurotransmitter to a receptor at a synapse exerts a more prolonged effect,
such as
increasing or decreasing the sensitivity of the target neuron to other
neurotransmitters. In
the perception of pain, for example, Substance P is released by nocioceptive
(pain-
transmitting) nerve fibers in the spinal cord, and is excitatory, while
enkephalins and
endorphins have the opposite effect, and inhibit transmission of pain signals.
The pain relief
provided by morphine arises from binding to endorphin receptors; however, the
use of
endorphins for the treatment of pain is not possible at present, because of
the difficulty in
safely delivering such peptides into the brain.
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Cytokines and Other Growth Factors
Some cytoldnes and other growth factors can function as neurotrophic factors,
by
acting locally to stimulate nerve growth. However, cytoldnes can be
distinguished from
neurotrophic factors by their ability to stimulate growth and mitosis of
various non-neuronal
and dividing cells relating to the nervous system. Examples include acidic and
basic
fibroblast growth factor (aFGF and bFGF), epidermal growth factor (EGF),
platelet derived
growth factor (PDGF), insulin-like growth factor-I and II (IGF-I and IGF-II),
tumor
necrosis factor-B (TGF-B), leukemia inhibitory factor (LIF), various
interleukins, etc.
These molecules are involved in normal physiological processes such as CNS
tissue
growth, remodelling and repair after injury, and immune responses within the
CNS. While
they have potential to be used to treat CNS injuries or infections, they pose
a risk of
stimulating cancer cells, especially if administered in a systemic fashion
throughout the
CNS. Nevertheless, they are of great interest to neurology researchers, and
development of
methods for delivering cytokines and similar molecules to discrete and limited
CNS regions
would be of great interest and potential benefit.
Table 1 provides a partial listing of known CNS-active polypeptides. To
illustrate
one area of application in slightly more detail, Table 2 provides examples of
some CNS
disorders, along with the polypeptides that are currently thought to have
great promise in
treating or preventing these disorders. Tables 1 and 2 contain only partial
listings, and
numerous research efforts (including the human, mouse, and rat genome
sequencing
projects) are rapidly leading to the identification of numerous other
neuroactive
polypeptides.
The biggest and most difficult obstacle that is hindering the evaluation of
neuroactive
polypeptides, and the use of such therapies in patients who need such help, is
the severe
difficulty of actually delivering neuroactive polypeptides to specific regions
of the brain and
spinal cord where those polypeptides must be able to contact specific and
targeted
populations of neurons or glial cells in order to provide a therapeutic,
preventative, or other
benefit. Accordingly, the purposes and goals of this invention are: (i) to
provide methods
for delivering foreign polypeptides into areas of brain and spinal tissue that
are protected by
the BBB, and (ii) to provide methods for delivering foreign polypeptides to
targeted,
specific, and limited regions of brain and spinal tissue, and/or to targeted,
specific, and
limited types or classes of cells inside the CNS.
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PRIOR ART CNS DRUG DELIVERY METHODS
Reviews of the drug delivery literature (e.g., Langer et at 1989; Langer 1990;
Madrid et at 1991; Thoenen et at 1994; Zlokovic 1995, Cloughesy et at 1995,
Davis et at
1995, Abbott et at 1996, Begley 1996, Kroll et at 1998, Pardridge et at 1998,
Rochat et at
1999, and Rapoport 2000) identify few currently available, effective, and
clinically practical
alternatives for delivering polypeptides across the blood brain barrier.
Various approaches
to delivering drugs into the BBB that have been tried, without great success,
are reviewed
in articles such as Zlokovic 1995 and Rapoport 2000. These efforts can be
categorized and
summarized as follows:
1. Invasive methods, which require physical intrusion, such as surgery, or at
a
minimum, use of a hypodermic needle to puncture cells and membranes. One
example is
intraventricular injection or infusion, which require a neurosurgeon to gain
access to one of
the "ventricles" that serve as production sites or gathering nodes for
cerebrospinal fluid, so
that a drug can be injected into the fluid that has accumulated in a
ventricle. Most of these
ventricles are buried deep inside the brain (the fourth ventricle is somewhat
accessible in
humans, but it is at an exit point for fluid leaving the brain, so it is not
useful for
delivering drugs to most of the brain). Injecting drugs into brain ventricles,
even if only
with a needle, is difficult and dangerous. Any penetration by a blade or
needle will damage
capillaries along the way, and if blood leaks out from damaged capillaries, it
will contact
neurons that must be protected from blood by the BBB. If cautery is used to
minimize
blood leakage, capillaries and small arteries and veins that are cauterized
will no longer
carry blood, and neurons served by those blood vessels may be killed.
Another invasive method involves transplantation of genetically selected
and/or
engineered cells, which can secrete neurotrophic molecules at high rates, into
target regions
of the brain (e.g., US Patent 5,082,670, Hefti et at 1992). The insertion of
such cells into a
target portion of the brain requires invasive access, using blades or needles,
and poses
serious problems and dangers. Because of these problems, these invasive
techniques are
cannot be used in humans except in very limited clinical trials, in patients
suffering from
terminal diseases, or diseases that are so severe and debilitating that a
patient cannot be
significantly helped by any other treatment.
2. Non-invasive methods, which use chemical, cellular, carrier-mediated, or
other
non-surgical methods to transport neurotrophic or similar molecules across the
blood-brain
barrier. These methods typically require fairly large quantities of vesicles,
complexes, or
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other macromolecules to be injected into the bloodstream, in the hope that
some quantity of
the injected "passenger" molecules will enter the brain and be transported
across the BBB,
into brain tissue. Examples of these approaches are described in items such as
US patent
5,154,924 (Friden 1992), a chapter by Thoenen et al in Racagni et at 1994, and
Zlokovic
1995.
One of the main problems with these approaches is that if a certain compound
can
pass through the BBB in a significant quantity, that same compound will also
pass through
the much more permeable capillaries in the rest of the body, faster and in
larger quantities.
This leads to two serious problems. The first involves side effects that occur
when large
quantities of neuroactive compounds are delivered into non-CNS tissue; such
compounds
can disrupt the peripheral nervous system, or other types of cells or tissue.
The second set
of problems relate to the very high expenses that arise when large quantities
of complex
drugs must be carefully synthesized, purified, and quality-tested before
injection into a
human. In addition, such injections or infusions usually must be repeated,
often numerous
times, and after an injection or infusion of a large quantity of a neuroactive
compound, a
patient must be monitored for at least a day or more, usually in a hospital or
clinic setting
rather than at home, so that emergency measures may be taken immediately if
the patient
suffers an adverse reaction.
Also, the prior methods do not allow foreign polypeptides to be administered
only to
selected neurons, classes of neurons, or brain regions. By contrast, this
invention offers a
number of promising approaches to administering foreign polypeptides to
targeted neurons
in a controllable and selective (or at least enriched) manner. By targeting
only certain
locations or regions inside the CNS, and/or by targeting only certain types or
clusters of
neurons, the risk and severity of side effects can be minimized.
GENE THERAPY
One approach that has potential for treating some CNS disorders involves gene
therapy. In prior efforts to genetically modify brain and spinal neurons, two
main
approaches have been used. In one approach, cells are genetically altered,
outside the body,
and then transplanted somewhere in the CNS, usually in an area inside the BBB.
In the
other approach, genetic "vectors" are injected into one or more regions in the
CNS, to
genetically alter cells that are normally protected by the BBB. Because
serious obstacles
arise when attempts are made to adapt procedures that work well, in other
parts of the
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body, to the special challenges of treating brain or spinal tissue, these two
types of
approaches are described in more detail below.
Transplant of Genetically Engineered Cells
In conventional tissue, cell transplants usually involve isolating a
population of cells,
manipulating those cells under cell culture (in vitro) conditions, and then
transplanting the
manipulated cells into the animal or patient. In some cases, the cells are
obtained from the
host, to minimize potential rejection problems after the cells are implanted
back into the
host. In other cases, they may be from other sources (such as an immortal cell
line, fetal or
stem cells, etc.). The genetic manipulation may involve precise and controlled
techniques,
or it may involve "shotgun" approaches followed by screening tests.
Using this general approach in CNS tissue is generally not practical, for a
number of
reasons, including the following brief listing. First: the CNS is
heterogeneous in the
extreme, and nervous tissue from one region can not be substituted for nervous
tissue from
another region. Second: CNS neurons in adults are post-mitotic, and will not
divide and
repopulate if a vacancy is created. Third: nerve cell processes or axons, once
broken, do
not readily regrow and reestablish synaptic and other connections. Fourth:
surgical or
similar extraction of CNS tissue poses great risks to a patient, even if the
surgery is done
with the highest level of skill and care.
For these and other reasons, in the vast majority of cases, it simply is not
feasible or
practical to remove a population of CNS neurons from a patient, genetically
manipulate the
neurons, and then return them back into the patients's brain or spinal cord.
Therefore, most
experiments to implant genetically engineered cells into CNS tissue have not
even attempted
to use cells taken from the same patient. Instead, early tests used
genetically engineered
cells (not necessarily neurons) that had been modified to secrete abnormally
large quantities
of some desired protein (such as nerve growth factor, for transplantation into
the brains of
patients suffering from Alzheimer's disease). These types of tests and
treatment efforts are
described in articles such as Blesch and Tuszynski 1996, Karpati et al 1996,
Fick and Israel
1994, Fisher and Ray 1994, and Friedman 1994, and in patents such as US
5,082,670
(Hefti et al 1992). Although this research is interesting and may become
useful some day,
major risks and dangers will be unavoidable, whenever foreign cells must be
implanted
inside CNS tissue. Any such implants must puncture or cut through brain or
spinal tissue,
and the process of implantation will necessarily injure the brain or spinal
tissue that must be
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)enetrated or displaced.
By contrast, genetic manipulation of CNS cells, without moving the cells
(called in
dtu treatment) offers better hope for correcting a problem without inflicting
invasive
lamage on brain or spinal tissue.
Genetic Alteration of CNS Cells In Situ
This approach aims to introduce into the CNS a source of a desirable
polypeptide,
by genetically altering cells within the CNS without moving those cells. In
the prior art,
this has been achieved, a number of times, by directly injecting genetic
vectors carrying
foreign genes into brain or spinal tissue. However, this approach is invasive;
brain or spinal
tissue must be punctured or cut, somehow, by a needle or blade. What is
needed, instead,
is a non-invasive method of genetically transfe,cting or transforming cells
that sit inside the
BBB, without having to puncture, cut, or otherwise invade or penetrate any
tissue that is
protected by the BBB.
The terms "transfect" and "transform" are used interchangeably herein. Both
terms
refer to a process which introduces a foreign ("exogenous") gene into one or
more
preexisting cells, in a manner that causes the foreign gene(s) to be expressed
to form
polypeptides. To some scientists, "transfect" implies that any foreign gene(s)
will be
expressed only transiently, in a manner analogous to an infection that lasts
only for a while
and eventually is stopped. By contrast, "transform" implies a permanent
genetic alteration
that will be passed on to any and all progeny cells, usually due to
integration of the foreign
gene(s) into one or more cell chromosomes. However, the boundary lines between
those
terms are blurred, and the distinctions between those terms are not used
consistently by all
scientists. Accordingly, "transfect" and "transform" are used interchangeably
herein,
regardless of how long a foreign gene might continue to be expressed after it
enters target
cell(s).
GENE VECTORS
There are two broad classes of genetic vectors. The viral vectors are derived
from
viruses, and make use of the lipid envelope or surface shell (also known as
the capsid) of a
virus. These vectors use a virus's ability to bind to one or more particular
surface proteins
on certain types of cells, and then inject the virus's DNA or RNA into the
cell. These have
become the dominant class of vectors that have been used in attempts at human
gene
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therapy, and they are described in numerous published articles. Gene transfers
into CNS
aeurons have been reported, using vectors derived from herpes simplex viruses
(e.g.,
European Patent 453242, Breakfield et al 1996), adenoviruses (La Salle et al
1993), and
adeno-associated viruses (Kaplitt et al 1997).
All other genetic vectors are generally grouped into a broad class of non-
viral
vectors. These vectors typically contain a "gene expression construct", which
is understood
by those skilled in the art. By way of illustration, such a construct might be
a plasmid,
which normally would contain (i) a bacterial origin of replication, so it can
be grown
quickly in host cells such as E. coli; (ii) at least one selectable marker
gene, to allow host
cells containing the plasmid to inactivate an antibiotic, grow on lactose
without requiring
any other carbon source, or turn color when certain chemicals are present,
and, (iii) at least
one passenger gene, containing a suitable promoter and a coding sequence that
will express
the desired polypeptide, after the gene expression construct has been inserted
into targeted
mammalian cells.
A non-viral genetic vector is created by adding, to a gene expression
construct,
agents that will enable and promote entry of the gene construct into target
cells. Several
commonly used agents include cationic lipids, positively charged molecules
such as
polylysine or polyethylenimine, and/or ligands that bind to receptors
expressed on the
surface of the target cell (such as, in some cases, a viral ligand that was
originally obtained
from a virus). Major categories of non-viral gene vectors include:
1. cationic gene vectors. DNA strands and cell surfaces are both negatively
charged.
Therefore, positively-charged agents can help DNA strands overcome that
electrical
repulsion, and enter target cells. Examples include polylysine,
polyethylenimine (PEI), and
various cationic lipids. These agents are described in articles such as Sahenk
et al 1993,
Nabel et al 1997, and Li et al 2000.
2. receptor-targeting gene vectors, in which a strand of DNA is bonded to a
molecule that normally has its own way to enter certain types of cells,
through receptor
molecules on the surfaces of certain cells. This approach was demonstrated in
liver cells in
US patent 5,166,320 (Wu et al 1992). By using this approach, DNA delivery can
be
targeted to a particular type of cell, by coupling DNA to a molecule that is
selectively taken
up by that type of target cell. This process depends on cell receptors, which
typically are
proteins that straddle the outer membrane of a cell, with part of the receptor
protein
exposed outside the cell, and part of the protein anchored inside the cell
cytoplasm. To be
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suitable for this type of uptake, the receptor must undergo a process called
"endocytosis",
which means that, when a suitable ligand binds to the receptor, a process is
triggered that
cause both the receptor, and the ligand that is bound to it, to be pulled into
the cell.
Since endocytosis is of substantial importance in one aspect of this invention
(which
involves a new type of in vivo screening method that uses nerve fibers to
identify ligands
that can activate and drive endocytosis), it is discussed in more detail
below.
3. Other non-viral genetic vectors. Since viral vectors suffer from problems
and
limitations, and since cationic compounds and receptor-targeting ligand
vectors currently are
not nearly as efficient as viral vectors in leading to expression of foreign
genes by target
cells, researchers have attempted to develop ways to enhance gene delivery
into cells. One
such effort involves the use of viral capsid proteins that can rupture the
"endocytotic
vesicle" that is formed when a foreign molecule or particle is drawn through a
cell's outer
membrane, through endocytosis. Before foreign DNA can begin acting as a gene,
it must
escape from the endocytotic vesicle, and some types of viruses (including
adenoviruses)
have evolved capsid molecules that rupture endocytotic vesicles, thereby
releasing the
foreign DNA inside the cell cytoplasm. An example of this type of effort is
described in
Curiel 1997.
Various other efforts are being made in other areas, but none of those efforts
have
yet managed to reach the mainstream of research on genetic vectors.
VIRAL CNS INFECTIONS AND TRACER STUDIES
The fact that some viruses (such as rabies and polio viruses) can spread from
neuron
to neuron, in CNS tissue, has allowed researchers to use viral spreading to
track neuronal
systems and pathways, using "trans-neuronal tracing"
There are important similarities between the patterns of motor neuron
infections by
polioviruses, and the patterns of motor neuron degeneration in patients
suffering from
amyotrophic lateral sclerosis. Similarly, the degeneration of basal forebrain
cholinergic
neurons that is an early feature of Alzheimer's disease shows important
similarities to
damage to certain neurons after administration of rabies virus to the
olfactory epithelium in
the nose. These previously unpublished observations are disclosed herein,
because the
similarities between patterns of viral infection in CNS tissue, and patterns
of
neurodegeneration in certain CNS disorders, suggests that the methods
disclosed herein
(i.e., non-invasive delivering of therapeutic polypeptides into the brain or
spinal cord, in a
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manner which relies on certain neurons to transport and deliver polypeptides
to other
neurons that share contact points with the carrier neurons) offers the promise
of both: (i)
powerful therapeutic treatments, and (ii) powerful experimental approaches
that can be used
by researchers to, for example, selectively alter patterns of gene expression
and/or nerve
cell function within brain or spinal tissue, and/or study various networks and
principles
within CNS tissue, including factors that govern the response of CNS tissue to
assaults
ranging from viral infections to neurodegenerative diseases.
ENDOCYTOTIC SURFACE MOLECULES
One aspect of this invention involves the discovery and disclosure of a new
method
for in vivo screening and identification of "ligand" molecules that can be
used to activate
and drive the process of endocytosis (i.e., transport of a foreign molecule,
into a cell
interior) These types of ligand molecules can be regarded as vehicles,
carriers, or transport
systems, that can be used to pull or carry useful "passenger" or "payload"
molecules into
specific targeted types of cells. These passenger molecules can be segments of
DNA
carrying foreign genes, if the goal is gene therapy or similar genetic
manipulation to deliver
polypeptides into brain or spinal tissue, or other targeted cells. However,
this type of
endocytotic transport is not limited to gene therapy, and also can be used to
deliver
therapeutic drugs, diagnostic compounds, or other useful agents into
specifically targeted
classes of cells.
This type of endocytotic transport (also referred to interchangeably as
internalisation) mainly involves a class of proteins known as "receptor"
proteins, which are
present on the surfaces of cells. However, at least some types of other
surface molecules
are known to also trigger the process of endocytosis (as examples, tetanus and
cholera
toxins are known to bind specifically to particular surface carbohydrate
molecules, in a
manner that activates and drives internalisation of the resulting ligand-
carbohydrate
complex). Therefore, although most references herein are to endocytotic
receptor proteins
as the main exemplary class that will be used to explain and illustrate the
invention, it
should be kept in mind that certain other types of endocytotic surface
molecules can also be
used as the targets of endocytotic ligands as discussed herein.
The structures of cell membranes and endocytotic receptor proteins, and the
process
and mechanisms of endocytosis, are discussed in various reference works, such
as Alberts
et al, Molecular Biology of the Cell. In Alberts et al's third edition, 1994,
the relevant
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ages include 478-488, 618-625, and 731-744 (which discuss cell membranes and
receptor
roteins), and pages 618-626 and 636-641 (which describe the proteins and
processes
wolved in endocytosis). It should also be noted that endocytosis can include
pinocytosis (if
ie ligand which is being taken inside the cell is relatively small) or
phagocytosis (if a
arger item, such as an entire cell or viral particle, is being taken into a
cell).
Not all candidate ligands that bind to an endocytotic receptor protein can
drive and
;omplete the process of endocytosis. As an example, an important class of "low
affinity"
ierve growth factor receptors, known as p75 receptors, has been identified in
mammals,
aid monoclonal antibodies that can bind to these receptors have been created
by numerous
=esearch teams. However, most of those monoclonal antibody lines cannot drive
and
3omplete the process of endocytosis, after they bind to p75 receptors. Only a
few particular
monoclonal antibody preparations are internalised by p75 receptors; this
includes a
particular line known as MC192 antibodies, initially disclosed in Chandler and
Shooter
1984, and later reported to be internalised by p75 receptor bearing cells in
Yan et al 1988.
Substantial work has been done to identify ligands that can enable endocytotic
transport into mammalian cells. Much of that work involves a class of genetic
research
tools called phages, and phage display libraries. Very briefly, phages (this
word is short for
bacteriophages) are viruses that can infect and reproduce in bacteria. Certain
types of
phages which have long shapes, known as "filamentous phages" (abbreviated as
"Ff"
phages) were studied and developed by genetic researchers, because they can be
engineering to express foreign proteins, that will be accessible (displayed)
on the surfaces
of phage particles that remain viable and infective.
One strain of these filamentous phages was manipulated to give it a bacterial
plasmid
origin of replication, thereby converting it into a "phagemid". Its DNA can be
reproduced
in large quantities in E. coil cells, either in double-stranded form, as
circular plasmids, or
in single-stranded form, which can be packaged in phage coat proteins for
secretion as new
and infective phage particles without killing the host cells. This phage line
was also given a
kanamycin resistance gene, as a selectable marker, allowing E. coil cells that
carry the
phage to grow in culture media that contains kanamycin, an antibiotic that
kills
non-resistant cells. This phage line, designated as the M13 line, is
commercially available,
and is widely used in genetic research.
Various researchers and companies have created entire "phage display
libraries", in
which a large population of phages (such as M13 phages) is manipulated so that
it will
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display a wide variety of different foreign proteins. Two such display
libraries were used
by the Applicants herein, in their work to create and test an in vivo
screening process to
identify endocytotic ligands that could bind to. targeted endocytotic
receptors on the surfaces
of neuronal fibers that extend outside the blood brain barrier. One of those
phage display
libraries, known as the scFv library, which contains roughly 13 billion
different phagemids,
each of which carries a foreign polypeptide sequence that normally appears in
the antigen-
binding "variable fragment" domains of human antibodies. The other phage
display library
known as the PhD-C7C library, carries small foreign polypeptide sequences
containing 7
amino acids each, sequenced together randomly using a process called
"combinatorial
chemistry". Both of these two phage display libraries are described in more
detail below, in
the Detailed Description section, and the Examples.
Phage display libraries offer powerful and useful tools for screening huge
numbers
of different "ligand candidates" or other polypeptide sequences. They can help
researchers
identify, isolate, and then reproduce those few phages, in a huge library,
that happen to
carry a foreign polypeptide sequence that is of interest, because of some
particular binding
or cellular activity.
However, because of a number of cellular and physiological factors, it was
very
difficult or totally impossible (under the prior art) to do any screening of
phage display
libraries in vivo (i.e., in the bodies of living animals). Instead, nearly all
such screening
was limited to affinity columns (which contain immobilized antigens or
antibodies, or
similar binding reagents), or in vitro conditions (using cells in culture
media).
As a result, when it came to efforts to identify and isolate ligand molecules
that
could activate and drive the process of endocytosis, the only real and
substantial progress
that had been created prior to this invention was in identifying ligands that
could enable
transport into cancer cells, and cells involving autoimmune diseases, because
those two
classes of cells can be grown and tested readily, in in vitro cell culture.
Examples of such
works include US patents 5,977,322 (Marks et al 1999), 6,113,898 (Anderson et
al 2000),
and 6,376,170 (Burton et al 2002), and PCT applications WO 2000-29004
(Plaksin), 2000-
38515 (Ferrone), and 2000-39580 (Christopherson et al). There was no
comparable
progress in successfully screening phage display libraries to identify ligands
that can drive
endocytotic transport into other types of tissue cells, such as neurons.
The Applicants herein were searching for ways to genetically transform neurons
(and, in particular, neurons that straddle the blood-brain barrier and have
fibers that extend
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autside the BBB). In addition, because they. were aware of the problems and
limitations that
have plagued and thwarted the medical use of viral vectors, they were looking
for other
types of transport and delivery systems, which could pull strands of foreign
DNA into cells
(and, in particular, into neurons that straddle the BBB, and have fibers that
extend outside
the BBB).
All of these efforts came together,, and have led to the invention disclosed
herein.
Prior to this invention, despite the huge advances in molecular biology and
neuroscience in recent years, there remains a major and unmet need for better
methods of
transporting useful polypeptides across the blood-brain barrier, so that they
can provide
therapeutic benefits to neurons in brains and spinal cords, without requiring
invasive
damage.
Accordingly, one object of this invention is to disclose an improved and non-
invasive method for delivering polypeptides to targeted cells inside the BBB.
Another object of this invention is to disclose a new method of transfecting
sensory
neurons or motor neurons that "straddle" the BBB, in a manner which causes the
BBB-
straddling neurons to deliver therapeutic polypeptides to neurons which are
located entirely
within the blood-brain barrier.
Another object of this invention is to use genetic engineering methods and
compounds to provide a method of transporting polypeptides into CNS tissue, in
a manner
that can be used to selectively affect targeted clusters, regions, or types of
neurons located
entirely within the blood-brain barrier.
Another object of this invention is to disclose new and practical in vivo
methods for
identifying and isolating ligand molecules that can be used to effectively
transport
"passenger" or "payload" molecules (including, but not limited to, DNA strands
that encode
foreign proteins) into selected, targeted, and limited. types and classes of
neuronal fibers,
and other selected and targeted classes of animal cells.
Another object of this invention is to disclose a new method for identifying
and
isolating ligand molecules that can be used to effectively transport
therapeutic drugs,
diagnostic or analytical compounds, or DNA sequences, into selected, targeted,
and limited
types and classes of animal cells.
Another object of this invention is to disclose a new method for in vivo
screening of
libraries, repertoires, or other assortments containing multiple candidate
polypeptides or
other compounds that have been created by combinatorial chemistry, to identify
and isolate
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those particular candidates that undergo endocytotic transport into cells.
These and other objects of the invention will become more apparent through the
following summary, drawings, and description of the preferred embodiments.
SUMMARY OF THE INVENTION
A method and compounds are disclosed for the noninvasive transport and
delivery of
polypeptides through the blood brain barrier (BBB) and into the central
nervous system
(CNS) of a mammal or other higher animal, in a manner which allows foreign
polypeptides
to contact cells located wholly within the BBB. This method enables previously
unavailable
forms of medical or veterinary treatment in humans and other animals, and it
can also be
used for commercial purposes, such as in livestock.
This method is accomplished by using a genetic vector to transfect a selected
type of
neuron which "straddles" the blood brain barrier. Examples of such BBB-
straddling neurons
include sensory neurons (such as olfactory receptor neurons, and nocioceptive
neurons), and
motor neurons; pre-ganglionic neurons of the autonomic nervous system may also
be
useful, in some treatments. Copies of the genetic vector are introduced and
administered to
the patient or animal in a manner that causes the vectors to contact and
transfect neuronal
"projections" that extend outside the BBB. After the vector enters a
peripheral projection of
a BBB-straddling neuron, a vector-borne gene encoding a CNS-active polypeptide
will be
transported by retrograde transport to the main cell body, where it will be
expressed by the
transfected neuron, to form therapeutic or otherwise useful polypeptide
molecules. These
polypeptides will be of a type that normally are secreted by cells, or they
can be provided
with leader sequences that can promote secretion; accordingly, they will be
transported
within the transfected neuron to one or more secretion sites located within
CNS tissue that
is protected by the BBB. The polypeptides that are secreted at such locations
inside the BBB
will then be able to contact targeted neurons that are located wholly within
(and are
therefore protected by) the blood-brain barrier.
Using this system, polypeptides such as neurotrophic factors can be
noninvasively
delivered to targeted types and classes of neurons that lie wholly within the
BBB. This
allows new methods of treating brain and spinal neurons that have been
injured, or that are
degenerating due to aging, disease, or other disorders. Alternately, this new
method allows
polypeptides that can suppress neuronal activity to be transported across the
BBB, to reduce
problems involving unwanted and excessive neuronal activity, such as
neuropathic pain.
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rills approach also allows new methods of delivering polypeptides that can
modulate
..ndocrine functions into the CNS, thereby allowing improved treatment of
various medical
?roblems in humans, and improved ability to modulate growth, maturation,
reproduction, or
Dther endocrine-related functions among animals, including livestock and
endangered
species.
In addition, a new in vivo screening method is disclosed, for identifying and
isolating endocytotic ligands that will be taken into, and transported within,
neuronal fibers.
This ligands can be used to prepare gene vectors that can deliver cells into
selected BBB-
straddling vectors. Briefly, this screening method uses the following steps:
(1) emplacing
multiple candidate ligands at a first location inside the body of a living
animal, where the
candidate ligands will directly contact nerve fibers (such as a sciatic nerve
bundle, in the
leg of a rat); (2) allowing enough time to pass for the nerve fibers to
internalise those
particular ligands which can activate and drive endocytosis; (3) harvesting
segments of the
nerve fibers, at a site (such as distal to a ligature that constricts the
sciatic nerve, in a rat
hip) that is sufficiently distant from the ligand emplacement site to avoid
collecting ligand
candidates that did not enter or were not transported by the nerves; and, (4)
removing the
internalised ligands from the harvested nerve segments.
This method can be carried out using phage display libraries (for polypeptide
ligands), or other ligand repertoires that have been created by combinatorial
chemical
synthesis. Since it offers a preferred mode of carrying out this invention, it
is disclosed and
claimed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 depicts the use of a genetic vector to transfect an exposed
projection
("mucosal tip") of an olfactory receptor neuron, for the purpose of delivering
therapeutic
polypeptides through the BBB to neurons that reside wholly within the BBB. The
transfected olfactory receptor neuron will express the vector-borne gene into
polypeptides,
which will then be transported through a neuronal axon which crosses the BBB.
The
polypeptides will then be secreted inside the BBB, by the BBB-straddling
neuron, where
they can contact CNS neurons that are located wholly inside the BBB, such as
cholinergic
neurons in the basal forebrain.
FIGURE 2 depicts a viral vector which can transfect neurons; this vector
includes a
capsid shell, binding ligands on the surface, and a genetically-engineered
genome which
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contains a useful "passenger" or "payload" gene.
FIGURE 3 depicts transfection of an olfactory receptor neuron, and depicts the
route, through the olfactory bulb, that the vector-encoded polypeptide will
travel to reach
and contact a terminus, located at the tip of a neuronal process which is part
of a
cholinergic neuron that has its main cell body inside the basal forebrain.
FIGURE 4 (which includes FIGS. 4A, 4B, and 4C) depicts the use of a genetic
vector for the purpose of delivering anti-neurotrophic polypeptides to
neurotrophic-factor-
producing cells that reside wholly within the BBB, to reduce an unwanted
excess of
neuronal connections or activity, as occurs in conditions such as neuropathic
pain. In this
method, an "NPC" (neuropathic pain control) vector is used to transfect
nocioceptive
neurons which innervate tissues such as skin. The nocioceptive neurons will
express vector-
encoded polypeptides having anti-neurotrophic activity, and will release those
polypeptides
into spinal tissue. As depicted in FIGS. 4B (which shows abnormally large
numbers of
unwanted pain-signalling neuronal connections, before the treatment) and 4C
(which shows
a reduced number of unwanted pain-signalling connections, after the
treatment), this type of
blockade or inhibition of neurotrophic receptors or factors will lead to
suppression or
atrophy of the excessive neuronal connections that were causing or aggravating
the
neuropathic pain condition.
FIGURE 5 (which includes FIGS. 5A, 5B, and 5C) depicts the use of a genetic
vector to transfect "spinal motor neurons" in a muscle for the purpose of
delivering
therapeutic polypeptides to a class of neurons (called "upper motor neurons")
that lie
wholly within the BBB. This type of treatment would be carried out, for
example, in a
patient with a limb that has become paralyzed or impaired due to stroke,
injury, or disease.
As shown in FIG. 5A, the gene vector will be injected into muscle tissue,
where it will
transfect spinal motor neurons that straddle the BBB; those neurons will
express vector-
encoded polypeptides having neurotrophic activity, and will release those
polypeptides into
spinal tissue, thereby delivering neurotrophic polypeptides to the upper motor
neurons that
are fully inside the BBB. FIG. 5B depicts a condition in a stroke-impaired or
similar
patient before treatment, in which some (but not all) of the neuronal
"processes" which
belong to upper motor neurons have degenerated and/or atrophied, and are no
longer able
to interact adequately with spinal motor neurons, leading to impairment or
paralysis of a
limb. FIG. 5C depicts an improved condition after treatment with a neuronal
growth
factor; in this condition, the still-functioning neuronal process which
belongs to one of the
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upper motor neurons has created ("sprouted") new and additional synaptic
connections,
which can interact with the spinal motor neurons that were not being
adequately innervated
prior to the treatment.
FIGURE 6 depicts the use of a genetic vector to transfect projections of lower
motor
neurons of the hypoglossal nucleus (a part of the brain stem), for the purpose
of delivering
therapeutic polypeptides through the BBB to neurons that reside wholly within
the BBB.
The exposed ends of these neuronal projections are present in the tongue;
therefore, this
class of neurons offers a relatively direct route for introducing foreign
polypeptides into the
brainstem portion of the CNS.
FIGURE 7 is a schematic display of the two ligatures that were emplaced around
a
sciatic nerve bundle in a rat, and of the phage-containing collagen gel that
was emplaced in
contact with the cut end of a sciatic nerve bundle, in a manner that enabled
endocytotic
uptake of phages into the sciatic nerve fibers.
FIGURE 8 is a photograph of fluorescent-labelled antibody-phage conjugates
that
accumulated within a sciatic nerve bundle, next to a hip ligature. The
ligature (a loop of
tightened suture material) prevented those antibody-phage conjugates, which
had been
internalised by the sciatic nerve fibers, from being retrogradely transported
beyond the
ligature constriction site.
FIGURE 9 schematically depicts a cyclic in vivo ligand selection process, in
which
ligand-displaying phages from a phage display library are selected for
endocytotic uptake by
nerve cells, using the in vivo method disclosed herein, and wherein the
selected phage
population that results from one cycle is used as the starting material for
screening in the
next cycle.
DETAILED DESCRIPTION
As suggested by the Summary section, above, this invention can be regarded has
having four distinct elements or components. All four must work together, in a
coordinated
and sequential manner, in order to carry out the invention successfully. In a
numbered and
abbreviated summary, those four elements are:
A (1) suitable genetic vector carries a DNA sequence which includes at least
one
desired "passenger gene", which may include a marker gene (to simplify
detection and
analysis, during research) and/or a payload gene (which encodes a therapeutic
or otherwise
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desired and useful polypeptide). This type of vector is used to genetically
transfect (2) one
or more selected types of neurons that straddle the blood-brain barrier
(examples include
olfactory receptor neurons, and lower motor neurons). After the passenger gene
has entered
a BBB-straddling neuron, the neuron will transport the gene to the main body
of the
neuron, by a natural process called retrograde transport. After it reaches the
main cell
body, the gene will be expressed, thereby forming (3) 'foreign" or "exogenous"
polypeptide
molecules, inside the transfected BBB-straddling neuron. These foreign
polypeptide
molecules (which, in some cases, may be identical to "endogenous" or "native"
polypeptides that are not being made in sufficient quantities by a patient)
will then be
transported, still within the transfected BBB-straddling neuron, to a
secretion site, located
inside the BBB. The polypeptide molecules will then be secreted by the
transfected BBB-
straddling neuron, at the secretion site inside the BBB. This secretion will
cause the foreign
polypeptides to contact (and to treat, modulate, or otherwise affect, in a
therapeutic or other
useful manner) other types of (4) "target" CNS neurons or other cells that lie
entirely within
the BBB.
In the discussion immediately below, a virus-derived vector is used to
illustrate this
method of treatment; however, non-viral vectors alternately may be used, as
discussed in
later sections.
A first example which illustrates this method of treatment is illustrated in
Figures 1
through 3, using consistent callout numbers in all three drawings. Figure 1
depicts this
treatment on a "macroscopic" level; a liquid containing numerous copies of
genetic vector
100 is introduced into the nasal sinuses of patient 80. The vector contacts
the exposed
"peripheral projection" 212 (see FIG. 3) of olfactory receptor neuron 200;
these neuronal
projections are accessible on the surfaces inside the nasal sinuses, and they
can be directly
contacted by various airborne compounds, as one of the biochemical processes
involved in
the sensation of smell.
As shown on a different scale (at a microscopic, cellular level) in FIG. 3,
once the
DNA carried by the genetic vector 100 has entered a neuronal projection 212,
it is carried
to the main cell body 224, by retrograde transport (discussed below). Because
the genetic
vector in this example has been derived from a type of virus that is fully
capable of
infecting nasal receptor cells, the vector DNA (which carries passenger gene
160, as shown
in FIG. 2, as merely one component inserted into an disabled but infective
virus genome) is
able to help promote and facilitate this process. Accordingly, the use of
infective viruses to
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create the vector delivery system will help ensure that the transcribed
portion of the
passenger gene 160 will be expressed into messenger RNA strands 299 in at
least some
transfected neurons, as shown in FIG. 3.
The mRNA strands which have been transcribed from the passenger gene 160 will
then be translated into multiple copies of foreign polypeptide molecules 300,
by the normal
cellular mechanism involving ribosomes 226 inside the transfected neuron 200.
Due to natural transport and secretion mechanisms (which can be enabled or
enhanced, if desired, by using genetic engineering techniques as discussed
below to add one
or more specialized transport or secretion sequences to an end of a
polypeptide chain), at
least some of the foreign polypeptide molecules 300 will then be transported
(using a
process called anterograde transport) to various synaptic terminals 242 (or
other peptide-
secreting locations) that belong to the BBB-straddling neuron 200.
At these locations, which are located entirely within BBB-protected CNS
tissue, the
foreign polypeptide molecules 300 will be released by the BBB-straddling
neuron 200. This
secretion process allows the secreted foreign polypeptide molecules 300 to
directly contact
(and to begin exerting therapeutic or other modulating effects on) other
classes of neurons
which are referred to herein as "target" neurons; these "target" neurons lie
wholly within
the BBB. In the schematic illustrations shown in FIGS. 1 and 3, the
polypeptides are
secreted by transfected olfactory receptor neuron 200 within a roughly
spherical globular
structure called a "glomerulus" 910, which contains various types of
"targeted" neuronal
structures, as discussed below.
To condense this process into short form, this overview and FIGS. 1-3 indicate
how
(1) a genetic vector carrying a passenger gene, is used to transfect (2) BBB-
straddling
neurons, which will then express (3) foreign polypeptides, which will then be
secreted into
CNS tissue inside the BBB, where they will contact and affect (4) "target" CNS
neurons
located wholly within the BBB.
Each of these four elements are described in more detail below. Since the
optimal
design of a genetic vector will depend on the particular type of BBB-
straddling neuron that
will be transfected, BBB-straddling neurons are discussed first.
TYPES OF BBB-STRADDLING NEURONS
Neurons that straddle the BBB can be divided into three main classes: sensory
neurons, motor neurons, and pre-ganglionic autonomic neurons. All of these
classes of
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BBB-straddling neurons discussed have been studied extensively, and genetic
vectors are
known that can be used to transfect each of these classes of neurons.
The first major class of BBB-straddling neurons addressed herein are the
sensory
neurons, which are the "front line" cells that are directly involved in sight,
smell, taste, and
various sensations involved in touch, pain, the sense of position of a limb,
etc.
Sensory neurons have specialized components (referred to herein as "peripheral
projections") that extend out to (or very near to) certain surface or tissue
regions outside the
BBB. As used herein, a "peripheral projection" is the portion of a BBB-
straddling neuron
that is most directly accessible to a genetic vector. A genetic vector will
not need to cross
the BBB, in order to contact a peripheral projection.
Conversely, the term "central projection" refers to a fibrous portion of a
neuron
which extends away from the main cell body, and which extends either: (i)
through the
BBB, if the main cell body is located outside the BBB, as occurs with many
types of
sensory neurons; or, (ii) closer to the center or the brain or the upper
spinal cord, if the
main cell body is located inside the BBB, as occurs with motor neurons and pre-
ganglionic
autonomic neurons.
The term "axon" also should be noted and understood, with regard to central or
peripheral projections. In most cases, the term "axon" refers to the single
largest and
longest projection that emerges from a neuron's main body. However, many
sensory
neurons can be regarded as having two axons, with one extending toward the
periphery,
while the other extends toward the central poition of the brain. Because one
of the primary
roles of most BBB-straddling neurons is to shuttle nerve impulses between the
peripheral
and central nervous systems, the portion of a BBB-straddling neuron that
actually crosses
the BBB can almost always be properly classified as an axon.
Four classes of BBB-straddling neurons deserve particular consideration, for
potential use as disclosed herein. Two of those classes are sensory neurons:
(i) olfactory
receptor neurons, and (ii) nocioceptive (pain-signalling) neurons. A third
class comprises
motor neurons, which are involved primarily with musculo-skeletal control;
this class
includes a subclass called tongue motor neurons, which are of special interest
because their
cell bodies are in the brainstem. The fourth class, called "pre-ganglionic
autonomic
neurons", are not highly promising candidates for most types of human medical
uses, since
genetic alteration of these neurons might affect the autonomic nervous system;
however,
they should be recognized, because they may be useful for treating certain
types of medical
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Problems, and for various types of research. Each of these four subclasses is
discussed
eparately, below.
Nfactory Receptor Neurons
One class of BBB-straddling neurons preferred for use herein includes
olfactory
receptor neurons. Because of the role they play in the sense of smell, their
peripheral
)rojections are exposed and accessible on the interior surfaces of the nasal
sinuses, where
they can be directly contacted and activated by various types of airborne
molecules that are
being drawn in through the nose.
These neurons are described in various textbooks on physiology, such as Guyton
and
Hall, Textbook of Medical Physiology, 9th edition (1996), pages 678-681, and
additional
references cited therein on pages 681-682. Very briefly, a human olfactory
membrane has a
surface area of about 2.5 square centimeters, and typically has about 100
million receptor
neurons. The exposed tip of each olfactory receptor neuron usually has about 6
to 12 tiny
hairs called cilia, which extend downward several microns into a layer of
mucus. It is
presumed that most molecules which trigger perceptions of odor interact with
the cilia
and/or receptor proteins that straddle the membrane of the exposed surface of
a receptor
neuron. Although most molecules which trigger sensations of smell do not enter
the neurons
themselves, it is known that a few specific types of molecules (such as wheat
germ
agglutinin, which binds in a non-specific manner to most glycoproteins) are
taken up and
transported into olfactory receptor neurons, presumably by some form of
endocytosis.
Several published reports have also stated that adenovirus vectors, when used
to contact the
olfactory membrane, can indeed transfect (i.e., insert foreign genetic
material into)
olfactory receptor neurons, as evidenced by subsequent expression of marker
genes by the
transfected neurons.
Olfactory receptor neuron 200, illustrated in a schematic manner in FIG. 3,
comprises a number of cell parts that will be mentioned because of how they
interact with
one or more parts of the genetic vector 100. While the nasal sinus membrane 90
is shown
schematically as a distinct layer, it is made up of the exposed apical
surfaces of the
olfactory support cells of the olfactory epithelium. A neuronal projection 210
passes
through the nasal sinus membrane 90, creating an exposed terminus 212 (also
called a
mucosal tip, end, or knob). This exposed neuronal tip 212, which sits inside
the nasal sinus
cavity, allows the neuronal tip 212 to be contacted by genetic vectors (as
disclosed herein)
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that are carried by a liquid nasal spray.
Olfactory receptor neuron 200 also comprises cell cytoplasm 220, a nucleus 222
which sits inside the main cell body 224, ribosomes 226 which translate mRNA
into
polypeptides, and an axon 240 which passes through the blood-brain bather
(BBB).
As noted in the Background section, the BBB is not a single membrane
structure;
instead, it is a network of capillary walls that have unusually tight
junctions between the
endothelial cells that form the capillary walls. While some scientists might
argue that the
olfactory epithelium (i.e., the mucous membrane surface that contains the
olfactory receptor
neuron projections, inside the nasal sinuses) can be considered an extension
of the brain,
and therefore presents a region of CNS tissue where the BBB does not exist, it
is clear
from physiological studies that olfactory glomeruli sit entirely within the
BBB, and are
protected by the BBB from unwanted molecules that might trigger spurious and
unhelpful
nerve impulses. To represent that fact, double-dashed line 900 is used in FIG.
1 to
schematically represent the BBB. The peripheral projection 210 and main cell
body 224 of
olfactory receptor neuron 200 sit outside the BBB, while axon 240 passes
through it, and
then branches into numerous synaptic terminals 242 which sit inside BBB-
protected CNS
tissue. Accordingly, olfactory receptor neuron 200 straddles the BBB.
The synaptic terminals 242 of olfactory receptor neuron 200 are located in a
roughly
spherical globular structure, shown as "glomerulus" 910 in FIG. 3. In a human,
there are
thousands of glomeruli, and each glomerulus contains the synaptic terminals of
roughly
25,000 axons from olfactory receptor neurons. Each glomerulus 910 is also the
terminus for
thousands of dendrites and projections 920 from large "mitral" cells and
smaller "tufted"
cells. While the cell bodies and dendrites of the mitral and tufted cells are
drawn as lying in
the glomerulus, their main cell bodies are located in bulb structures
positioned above the
glomeruli. In addition, each glomerulus (or surrounding regions that are
closely proximate
to the glomerulus) also contains termini 934 located at the tips of long
fibers (called
processes) 932 which extend down from neurons 930, called "basal forebrain
cholinergic
neurons", since they are located in the basal forebrain, and since they are
activated mainly
by the excitatory neurotransmitter acetylcholine.
The schematic representations in FIG. 3 are selective illustrations, and do
not
attempt to illustrate all targeted neuronal structures. For example, also
found in or in the
near the glomeruli are termini of: (i) fibers extending from "serotonergic"
neurons in a
brain region called the raphe nucleus, which have their main cell bodies in a
part of the
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midbrain; and, (ii) fibers extending from "noradrenergic" neurons which have
their main
cell bodies in a part of the brain called the locus coeruleus nucleus.
Depending on the
therapeutic polypeptide being delivered, these or other neurons residing
wholly within the
BBB might be targeted by a treatment as disclosed herein.
In general, it is anticipated that polypeptides released by a transfected
olfactory
receptor neuron 200 are likely to be able to contact and exert modulating
effects on any (or
nearly any) type of neuron that has been shown, using so-called "trans-
synaptic tracer
studies" (e.g., Lafay et al 1991; Barnett et al 1993), to be infected by virus
particles
released by virus-infected olfactory receptor neurons. Such neurons include
mitral cells and
tufted cells (these are infected in large numbers, since very large numbers of
their termini
are present in the glomeruli), and at least some basal forebrain cholinergic
neurons, raphe
nuclei serotonergic neurons, and locus coeruleus noradrenergic neurons (which
have also
been shown to be infected, in tracer studies).
It should also be recognized that various types of "glig cells" are also
likely to be
contacted by polypeptide molecules 300 that are secreted by olfactory receptor
neuron 200.
As described in more detail below, glial cells (also called neuroglial cells)
include various
types of cells which cannot receive or transmit nerve signals, and which
instead support and
serve the neurons located inside BBB-protected CNS tissue.
Accordingly, the term "target cells" is used herein to refer to cells which
sit entirely
within BBB-protected CNS tissue, and which are the intended "targets" of the
foreign
polypeptide molecules 300 that are encoded by the passenger gene(s) in a
genetic vector
100 as described herein. A BBB-straddling neuron which is actually contacted
and
transfected by a genetic vector is not regarded herein as a target cell;
instead, that type of
BBB-straddling neuron should be regarded as part of the delivery mechanism,
and can be
referred to by terms such as "delivery cell", "transfection conduit", or as a
"primary"
transfected neuron.
If olfactory receptor neurons are used as the delivery route, another
physiological
factor is potentially important, and should be recognized. Olfactory receptor
neurons
gradually die off, and are constantly being replaced by newly-created neurons.
In mice, the
"half-life" of olfactory receptor neurons is about 3 months, and the half-life
in humans is
presumed to be roughly comparable.
Therefore, if olfactory receptor neurons are transformed in a stable manner by
genetic vectors (i.e., if the genetic vectors cause one or more foreign genes
to be inserted
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into the chromosomes of the olfactory receptor cells), the transformed cells
will
nevertheless gradually die off over the next weeks and months. As a result,
depending on
the type of vectors and polypeptides that are involved and the effects that
are desired in a
particular patient, it may be necessary to readminister additional vectors to
the patient,
every few weeks.
Nocioceptive Neurons
Another class of BBB-straddling sensory neurons transmits pain signals from
the
skin and body, into the spinal cord. These neurons generate nerve impulses in
response to
impinging substances, signals, or events from the environment; accordingly,
these are the
neurons that commence the process that generate feelings of pain or discomfort
when the
skin has been cut, scraped, or hit, or exposed to intense heat or cold. These
pain-signalling
neurons are usually called "nociceptive" or "nocioceptive" neurons, and are
sometimes
called nociceptors or nocioceptors. Nocioceptive neurons are just one of a
number of
different functional types of BBB-straddling sensory neurons that form part of
the dorsal
root ganglia.
FIG. 4 depicts, in a highly schematic fashion, a nocioceptive neuron 400. Like
most
other sensory neurons, this neuron 400 has its main cell body 402 in a tissue
region which
is outside of, and not protected by the BBB; in the case of nocioceptive
neuron 400, the
main body 402 is located relatively close to spinal cord 480, in a dorsal root
ganglion. This
neuron has a peripheral projection 410 which extends outwardly, i.e., away
from the spinal
cord 480, and toward the skin (other projections also extend to other regions
deeper within
the body). This peripheral projection 410 branches out into numerous "near-
surface
terminals" 412, located in shallow layers of tissue just beneath the skin
surface 405.
In the other direction, nocioceptive neuron 400 also has an axon 420, which
passes
through the blood-brain barrier. As indicated in FIG. 4B, axon 420 branches
into processes
422, 424, and 426 (shown in FIG. 4B), inside BBB-protected CNS tissue in
spinal cord
480. Each process terminates in one or more synaptic junctions that allow the
nocioceptive
neurons to transmit its pain signal to "second order" neurons, inside spinal
cord 480.
The surface of spinal cord 480, which is shown as a cross-sectioned segment,
has:
(i) a ventral median fissure 482 (also called an anterior median fissure),
positioned toward
the front of the patient; and, (ii) a dorsal median fissure 484 (also called a
posterior median
fissure), flanked by two smaller dorsal-lateral (or postero-lateral) fissures.
The mass of
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spinal cord 480 comprises "white matter" 486 which surrounds "gray matter"
490. The
gray matter 490 comprises left and right ventral (anterior) horns 492, and
left and right
dorsal (posterior) horns 494. These four horns are. all connected to a central
portion, called
the gray commissure. The large bundles of nerve fibers which emerge laterally
from the
spinal cord, as shown in FIGS. 4 and 5, usually are called the posterior (or
dorsal) roots,
and the anterior (or ventral) roots. The term "ganglion" generally refers to
aggregations of
neuronal cell bodies outside the CNS, and can also be used to refer to these
nerve bundles.
If skin surface 405 is cut or scraped, the nerve signals that commence at the
near-
surface terminals 412 of nocioceptive neuron 400 travel through the peripheral
projection
410, through the cell body 402, through the axon 420 which crosses the BBB,
and into the
branched dendrites 422-426 inside the dorsal horn 494 of spinal cord 480. The
synapses at
the tips of dendrites 422-436 release neurotransmitters, which trigger nerve
impulses in the
spinal neurons. Those nerve impulses travel up the spinal cord, to centers
inside the brain
which process the arriving nerve signals in ways that the brain interprets as
pain.
Various medical problems have been grouped together under name, "neuropathic
pain." As indicated by the name, neuropathic pain involves a pathological
condition that
affects neurons, in a manner that generates unwanted and excessive pain
signals. This often
involves some anatomical reorganization of the nerve connections within the
BBB, such that
there is a chronic and inappropriate pain response. The term "hyperalgesia" is
also used, as
a descriptive term that translates directly into "excess pain", and the term
"allodynia" is
also used for this condition.
Neuropathic pain is a well-known cluster of medical problems, and this broad
category includes diabetic neuropathy, "phantom pain" from limbs or
extremities that have
been amputated, arachnoiditis, trigeminal pain, post-infective pain (such as
outbreaks of
"shingles", caused by herpes zoster viruses), and lingering chronic pain that
arises after a
traumatic injury or surgery and then will not recede, even after the normal
timespan of
recuperation has long passed. Causalgia is another type, and involves burning
sensations
(the root word "caus-" arises from the same root as "cautery", and has nothing
to do with
causation). Other types of neuropathic pain are also known, and it should be
recognized that
neuropathic pain can range over a very wide span of intensity, starting at
annoying, up to
excruciating, debilitating, and unbearable. Indeed, neuropathic pain is
involved as a major
factor in many suicides; chronic and incurable pain can be so intense and
relentless that it
drives many sufferers to commit suicide.
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A cellular mechanism that is believed to be widely involved in many types or
cases
of neuropathic pain is illustrated in FIG. 4B. In this schematic drawing, axon
420NP has
become involved in a neuro-pathological problem that has caused it to sprout
too many
dendrites 422, 424, and 426, which have begun to interact with spinal neurons
432, 434,
and 436 (some of which may be inappropriate) located in the dorsal horn of
spinal cord
480NP. This sprouting and chronic activation of too many dendrites from a
single
nocioceptor axon causes or aggravates the transmission of too many pain
signals into and
through the pain-plagued spinal cord 480NP. This condition, of too many
neuronal
connections involving pain-transmitting neurons, as shown in FIG. 4B, is
referred to as
"hyper-innervation".
This invention offers a method of controlling and reducing neuropathic pain,
by
administering a genetic vector that can transfect nocioceptor neurons in or
near an affected
area, as further described herein and illustrated in FIGS. 4A-4C. This type of
vector,
shown in FIG. 4A as vector 100NPC (the letters "NPC" refer to "neuropathic
pain
control"), will carry an "NPC" passenger gene which is designed to suppress
(rather than
increase) neuronal activity, as discussed below.
Since the nocioceptive receptors are likely to be located in shallow regions
beneath
the skin, subcutaneous, intramuscular, or other relatively shallow injection
is a preferred
route of administration. Alternately, topical and other modes of
administration also can be
evaluated, including: (i) topical application of a vector-carrying ointment,
cream, or other
solution or suspension, which can also contain an agent that promotes
permeation through
tissue, such as dimethylsulfoxide, methylsalicylate, etc.; (ii) topical
application of a vector-
carrying formulation to a roughened, abraded, or otherwise physically-treated
area of skin;
and/or (iii) topical application of a vector-carrying formulation to skin that
has been
chemically treated, such as by the types of chemicals that are used for "skin-
peeling"
treatments.
For purposes of further discussion, it is assumed that a liquid containing a
genetic
vector 100NPC, for neuropathic pain control, will be injected in a shallow
subcutaneous
manner into a region of skin at or near the location of an apparent "focal
point" (also called
a locus, seat, hot spot, etc.) where neuropathic pain is perceived most
intensely by a
patient. As mentioned above, vector 100NPC carries a gene designed to suppress
(rather
than increase) neuronal activity. Such suppressor genes can encode, for
example: (i)
monoclonal antibodies (or antibody fragments) that will bind to and inactivate
one or more
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types of neuro-trophic or other neuro-stimulatory peptides or compounds; or,
(ii) peptide
fragments that will competitively bind to, occupy, and block one or more types
of neuronal
cell receptors that are involved in neuro-stimulatory processes. The NPC gene
will be
transported (using retrograde transport, as noted above) to the cell body 402
of nociceptor
neuron, where the gene will be expressed to form copies of an NPC polypeptide
as
described above. The NPC polypeptides will then be transported (using
anterograde
transport) by the neuronal axon 420, across the BBB and into a spinal cord
480NP which is
being plagued by neuropathic pain, caused or aggravated by a hyper-innervation
condition
that has a component within the spinal cord, as shown in FIG. 4B.
The NPC polypeptides will be released into the BBB-protected spinal tissue, at
or
near the site of the hyper-innervation condition that exists inside the spinal
cord. By
exerting their suppressive effects (such as by binding to, blocking, competing
against, or
otherwise suppressing cellular agents or processes that stimulate or sustain
higher levels of
neuronal activity), the NPC polypeptides will help reduce and mitigate the
neuropathic pain
condition, either on a permanent basis, or on a basis that may last for days
or weeks,
depending on what type of treatment is used.
Motor Neurons
Another class of BBB-straddling neurons is usually called motor neurons. These
mainly transmit instructions from the CNS to the muscles of the body, to
"innervate"
skeletal musculature and place the muscles under the control of the CNS. One
major
subclass of motor neurons, usually called "spinal motor neurons", have their
cell bodies in
the spinal cord, and projections that extend out through the BBB to contact
peripheral nerve
cells and muscle.
FIG. 5 (which comprises FIGS. 5A-5C) schematically depicts use of a spinal
motor
neuron as a "transfection conduit" (as described above) to stimulate and
increase neuronal
control over a muscle, in a patient who has a weakened limb due to a stroke,
traumatic
injury, neurodegenerative disease, or similar cause, by delivering therapeutic
polypeptides
to upper motor neurons that lie wholly within the BBB. FIG. 5A depicts spinal
motor
neuron 500, which has a cell body 502 located inside BBB-protected tissue in
spinal cord
520. Spinal cord 520 has the same structure shown in FIG. 4A, and motor neuron
cell body
502 is located inside the gray matter, in ventral horn 522. The axon of the
spinal motor
neuron 500 passes through the BBB, and forms a long "process" 504 which
extends to
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muscle fiber bundle 550. Inside the muscle fiber bundle 550, the neuronal axon
or process
504 branches into dendrites and terminals 506, which (in a healthy person)
interact with the
muscle tissue to trigger muscle contractions at desired times, thereby
providing "voluntary
motor control" of the arms, legs, etc.
FIG. 5B is a schematic depiction of the cell bodies of three distinct spinal
motor
neurons, designated as 512, 514, and 516, in a patient who has suffered a
stroke or a head
or spinal injury, or who is suffering from a neurodegenerative disease (such
as amyotrophic
lateral sclerosis) or similar problem that has impaired his voluntary motor
control over an
arm or leg. This impaired condition is due, at least in part, to dead or
damaged upper
motor neurons in the brain or brainstem, above the location of the three motor
neuron cell
bodies. Some of the dead or damaged upper motor neurons in the brain or
brainstem, above
the location of spinal motor neuron cell bodies 512-516, had supplied nerve
impulses to
spinal motor neurons 512 and 516; as depicted schematically in FIG. 5B, when
various
upper motor neurons were damaged or killed, their processes 532 and 536
(indicated by
dotted lines in FIG. 5B) fell silent and began to degenerate, leaving only one
of the three
spinal motor neurons (illustrated as the center process 534) with an active
supply of
incoming nerve signals, from its upper motor neuron. Since the two motor
neurons 512 and
516 are no longer receiving any incoming nerve impulses, they have fallen into
silence and
disuse, and are in danger of atrophying, deteriorating, and dying over time.
FIG. 5C schematically depicts the result of treatment of this condition,
following
injection of a liquid containing copies of genetic vector 100 into the muscle
bundle 550 that
is no longer adequately functioning. Vector 100 carries a therapeutic gene
which encodes a
neurotrophic factor or other polypeptide that stimulates neuronal activity or
replication. The
gene carried by this vector is transported into the cell bodies 512-516, where
the gene is
expressed into neuro-stimulating polypeptide molecules. These polypeptides are
then
secreted by the three motor neurons, and they act as signals which stimulate
and attract the
growth and/or activation of additional dendrites from any nearby neurons which
are still
viable and active, including the upper motor neuron having the active process
534. This
causes the active process 534 to sprout additional processes 534a and 534c,
which can form
synapses with other neurons. The additional newly sprouted synaptic junctions
thereafter
begin activating the processes from spinal motor neurons 512 and 516 once
again. When
coupled with a physical therapy and exercise program, this restores to the
patient a greater
degree of voluntary control over his arm or leg.
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Tongue Motor Neurons
One subclass of motor neurons that may be highly useful in this invention has
projections which extend through the BBB, to innervate the muscles of the
tongue. These
"tongue motor neurons" control the movement of the tongue, for both eating and
talking.
Their cell bodies are located within the brainstem; therefore, they offer a
promising route
for delivering foreign polypeptides into the brainstem portion of the CNS.
This is depicted in FIG. 6, which depicts a tongue motor neuron 600, having a
cell
body 602 located in the brainstem 950, and a long peripheral projection 604
which passes
through the BBB and terminates inside the tongue 62 of patient 60. The
accessible tip of
neuronal projection 604 is contacted by a genetic vector 100A, by means of a
carrier liquid
injected into the tongue 62. The vector DNA will be retrogradely transported
through the
neuronal projection 604, into the cell body 602. The passenger gene will be
expressed into
a therapeutic polypeptide, which will be secreted by the tongue motor neuron
600, at
locations in the brainstem. These secreted polypeptides will contact and exert
their effects
on various other neurons 952 and 954, located in brainstem 950.
PRE-GANGLIONIC AUTONOMIC NEURONS
The last major class of BBB-straddling neurons that will be specifically
discussed
herein is usually called pre-ganglionic autonomic neurons. Like motor neurons,
their cell
bodies are located inside the BBB. Their axons extend out through the BBB, and
connect
with nerves of the sympathetic and parasympathetic nervous systems. As part of
the
"autonomic" system, these neurons are involved in the control of various
functions that are
not under conscious control (such as blood pressure, digestion, excretion,
sweating, etc.).
The term "ganglion" implies a bundle of neurons. Accordingly, "pre-ganglionic"
neurons include neurons whose cell bodies are found within the CNS in clusters
(also called
nuclei) and whose axons project through the BBB to innervate and make contact
with the
neurons found in the ganglion lying outside the BBB.
It is believed that the methods and genetic vectors of this invention can be
adapted
and used, if desired, to genetically transfect pre-ganglionic autonomic
neurons, and it is
believed that in at least some cases, such transfected pre-ganglionic
autonomic neurons will
subsequently transport and deliver the foreign polypeptides into the brainstem
and/or spinal
cord, and possibly other CNS neurons lying wholly within the BBB (for
generally
supporting data, see, e.g., Pickard et al, 2002).
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However, it should be recognized that sensory neurons and motor neurons are
likely
to be somewhat easier to work with and evaluate, at least during the early
stages of
development of this invention. This is due to several factors. On one hand,
the projections
of sensory and motor neurons actually reach (or closely approach) exposed and
accessible
surface location's (in the case of olfactory neurons), and relatively shallow
muscle and
subcutaneous regions (in the case of pain-transmitting and motor neurons). By
contrast,
pre-ganglionic autonomic neuron terminals are buried substantially deeper
beneath the skin
surface, within the autonomic neuronal ganglions. Therefore, pre-ganglionic
autonomic
neurons are likely to pose somewhat greater technical challenges (and somewhat
greater
risks) for accurate vector delivery than sensory or motor neurons, if used as
BBB-straddling
"transfection conduits".
Therefore, the examples and most of the discussion herein focus on using
sensory
neurons or motor neurons, as BBB-straddling neurons that can be transfected by
genetic
vectors, to describe and illustrate the invention herein. Those classes of BBB-
straddling
neurons are believed to provide generally preferred candidates for initial
development and
testing of this invention. Nevertheless, pre-ganglionic autonomic neurons
should be
recognized as having potential use as BBB-straddling "transfection conduits"
as disclosed
herein, and may eventually become highly useful for various types of
therapeutic or other
treatments.
GENETIC VECTORS
As summarized above, genetic vectors (such as viral vectors, liposomes, and
ligand
vectors that target endocytotic receptors on BBB-straddling neurons) that
carry one or more
useful "passenger" genes (which can include marker genes and/or payload genes)
are
contacted with the peripheral projections of BBB-straddling neurons, in a
manner which
promotes transfection of vector DNA (including the useful passenger gene) into
one or
more BBB-straddling neurons. Once inside such neuron(s), at least some copies
of the
passenger gene(s) will be transported through the projection to the main cell
body, by the
natural process of retrograde transport. Once inside the cell body, the
passenger gene(s)
will be expressed by the normal intracellular mechanism, to create the gene-
encoded
polypeptide.
Since most polypeptides of interest herein (such as neurotrophic growth
factors) act
as hormones or growth factors, which normally and naturally must be secreted
by cells in
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rder to carry out their necessary functions, such polypeptides, when expressed
by
ran sfected BBB-straddling neurons, will be well-suited for secretion by the
transfected
ieurons.
Accordingly, this invention discloses a class of genetic vectors that can be
used to
Tansfect certain types of selected CNS neurons which straddle the BBB, in a
manner which
auses the transfected neurons to express and then secrete useful and
therapeutic
polypeptides, into CNS tissue that normally is protected from foreign
polypeptides by the
blood-brain barrier.
FIG. 2 shows a schematic depiction of a viral vector 100. This vector can be
regarded as having three primary components: an encapsulating portion 110,
binding ligand
proteins 120, and genome 150 (which, in FIG. 2, is shown as double-stranded
DNA, or
dsDNA).
In viral vectors (such as vectors derived from adenoviruses) that do not have
lipid
"envelopes", the encapsulating portion 110 is made of capsid proteins 112,
which fit
together in a semi-interlocking manner. In such vectors, the protein "shell"
is usually called
a capsid, and the binding ligand proteins 120 usually are nothing more than
capsid protein
domains which are exposed on the exterior surface of each viral particle.
In other viral vectors (such as vectors derived from herpes viruses), the
encapsulating portion (usually called an "envelope") is made of lipids,
usually arranged in a
bilayer form which is comparable to the lipid bilayers that make up the outer
membranes of
most mammalian cells. In such vectors, the binding ligand proteins 120 usually
straddle the
envelope layer, and a protruding external portion of each protein extends
outwardly, so it
can contact and bind to cell surface proteins.
Regardless of whether a lipid envelope is present, the viral binding ligands
120
adhere (in a non-covalent manner, comparable to a binding reaction between an
antibody
and an antigen) to complementary proteins on the surfaces of cells that can be
infected by
that type of virus. Some types of viruses (and vectors derived from them) can
have binding
ligands that are highly specific; these types of viruses can infect only
certain types of cells
which have complementary surface proteins. Other types of viruses (and vectors
derived
from them) have binding ligands that are much less specific, and can bind to
and infect a
much wider variety of cells.
The vector genome carried by the vector 100 shown in FIG. 2 comprises double-
stranded DNA (dsDNA). Other types of viral vectors can carry single-stranded
DNA, or
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single- or double-stranded RNA. Various genetic vectors with all four
categories of
genomes are known, and any such vector can be evaluated for use as disclosed
herein. In
general, viral vectors carrying dsDNA genomes are regarded as likely to offer
preferred
candidates for early evaluation of this invention. During laboratory
manipulation of
synthetic or isolated genes, it tends to be easier to work with dsDNA, than
with ssDNA
(which tends to be "sticky" due to the innate attraction of the bases for each
other) or RNA
(which tends to be somewhat less stable than DNA). Accordingly, most of the
viral vectors
that have been developed to date (including vectors derived from herpes
viruses or
adenoviruses) contain dsDNA genomes.
Using conventional terminology that has previously been developed for use with
viral vectors, genome 150 carried by a viral vector (and by various other
types of genetic
vectors as well) can be regarded as comprising a number of "domains". One or
more
"passenger genes" 160 will most commonly be inserted somewhere into the middle
of the
viral genome, in order to ensure that both ends of the viral genome can
function properly
once the viral genome enters a transfected cell. Accordingly, the insertion of
passenger
gene 160 into the center of a viral genome results in creating two "flanking
sequences" 155
and 157, containing native or modified viral DNA sequences which flank both
ends of the
passenger gene. The tips of the flanking sequences 155 and 157 frequently will
be coupled
to viral polypeptides 158 and 159; in general, these types of polypeptide
"caps" evolved to
help viral DNA remain relatively stable and resistant to the defensive
mechanisms inside a
cell that normally attempt to chew up and dismantle viral DNA after it has
invaded a cell.
If desired, either or both these polypeptide "caps" can be selected and/or
modified to
promote and speed up various cellular processes (such as retrograde transport,
active
transport of the viral DNA into the cell nucleus, etc.).
The "passenger gene" 160 can also be regarded as having three distinct
domains.
The promoter region 162 contains a signalling sequence, which directs
"transcriptase"
enzymes to get ready to begin transcribing that strand in the DNA double
helix, to form
strands of messenger RNA. This promoter region normally contains a so-called
"TATA"
box or similar signal, which directs the enzymes to begin transcribing mRNA
from the
DNA sequence, at a location which is usually about 25 bases downstream from
the TATA
box. For simplicity of discussion herein, that sequence of about 25 bases,
located between
the TATA box and the first base which is transcribed into mRNA, is regarded
herein as
part of the gene promoter. Alternately, if preferred, it can be referred to as
part of a "non-
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transcribed leader sequence", which would not need to be regarded as part of
the actual
gene promoter.
The gene promoter is then followed by a "coding" region 164, which determines
the
sequence of the bases in the mRNA strand that will be transcribed from that
gene. This
region includes the DNA sequence that specifies an AUG "start" codon in the
mRNA strand
(which specifies a methionine residue as the first amino acid at the N-
terminus of the
polypeptide), and a "stop" codon (which truncates the translation of the mRNA
strand by
the ribosomes). In some vectors, the coding region in a passenger gene may
also contain
introns, which will be deleted from the final mRNA strand by "editing"
processes inside the
host cell.
The coding region is then followed by a "non-translated" sequence 166. This
domain
will be transcribed and will be part of the mRNA strands that are created
inside a host cell.
These domains, when present on mRNA strands, help stabilize the mRNA strands
inside
host cells, and protect them against rapid degradation by enzymes. However,
this non-
translated sequence is not translated into a polypeptide sequence, by
ribosomes.
Variations on this general genetic structure are possible and may be used in
this
invention. As just one example, after the coding region 164 for one
polypeptide and before
the non-translated sequence 166, an "internal ribosome reentry site" (TRES),
such as
derived from a RNA virus such as encephalomyocarditis virus, may be inserted
with a
second coding sequence to allow co-expression of a second polypeptide (e.g.,
Wong et al
2002). The IRES instructs ribosomes to bind to the mRNA segment in a second
location,
and to commence expressing a second polypeptide in parallel with the first.
Viruses have evolved various different ways of introducing their DNA (or RNA)
into cells, and genetically engineered viral vectors can make use of the same
types of
infective processes to deliver their DNA "payload" into susceptible cells.
As one example, in a process that is fairly common process among viruses that
do
not have lipid envelopes, a virus's capsid proteins will attach to protein
molecules that are
displayed on the host cell's outer membrane. This attachment process initiates
a process
called "invagination", which results in the virus being drawn into and
becoming packaged
within a lipid bilayer envelope, or vesicle, that is formed entirely of lipids
from the cell's
membrane, but which is now located inside the cell, suspended in its
cytoplasmic fluid.
This bubble-like vesicle, often called an "endosome," subsequently ruptures,
either with the
aid of the virus capsid proteins, or due to digestive processes or organelles
inside the cell's
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;ytoplasm. This rupturing of the endosome liberates the viral DNA into the
cell's
.:ytoplasm.
In another example, in a process which is fairly common among herpes viruses
and
)ther viruses that have lipid bilayer envelopes, the different lipid bilayer
membranes which
enclosed the virus and the cell become fused together, and effectively merge
with each
Dther. This results in the entire contents of the virus's lipid envelope being
transferred into
the cell cytoplasm.
Regardless of which infective method is used by a selected type of viral
vector, the
result is that an effective viral vector transfers some or all of its genetic
material into the
cytoplasmic fluid of a cell. Returning to the example illustrated in FIG. 1,
viral vector 100
inserts its DNA 150 into the exposed mucosal tip 212 of olfactory receptor
neuron 200.
In some cases, after a genetic vector carrying a passenger gene has
transfected
neurons which straddle the BBB, any of several other fates and effects can
result,
depending on the replication and transmission traits of the vector system that
was used. In
most cases, it is assumed that the vector and passenger genes will remain
inside transfected
BBB-straddling neurons, and polypeptides expressed by the passenger genes in
those
transfected neurons will simply be secreted by the transfected neurons.
However, some
types of vectors (referred to as "trans-neuronal" vectors) may themselves be
secreted by the
BBB-straddling neurons, at locations inside the BBB; this would allow such
trans-neuronal
vectors (or their genetic material) to contact and transfect other neurons
which are entirely
inside the BBB.
It also should be noted that genetic vectors and gene constructs developed for
use as
disclosed herein can use any of various techniques and DNA sequences that are
known for
expressing higher quantities of an encoded polypeptide. Several such
techniques and
sequences are discussed below, under the subheading, "Gene and Vector
Constructs;
Expression, Transport, and Secretion Enhancers".
It also should be recognized that "marker genes" (also called "reporter
genes"),
which can allow easier, faster, less expensive, more reliable, or otherwise
enhanced
detection, quantification, and/or isolation of transformed cells and cellular
pathways and
fates, offer an important category of passenger genes which can be carried by
genetic
vectors as disclosed herein. Such genes, which are well known in genetic
engineering, can
allow facilitated and improved research, evaluation, and development in
various fields of
industry, commerce, and medicine, in various ways that will be readily
apparent to those
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skilled in the art.
Additional comments on various types of genetic vectors are contained in the
Examples, below.
CANDIDATE THERAPEUTIC POLYPEPTIDES
The genetic vectors disclosed herein should be regarded essentially as
vehicles,
carriers, or delivery systems. As such, the particular choice of passenger and
payload
molecules that can be transported and delivered, by these vehicles, is largely
up to the
choice of a particular user who intends a particular use.
As a general rule, the genetic vectors disclosed herein will be able to
transport and
deliver essentially any chosen gene, having any nucleotide sequence, in a
manner that will
allow the corresponding polypeptides to be expressed by that gene, in
transfected neurons.
As such, most polypeptides that will be of interest to most physicians and
researchers will
be polypeptides that can be secreted, by the transfected BBB-straddling
neurons, at sites
located within the BBB. However, the requirement for secretion, in most cases
that will be
of interest to physicians and researchers, should not be regarded as a severe
limitation, for
two reasons. First, nearly all types of "neuroactive" polypeptides are
inherently secreted; as
a general rule, their neuroactivity was discovered and recognized due to their
ability to be
secreted by one type of cell within the CNS, and to act upon other types of
cells within the
CNS. And second, if some particular candidate polypeptide that is of interest
is not
naturally and normally secreted by cells, that polypeptide sequence can be
coupled to any of
numerous known "leader" polypeptide sequences that cause cellular secretion.
It should also be recognized that the endocytotic ligands disclosed herein do
not
address and are not directly concerned with secretion of polypeptides or other
compounds,
by transfected neurons. Instead, these ligands are designed and intended to
enable targeted
delivery of gene vectors to selected BBB-straddling neurons. It also should be
noted that
these same types of endocytotic ligands can also function as transport and
delivery systems
for other classes of passenger molecules, including therapeutic drugs,
diagnostic
compounds, or other compounds, into BBB-straddling neurons (and other classes
of targeted
cells) having endocytotic surface molecules to which the ligands will
specifically bind).
Therefore, it may be that in some cases, drugs or other non-DNA compounds that
can be transported into BBB-straddling neurons, by means of entry through
peripheral
projections that are accessible outside the BBB, may well be secreted by those
same
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neurons, at central projections that are located inside the BBB (as has been
demonstrated
with certain trans-synaptic tracer molecules, such as wheat germ agglutinin).
This route of delivery for drugs and other compounds may be especially
promising,
if the coupling component used to connect the passenger component to the
endocytotic
ligand can enable the passenger to be detached from the ligand, after the
ligand-passenger
complex has entered a BBB-straddling neuron. Such coupling components are
often referred
to as "labile" agents, which indicates that they are not especially tight or
durable, and can
be broken apart under various conditions. Various types of labile coupling
components are
known to those skilled in the art (the acidity-sensitive spacer molecules
described in US
patents 4,631,190 and 5,144,011, by Shen et al, offer one example). Any such
labile
coupling component can be evaluated, if desired, to determine whether it can
be used with
a particular type of therapeutic drug, analytical compound, or other passenger
component
that is of interest, in conjunction with a particular endocytotic ligand (or
class of ligands),
to form molecular complexes that can: (1) deliver the passenger components
into BBB-
straddling neurons, via accessible peripheral projections, and (2) enable the
passenger
components to be detached from the endocytotic ligands, after the complex has
entered a
BBB-straddling neuron, in a manner that (3) will allow secretion of the
released passenger
component, by the BBB-straddling neuron, at a secretion site located inside
the BBB.
Returning to the issue of the range and variety of polypeptides that can be
introduced into CNS tissue by using genetic vectors to introduce foreign genes
into
BBB-straddling neurons as disclosed herein, Tables 1 and 2 list some of the
options that are
available in just one area of application (i.e., in therapeutic treatment of
various
neurodegenerative or neuropathic diseases, in humans). To provide additional
information
on items that are listed without any narrative support in the Tables, the
following summary
and overview provides a somewhat expanded listing of various types of
polypeptides that
can be used in human medical therapy, and of the specific classes of disorders
that the
various polypeptide types can modulate. This discussion is not exhaustive, and
those skilled
in neurology or neuropharmacology will recognize other potential uses and
therapies, after
the methods disclosed herein have been made known to the public.
1. Various neurotrophic factors, growth factors, or neurite inhibitory
factors, such
as listed in Table 1, may help prevent or repair various forms of neuronal
damage caused
by CNS disorders such as neurodegenerative diseases, or by ischemic or hypoxic
crises
such as stroke, cardiac arrest, suffocation, blood loss, or other types of
physical injury or
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rauma.
2. Various neurotrophic hormones, growth factors, or neurite inhibitory
factors can
ielp stimulate the formation of new synaptic connections between existing
neurons and/or
;uide the outgrowth of neuronal processes to facilitate some connections and
discourage
)thers. In some patients, this type of treatment can help facilitate the
recovery of nervous
function loss due to aging or various diseases. It may also help patients
regain muscular,
-ipeech, and other functions after a stroke, head injury, or other ischemic,
hypoxic,
axcitotoxic, or similar crisis.
3. Various types of endocrine, paracrine, and related or similar polypeptides
can
help treat various glandular, growth-related, maturation-related, sexual, and
other disorders.
4. Polypeptides that can increase the quantities of certain neurotransmitter
molecules
inside the BBB can treat various neurodegenerative diseases. For example,
polypeptides that
can increase dopamine levels inside the brain (by acting as enzymes, hormones,
or release
factors, or through various other mechanisms) can be used to treat Parkinson's
disease.
Alternately, polypeptides that can increase acetylcholine levels may be useful
for treating
Alzheimer's disease.
5. Cytotoxic or growth-suppressing polypeptides can be used inside the BBB to
treat
certain types of cancer or other diseases.
6. Various types of receptor antagonists, antibodies, and other polypeptides
that can
block or suppress one or more types of neuronal activity can be used to help
control and
reduce neuropathic pain, hyperalgesia, and similar problems.
7. Lysosomal storage diseases due to lack of a particular polypeptide in the
CNS
may be treated by delivery of that polypeptide into the CNS.
8. Infections of the CNS by viruses, prions, or bacteria may be treated by
delivering
into the CNS that help control or reduce the spread of the infection. For
example, delivery
of polypeptides that bind to the receptors and inhibit virus docking may be
able to reduce
the spread of viruses such as HIV within the CNS.
9. Delivery of recombinant antibodies to antigens within the CNS can be used
to
modulate physiological processes in a beneficial or useful way. For example,
delivery of
recombinant antibodies to myelin associated neurite inhibitory molecules such
as No-Go
may be able to enable regrowth and regeneration of CNS nerves, following
spinal cord
injury and other traumatic injuries.
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TARGETED CNS NEURONS OR GLIAL CELLS
The term "target cell" is used to refer to a neuron or glial cell that: (i) is
part of the
CNS, and lies wholly within the BBB; and (ii) is contacted by, or is intended
to be
contacted by, an exogenous polypeptide that has been delivered into BBB-
protected brain or
spinal tissue by a method and vector as disclosed herein.
It should be noted that this term deliberately excludes BBB-straddling
neurons, even
though such neurons can be regarded and referred to by terms such as "primary"
or
"initial" targets of the genetic vectors that will be used to contact and
transfect such cells.
In the overall scheme of the invention disclosed herein, the transfection of
such BBB-
straddling "initial targets" will be valuable and useful, only insofar as that
step in the multi-
step process will later lead to the subsequent delivery of vector-encoded
polypeptides into
CNS tissue that is protected by the BBB. Accordingly, the real "targets" of
this invention
are neurons or glial cells that are entirely within, and protected by, the
BBB, and the BBB-
straddling neurons that are used in the delivery mechanisms disclosed herein
should be
regarded as conduits, or passageways, rather than as the real target cells.
Any references herein to "glial" cells arises from the fact that, within BBB-
protected
brain and spinal tissue, cells are divided into two categories, referred to as
neurons, and
glial cells (also called "neuroglia cells" in some medical texts). By
definition, the term
"neuron" is limited to cells that can receive and transmit nerve signals. The
term "glial
cells" is a broader residual term, and it includes all types of CNS cells that
cannot receive
or transmit nerve signals. These glial cells perform. various activities that
can be regarded
as supporting, housekeeping, and "nursing" functions within the CNS; this
helps neurons do
their essential work. The word "glia" comes from the same root word as "glue";
glial cells
were initially thought of as the "glue" that holds CNS tissue together. Glial
cells are
divided into various categories, including oligodendroglia cells, astrocytes,
ependymal cells,
and microglia cells. They are discussed in nearly any textbook on neurology,
and are a
crucial part of the CNS.
For several reasons, the initial work to develop this invention is likely to
focus upon
using exogenous polypeptides to contact and modulate neurons, rather than
glial cells. One
major reason driving that trend is that it is likely to be much easier to
confirm and quantify
CNS responses that directly involve neurons, as compared to effects and
responses that
involve glial cells first, and affect neurons only as a secondary effect.
Accordingly, while some researchers will prefer to evaluate this invention by
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bcusing on "target cell" neurons lying entirely within the BBB, it should be
recognized
hat: (1) the methods and vectors of this invention may also be useful for
providing ways to
Teat CNS tissue by using exogenous polypeptides to contact and treat glial
cells; and (2)
ertain specific conditions involving glial cells are likely to merit
relatively early research
and evaluation, such as potential treatments for certain types of glial cell
cancers (including
glioblastomas and astrocytomas), and methods for modulating the responses of
glial cells to
traumatic injury, or hypoxic or ischemic insult.
As implied by the term "target", not all neurons or cells wholly within the
BBB will
necessarily make contact with polypeptides delivered using the invention.
Where a BBB-
straddling neuron (or cluster of neurons) is transfected by a genetic vector,
the "target"
neurons or glial cells lying wholly within the BBB will be either: (i)
positioned in close
proximity to the BBB-enclosed synapses or other terminals of transfected
neurons; or, (ii)
have cell processes or extensions (such as dendrites, axons, or terminals)
that are in close
proximity to the BBB-enclosed synapses or other terminals of transfected.
neurons. Where a
"transneuronal" vector is used (i.e., where the genetic vector itself will be
able to travel
through a BBB-straddling "primary" neuron, to a second- or third-order neuron
that is
located inside the BBB, and that will be transfected by the vector so that it
will
subsequently express the encoded polypeptide), "target" neurons or glial cells
may be
located in close proximity to second-order or third-order transfected neurons
which will
express and secrete polypeptides encoded by vector-borne foreign genes.
As examples of how one or more classes of neurons inside the brain can become
"target" neurons inside the BBB, basal forebrain cholinergic neurons,
serotonergic raphe
neurons, and noradrenergic locus coeruleus neurons do not have direct synaptic
junctions
with BBB-straddling olfactory receptor neurons; nevertheless, these classes of
cholinergic,
serotonergic, and noradrenergic neurons inside the BBB can be target neurons,
if olfactory
neurons are transfected by a genetic vector as disclosed herein, because they
can take up
polypeptides released by transfected olfactory receptor neurons. This is
illustrated by the
fact that these classes of cholinergic, serotonergic, and noradrenergic
neurons inside the
BBB can be infected by rabies or herpes simplex viruses released from virus-
infected
olfactory receptor neurons (Lafay et al 1991, Barnett et al 1993).
The principle that CNS neurons or glial cells can be "targeted" for contact
and
treatment by exogenous polypeptides, if they are positioned inside the BBB
adjacent to or
within fairly close proximity to a central projection of a BBB-straddling
cell, can be used
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nost advantageously if supported by the best currently available information
on the
matomical relationships between transfected and targeted cells. This type of
anatomical
knowledge can be found in the technical literature where investigators have
made use of
viruses or other "transneuronal labelling" molecules. For example, if a
particular class of
CNS neuron or glial cell has been shown to become infected with a
transneuronal virus,
following application of that type of virus to BBB-straddling neurons in lab
animals, the
subsequently-infected class of CNS neurons or glial cells can be used as
target neurons or
glial cells for the purposes of this invention. This type of neuronal mapping
has already
been carried out to some degree by various researchers, and is described in
references such
as Loewy 1998 and Norgren et al 1998. In addition, this type of work continues
to this day,
and as it continues to provide more information on neuronal circuitry, that
additional
information can be taken into account by anyone practicing this invention.
The preferred method for contacting a genetic vector with a peripheral
projection of
a sensory or motor neuron will depend on the structure and location of the
targeted
peripheral projection. For example, administration to olfactory receptor
sensory neurons
can be via nasal instillation, such as by using a nasal spray, or by using a
liquid-saturated
packing material that can be placed in the nasal sinuses, in direct contact
with the nasal
surface area which contains olfactory projections, for some period of time
(such as 30 to 90
minutes).
If desired, direct and sustained contact between a gene vector and the
olfactory
neuron projections can be further promoted by steps such as (i) using a nasal
decongestant
to reduce and minimize any mucous covering the nasal sinus surfaces; (ii)
using a
preparation of a cleaning or similar agent, such as dilute isopropyl alcohol,
acetone, etc., to
further clean and prepare the area to be contacted; and/or (iii) using a
mechanical scraping
procedure, as is commonly performed by otolaryngologists to treat patients
with recurrent
nasal sinus infections. In general, neuronal projections in a surface area
which has become
irritated to a point of mild inflammation tend to be more receptive, to
cellular uptake of
foreign molecules, than cells which can be regarded as being in a quiescent or
resting state.
It may also be possible to further stimulate entry of genetic vectors into
neuronal
projections by means of an electrical charge or surge applied to the nasal
surface, in a
manner comparable to the in vitro cell transfection technique of
"electroporation". If
desired, any or all of these steps can be carried out while a patient is under
general
anesthesia, or under a mild local anesthesia.
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If desired, neuronal contact and uptake can be further promoted and increased
by
:.ompounds referred to as "muco-adherents". Such compounds are being developed
to
increase the delivery of various drugs into the bloodstream, via transmembrane
modes such
as nasal sprays. Examples of muco-adherents include chitosan (discussed in
articles such as
Schipper et al 1999) and various polysaccharide colloidal preparations
(discussed in articles
such as Janes et al 2001); also see Rillosi et al 1995 and Lim 2000 for
further discussion of
muco-adherents.
Administration to projections which are part of nocioceptive neurons can be
via
cutaneous or sub-cutaneous injection, by various controlled skin abrasion
techniques, and
possibly by topical application if adequate penetration can be achieved (skin
penetration can
be promoted by agents such as dimethyl sulfoxide, if desired). If motor neuron
projections
are used as the access route, administration usually will require sub-
cutaneous or
intramuscular injection. For example, projections which are part of the lower
motor
neurons of the hypoglossal nucleus can be accessed by injecting a genetic
vector into the
muscles of the tongue. In some cases, gene vectors may be administered via an
intravenous
route (especially if a particular nerve-targeting component is included);
however, it is
generally believed preferable to administer gene vectors by intramuscular or
similar
injections that will establish and sustain high concentrations of vectors at
desired targeted
locations.
This also points out a significant difference between this mode (involving
targeted
transfection of selected neurons), and delivery of genes, polypeptides, or
other compounds
via injection into cerebrospinal fluid. Physical delivery into the
cerebrospinal fluid system
(such as via catheter into the ventricles of the brain or intrathecal space of
the spinal cord)
will distribute a neurologic agent to a large number and array of CNS cells.
In contrast,
this invention describes how delivery of desired therapeutic polypeptides can
be targeted to,
or result in controlled and preferential delivery to, only limited number of
cells or neuronal
processes, which are in close contact with (or synapsing with) transfected BBB-
straddling
neurons.
It is anticipated that the same procedures and classes of genetic vectors
disclosed
herein can be adapted and used, if desired, to introduce polypeptides into the
brains and
spinal cords of non-human mammals, and into other classes of animals that have
blood-
brain barriers, including reptiles and birds. As such, this invention may well
become useful
for controlling and regulating the rate of growth and/or reproductive status
of livestock,
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pets, and other animals. For this and other purposes, hypothalamic releasing
factors can be
delivered via the olfactory receptor neurons to regulate the release from the
pituitary gland
of potent hormones. For example, GHRH may be delivered to stimulate release of
growth
hormone, to accelerate an animal's growth rate. Also, sustained delivery of
GnRI4 at
supramaximal dose may be used to inhibit normal release of LH and/or FSH
release, in a
manner which may achieve or promote contraception, including reversible
contraception. If
the methods disclosed herein are being adapted to other mammalian species, or
to other
classes of animals such as birds and reptiles, the primary adaptations that
will need to be
made include (i) selection and use of genetic vectors that are well-suited for
transfecting the
projections of BBB-straddling neurons in the chosen animal type, and (ii)
selection and use
of a polypeptide type which has the desired activity in that particular
species of animal.
Without disregarding these and other potential applications, the remaining
discussion
herein of genetic vectors focuses solely on animal tests to prove the methods
and
procedures disclosed herein, and on treating humans for medical purposes.
VIRAL VECTORS
Review articles that describe various types of mammalian viral vectors include
Karpati et al 1996, and Kaplitt and Maldmura 1997. At least four types of
viral vectors
have been used to transfect neurons. Those virus types are:
A. Adenoviruses
These are double-stranded DNA viruses that are often found in various glands;
wild-
type viruses cause respiratory infections, conjunctivitis, and various other
problems.
Genetically-engineered adenoviruses that have been rendered incapable of
replicating
(except in special types of cells and/or culture media that exist only in
laboratory
conditions) have become the main class of viral vectors used in in vivo
studies on
mammals, including gene therapy efforts on humans. Adenovirus vectors have
been used to
transfect various types of neurons, as reviewed in Smith and Romero 1999.
B. Adeno-Associated Virus (Dependovirus; adenosatellite virus)
These are single-stranded DNA viruses that depend on Adenoviruses for
replication.
Methods for preparing adeno-associated virus vectors can be found in the
chapter by
Bartlett and Samulski, in Robbins (ed.) 1997.
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C. Hapes Simplex Viruses (HSV)
These are double-stranded DNA viruses, with capsids that are surrounded by
lipid
envelopes. Use of engineered HSV vectors to transfect neurons is discussed in
Staecher et
al 1998.
D. Retroviruses, including Lentiviruses
These viruses contain RNA, rather than DNA. Use of a lentivirus vector to
deliver
and express the GDNF gene into lower motor neurons of mice was described in
Hottinger
et al 2000.
These four classes of viruses appear to be receiving the most effort and
attention at
this time, in attempts to create viral vectors that can transfect cells but
which are generally
nonpathogenic after they enter their target cells (usually due to deletions or
defects in one
or more genes which encode proteins required for replication).
However, other classes of known neurotropic viruses (which includes numerous
types of viruses that cause viral encephalitis, including some RNA viruses)
offer promising
candidates for vectors that can selectively (or at least preferentially)
transfect neurons. If
any of those classes of viruses can be rendered safe and non-pathogenic by
means of
genetic manipulation comparable to the steps used to render other viral
vectors non-
pathogenic, they may be well-suited for use as disclosed herein.
Alternately or additionally, using genetic engineering techniques, various
types of
viral vectors that do not have a strong affinity for neurons can be provided
with selected
and/or modified surface proteins (including chimeric surface proteins) that
bind
preferentially to surface proteins on the projections of olfactory neurons,
motor neurons, or
other types of neurons that straddle the BBB. If properly developed and used,
modified
viral surface proteins with increased neuron-binding affinity may increase the
speed,
efficacy, and other benefits of viral vectors when used as disclosed herein.
NON-VIRAL VECTORS
As briefly summarized in the Background section, and as discussed in more
detail in
various examples below and in numerous published articles, several types of
non-viral
genetic vectors are known, and can be evaluated for use as disclosed herein.
Primary
candidates for classes of nonviral vectors which can transfect neuronal
projections include
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(1) cationic materials, including cationic liposomes, and (2) protein-DNA
complexes
containing polypeptide ligands that bind to endocytotic receptors on neurons.
On the subject of ligands that can bind to endocytotic receptors on neurons,
it should
be noted that a iiighly useful and potentially very powerful in vivo screening
method has
been developed by the Applicants herein, for identifying and isolating ligands
which can
bind to endocytotic receptors on the surfaces of neuronal fibers outside the
BBB. Briefly,
this new method of in vivo screening uses the following steps:
(1) emplacing multiple candidate ligands at a first location inside the body
of a
living animal, in a manner that causes the candidate ligands to directly
contact neuronal
fibers (such as a sciatic nerve bundle, in the leg of a rat);
(2) allowing enough time to pass for the neuronal fibers to internalise those
particular ligands that activate and drive the process of endocytotic
internalisation and
retrograde transport;
(3) harvesting segments of the neuronal fibers, at a harvesting site that is
sufficiently
distant from the ligand emplacement site to avoid collecting ligand candidates
that did not
enter the neuronal fibers or that were not retrogradely transported; and,
(4) removing the ligands from the harvested segments of neuronal fibers, for
reproduction and further processing.
This new method of in vivo screening offers a powerful tool for developing
improved non-viral vectors (and targeted viral vectors as well) that can bind
specifically to
any particular endocytotic receptor or other endocytotic surface molecule.
Accordingly,
since it offers a preferred mode of carrying out this invention, it is
described in detail
herein, under a separate subheading and in various Examples, below.
It should also be recognized that numerous methods and tricks are known to
those
skilled in the art, for increasing the likelihood that a non-viral vector will
succeed in
accomplishing its intended results. As just one example, a neuron-targeting
non-viral vector
has been reported which makes use of neurotensin, to target plasmid delivery
to neurons of
the nigrostriatal and mesolimbic dopaminergic systems (Martinez-Fong et al
1999). Various
other examples are discussed in the following sections.
In general, plasmid vectors constructed for use with a cationic or endocytotic
delivery mechanism will contain both: (i) sequences to enable replication of
the plasmid in a
host cell, such as E. coif, and (ii) sequences that enable expression of the
gene for a
therapeutic polypeptide in target cells. For replication, the vector
ordinarily carries a
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replication site, as well as marker genes that allow simple selection of
transformed cells.
For example, for replication in E. coil, the plasmid pBR322 (Bolivar et al
1977) contains
genes for ampicillin and tetracycline resistance, which allow quick and simple
identification
of transformed cells by using those antibiotics.
The sequences that enable expression of the gene for a therapeutic polypeptide
in a
target cell ordinarily include an origin of replication (if necessary), a
promoter located in
front of the gene to be expressed, any necessary ribosome binding sites and/or
RNA splice
sites, a polyadenylation site, and transcriptional terminator sequences.
GENE AND VECTOR CONSTRUCTS; EXPRESSION, TRANSPORT AND SECRETION
ENHANCERS
A variety of known genetic engineering techniques and DNA or polypeptide
sequences can be used to improve and increase: (i) the likelihood that this
method will
successfully accomplish a detectable level of a desired and intended result,
in any particular
animal or patient; (ii) the potency, efficacy, duration, or other desired
aspects of the
treatment, in treated animals or patients; and, (iii) the ability of
researchers and physicians
to track and monitor the status, progress, and results of a treatment, and the
locations and
concentrations of exogenous genes and/or polypeptides.
This section describes various examples of such known techniques and
sequences,
along with a brief indication of how they can be applied to the methods and
vectors of this
invention. This listing of illustrative examples is not exhaustive or
exclusive, and those
skilled in the art will recognize various other genetic engineering
techniques, reagents, and
gene and peptide sequences and fragments that can also be adapted for use as
disclosed
herein.
In addition, it should be understood that the techniques and/or sequences
disclosed
herein can be combined with each other, in various ways that will be apparent
to those
skilled in the art. As just one example, a gene construct can be developed,
for expressing a
gene encoding for a mature neurotrophin in BBB-straddling neurons, which
encodes a
polypeptide that contains both: (i) a pre-pro-BDNF leader sequence, at the N-
terminus of
the sequence encoding the mature neurotrophin, and (ii) an "epitopic tag"
placed elsewhere
in the sequence encoding the mature neurotrophin. Similarly, it should be
recognized that
other, additional genetic engineering techniques or sequences that are now
known or
hereafter discovered may also be adapted for use as disclosed herein.
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As one example of a technique for increasing the expression of a passenger
polypeptide carried by a genetic vector, the polypeptide coding sequence can
be placed
under the control of a powerful "gene promoter" sequence that will drive high
levels of
transcription of mRNA strands containing the coding sequence. Various promoter
sequences
that act as strong promoters in human cells (including human neurons) are
known, and
include, for example, promoter sequences derived from various types of
pathogenic viruses,
such as a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) long
terminal
repeat (LTR) promoter, and a simian virus 40 (SV-40) "early" promoter.
Alternately, in some cases, it may be preferable to place one or more genes
(such
as, for example, a gene which encodes a marker polypeptide) under the control
of a so-
called "inducible" promoter, which will be active only under certain
conditions or when a
certain compound is present.
Also of interest herein is a class of gene promoters usually referred to as
"tissue-
specific" promoters. These types of promoters cause a polypeptide coding
sequence to be
efficiently expressed into mRNA, only in specific types of tissues or cells.
If a gene
construct containing a tissue-specific promoter is delivered into the wrong
cell or tissue
type, those "non-intended" cells usually will not have the transcription
factors or enzymes
which can recognize that particular tissue-specific promoter; therefore, the
gene construct
will not be expressed (or will be expressed only at low rates) in those types
of "non-
intended" cells. Accordingly, tissue-specific promoters offer potentially very
useful
candidates for evaluation as described herein.
From published transgenic animal studies, a number of tissue-specific
promoters
have already been identified and published, and their number is increasing as
additional
studies on transgenic animals are published. The following non-exhaustive, non-
exclusive
list contains several examples of tissue-specific promoters that may be useful
in this
invention:
(i) tissue-specific promoters that appear to be much more active in olfactory
receptor
neurons than in other classes of neurons, include an olfactory marker protein
promoter
described in Servenius et al 1994, and an M4 olfactory receptor protein
promoter described
in Qasba and Reed 1998;
(ii) a tissue-specific promoter that appears to be much more active in
nocioceptive
sensory neurons than in sympathetic neurons, as described in Watson et al
1995. This gene,
which expresses "minimal calcitonin gene related peptide" (CGRP), may also
offer a
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:liemically-inducible gene promoter as well, since it appears to be much more
active in the
?resence of nerve growth factor (NGF). Therefore, if the CGRP gene promoter is
used,
;ene expression in sympathetic neurons could be minimized, which can help
minimize
undesired side effects. This type of gene promoter .may be especially useful,
since
aocioceptive sensory neurons as well as sympathetic neurons are known to
internalize NGF,
via receptor-mediated endocytosis. Since both nocioceptive and sympathetic
neuronal types
will likely be transfected, if peripheral administration is used with a non-
viral vector that
exploits NGF-receptor-mediated endocytosis to target gene delivery to
accessible neurons,
the ability to create a gene construct that will not be expressed at
substantial levels inside
transfected sympathetic neurons, by using a gene promoter such as the CGRP
promoter,
may be highly advantageous.
(iii) a tissue-specific promoter which appears to be much more active in
spinal motor
neurons than in nocioceptive or other sensory neurons, and which drives
expression of the
alpha-1 subunit of glycine receptors, as described in articles such as Bechade
et al 1994,
Rajendra et al 1997, and Zafra et al 1997.
In general, where a transgene construct has been shown to restrict expression
of a
selected gene to a particular class of neurons in a line of transgenic
animals, the same
pattern of restricted gene expression can be expected when an appropriately
prepared gene
construct (containing the same promoter and associated gene expression
elements) is
delivered in vivo by viral or non-viral vectors. Thus, reference should be
made to
transgenic animal protocols (e.g., Causing and Miller 2000) when designing
gene
expression. constructs.
In a related and similar manner, it also may be possible and desirable to use
naturally-occurring or synthetic promoters and/or enhancers to further
stimulate, restrict, or
control expression of a foreign gene in various ways. For example, it may be
possible to
establish or increase foreign gene expression in injured neurons, without
substantially
affecting other neurons, by using a gene promoter which appears to be
specifically activated
after a nerve lesion, but which appears to be otherwise silent (see Funakoshi
et al 1998).
Alternately, a glucocorticoid-responsive promoter may be used to drive gene
expression
after neurological insult when, as a result of the stress response,
glucocorticoid-adrenal
stress steroids are present in high levels (e.g., Ozawa et al 2000).
Alternately, the latency-
associated transcript promoter from herpes simplex viruses (Lachmann et al
1997) is likely
to drive gene expression in a manner that may be more prolonged than can be
achieved by
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various other promoters.
As another example of a technique that can be used to increase protein
expression,
scientists who create engineered genes for transfection often delete one or
more
"non-preferred" codons and replace them with ="preferred" codons. The
distinction between
preferred versus non-preferred codons arises from the fact that most species
(and many
different cell types, within a certain species) have evolved with a repertoire
of both: (i)
preferred codons, which will pass quickly and without delay through the
translation system
inside ribosomes, and (ii) non-preferred codons that will slow things down,
inside
ribosomes. This system of preferred and non-preferred codons provides a
cellular
mechanism that is highly useful for regulating gene expression, so that
properly balanced
quantities of thousands of different proteins can all be present at suitable
concentrations,
inside a single cell. However, in genetic engineering, by getting rid of non-
preferred
codons and replacing them with preferred codons, a genetic vector can bypass
the normal
control system, and drive the production of unusually high levels of a foreign
protein. This
type of approach is described in numerous published works, such as US patent
5,795,737
(Seed et al, 1998) and various articles cited therein.
Another known genetic engineering trick involves "cysteine-depleted" variants
of a
polypeptide. As is well known, residues of cysteine (an amino acid with a
highly reactive
sulfhydryl group, --SH) in a polypeptide chain tend to react with other
cysteine residues.
When two cysteine residues react with each other, they form a disulfide bond.
Disulfide
bonds are very important in ensuring that, when a polypeptide is being
synthesized inside a
cell and is being subjected to "post-translation processing", it will be
folded into its proper
three-dimensional configuration. However, when genetic engineering is used to
create
proteins, cysteine residues that are not involved in disulfide bonds may
generate problems
and impede the desired result. Such problems often can be avoided and overcome
by
replacing one or more cysteine residues, at certain locations in a polypeptide
sequence, with
other residues. This is done by replacing the codon which specifies a cysteine
residue, at
some particular location in a gene, with a different codon that will specify
some other
non-cysteine amino acid residue. These types of cysteine-depleted "muteins"
are described
in various US patents, such as 4,737,462.
Genetic engineering techniques also can be used to add a so-called "leader
sequence"
and/or "signal sequence" to a foreign polypeptide that is encoded by a genetic
vector as
disclosed herein. Although the term "leader sequence" is not always used
consistently or
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precisely, it generally refers to a polypeptide sequence which has either or
both of the
following traits: (i) it causes or promotes the transport of the resulting
leader-plus-
polypeptide molecule to a certain location in a cell, or the secretion of the
leader-plus-
polypeptide by host cells, as can be demonstrated when the leader sequence is
added to
foreign polypeptides that normally do not undergo that type of transport or
secretion;
and/or, (ii) it is the portion of an initial polypeptide which is subsequently
cleaved off, by
natural "post-translational processing," from a smaller version of the final
(mature)
polypeptide. In nearly all cases, a leader sequence will be positioned at the
N-terminus of a
polypeptide; that is the end (often called the "head") which is created first,
and which
emerges first from the ribosome as the polypeptide is created.
Similarly, the term "signal sequence" is not always used consistently or
precisely. In
general, it refers to a polypeptide sequence that leads to some particular
type of result,
effect, or process, as can be demonstrated by the ability of that signal
sequence to impart
the same result or process to other peptide sequences that normally do not
undergo that
result or process. As examples, signal sequences can lead to (i) enclosure,
sequestering, or
other processing or "packaging" of a polypeptide into vesicles or other
compartments; (ii)
transport of a polypeptide to a particular component or region of a cell; or
(iii) secretion of
a polypeptide by the host cell. Accordingly, "signal sequence" is used more
broadly than
"leader sequence", and generally includes leader sequences. In addition, there
is no
implication that a signal sequence must be cleaved off of an initial
polypeptide, to create a
mature final version of the polypeptide; signal sequences are often retained
by fully mature
polypeptides.
Various neuronal leader and/or signal sequences are known that are believed to
increase either or both of two processes that are highly useful in this
invention. Those two
processes are: (i) anterograde transport of a polypeptide, out of the main
body of a neuron
and through a "central projection" which will transport the polypeptide, while
still inside
the cell where it was synthesized, across the blood-brain barrier and/or
closer to a desired
targeted area of the brain; and/or, (ii) secretion of a polypeptide, by a
neuron, at synaptic
or other terminals belonging to the neuron.
One such type of leader sequence which may be highly useful, in various
settings, is
a leader sequence that is initially found in a polypeptide called "pre-pro-
BDNF", where
BDNF is the acronym for brain-derived neurotrophic factor. BDNF is a homologue
of
nerve growth factor (NGF), but unlike NGF, pre-pro-BDNF is known to be
efficiently
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transported, in an anterograde direction, by nocioceptive neurons. This has
been confirmed
by animal tests, showing that BDNF that was expressed inside nocioceptive
neurons can be
detected later in spinal tissue. The shorter, final (or "mature") form of BDNF
is created
when the leader sequence is cleaved off of the initial longer polypeptide. The
BDNF
polypeptide, and the pre-pro-BDNF leader sequence, are described in a number
of
published articles, including Conner et al 1998, Altar et al 1999, and Tonra
1999. Anyone
skilled in this particular art who evaluates what is known about the BDNF
polypeptide and
the pre-pro-BDNF leader sequence, will recognize that this leader sequence,
and any other
peptide leader sequence which is known to enable or increase anterograde
transport within
neurons and/or secretion by neurons, may be highly useful in increasing the
success rates
and efficacy levels of this invention.
EPITOPIC TAG SEQUENCES
Another genetic engineering technique also deserves attention, since it may be
highly
useful in this invention. This technique involves "epitope" sequences, tags,
and constructs.
The prefix "epi-" refers to an exposed and accessible surface; as examples,
the
epidermis is the outermost dermal (skin) surface, and the epithelium is the
exposed surface
of a mucous membrane.
Similarly, the term "epitope" is used to refer to a surface-exposed domain of
a
protein which can be firmly bound by an antibody. An antibody does not wrap
itself around
the entire surface of an antigenic protein; instead, a localized binding
domain which is part
of the antibody will bind to a localized binding domain which is part of the
antigen, in a
manner that is analogous to two jigsaw pieces fitting together along one (and
only one) edge
of each piece.
By using tests in which digested (cleaved) fragments of an antigenic protein
are
passed through a column in which monoclonal antibodies have been affixed to
tiny beads, it
is not difficult to identify the epitopic region of an antigenic protein, with
respect to a
particular line of monoclonal antibodies. However, different lines of
monoclonal antibodies
will bind to different surface areas of an antigenic protein; accordingly, a
protein generally
has a number of different epitopic regions, and the common factor that
determines which
regions are epitopic is whether a region is exposed, and accessible to
antibodies, on the
surface of the protein. For this reason, epitopic analysis is often used by
researchers to help
them determine the three-dimensional structure of a complex protein; as a
general rule, the
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amino acid sequences which show up as epitopic sites in a protein are the
amino acid
sequences that are exposed, and accessible to antibodies, on the surface of
the protein.
The practice of using epitopic "tag" sequences in genetic engineering arises
from the
following fact: it is relatively easy to create and identify monoclonal
antibodies that will
bind, with very high levels of selectivity, to known epitopic sequences.
Several such
monoclonal antibody lines (and the corresponding high-affinity antigenic amino
acid
sequences to which they bind) are well-known; examples include monoclonal
antibody lines
that will bind to known amino acid sequences derived from polypeptides such as
c-myc, and
a hemagglutinin protein from the influenza virus.
Because polypeptide expression in cells starts at the N (amino) terminus, and
ends at
the C (carboxy) terminus, there is a generally high probability that amino
acid segments
located at or near the C terminus will be exposed on the surface of the
protein. This arises
from the fact that the folding and shaping process which generates the final
three-
dimensional polypeptide will begin taking place as the strand is being created
and extended,
since various amino acid residues or domains located along the strand in
specific locations
will begin attracting or repelling each other as the strand is being formed.
Although this
depiction is highly simplified and does not adequately address the subtleties
of protein
folding, one can envision the first part of the strand that emerges from a
ribosome as
forming the "core" of the emerging polypeptide, and the rest of the strand
generally being
wrapped around or otherwise added to the initial core.
Because of this factor, amino acid sequences that are at or near the "tail
end" (the
carboxy terminus) of a polypeptide have a relatively high likelihood of being
exposed and
accessible, as epitopic sites, on the surface of the polypeptide. To take
advantage of this
factor, and to reduce the risk that insertion of an epitopic site into a
central domain might
disrupt or degrade an essential activity or trait of the polypeptide, the
normal practice is to
position epitopic tag sequences at or near the tail end (the carboxy terminus)
of a
polypeptide that is being modified to include an epitopic tag. This generally
offers the best
approach to initial research using epitopic tags, and experimental data
obtained using this
approach can be used to optimize subsequent efforts, if necessary.
Alternately or additionally, if the full three-dimensional structure of a
polypeptide
molecule is known, and if the polypeptide is known to have an "active" site
that is crucial
for catalytic activity or receptor binding, it may be possible to insert a
surface-accessible
epitopic site into the polypeptide, on the opposite side of the molecule.
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By using relatively simple in vitro screening tests, any such modified
"tagged"
protein can be tested to determine whether it still has the desired activity,
and tagged
proteins which pass those initial tests can be tested more extensively using
in vivo animal
models.
Accordingly, the genetic vector system disclosed herein allows a gene
construct to
be created, in which a unique or at least highly uncommon and detectable
"epitopic tag
sequence" can be added to (or inserted into) nearly any type of known protein
(such as, for
example, nerve growth factor). Using known genetic engineering techniques, it
is a
straightforward procedure to modify the coding sequence of a gene construct
carried by a
genetic vector, in a manner which will add a relatively small number of
additional codons
to the "native" coding sequence, or which will substitute and replace a few
codons in the
native coding sequence by other codons that do not normally appear in the
native protein.
In various forms of research and treatment that are taught herein, genetic
vectors
carrying gene constructs that will express epitope-tagged polypeptides can be
highly useful,
since they can make it much easier (and in some cases, they may be required to
make it
possible) for researchers and physicians to monitor and quantify the results
and effects of
the genetic treatments disclosed herein. As one example of the types of
problems that can
be encountered when naturally-occurring polypeptides must be analyzed, under
the current
state of the art, naturally-occurring nerve groWth factor (NGF) tends to be
degraded by
most types of tissue fixation methods; in addition, NGF is very difficult for
even highly
skilled researchers to measure accurately, and high degrees of homology and
antibody
cross-reactivity exist, in the different versions of NGF that are found in
animals as widely
different as rats and humans. Therefore, epitope-tagged versions of NGF (and
of various
other neuroactive polypeptides) can be used to avoid or at least minimize such
difficulties.
It also should be noted that epitope-tagged polypeptides are used much more
frequently and widely in research, than in clinical practice on humans. In
general, epitope-
tagged polypeptides are used most commonly for developing, optimizing, and
proving the
effects and efficacy of a certain treatment approach, in cell culture tests
and animal tests. In
addition, they sometimes are used in small-scale "Phase 1" or "Phase 2" human
clinical
trials, which are authorized under "Investigational New Drug" applications in
the U.S., and
under similar governmental review and approval mechanisms elsewhere. By the
time a
treatment is ready to be tested in larger multi-site Phase 3 clinical trials,
any epitopic or
other "foreign" sequences usually will be removed, to reduce the risk of
generating an
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immune response involving antibodies that would recognize and attack the
epitopic
sequence.
IN VIVO IDENTIFICATION OF ENDOCYTO TIC LIGANDS, FROM PHAGE
LIBRARIES AND COMBINATORIAL CHEMISTRY
As mentioned in the Summary section, a new type of in vivo screening method
has
been created by the Applicants, for identifying and isolating endocytotic
ligands that will be
taken into, and transported within, neuronal fibers. Since this new method
offers a
preferred mode of carrying out this invention, it is disclosed herein in
detail, to enable
those skilled in the art to use this method to create improved genetic vectors
that can be
used to delivery polypeptides into BBB-protected CNS tissue.
To fully understand the in vivo screening method that is involved, and the
endocytotic ligands that can will result from this screening method, some
background
information needs to be provided on endocytotic ligand receptor proteins. Like
nearly all
cell surface receptors, endocytotic receptors straddle the outer membrane of a
cell.
Accordingly, they have three distinct domains: (i) an extracellular portion,
which is exposed
and accessible to molecules carried by the aqueous fluids that surround and
contact the cell;
(ii) an interior portion which is embedded in the lipid bilayer membrane that
encloses the
cell; and, (iii) an intracellular portion, which is exposed to the aqueous
cytoplasm inside the
cell.
In general, ligand receptors have two traits that distinguish them from
various other
types of non-ligand receptors. The first trait is that each particular type of
endocytotic
ligand receptor will interact with, and will be triggered and activated by,
only a single type
of extracellular molecule (or, in some cases, only a very small and limited
group of
structurally similar extracellular molecules). This binding mechanism can be
regarded as
being directly comparable to a "key-in-lock" arrangement; only a small number
of keys,
having certain exact and limited sizes and shapes, can fit into a certain
lock. This trait
distinguishes ligand receptors from, for example, various types of transport
proteins that
will grab nutrients floating outside the cell, and transport those nutrients
into a cell interior.
The second distinguishing trait is that, when a binding reaction does occur
between a
ligand and an endocytotic ligand receptor protein, the two molecules will
remain bound to
each other for an extended period of time. This distinguishes ligand receptors
from
neurotransmitter receptors that are triggered by acetylcholine, glutamate, and
other classical
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neurotransmitters. Instead of being measured in milliseconds (as occurs with
most
neurotransmitter receptors), ligand binding reactions usually can be measured
in minutes,
hours, or even days.
In an "endocytotic" ligand binding reaction, the ligand binds to the ligand
receptor
in a way that forms a ligond-receptor complex, which will then be taken inside
the cell,
where both the ligand and the receptor typically are eventually digested, so
that their
building blocks can be recycled. To understand how and why this occurs, one
should
consider polypeptide hormones such as growth hormones, nerve growth factors,
etc., that
can cause permanent alterations in the size, health, nerve or reproductive
system, or other
traits or organs of an animal. If these types of hormones were released by
their receptors,
they would float back into the extracellular fluid, and they could then
contact and activate
additional receptors on other cells, in a manner that could lead to
unregulated and
potentially uncontrollable effects.
As used herein, the terms endocytosis and internalization are used
interchangeably,
to refer to a cell-driven process in which an extra-cellular molecule (other
than a nutrient or
oxygen) which has become bound in a specific manner (usually referred to as
"affinity
binding") to a molecule on the cell surface, is drawn into the cell interior.
This process
includes receptor-mediated endocytosis, involving ligands that bind to
receptor proteins. It
also includes the process in which ligands that bind specifically to other
types of cell
surface molecules (including surface carbohydrates) form other types of ligand
complexes
that are drawn into a cell. The terms endocytosis and internalization also
include the
processes called pinocytosis (which involves the intake of very small
particles, or soluble
molecules) and phagocytosis (which involves the intake of larger particles,
such a virus
particles or bacterial cells), provided that such processes involve specific
binding of a
ligand molecule to a cell surface molecule, in a manner that forms a complex
which is
subsequently internalized by the cell.
The processes ino\volved in endocytosis are well-known, and are discussed in
numerous reference works, such as Alberts et al, Molecular Biology of the Cell
(third
edition, 1994), pages 618-626 (which describe and illustrate endocytosis) and
pages 636-641
(which describe clathrin proteins and triskelions, which aid and facilitate
the process of
endocytosis), and pages 731-734 (which describe and depict internal
conformation changes
that occur when a ligand binds to a membrane-straddling protein).
In most types of animal cells, endocytosis of ligand-receptor complexes
involves
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transport of the lipid vesicle only over very short distances, since the
typical diameter of
most animal cells is in the range of about 10 microns, or 1/100 of a
millimeter.
However, ligand-receptor complexes in neurons can travel much longer
distances.
Many neurons in the brain and spinal cord have long fibers (including axons,
dendrites, and
"processes") that extend for multiple centimeters, and some types of neurons
that carry
nerve signals in humans from a hand or a foot to the spinal cord (or vice-
verse) have fibers
that extend more than a meter. Accordingly, endocytosis that occurs within
these types of
long fibers must be able to carry a ligand-receptor complex all the way from
the most
distant tip of the neuronal fiber, where some ligand-receptor complexes will
first enter a
neuron, to the main cell body of the neuron, regardless of how far that
distance may be.
As mentioned previously, "retrograde" transport occurs when a molecule is
transported from an extremity (or "terminal") of a neuron, along an axon or
dendrite,
toward the main body of the cell, where the nucleus is located. Transport in
the opposite
direction is called "anterograde" transport, and the term "axonal transport"
should also be
recognized and understood. Many types of nerve cells have a single main fiber,
which is
the largest fiber that extends out from the main cell body. That largest fiber
is usually
called the axon. The term "axonal transport" includes and refers to any form
of transport of
molecules within an axon, regardless of which direction the molecules are
travelling (i.e.,
toward the cell body, or away from the cell body). Therefore, retrograde
transport that
occurs within an axon is a form of axonal transport. However, the term
"retrograde
transport" is preferred herein, since it also indicates the direction of
travel.
Not all candidate ligands that can bind to an endocytotic ligand receptor can
activate
and drive the internalisation process to full completion. As an example,
monoclonal
antibodies can be generated, rather easily, that will indeed bind to known
endocytotic
receptors, but there are few reports of such antibody preparations that can
successfully
trigger and then complete the entire process of receptor-mediated endocytosis.
One example
of an antibody preparation that reportedly can trigger and then complete the
process of
receptor-mediated endocytosis in rat neurons is a monoclonal antibody,
initially designated
as IgG-192 and subsequently called MC192, described in Chandler and Shooter
1984. The
MC192 antibody binds specifically to a rat neuronal receptor that was known in
the mid-
1980's as the "low affinity nerve growth factor receptor", and that was
subsequently
designated as the p75 receptor. Years after the MC192 monoclonal antibody was
created,
other researchers reported that when it was radiolabelled and injected into
rats, it was
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internalised and retrogradely transported into the cell bodies of neurons that
express the p75
receptor on their surfaces (Yan et al 1988).
That example may prove that it is theoretically possible to develop an
antibody that
can trigger and then complete the process of ligand-receptor endocytosis;
however, major
obstacles still remain before that type of research discovery can be used in
human medicine,
and two crucial sets of questions immediately arise.
The first set of questions center on the difficulties of extending those types
of
findings, to human medicine. Obviously, it is highly problematic and in many
cases illegal
to inject antigens or unproven antibody fragments into humans. Even more
importantly, the
screening tests that would need to be performed, in order to prove that some
particular
antibody type that works well in animal tests can also work well in humans,
would be very
difficult and potentially impossible, unless they are done in ways that
currently are not
acceptable in human research. If one begins to seriously contemplate the
obstacles that
would confront such research tests (which are likely to require samples of
spinal tissue to
be removed and then analyzed to determined whether radioactively-labelled
tracer molecules
actually reached the spinal cord), one ends up pondering tests on murderers
who are
condemned to be executed within the next few days, or on people who are going
to die of
cancer or other terminal diseases within the next few days. However, tests
involving
removal of solid tissue from a human spinal cord, so the tissue can be
analyzed, are not
allowed in any industrial nation where modern medical practices are used.
The second set of difficult questions centers on the issue of what types of
molecules
an "endocytotic receptor antibody fragment" might be able to carry along with
it, into a cell
interior, if the antibody fragment itself can work as hoped as an endocytotic
trigger.
Clearly, there is no therapeutic value in having antibody fragments, with
nothing else
attached, pulled into the interiors of neurons. Instead, such antibody
fragments must be
regarded merely as vehicles (or as locomotives, which could be used to pull a
train). They
will not be useful unless they can carry or pull some type of "passenger" or
"payload"
molecule into the neurons they are entering.
However, it must be recognized from the outset that coupling any additional
molecular fragment to an endocytotic antibody fragment will necessary enlarge
the resulting
conjugate. Depending on how much larger the conjugate will be, this
enlargement may
substantially reduce the ability of the conjugate to be pulled into neurons
with the same
level of efficacy as the antibody fragments alone. There is no good way to
answer that type
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of question at an early stage, during the research that will be necessary on
any such
antibody fragment. Instead, the transport vehicle must be evaluated and proven
to work, on
its own, before it becomes worthwhile to test that vehicle's ability to carry
(or pull)
passenger or payload components.
Accordingly, since the reports published to date indicate that only a small
subset of
the antibodies generated against endocytotic receptors may be capable of
mimicking a
natural ligand's ability to trigger and then complete the process of receptor-
mediated
endocytosis, the process of testing each of dozens or even hundreds of
monoclonal antibody
candidates, to identify rarely-occurring internalising antibodies, renders
those problems
even more difficult and expensive.
It should also be noted that in vitro screening processes, to evaluate
candidate
endocytotic ligands by using cell culture conditions, suffers from two major
problems.
Those two problems have effectively limited progress in that field to work on
cancer cells,
and on blood cells that are involved in autoimmune diseases.
To understand these two problems, one must first note that most evaluations of
"libraries" or "repertoires" of candidate ligands, in an effort to identify
and isolate the rare
"needle-in-a-haystack" ligands that can activate and drive endocytosis, will
usually begin
with either: (i) a phage display library, or (ii) a preparation that was
created by
"combinatorial chemistry". Phages (which are viruses that can infect bacterial
cells) and
phage display libraries are described in articles such as Koivunen et al 1999,
Cabilly 1999,
Shusta et al 1999, Larocca et al 1999, 2001, and 2002, Rader 2001, and
Manoutcharian et
al 2002. Combinatorial chemistry is described in articles such as Lockhoff et
al 2002,
Flynn et al 2002, Edwards et al 2002, Ramstrom et al 2002, Ley et al 2002,
Lepre et al
2002, Liu et al 2003, Edwards 2003, and Geysen et al 2003.
When researchers try to use in vitro cell culture preparations, in their
efforts to
screen phage display libraries to identify endocytotic polypeptide ligands,
they encounter
two main problems: (i) multiple different phages will usually bind to multiple
different
proteins and other molecules, on the surfaces of cells, without being taken
into the cells;
and, (ii) it is very difficult to rinse off, wash off, or otherwise reliably
remove any and all
phages that are clinging to the surfaces of cells, and that have not been
taken inside the
cells, without killing and lysing the cells or otherwise creating severe
problems that will
interfere with other desired processing of the cells and/or internalized
phages. Because of
these two factors, it is very difficult to prevent "false positives" from
being selected.
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Accordingly, in vitro screening of phage libraries for endocytotic ligands has
succeeded to a
significant degree only in working with cancer cells or blood cells, which can
be grown
readily in liquid cell culture solutions. By contrast, in vitro screening of
phage libraries for
endocytotic ligands has not succeeded, to any substantial degree, in cell
cultures that
involve anchorage-dependent cells of the type that generate cohesive tissues.
The problems summarized above apply to even the simplest tissue culture
systems,
where all of the cells can be clonal duplicates and have exactly the same
receptor types.
The notion of attempting to carry out phage library screening tests in an
intact and still-
living animal (where multiple different tissue and cell types, each with their
own specialized
set of receptors and other surface molecules, must coexist in close contact
with each other,
and with blood and lymph constantly circulating through and between the
different tissues
regions and cell types) simply is not within the mindset of ordinary artisans
who are skilled
and practiced in the art of phage library screenings, and who understand the
considerable
difficulties of doing it successfully even in the simplest cell culture
conditions.
Accordingly, there have been many efforts, and much progress, in using phage
display libraries to develop improved genetic engineering methods and vectors
that can be
used to genetically transform and treat cancers, and autoimmune diseases.
However, there
have been few efforts, and only very paltry and limited progress, in using
phage display
libraries to treat other diseases, or to create genetic vectors that can
enable the
transformation of neurons and other cells that are present in cohesive tissue,
inside the
body. Under the prior art, the challenges and difficulties of eliminating
false positives,
when phage display libraries are screened for endocytotic uptake into neurons
and other
cohesive tissue cells in intact animals, in in vivo tests, have been so
severe, and so
formidable, that they have effectively blocked and prevented any substantial
progress in that
field of research. Prior to this invention, no one had figured out how to make
practical use
of phage display libraries, to accomplish the results that can now be achieved
by the in vivo
screening method of this invention.
Accordingly, this newly developed in vivo screening method can be described as
follows, with reference to Figures 7-9. Additional information is contained in
Examples 22
through 31, below.
In a preferred embodiment, this in vivo screening process can use the sciatic
nerves
of rodents, such as rats or mice. Both of these species are inexpensive and
easy to breed
and raise, and they have become the standard animal models used in most
genetic research
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in small mammals. A huge foundation of information, species-specific
biomolecules
(including gene promoter sequences, gene coding sequences, monoclonal
antibodies, etc.)
and specialized animal strains, have been developed for genetic work with
mice, and
gateways that can be used to access that information are freely available on
websites.,
Although the corresponding information, reagents, and strains for rat genetics
are somewhat
less, they are still enormous and quite useful, and can be accessed through
websites.
Because of the larger size of rats, it is easier to work with their sciatic
nerves
(which pass, on each side of the animal, from the spinal cord, through one hip
and leg,
down to the foot) than with mice. This can be done by known methods, such as
discussed
below.
However, even in mice, the sciatic nerves are long enough and sufficiently
distinct
to enable the required surgical manipulations, using the procedures disclosed
herein
(especially if such manipulations are carried out by researchers who have done
such work
before). In addition, it should be kept in mind that surgical manipulations in
mice can be
done with the aid of binocular microscopes, and surgical tools that are
commonly used by
ophthalmologic surgeons and neurosurgeons. Additional comments on surgical
methods are
contained in Example 25, below.
It should also be noted that other types of laboratory animals (which may
include
primates, non-mammalian vertebrates, or even some types of invertebrates) can
also be
evaluated for potential use in this type of in vivo screening, if desired. In
particular, some
animals are known to have exceptionally large neuronal axons; as one example,
some types
f, ,= . . ":=.e . a IN . III = I. I I
IP .01 he e
way that Chinese hamster ovary (Cl-JO) cells became widely used in research
laboratories
because they contain unusually large cell components, squids or other animals
that have
unusually large nerve fibers or bundles can be used as disclosed herein, if
desired.
The placement of candidate ligands in contact with a sciatic nerve bundle can
be
done in a manner that is schematically illustrated in MG. 7, and discussed in
more detail in
Examples 25 and 26, below. Briefly, if an inducible receptor (such as the low-
affinity p75
nerve growth factor receptor) is going to be.targeted, a first ligature 1102
is emplaced and
then tightened around the sCiatic nerve bundle 1090. This ligature 1102 is
created by
placing a strand of suture material around the nerve bundle, and then
tightening the loop
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md tying it off, in a manner that creates a constriction that acts as a
tourniquet, by
iindering the normal flow of fluids and molecules inside the nerve fiber.
As shown in FIG. 7, ligature 1102 can be placed adjacent to the "tibial branch
)ifurcation" 1092, where the sciatic nerve bundle 1090 divides into two major
branches,
which serve different parts of the leg and foot. Ligature 1102 preferably
should be placed
above, and fairly close to, the tibial branch bifurcation 1092. As mentioned
in the
Background section, the terms "above" and "proximal" indicate a location
closer to the
animal's spinal cord (toward the right side of the drawing shown in FIG. 7).
By contrast,
the terms below and distal indicate a location farther away from the spinal
cord, and closer
to the leg or foot (toward the left side of the drawing in FIG. 7).
The purpose of ligature 1102 is to increase the number of p75 receptors that
will be
expressed on the surfaces of the sciatic nerve bundle. The p75 receptor
interacts with
certain neurotrophic factors (also called nerve growth factors) which are
polypeptides that
have hormone-like effects on nerve cells. The best known such molecule was
called nerve
growth factor, since it was discovered fairly early in the process; as
additional such
molecules were discovered, they were given names such as brain-derived
neurotrophic
factor (BDNF), neurotrophin-3, and neurotrophin-4/5. The neurotrophins
effectively
stimulate neurons in ways that generally lead to increased metabolic
activities, the
formation of additional synaptic connections with other neurons, etc. If a
neuronal fiber of
a motor neuron is injured or distressed, one of the ways the motor neuron
responds is by
increasing the number of p75 receptors on its neuronal fiber, which may give
it a better
chance to grab and bind any nerve growth factor molecules that happen to be in
the
surrounding extracellular liquids. This process of "upregulating" certain
types of neuronal
receptors on the surfaces of nerve fibers has been discovered and shown to
occur in certain
neurodegenerative diseases, notably including amyotrophic lateral sclerosis,
also called
ALS, Lou Gehrig's disease, and motor neuron disease. It also occurs after
various types of
trauma.
Accordingly, ligature 1102 is designed to exploit that type of neuronal
response. By
emplacing and tightening a loop of suture material around the nerve fiber, in
a manner
which creates a tourniquet that blocks the flow of fluids through the fiber,
it is possible to
increase the number of p75 receptors along the length of the nerve fiber,
between the spinal
cord and the ligature site. Based on various tests, including staining tests
that use
monoclonal antibodies that bind to p75 receptors, the increase in p75
receptors is estimated
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,o be about 10 to 15-fold.
This receptor expression response, by a nerve fiber, to a constrictive
ligature, occurs
bver a span of roughly a week. Therefore, after ligature 1102 is placed and
tightened
around the sciatic nerve bundle, the wound should be closed and sutured, and
the animal
should be allowed to recover, for at least several days and preferably for
about a week,
before the next surgical procedure is performed.
During the second procedure, two different sites will be surgically opened,
roughly
2 to 3 centimeters apart from each other. One site will be close to the same
site where the
ligature 1102 was placed; indeed, the surgical opening can be located at the
same site as
before. At this site, the sciatic nerve bundle is cut (i.e., transected), by
using a scalpel or
scissors, in a manner that generates two ends (which can be blunt, angled,
etc.). The cuts
are made just above and below ligature 1102, and a small portion of the nerve
bundle
which contains the ligature can be excised and discarded. These cuts will
create two ends of
the nerve bundle, designated as distal end 1094 (which will no longer be
active), and
proximal end 1130 (on the side that is toward the spinal cord).
At the site where the sciatic nerve bundle 1090 is cut, a bolus of material
1150 is
emplaced. This bolus 1150 is made a porous and permeable material (such as a
collagen gel
foam) that contains a large number of phage particles (preferably in the
millions or billions
of "colony forming units" (cfu)). This bolus 1150 should be emplaced and
secured at this
site, in a manner that will promote sustained intimate contact between (i) the
phage particles
that are contained in bolus 1150, and (ii) the cut end 1130 of the sciatic
nerve bundle 190.
This type of emplacing and securing can be done by means such as:
(a) using a strand of suture material 1132 to tie together the two cut ends
1094 (i.e.,
the distal cut end) and 1130 (i.e., the proximal cut end) of the sciatic nerve
bundle 1090;
and,
(b) wrapping and securing a small sleeve or cuff 1136, made of a watertight
material
such as silicone rubber, around bolus 1150 and around the two ends 1094 and
1130 of the
sciatic nerve 1090, in a manner which encloses the bolus and the two nerve
ends inside a
small watertight cylindrical volume. If desired, the sleeve 1136 can be
secured by wrapping
and tying one or more suture strands around it, by placing a droplet or bead
of adhesive
material on the outer surface of the sleeve, or by any other suitable means.
Accordingly, this site, where the bolus of material containing phage particles
is
emplaced, can be referred to as either the phage placement site, or the phage
contact site.
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This is the location where the library or repertoire of phage particles will
contact the cut
end of the sciatic nerve. Phage particles that happen to display, on their
surfaces,
polypeptide sequences that will trigger endocytosis (such as through a p75
receptor on the
surface of a nerve fiber) can be internalized by the nerve fibers, at this
site.
At a separate and distinct site, preferably located roughly a centimeter or
more away
from the phage placement site, a second site is surgically opened, and a
second ligature
1202 (formed by a loop of suture material) is emplaced and then tightened and
tied around
the sciatic nerve bundle. Since the rat hip offers a convenient location, far
enough away
from the phage placement site to eliminate any significant risk of false
positives caused by
phages clinging to the outsides of sciatic nerve fibers, this site preferably
should be in the
hip region, and it is referred to herein as the hip ligature site. The hip
ligature 1202 will act
as a constriction or tourniquet around the sciatic nerve bundle 1090, and must
be tight
enough to substantially hinder the travel of fluids or molecules, inside the
nerve fibers,
across that blockage point. Accordingly, this constriction will generate a
phage
accumulation zone 1204, inside the nerve bundle and distal to the hip ligature
1202.
The rat wounds are closed and sutured, and a suitable span of time (such as
about
18 hours) is allowed to pass, to give phage particles that happen to be
carrying ligands that
can effectively activate and drive the process of endocytosis, enough time to
enter the nerve
fibers, and then be retrogradely transported through a significant length of
the nerve fibers,
toward the spinal cord).
After that span of time has passed, the rat is painlessly sacrificed, the site
of the hip
ligature is opened, and a segment of the sciatic nerve bundle immediately
adjacent and
distal to the hip ligature is removed (harvested). This short bundle of nerve
fibers is then
divided into small pieces, and processed using chemicals that will partially
digest cell
membranes (which are made of lipid bilayers) without damaging the phage
particles. This
processing allows the collection and isolation of viable phage particles that
had been
internalised into the nerve fibers.
The phage particles that are selected by a round of in vivo screening as
disclosed
above can be reproduced and/or manipulated in any way desired. As examples,
any and all
of the following procedures can be carried out, using phage populations
selected by the in
vivo screening process disclosed herein:
(1) if the phages are "phagemids" (which merely requires the phage to contain
a
bacterial origin of replication), they can be amplified (reproduced), by using
E. con cells
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vithout helper phages, in ways that will generate double-stranded DNA in
plasmid form. It
;hould be noted that nearly all modern phage display libraries use phagemids,
since they
:.nable various useful procedures, including the synthesis of circular plasmid
DNA in any
lesired quantity. All phage display libraries used herein were phagemid
libraries.
(2) by using E. coli cells plus helper phages, the selected phages can be
amplified in
ways that generate new and fully infective phage particles containing ssDNA.
These phage
particles can be used as the starting reagents in anbther cycle of in vivo
screening, which
(during the early cycles) can be used to refine an "enriched" population of
endocytotic
phages into an "elite" population that is likely to contain ligands that are
even more
effective at triggering and driving endocytosis.
(3) when enough selection cycles have been completed to suggest that a
suitable
point for phage analysis has been reached (in most cases, this is likely to
happen after at
least one, up to about three cycles of screening, or possibly more in some
cases), the
phages selected by the last round of in vivo screening (or, indeed, by any
round of in vivo
screening) can be used to create either or both of the following: (i) any
desired quantity of
double-stranded or single-stranded DNA, for nucleotide sequencing to determine
the exact
sequence of the gene that encoded a particular endocytotic ligand; and/or,
(ii) any desired
quantity of the coat protein which carries an endocytotic ligand, in a soluble
form that can
be processed and sequenced, to determine the amino acid sequence of the ligand
domain in
that particular coat protein.
Photographic Confirmation of In Vivo Screening Results
The efficacy and success of the in vivo screening process disclosed herein is
depicted, visually, by the photograph in FIG. 8. This photograph was created
during an
actual test of this in vivo selection process, using fluorescent reagents to
indicate the
locations and concentrations of phages that were internalised within the
sciatic nerve bundle
(the phage preparation and staining reagents that were used in this test are
described in
Example 27, below). The left side of the photograph in FIG. 8 shows fairly
high
concentrations of fluorescent-labelled phages, in the nerve portion that
corresponds to phage
accumulation zone 1204 as shown in FIG. 7. The choked and narrow zone in the
center of
the photograph was created by the hip ligature 1202. The right side of the
photograph
shows the sciatic nerve on the proximal side of the hip ligature (i.e., in the
direction of the
spinal cord). Since very few or no phages were able to squeeze past the hip
ligature 1202
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Ind reach that part of the sciatic nerve, it shows almost no fluorescent
labelling.
Use of In Vivo Screening with Combinatorial Chemistry
The general approach disclosed herein, for in vivo screening and
identification of
endocytotic ligands, can be adapted for use with candidate ligand molecules
created by
"combinatorial" chemical synthesis. Over the past 20 years, this branch of
chemical
synthesis and screening has become a highly active field, and an April 2003
search of the
National Library of Medicine database revealed more than 900 review articles
on this field
of research. Recent review articles include Lockhoff et al 2002, Flynn et al
2002, Edwards
et al 2002, Ramstrom et al 2002, Ley et al 2002, Lepre et al 2002, Liu et al
2003,
Edwards 2003, and Geysen et al 2003. The synthesis methods and approaches
described in
those review articles can be used to provide a wide range of highly diverse
combinatorial
libraries.
One of the essential traits of any such combinatorial library is that it must
be
adaptable to at least one or more types of screening tests. Otherwise, a
mixture of
thousands or millions of different candidates would be totally worthless,
since no one would
be able to tell which particular compounds, in the mixture of thousands or
millions of
candidates, would be useful for some particular purpose.
Therefore, any type of combinatorial library will be created in a manner that
provides the candidate compounds with some type of "handle" that can be used
to identify
or manipulate the candidates (or that can identify or manipulate those
particular compounds
that were modified, isolated, or otherwise distinguished by a reaction or
screening process)
in one or more useful ways.
A fairly generous variety of these types of "handles" are known, and the
variety of
known approaches will enable at least one and usually more of these "handles"
to be
adapted to in vivo screening of combinatorial libraries, using nerve fiber
manipulations as
disclosed herein. As examples, well-known classes of "handle" approaches that
can be used
to process and control combinatorial chemistry repertoires can include any and
all of the
following:
1. microscopic beads, tubes, or other solid surfaces, usually made of a
plastic,
starch, or similar compound. These beads or other solid surfaces usually serve
as a
substrate or "anchor", and provide (on their surfaces) reactive groups that
will become
attachment points for chemical chains that will be added to those reactive
groups. If
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lesired, these types of microscopic beads can be created with diameters that
are well suited
or phagocytotic intake by mammalian cells.
2. special reactive moieties that occur only once in each candidate compound
in a
:ombinatorial library. These unique reactive moieties can be used to enable
attachments,
themical reactions, or other manipulations, that can be used at any stage
during or after a
;creening process is carried out, to precipitate, condense, or otherwise
gather, isolate,
;onjugate, label, or manipulate particular candidate compounds that were
transported to a
arget location, or that became involved in a chemical or cellular reaction of
interest, or
:hat otherwise acted differently from the unsuccessful candidates, during a
screening test.
3. non-toxic fluorescent "labels" or "tags" that will emit light at one
wavelength,
when excited by light having a different wavelength. This enables the use of
equipment
called "flow cytometers" (also called cell sorters, and similar terms), to
segregate cells or
particles that have fluorescent activity. In a typical flow cytometer with
sorting capability,
millions of cells or particles can be passed through a narrow tube, one at a
time, at a
known and controlled velocity. At one location in the pathway, each cell or
particle passes
through a light with an excitatory wavelength. Individual cells or particles
that contain or
are attached to a fluorescent label or tag will respond by emitting light at
the different
wavelength. This fluorescence, which occurs within nanoseconds, is detected by
an optical
sensor which is tuned to the fluorescent wavelength. When that optical sensor
detects
fluorescent light emitted by a certain cell or particle, it triggers a tiny
jet of gas or liquid,
at a location slightly downstream in the flow path of the cells or particles.
That jet of gas or
liquid is timed to coincide with the passage of the fluorescent cell or
particle, through a
junction in the pathway. If the jet of gas or liquid pushes a fluorescent cell
or particle to
one side, in the flow path, it will enter a separate collection tube, which
will carry it to a
collection vessel. In this way, a flow cytometer can process millions of cells
or particles
within a span of hours or even minutes, and it can isolate even a single
individual
fluorescent cell or particle, out of a population of millions.
4. other types of labels or tags, such as compounds that include radioactive
isotopes,
or specialized molecular structures that can be located and tracked by
sophisticated
analytical methods such as magnetic resonance imaging, Raman scattering, etc.
5. whenever an assortment of candidate ligands includes or involve
polypeptides,
phage display libraries offer exceptionally powerful, flexible, and adaptable
"handle"
systems for working with such polypeptides. If even a single phage particle is
isolated
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which carries a highly effective and potentially useful ligand polypeptide,
then that single
phage particle can be grown rapidly into an entire clonal colony, which can
provide an
unlimited supply of both the polypeptide, and the gene which encodes that
polypeptide,
using procedures as described herein or as otherwise known to those skilled in
the art.
Indeed, the PhD-C7C phage display library offers an example of a combinatorial
approach that has been adapted for use with polypeptides. In this library,
essentially random
segments of short polypeptides, seven amino acids long, were created by
combinatorial
chemistry. These randomly-created short polypeptide sequences were
incorporated into
phage particles, and those phage particles provide the "handles" which can be
used to
manipulate, reproduce, and screen the combinatorial assortment of polypeptides
in the PhD-
C7C library.
As mentioned above, any combinatorial library must necessarily be created in a
manner that will render it susceptible to at least one type of system or
mechanism that
enables researchers to handle and manipulate the candidate compounds in the
library.
Otherwise, it would be useless to generate such libraries, if they could not
be screened by
effective and logical methods. Therefore, the range and variety of methods
that have been
developed over the past 20 years, for screening combinatorial libraries, have
become quite
sophisticated and powerful. Accordingly, the in vivo screening methods
disclosed herein can
be regarded as merely providing one more new (and potentially powerful, and
useful)
method for screening candidate compounds that have been created by
combinatorial
chemistry.
Genetic Engineering Methods to Extend In Vivo Screening to Other Receptors,
Other
Species, and Other Classes of Neurons
Those skilled in certain related arts will recognize various ways in which
this
invention, initially developed and tested using the motor neurons of the
sciatic nerve in rats,
can be expanded and extended in several particular directions that will be of
interest to
research, physicians, and others.
As one example, those skilled in neuroanatomy and neuronal tracing studies
will
recognize ways in which this invention can be expanded beyond sciatic motor
neurons, to
enable its use: (i) with sympathetic processes emanating from the superior
cervical ganglion
and sensory processes emanating from the trigeminal ganglion sensory nerves,
following
injection of phage libraries into the anterior eye chamber; (ii) with
olfactory receptor
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sensory neurons (harvesting olfactory bulb tissue), trigeminal ganglion
sensory and superior
cervical ganglion sympathetic nerves, following administration of test
libraries into the nasal
cavity; (iii) with retinal ganglion cell sensory neurons, following injection
into the posterior
chamber of the eye; and, (iv) with various neurons of the central nervous
system, following
injection into the lateral or other ventricles of the brain. By such means,
ligands targeted at
endocytotic receptors that are naturally expressed by these particular
neuronal populations
can be identified and isolated, for use in diagnosing and treating disorders
that involve those
particular classes of neurons.
Similarly, those skilled in genetic engineering and molecular biology will
recognize
ways in which this invention, initially developed and tested using p75
receptors in rats, can
be expanded and extended to enable its use: (i) with endocytotic receptors
other than the
p75 receptor; (ii) with endocytotic surface molecules other than receptors;
(iii) with
endocytotic receptors that are present in species other than just rats,
including human
receptors; and, (iv) with endocytotic receptors that are present on specific
types of cells and
tissues other than neurons (such as, for example, receptors that normally are
found in
significant numbers only on the surfaces of cells in kidneys, livers, lungs,
hearts, etc.).
This type of work has been done before in a number of cases, because once the
DNA sequence that encodes a particular type of human receptor protein is
known, that
human gene sequence (or any portion thereof) can be used to transform animals
of a
different species, such as mice or rats. The genetically transformed animals
will then
express the human receptor protein, having the exact same human amino acid
sequence. As
just one example, the human receptor protein that enables polio viruses to
infect certain
types of motor neurons, in humans and certain other primates, was used to
transform mice.
This allowed the transformed mice to be used as inexpensive animal models, for
studying
polio and polioviruses.
In a similar manner, as an example of how that type of genetic engineering can
be
adapted to enable in vivo screening as disclosed herein, the following series
of steps can be
carried out, by skilled artisans, using DNA sequences and other reagents and
methods that
are already known and available in the art:
1. The human homologue of the p75 gene, which has been fully sequenced
(Johnson
et al 1986) can be placed in a chimeric gene, under the control of the p75
gene promoter
normally found in mice or rats;
2. This chimeric (mice or rat promoter/human coding) version of the p75 gene
can
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then be used to genetically transform selected types of mice or rats, such as
strains of mice
that have a "knockout" mutation which prevents them from properly expressing
the p75
receptor; such strains are available from Jackson Laboratories as stock number
002213
(strain name B6.12954-Ngfrun"").
3. The transformed mice or rats will express the human version of the p75
receptor
protein, and human p75 receptors will appear in the same locations where the
rat p75
receptor protein normally exists, including on the surfaces of sciatic nerve
fibers that extend
outside the blood-brain barrier;
4. A ligand library (such as the scFv phage display library, or the PhD-C7C
phage
display library) is then screened, using the same procedures disclosed herein,
to identify
candidate phages that will undergo endocytotic uptake and retrograde
transport, through a
process that is mediated by binding of the ligand domain of a phage particle
to the human
version of the p75 receptor.
5. Alternately or additionally, ligands that have been discovered and
identified to
enter cells through the rat p75 receptor protein can be tested, in vitro, to
determine whether
= they will also enter cells that have human p75 cell receptors (such as on
a human
neuroblastoma cell line that grows readily in suspension culture and that
expresses the
natural version of human p75;
6. Alternately or additionally, if the endocytotic efficiency of a ligand that
readily
enters rat neuronal fibers through rat p75 receptors is tolerable but not very
high, when it
interacts with human p75 receptors, then that particular ligand can become the
starting
compound in a process that will (i) use site-directed or random mutagenesis to
create
numerous analogues of the rat-p75-binding ligand, and (ii) use in vitro
screening to identify
and evaluate promising analogues that can readily enter cells through human
p75 receptors.
These are just a few examples of how the in vivo screening methods disclosed
herein
can be adapted for use in discovering, isolating, and analyzing ligands that
will enable
efficient transport of passenger or payload molecules into human cells, for
use in human
medicine, diagnostics, analysis, and research.
Molecular Complexes and Methods Enabled by This Invention
The true value of the screening methods disclosed herein comes not from the
act of
identifying particular ligands that can activate and drive endocytotic
internalisation, but
frOrn the subsequent ability to incorporate and use those selected ligands, in
'molecular
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;omplexes" that can be used for medical, diagnostic, and similar purposes.
As used herein, the term "molecular complex" refers to a molecular assemblage
that
ncludes at least two distinct components: at least one ligand component, and
at least one
passenger or payload component.
In order to fall with the claims that refer to such ligands or to molecular
complexes
which include such ligands, a ligand component must meet two criteria, as
follows.
First, the ligand component must have been identified by an in vivo selection
process
as disclosed herein (i.e., to be covered by a claim such as claim 1, the
ligand component
must have been identified by a process of in vivo selection that required, at
a minimum,
endocytotic uptake into neuronal fibers, for such selection to occur). This
type of
identification is an essential step, in the screening methods of invention,
and in the
molecular complexes that can be formed using ligands that were in fact
identified by this
method. To illustrate this fact, it can be presumed that a phage library
containing billions of
candidate ligand polypeptides (such as the scFv library, which was used and
screened as
described in Examples 28 and 29) does indeed contain hundreds, thousands, or
possibly
even millions of phage particles that are indeed carrying candidate ligand
sequences that are
quite capable of serving as potent, specific, effective endocytotic ligands,
which could be
used to carry passenger molecules into cells having p75 receptors on their
surfaces.
However, those hundreds, thousands, or even millions of phage particles which
have that
theoretical potential, in that huge library, are surrounded by billions of
other phages that
would be totally useless for that purpose, and that would provoke all kinds of
unwanted
responses if coupled to passenger molecules and injected into an animal or
human that
needs medical treatment.
Obviously, the screening and processing steps that are required to identify
and
isolate those phages which carry ligand sequences having a known and useful
endocytotic
activity is an absolutely critical step, in creating molecular complexes which
can actually
accomplish desirable and useful medical, analytical, or similar results.
The second requirement that applies to the ligand components that are used to
transport passenger or payload components in molecular complexes, as described
and
claimed herein, is this: the molecular complex, which includes a ligand
component that was
initially identified by the in vivo screening process disclosed herein, must
be able to actually
enter targeted types and classes of cells which have endocytotic surface
molecules to which
the ligand component will bind. If such a molecular complex cannot enter at
least one class
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of such cells, then that molecular complex is not covered, and is not intended
to be
covered, by the claims herein.
It should be clear, however, that once a particular ligand has been identified
which
can enter cells through a particular and targetable class of endocytotic
surface molecules
(and once the amino acid sequence of that particular ligand is known, if that
ligand is a
polypeptide), then that particular ligand can be synthesized in any desired
quantity, and it
can be used as an endocytotic transport system to carry a wide range of useful
"passenger"
or "payload" molecules into targeted cells. Accordingly, after such a ligand
component has
been identified by means of the new and powerful in vivo screening methods
disclosed
herein, then the use of that ligand, as an endocytotic transport component, in
molecular
complexes that also contain "passenger" or "payload" molecules, is not limited
to any one
specific type or class of passenger or payload molecule.
The terms "passenger molecule" and "payload molecule" are used interchangeably
herein, to refer to the portion of a molecular complex (i.e., containing a
ligand as set forth
above) that will perform one or more useful functions, or exert one or more
useful effects,
after the passenger molecule has entered a targeted cell containing an
endocytotic surface
molecule to which the ligand component Will bind. These terms are intended to
be
construed broadly, and in general, a passenger or payload molecule must be
interpreted by
recognizing how these same terms are used in other modes of transportation.
Cars, buses,
trains, airplanes, and bicycles are all useful, because they can carry
passengers, at speeds
and over distances which simply cannot be achieved, on a practical level, by
other means of
transportation. Similarly, freight trains, 18-wheelers, and tanker trucks and
boats are useful
because they can carry freight, which can be regarded as the payload whenever
a trip is
being made to an intended destination. Cars, buses, trains, airplanes,
bicycles, and boats
are highly useful and valuable modes of transport, not just because they can
carry one
particular person to one particular place, but because they can be adapted and
used to carry
numerous types of passengers or freight to numerous selected and targeted
destinations.
Accordingly, passenger or payload molecules, as disclosed and contemplated
herein,
should be interpreted broadly, and include but are not limited to each of the
following
major classes:
(1) DNA segments that are part of genetic vectors that are intended to
genetically
transform animals, or to medically treat humans in need of genetic therapy.
Indeed, one of
the first and foremost goals of this entire line of research was to identify
and create cell-
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targeting components that could be used to create new classes of genetic
vectors that can be
used to specifically target and transform only certain particular types of
cells, without
disrupting the status or activities of other cell types in ways that would
greatly increase the
risk and severity of unwanted side effects.
(2) therapeutic and/or diagnostic compounds (including pharmaceuticals,
imaging
compounds, etc.), for use in human or veterinary medicine.
(3) analytical compounds, reagents, and other substances that would be more
useful,
in industrial research and similar endeavors, if they could be transported
efficiently into
targeted classes of cells.
Finally, it should also be noted that a molecular complex which contains both
a
ligand component, and a passenger or payload component, must also have some
effective
means for coupling and holding those two components together, to form a
complex that will
hold together at least until the passenger or payload component has been
successfully pulled
inside a targeted cell. In some cases, depending on the passenger or payload
component, it
may be possibly to couple the passenger or payload component directly to the
ligand
component, by means of a direct covalent bond, or by means of a "coordinate"
bond (this
term refers to a class of molecular bonds having levels of strength and/or
stability that fall
somewhere between covalent bonds, and ionic attractions). However, if a
passenger
component is bonded directly to a ligand component, it is likely that this
type of molecular
complex may not be optimal, for at least some types of intended uses, because
it often will
be necessary to release the passenger component from the ligand component,
after the
molecular complex has entered a targeted cell, so that the passenger component
can then
carry out its intended function without having the ligand component still
attached to it. By
way of analogy, this is comparable to saying that a car, truck, or bus will be
substantially
more useful, if is it provided with doors that will allow passengers to leave
the vehicle,
once the vehicle arrives at an intended destination.
Accordingly, most types of molecular complexes that contain ligand and
passenger
components as disclosed herein preferably should also contain a suitable
"coupling
component", which will attach the ligand and passenger components to each
other by a
suitable means that will balance two different needs: (i) it must be
sufficiently strong and
stable to enable the molecular complex to remain intact, while the ligand
component is
performing its role and helping pull the passenger component into a cell
interior; and, (ii)
in many cases, unless the passenger component can exert its desired effects
while still
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oupled to the ligand component, the coupling means should provide some type of
structure
N mechanism that can allow the passenger component to eventually be released
or detached
from the molecular complex, after the molecular complex has successfully
entered a cell.
This patent application is not an appropriate forum for an exhaustive review
of
candidate coupling components that can achieve and/or balance those two
competing goals.
A variety of such candidate coupling components are known to those skilled in
the art of
drug delivery, and any such candidate coupling component can be evaluated for
use in a
particular molecular complex as disclosed herein, after the complete details
of the ligand
component, the passenger component, and the targeted cell type are all known.
Some of the broad classes of coupling compounds should be briefly mentioned,
to
provide an overview and working introduction to the range of options that will
be available
when a particular type of molecular complex is being designed to optimize a
combination of
a known ligand, a known passenger molecule, and a known targeted cell type:
1. crosslinldng agents that form covalent bonds by using relatively non-
specific
reactive groups, such as glutaraldehyde and other compounds that contain two
aldehyde or
other non-specific reactive groups at opposite ends of a spacer chain having a
controlled
length.
2. crosslinking agents that form covalent bonds, but only with specific
molecular
groups. These include "sulfo-SMCC", described in Example 24, which is used to
crosslink
an end of a DNA strand to a lysine residue in a polypeptide, for purposes such
as genetic
vectors and affinity purification.
3. affinity binding agents, which can have very high levels of tightness and
avidity
(as occur between two polypeptides called biotin and avidin, mentioned in
Example 27),
and which can have virtually any desired lower but still substantial level of
tightness and
avidity (which can be controlled by various means, such as by controlling the
elution
conditions during an affinity purification procedure).
4. coupling agents that use ionic attraction and/or hydrogen bonding to hold
two
components together. Since ionic attraction and hydrogen bonding are not
especially strong,
these types of agents typically involve compounds that contain multiple ionic
charges, all of
the same polarity, packed together in a fairly close arrangement. Examples
include
polylysine, polyethylenimine, and other positively-charged compounds that will
attract and
associate with negatively-charged phosphate groups in the backbones of strands
of DNA and
RNA.
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5. special types of connector molecules that are designed to weaken and break,
when
a molecular complex is subjected to acidity (such as occurs in lysosomes,
which are acidic
digestive organelles inside cells). These types of connector molecules are
described in US
patents 4,631,190 (Shen et al 1986) and 5,144,011 (Shen et a1 1992).
This is just a brief overview, and other types of connector molecules are also
known
to those skilled in the art. Nevertheless, it should be adequately clear that
various known
options with a wide range of strength, stability, and other traits are
available, for coupling
passenger components to ligand components.
Overview of Examples 22 - 32 (In Vivo Screening Methods)
Because of the complexity of the methods that are described in Examples 22-31,
and
of the specific types of phages and cells that were used as reagents in those
tests, and
because some of the test procedures that were eventually settled upon were
chosen after the
Applicants analyzed prior efforts that did not succeed, this section is
intended to offer an
overview and a narrative summary of the examples, and of how their information
is
organized.
Example 22 describes three types of bacteriophages that were used. These
included:
(1) M13K07 helper phages, which carry no endocytotic ligands; (2) a phage
display library
known as the scFv library, which contains roughly 13 billion different
phagemids, each of
which carries a candidate ligand that normally appears in human antibodies;
and (3) a phage
display library known as the PhD-C7C library, which carries small foreign
polypeptide
sequences that contain 7 amino acid residues, which were sequenced together
randomly,
using "combinatorial chemistry".
Example 23 describes the host cells (mainly the TG1 strain of E. coli cells)
that
were used, and it describes several techniques that were used with numerous
phage
populations, to amplify and titer those particular phage populations.
M13K07 helper phages are described first, in Example 22, even though they do
not
carry ligand polypeptides, because they were used to create antibody-phage
conjugates.
These conjugates were prepared to display copies of the MC192 monoclonal
antibody,
which is known to be internalised by rat p75 receptors, crosslinked to the
surfaces of the
helper phages. These antibody-phage conjugates were tested first, and shown to
be
internalised by sciatic nerve fibers. Accordingly, these established a set of
tools
(comparable to probe drugs) that enabled the Applicants to work out the
concepts and
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clethils of an effective approach that allows in vivo screening for
endocytosis, in rats by
manipulating sciatic nerve fibers. The methods that were used to crosslink the
antibodies to
the helper phages are described in Example 24, and the methods that were
eventually
developed to achieve endocytosis and retrograde transport of the antibody-
phage conjugates,
in sciatic nerves, are described in Examples 25 and 26. The methods and
reagents that were
used to create photographic proof of endocytosis and retrograde transport of
those antibody-
phage conjugates, as shown in FIG. 8 of this application, are described in
Example 27.
After the antibody-phage conjugates were used to develop a set of consistent
procedures for achieving reliable uptake of phage particles via p75 receptors,
the Applicants
began testing a phage display library known as the scFv library. The major
traits of that
library are described in Example 22. Very briefly, it contains a huge number
(roughly 13
billion) of foreign gene inserts that were initially obtained from human B-
cells. These gene
inserts encode the "variable fragments" of a wide range of human antibodies,
from people
of different ancestries. The initial in vivo screening tests that were done
with this library did
not provide consistent results. Therefore, the Applicants wrestled with those
problems, and
eventually settled on a process of pre-screening the scFv library, in vitro,
using a technique
called "biopanning", described in Example 28. Very briefly, this pre-screening
involves p75
polypeptides that have been immobilized on a hard plastic surface. Phage
particles that bind
to these immobilized p75 polypeptides were selected by this step, and used for
subsequent
in vivo screening, which provided much more consistent results that could be
understood
and interpreted. These procedures, and the results that were obtained, are
described in
Example 29.
Subsequently, the Applicants also tested their in vivo screening method on a
second
phage display library, called the PhD-C7C library. As summarized in Example
22, this
library was created by combinatorial chemistry, and contains short polypeptide
segments
(with 7 amino acid residues) that were randomly generated, and inserted into
the pill coat
proteins of the phages. These tests, and their results, are described in
Example 30.
The results of all of these screening tests confirm that in vivo screening of
phage
libraries, to select particular phages that are internalised and transported
by neuronal fibers,
is indeed a practical and effective way of identifying and isolating, from a
large display
library, particular phages that happen to carry ligand components that can
activate and drive
the process of endocytosis into nerve fibers.
These results, taken together, also confirm that stochastic processes are
involved,
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which rely on probability, and on the sizes of the populations that are being
challenged and
tested in a highly specialized set of tests. The assertion and claim herein is
not that this type
of selection process will succeed, in each and every screening attempt or
round. Instead,
the assertions and claims made herein center on the fact that this selection
process, which
uses in vivo tests on living animals in ways that were not previously known or
possible, can
be used (in repeating cycles, if desired) to identify, select, and isolate a
small number of
clonal display phages that will successfully enter into and be retrogradely
transported by
neuronal fibers, from among a potentially huge and/or random starting library
or repertoire.
These titering and photographic data also clearly demonstrate that this
approach
enables an in vivo screening method that can effectively identify ligand
molecules and
ligand fragments that can activate and drive the process of endocytosis, even
when coupled
to large molecular complexes. Based on those results, this type of in vivo
screening method
can enable researchers to identify such molecules (referred to herein as
"endocytotic
ligands"), and use them as part of a molecular transport system (which can
also be called a
carrier, vehicle, etc.) that can be used to transport "passenger" or "payload"
components
into cells. Such passenger components can include, for example, DNA segments
that are
part of genetic vectors, drug or diagnostic molecules that can provide
therapeutic,
diagnostic, or other medical benefits, and analytical compounds that would be
more useful,
in industrial research and similar endeavors, if they could be transported
efficiently into
cells.
It should also be disclosed that, as this patent application is being written
and filed,
none of the nucleotide gene sequences that encoded the polypeptide ligands
that performed
well in the in vivo screening tests are yet known, and none of the amino acid
sequences of
those polypeptide ligands are known. The laboratories of the Applicants herein
(which are
located in Australia) do not have the types of machines that are used to
determine
nucleotide or amino acid sequence information. Accordingly, the Applicants
shipped copies
of a number of selected phages that performed well in their in vivo screening
tests, to an
outside contract laboratory which is equipped to determine those sequence
data. However,
the resulting data have not yet been received, and those sequences are not yet
known, as of
the day this patent application is being filed.
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USING GENETIC VECTORS FOR DRUG ADMINISTRATION/DELIVERY INSIDE
BBB
The invention may be used by administering, into or onto tissue which is not
protected by the BBB, one or more copies of a gene vector that can transfect
one or more
types of BBB-straddling neurons. Clearly, the range of preferred and potential
modes of
administration will depend on the type of BBB-straddling neuron that is to be
transfected by
the vector, and the type and location of the peripheral projection that is to
be contacted by
the vector. As described above and in the examples, administration to
olfactory receptor
sensory neurons can be via nasal instillation; administration to nocioceptive
neurons can be
via cutaneous, subcutaneous, or possibly intramuscular injection, and possibly
by topical
administration (which can be accompanied by one or more agents or techniques
that will
increase epidermal penetration and tissue permeation, as described above);
administration to
lower motor neurons innervating the skeletal musculature can be via
intramuscular
injection; and, administration to the lower motor neurons of hypoglossal
nucleus can be by
injection into the muscles of the tongue.
It will be apparent to ,those skilled in the arts of medicine or neuroscience
that there
exist other ways to administer gene vectors to peripherally-projecting
neurons. It also will
be apparent to such persons that: (i) other types of neurons that straddle the
BBB (including
various types of sensory and motor neurons, as well as pre-ganglionic neurons
of the
sympathetic and parasympathetic systems) offer potentially useful targets for
transfection;
and, (ii) the known features and traits of the anatomy and structure of any
such class of
neuron will allow skilled neurologists and researchers to develop various
methods for
administering gene vectors to such neurons.
MODULATION OF ENDOCRINE AND PARACRINE HORMONAL SYSTEMS
It should also be recognized that, by enabling non-invasive delivery of
specific gene-
encoded polypeptides to cells, systems, and regions within the brain, this
invention may
also be able to provide new and previously unavailable methods and approaches
to
controlling or modulating various types of "downstream" effects or activities,
such as by
increasing or suppressing the release of various types of endocrine and/or
paracrine
hormones by various glands or organs, either in the CNS-protected brain tissue
(such as the
pituitary and pineal glands), or in other parts of the body (such as the
thyroid, thymus,
adrenal, or other glands, or in the pancreas, reproductive organs, etc.).
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This patent application is not the appropriate location for a detailed
analysis of the
endocrine or paracrine systems, and the comments below are intended solely as
a very brief
introduction and overview. For more information on the endocrine and paracrine
systems,
good overviews are provided in nearly any good textbook on physiology, and
more
information is contained in numerous full-length textbooks, such as Wilson &
Foster 1992,
Barrow & Sleman 1992, Brown 1993, DeGroot 1994, etc. Recent review articles
are not
especially helpful in establishing a working knowledge of the endocrine or
paracrine
systems, since they focus mainly on problems (such as glandular tumors,
hormone
disruptors such as pesticides, etc.), interventions (surgical or drug), or
interactions between
hormone systems and other systems such as immune responses; however, review
articles
offer a good base of information when the goal is to move beyond a working
knowledge of
the endocrine or paracrine systems, and into the realm of potential
interventions for
purposes such as therapy of human disorders or malformations, or livestock
breeding.
One of the crucial components of the endocrine system is the pituitary gland,
which
sits at the base of the brain, suspended from a region of brain tissue called
the
hypothalamus. The anterior lobe or gland of the pituitary is known to release
at least six
different hormones, and the release of each of these hormones is either
triggered or
suppressed by an "upstream" hormone, called a hypothalamic hormone. These
hormonal
systems (or pairings, relationships, etc.) include the following:
1. a hypothalamic hormone called thyrotropin-releasing hormone (abbreviated as
TRH; formerly called thyroid-stimulating hormone releasing hormone) causes the
pituitary
to release a hormone called thyrotropin (formerly called thyroid-stimulating
hormone, or
TSH);
2. a hypothalamic hormone called corticotropin-releasing hormone (CRH) causes
the
pituitary to release adrenocorticotropin;
3. a hypothalamic hormone called growth hormone releasing hormone (GHRH)
causes the pituitary to release growth hormone (also called somatotropin, and
often referred
to as hGH or HGH in the case of human growth hormone);
4. a hypothalamic hormone called growth hormone inhibitory hormone (GHIH, also
called somatostatin) inhibits the release, by the pituitary, of growth hormone
(somatotropin);
5. a hypothalamic hormone called gonadotropin releasing hormone (GRH or GnRH)
causes the pituitary to release two types of "gonadotropic" hormones, called
luteinizing
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lormone, and follicle-stimulating hormone; and,
6. a hypothalamic hormone called prolactin inhibitory hormone (PIH) inhibits
the
release, by the pituitary, of a hormone called prolactin.
By administering a genetic vector that will cause an increase in the
concentration of
one of the above-listed hypothalamic hormones inside BBB-protected brain
tissue (it should
be noted that a transient rather than permanent increase can be achieved by
the methods
disclosed herein, and transient increases are generally presumed to be
preferable in
therapeutic treatments of human medical or developmental disorders), it is
likely to be
possible to cause, in a controllable manner, either stimulation or inhibition
of the release of
a "downstream" or "dependent" pituitary hormone. Accordingly, as a result of
that type of
triggered and/or targeted pituitary stimulation or inhibition via a genetic
vector, it is
possible to stimulate, inhibit, or otherwise modulate the same types of
physiological effects
that are caused by the release, or inhibition, of the pituitary hormones.
As an alternate approach, it may be possible in at least some cases to
stimulate or
inhibit a targeted endocrine or paracrine system by administering a genetic
vector that
directly encodes a pituitary hormone, rather than its "upstream" hypothalamic
hormone.
Along these lines, radiolabelled tracer studies have shown that at least some
types of
proteins which have been delivered into the CNS by direction injection into a
brain
ventricle are cleared fairly rapidly from the CNS into the blood circulation
(e.g., Ferguson
1991). Therefore, if the rate of delivery of a hormone-type polypeptide into
BBB-protected
CNS tissue is sufficiently high, some of those hormone polypeptide molecules
will diffuse
into circulating blood, and will be distributed systemically.
With regard to delivering hormonal or other polypeptides into the brain with
the
intent of causing the polypeptides to contact specific regions, cells, or
structures within the
brain, it should be borne in mind that this invention may be able to offer, in
at least some
cases, an approach which will allow targeted delivery in ways that have not
previously been
available. Most prior art methods of delivering neuroactive molecules (such as
neurotrophic
factors) into the CNS appear to assume that the endocrine model of drug
delivery is the
appropriate method for delivering such molecules. Evidence that this
assumption is
prevalent is seen in numerous animal studies, as well as limited human
clinical studies in
which recombinant neurotrophins are injected or infused into cerebrospinal
fluid (typically
into the lateral ventricles). These drug administration approaches are based
on the
assumption that the flow of cerebrospinal fluid within the brain and spinal
cord represents
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an internal CNS circulation, analogous to the circulation of blood within the
periphery.
However, that assumption is unwarranted, because the flow of CSF in the CNS is
more analogous to the lymphatic system, in the periphery, than to blood
circulation. Like
the flow of lymph fluid, the flow of CSF is more of a uni-directional
drainage, rather than
a re-circulation of fluid.
Accordingly, when neuroactive molecules (such as neurotrophic factors) exert
their
physiological effect, not in an endocrine (system-wide) manner but rather in a
paracrine
(localized) manner, an appropriate drug delivery method preferably should not
involve
systemic administration, such as by intravenous infusion or injection, both
because of (i)
high levels of wastage of highly expensive drug compounds, and (ii) the
potential for
unwanted adverse effects, when systemically-injected molecules react with
various cells or
organs other than the desired targets. Instead, a more narrowly focused and
targeted system
should be used, if available.
This invention appears to offer a substantially improved method of drug
delivery,
which in some respects emulates paracrine delivery. This method can achieve or
at least
promote localized and focused delivery of drug to a target cell or region
(especially when
compared to other methods, such as intravenous injection) by transfecting only
certain
selected populations of BBB-straddling neuron(s), which will subsequently
release
polypeptides in a limited and desired "secretion zone".
The ability to use gene therapy to achieve sustained drug delivery has been
recognized in the art, as evidenced by numerous studies involving transplant
into the CNS
of cells that have been genetically engineered to secrete particular
recombinant molecules,
such as neurotrophins. However, nearly all such studies illustrate or imply
that the desired
intention is to achieve an endocrine-like form of drug delivery, with the
transplanted cells
secreting drugs in a manner that causes or allows systemic distribution. By
contrast, this
invention discloses an entirely different form of genetic therapy, which can
achieve a
paracrine-like, localized delivery of therapeutic polypeptides to a specific
cluster and/or
type of neuron within the BBB, by transfecting a limited number of neighboring
cells that
straddle the BBB.
SELECTIVE MODULATION OF NEURONAL SYSTEMS
The invention enables development of completely new approaches to treating
disorders of the nervous system, and the disclosed paracrine-like method of
drug delivery
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enables development of new processes or methods for selectively modulating the
TithCtjöiTöf
particular systems of neurons within the CNS.
Physiologically, the function of qrstems of neurons within the central nervous
system can change, in response to functional changes in the neurons that
penetrate through
the BBB. For example, changes in the electrical activity of nocioceptive
neurons that
penetrate through the BBB cause changes in the electrical activity of second
order sensory
neurons and the associated system of CNS neurons involved in sensation of pain
and
response to that pain. The anatomical connections between the neurons that
project through
the BBB, and neurons that reside wholly within the BBB, are not fixed and
static; instead,
the nature, number, and distribution of these connections can change, both
over time and in
response to various types of events (and often resulting in downstream changes
in still other
systems of neurons within the CNS). In other words, the systems of neurons
within the
CNS are plastic, changeable, and responsive to functional changes in the
neurons that
penetrate through the blood brain barrier.
Based upon that physiological fact, it is believed that in at least some
cases, this
invention may render possible to selectively modulate and alter the structure
and functioning
of at least some types of neuronal systems within the CNS, by altering the
patterns of
innervation (including strength, number, and distribution) of synaptic
connections between
transfectable peripherally-projecting systems, and targeted neuronal systems
within the
CNS.
As an example, it is well known in the field that neurotrophic factors are
intimately
involved in the plastic changes in synaptic density and innervation pattern,
in CNS systems,
in response to changes in electrical activity, such as the number and
frequency of nerve
impulses that arrive from other neurons located closer to the periphery.
Physiologically,
these neurotrophic factors act in what has been described above as a paracrine-
like manner.
It follows that the paracrine drug delivery approach disclosed herein can be
used to
selectively modulate the nature and extent of connections between one or more
peripherally
projecting neurons, and the CNS neurons or neuron systems with which they
interact. That
is, by using gene vectors to transfect BBB-straddling neurons, it will be
possible (in at least
some cases) to alter the nature and extent of the connections between those
BBB-straddling
neurons, and CNS neurons lying wholly within the BBB, and thereby selectively
modulating
one or more densities, functions, or other traits of the interacting CNS
neuronal systems.
As examples, this principle can be illustrated by two preferred embodiments:
(i)
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administration of recombinant anti-NGF, via transfection"ornomeeptiVe
htutonallial
straddle the BBB, can modulate the BBB-protected neuronal systems that are
involved in the
perception of pain; and, (ii) administration of recombinant NT-3 or GDNF, via
transfected
BBB-straddling spinal motor neurons, can modulate the CNS neuronal systems
involved in
voluntary control over motor function.
TREATMENTS FOR NEUROLOGICAL DISORDERS
This invention is likely to become useful for treating neurodegenerative
disorders of
the CNS (such as Alzheimer's disease), by methods which include delivering
neurotrophic
or neuroprotective factors to the neurons at risk of degenerating. It is
believed, for
example, that Alzheimer's disease probably can be treated in a useful manner
by
administering neurotrophic factors (such as nerve growth factor) into the CNS.
In accord
with that goal, this invention allows neurotrophic factors to be delivered, in
a relatively
focused and targeted manner, to basal forebrain cholinergic neurons that are
at risk of
degenerating in patients with Alzheimer's disease. In at least some patients,
this type of
treatment may be able to help slow, and potentially even halt, the
neurodegenerative
process.
This invention is also likely to become useful for treating trauma or injury
to the
CNS (such as that which occurs in head injury), by delivering neuronal growth
factors
(such as neurotrophic factors) to injured and surviving neurons. As just one
example,
GDNF, which acts on cortical motor neurons (which are frequently damaged in
stroke or
sometimes head trauma), can be delivered to these neurons by methods disclosed
herein. Its
potential benefits are described in various articles such as Schacht et al
1996.
This invention may also become useful for treating some cases of learning or
memory dysfunction, such as occur in aging, dementia, after brain trauma or
injury, and
after various types of major surgery, especially surgery involving a
cardiopulmonary bypass
machine. Such trauma and insults often lead to loss of function within one or
more regions
of the CNS. Restoration of function, if it can be achieved, apparently
requires and involves
compensatory changes in the organization of the CNS, typically including the
sprouting and
outgrowth of various affected neurons, and the establishment of functional
synapses on
other neurons. These types of "neuroplastic" processes have been demonstrated
both in
animal studies, where a digit or limb was severed, and in human cases, where a
stroke
victim regained substantial use of a paralyzed limb by undergoing therapy in
which one or
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two healthy limbs were strapped to the body and immoliihied, 'while the
patiefit did
repetitive exercises which actively and aggressively challenged the patient to
begin moving
and using the impaired limb once again, building upon and expanding the range
of motion
and control that remained in the limb after the stroke.
Neurotrophic factors can play important roles in such processes (e.g., Lo
1995), and
administration of NGF into the brain has been shown to enhance various CNS
activities and
functions, such as memory and learning (e.g., Fischer et al 1987 and 1991).
Accordingly, this invention provides a major avenue for expanding and
enlarging
upon that type of highly useful therapy, by allowing NGF and other
neurotrophic and
neurostimulatory factors to be delivered through the CNS and into the brain,
using
improved delivery systems.
This invention is also likely to become useful for treating disorders due to
excitotoxic damage of neurons, or resulting from diseases or injuries that
involve ischemia
(inadequate blood flow, as occurs during a stroke or cardiac arrest) or
hypoxia (inadequate
oxygen supply, as occurs during drowning, carbon monoxide poisoning, etc.) or
traumatic
head injury. In animal studies, NGF infusion can slow or reverse the
retrograde atrophy of
cholinergic cell bodies and fiber networks and other changes in the
cholinergic system that
are caused by infarction or measured by infarct volumes or severity (e.g.,
Cuello et al
1992). Administration of NGF (or induction of NGF synthesis in vivo by
clenbuterol) has
been shown to reduce infarct volume in rat models of permanent middle cerebral
artery
occlusion (Semkova et al 1999). Other animal data suggests that NGF is able to
act after a
brain insult to block progression of neuronal damage (e.g., Guegan et al
1998). Since this
invention is likely to prove useful for administering NGF after a stroke or
other brain
injury or insult, it likely will be able to reduce the extent and severity of
subsequent
neuronal loss.
The present invention further relates to methods for treating disorders of
sensory
function by modulating the function of the sensory neuron and/or the nerve
cells which
make synaptic contact with it in the CNS. For example, severe and persistent
pain involves
both nocioceptive neurons and CNS (spinal or brain) changes, and it has been
reported that
intrathecal administration of anti-NGF can reverse these types of changes, and
alleviate the
pain (e.g., Christensen et al 1996 and 1997). This invention allows the
function of the
sensory system to be modulated by delivering polypeptides (such as antibodies
which will
bind to and inactivate NGF, or which will occupy and block NGF receptors)
which act on
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)ne or more components of the sensory system, to alter iel fioldigtiMagqi9:
_
This invention can also be used for modulating neuronal physiology by delivery
and
mpression of neuropeptide genes, such as genes that express polypeptides that
can block
and suppress pain (such as so-called "endorphins"); genes that express growth
factors;
genes that express polypeptides to promote regeneration or prolong the life-
spans of cells;
and genes that express toxic polypeptides, such as to kill tumor cells.
Beyond that, this invention provides an approach that can be adapted to
treatment of
various types of CNS-related neurological disorders or deficiencies which are
correlated
with either too little or too much of some particular polypeptide. This can be
accomplished
by using this method to deliver, into BBB-protected CNS tissue, either: (i) a
polypeptide
which provides an additional quantity of a polypeptide, to reduce or eliminate
a deficiency;
or, (ii) a polypeptide which blocks, antagonizes, or otherwise suppresses a
certain
molecule, receptor, or reaction, thereby helping to controlling a CNS disorder
that is
caused or characterized by too much of a particular molecule.
EXAMPLES
The Examples below are organized as follows:
Examples 1-8 relate to delivering neuron-stimulating polypeptides to neurons
which
lie wholly within the BBB, by transfecting olfactory receptor neurons, using
vectors that
carry genes which encode such polypeptides. For purposes of illustration,
human NGF is
used as the prototypic polypeptide; as will be recognized by those skilled in
the art, genes
which encode other forms of NGF (such as mouse, other rodent, or simian NGF,
or any
mutated, epitope-tagged, fragmented, or other form of NGF which may be of
interest in
medicine or research) may alternately be used.
Examples 1-7 describe how NGF (or other neurotrophic or similar polypeptides)
can
be delivered to cholinergic neurons in the basal forebrain of a laboratory
animal, such as a
rat. Examples 1-4 contain a complete embodiment, divided into various
sequential steps.
Example 1 describes the assembly of the vectors; Example 2 describes
administration of
those vectors to the nasal sinuses; Example 3 describes methods of monitoring
delivery of
the polypeptide through the blood brain barrier; and Example 4 describes
methods of
measuring the physiological and behavioral effects of such treatments on lab
animals.
Following that "start to finish" description, Examples 5-8 describe delivery
of an
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NGF-encoding or similar genes into olfactory receptof naiibra, if
ifirdiffetent '
vectors. Example 5 describes vectors derived from herpes viruses; Example 6
describes
liposome vectors; Example 7 describes vectors with ligands that bind to
endocytotic
receptors on neuron surfaces; and Example 8 describes vectors that use
"transneuronal"
polypeptides that can promote transport of the entire vector from one neuron
to another.
Examples 9-13 involve a different approach, using gene vectors that encode
nerve
suppressing (rather than stimulating) factors; a polypeptide called "anti-
NGF", which binds
to and inactivates NGF, is used as illustration. These vectors are injected
into skin or
muscle regions, in order to transfect nocioceptive (pain-signalling) neurons,
in areas that
suffer from unwanted and excessive pain signals (often called neuropathic
pain, or
allodynia). By suppressing over-active pain signalling circuits, this approach
can help
reduce and control neuropathic pain.
Examples 14-20 describe a third major line of approach, in which genetic
vectors
carrying genes that encode nerve-stimulating factors are injected into muscle
tissue that is
impaired due to a stroke, spinal injury, etc. These types of impaired muscles
often suffer
from a lack of (or impairments in) voluntary control, caused or aggravated by
a loss of
properly functioning connections between upper motor neurons (which lie wholly
within the
BBB) and lower motor neurons (which straddle the BBB). Transfection of lower
motor
neurons in such impaired muscles, using genes that encode nerve-stimulating
factors, can
help expand, repair, and reconnect the damaged motor control networks, by
means such as
establishing new and additional connections between the lower and upper motor
neurons,
and/or by increasing synaptic activity levels between those classes of
neurons, thereby
reestablishing proper innervation and CNS control over such muscle systems.
Example 21 describes transfection of certain types of motor neurons located
inside
the tongue. This route of administration deserves special attention, since it
offers a route for
delivering polypeptides into certain portions of the brainstem.
EXAMPLE 1: CONSTRUCTION OF ADENOVIRAL VECTOR FOR
TRANSFECTING OLFACTORY RECEPTORS WITH NGF GENE, TO DELIVER
NGF TO CHOLINERGIC NEURONS IN THE BASAL FOREBRAIN
Methods for preparing non-pathogenic vectors derived from adenoviruses that
cannot
replicate, except in genetically engineered host cells that exist only in
laboratories, have
been published in articles such as Graham and Prevec 1995. Methods for
creating gene
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constructs which are small enough to be carried and defivefed
which will express NGF (or some other CNS-active polypeptide, such as GDNF, NT-
3,
CNTF, or BDNF, or any of numerous other polypeptides such as listed in Table
1) at
significant levels, inside transfected human neurons, are described in
articles such as
Romero et al 2000, Baumgartner and Shine 1998), Dijkhuizen et al 1997, and
Gravel et al
1997. Methods for propagating, purifying, concentrating, and titering
adenoviral vectors
carrying such gene constructs can be found in publications such as the chapter
by
Engelhardt (pp. 169-184) in Methods in Molecular Medicine: Gene Therapy
Protocols (P.
Robbins, ed., 1997).
The gene construct that will drive expression of the NGF polypeptide (or other
selected CNS-active polypeptide) will require proper selection of all relevant
portions of the
gene. As used herein, terms such as "gene" or "gene construct" normally refer
to a
complete and functional transcription unit, which can be manipulated under
laboratory
conditions using known procedures, and which, if transfected into a BBB-
straddling neuron,
is capable of: (i) causing the normal transcription and translation mechanisms
inside the
neuron cell body to synthesize the polypeptide encoded by the gene, and (ii)
instructing the
cell to appropriately process and secrete the mature polypeptide from
dendrites, processes,
synapses, or other terminals located inside the BBB.
A number of variants and enhancements of the simple "plain vanilla" type of
gene
constructs are known to those skilled in the art, including a number of
variants and
enhancements described in the Detailed Description section. Any such variants
or
enhancements are included within the terms "gene" or "gene construct" as used
herein.
The complete amino acid encoding sequences (without introns) of human NGF,
mouse NGF, and certain other forms of NGF, have been published and are known
and
available, in easily manipulated plasmid form, from various researchers;
alternately, they
can be created using well-known techniques and published information.
Also, if a polypeptide having an unusual "epitopic tag" sequence (such as c-
myc) is
desired, then the coding portion of the gene construct must include the DNA
sequence that
will encode the "epitopic tag" sequence. The inclusion of an epitopic tag may
assist in
demonstrating, refining, and otherwise enhancing the invention, in situations
where epitopic
tag sequences can assist researchers to distinguish between exogenous
polypeptide
molecules that are encoded by a genetic vector, and endogenous (native)
polypeptides that
are present naturally within the BBB of test animals or patients.
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The other crucially important part of a gene construqpirthp,gempyppoter_ and
numerous known gene promoters are known and available which can drive gone
expression
in olfactory receptor neurons. Examples of "selective" gene promoters that are
known to
drive gene expression specifically in olfactory receptor neurons include
promoters identified
in the olfactory marker protein gene (Servenius et al 1994) and the M4
olfactory receptor
gene (Qasba and Reed 1998). Use of such selective promoters can help minimize
the chance
that undesired or uncontrolled expression of the foreign gene will occur in
surrounding cells
or tissue.
Alternately, various types of viral and other promoters which are known to be
unusually strong promoters in mammalian cells can be used if desired, for
purposes such as
inducing the highest practical levels of expression of NGF (or other CNS-
active)
polypeptide in transfected cells. Examples of such strong promoters include
the early gene
promoter from cytomegalovirus, and the late gene promoter from simian virus-
40. Inducible
gene promoters can also be used if desired, so long as the inducing factor
which activates
the selected promoter can be administered in a way which ensures that it will
be transported
into transfected neurons in adequate quantities.
As noted above, for simplicity of discussion herein, the term "gene promoter"
as
used herein includes the so-called "TATA box", and the sequence of about 25
bases
between the TATA box and the actual start of transcription.
Once the promoter and coding sequence of the gene construct have been chosen,
various other portions of the gene construct can be chosen to enhance
expression,
packaging, transport, secretion, and/or performance of the vector-encoded
polypeptide.
These gene portions can include, for example: (i) a DNA sequence which will
encode a
"leader" or "signal" peptide sequence, which will instruct the transfected
neuron to
appropriately process and secrete the vector-encoded polypeptide (or a
"mature" form from
which the leader or signal sequence may be removed); (ii) a DNA sequence that
will be
transcribed into an mRNA sequence that will serve as a non-translated "tail"
region, which
will follow the "stop" codon in the mRNA, to facilitate translation and
release by
ribosomes; and, (iii) a transcription terminator sequence, which will direct
RNA
polymerase to truncate the mRNA strand when the DNA sequence is being
transcribed into
mRNA. These types of functional sequences are well-known, and can be obtained
or
adapted from nearly any mammalian gene cloning vector that allows a cloned
gene to be
expressed at high levels by transfected mammalian cells. By making use of the
pre-pro-
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BDNF sequence that precedes the mature BDNF polypeptide, in place of the pre-
pro-NGF
sequence that precedes the mature NGF sequence, anterograde transport and
release from
the sensory neuron terminals within the BBB can be facilitated, in at least
some types of
neurons.
EXAMPLE 2: ADMINISTRATION OF ADENO VIRAL NGF VECTOR TO
OLFACTORY EPITHELIUM, TO DELIVER NGF TO CHOLINERGIC NEURONS
IN THE BASAL FOREBRAIN
Adenovirus-derived (or other virally-derived) vectors which carry a gene
construct
that encodes NGF (or some other CNS-active polypeptide) may be administered to
the
olfactory epithelium, of either a human patient (for medical purposes) or a
test animal (for
test or other research purposes), by using nasal instillation of an aqueous
suspension of the
vector, at a suitable titer concentration.
The aqueous carrier liquid should be compatible with adenovirus and olfactory
epithelium vigor. Physiological saline (with buffering agents, if desired) can
be used, and
hypotonic or hypertonic solutions, or solutions containing any other component
that may
induce higher levels of viral transfection, can also be tested using routine
experimentation.
Articles such as Holtmaat et al 1996 provide information on dosage and
administration
techniques for efficient administration of adenoviral vectors via nasal
instillation in mice;
such procedures may be adapted for use in larger rodents such as rats or
rabbits, or in other
mammals, using methods known to those skilled in the art.
If desired, steps can be taken to increase (i) the extent and/or duration of
contact
between the fluid containing the gene vector and the olfactory neurons, and
(ii) the
receptivity of the neurons for taking in such genetic vectors. This can be
done by, for
example, administering a nasal decongestant to test animals (or human
patients) a few hours
prior to administration of the gene vectors, and by using an aqueous solution
(and possibly
a swabbing step, using a diluted solvent, such as isopropyl alcohol or
acetone, that can help
remove any mucous, oleaginous, or other viscous coating) to rinse and/or clean
the nasal
sinuses immediately prior to vector administration. The receptivity of the
olfactory neurons
may also be increased by using mild mechanical abrasion, using a small wire
loop, rounded
spatula tip, or similar tool. In general, neuronal projections in a surface
area which has
become irritated to a point of mild inflammation tend to be more receptive to
cellular
uptake of foreign molecules than cells which can be regarded as being in a
quiescent or
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resting state.
After allowing sufficient time for gene expression (usually in the range of
about 24
to 72 hours), the effectiveness of gene vector delivery can be assessed by
sacrificing some
of the test animals, removing the olfactory epithelium, olfactory bulb, and
basal forebrain,
and processing each tissue type separately, to measure for the locations and
concentrations
of the vector-encoded polypeptide within that type of tissue.
The tasks of measuring and monitoring can be relatively simple where the
vector-
encoded polypeptide is distinguishably different from endogenous (presumably
rodent)
polypeptides. These types of analyses can use methods such as: (i)
hybridization of cellular
mRNA with DNA probes that are complementary to the vector mRNA sequences, but
not
to endogenous mRNA sequences, using procedures as described in articles such
as Xian and
Zhou 2000; (ii) techniques which use "polymerase chain reaction" (PCR)
reagents and
methods to detect DNA or mRNA sequences from the vector, as described in
articles such
as Chie et al 2000; and, (iii) immunostaining or similar methods which use
monoclonal
antibodies that selectively bind to the polypeptide or epitope tag encoded by
the vector that
was used, and do not bind to endogenous polypeptide in the test species (such
antibodies are
commercially available; they also can be prepared if desired, using methods
disclosed in
articles such as Conner 2000, Rush et al 2000, and Zhang et al 2000).
If desired, time-dependent levels of exogenous mRNA and/or polypeptide
expression
by transfected olfactory receptors can be measured by repeating one or more
tests, over a
range of times after administration of the genetic vector.
If desired, to provide control populations of cells and animals for purposes
of data
analysis, a genetic vector carrying a human (or epitope-tagged animal)
polypeptide gene
construct can be administered to the olfactory epithelium on one side of a
test animal's
nasal sinus, and a control vector which carries some other gene (such as a
marker gene that
encodes an easily-detected polypeptide) can be administered to the olfactory
epithelium on
the other side of the animal's nasal sinus. After the animal is sacrificed,
histological and
immunological examination of the left and right olfactory receptors, olfactory
bulbs, and
basal forebrain regions can be used to evaluate (i) polypeptide expression by
the transfected
olfactory receptors, and (ii) polypeptide transport and delivery by the BBB-
straddling
receptor cells to other classes of neurons located inside the BBB.
EXAMPLE 3: MONITORING NGF POLYPEPTIDE DELIVERY TO CHOLINERGIC
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NEURONS IN THE BASAL FOREBRAIN
The quantity of NGF polypeptide (or any other vector-encoded CNS-active
polypeptide) which is delivered via transfectexl olfactory receptor neurons
(or any other type
of sensory neurons which straddle the BBB and which are transfected by a
genetic vector as
disclosed herein), into CNS regions that are entirely within the BBB, can be
monitored in
any specific class or cluster of neurons of interest, using histological and
immunological
analysis of cells and tissues from sacrificed test animals.
Well-established immunological procedures, such as immunohistochemistry and
"enzyme-linked immunosorbent assay" (ELISA) tests, can be used to monitor and
quantify
the production, release, and distribution of human NGF (or any other CNS-
active
polypeptide encoded by a genetic vector that has been administered to an
animal), at any
location of interest within the CNS, following administration of a genetic
vector to olfactory
receptor neurons, or other type of sensory neuron. If human NGF is the vector-
encoded
polypeptide, monoclonal and polyclonal antibody preparations which recognize
and bind to
human NGF, but not to mouse, rat, or other rodent NGF, are commercially
available, or
may be generated using procedures that are well documented in the literature
(e.g., Conner
2000; Rush and Zhou 2000). Methods for measuring NGF levels using
immunohistochemistry are described in articles such as Conner et at 1992, and
methods for
measuring NGF levels using ELISA are described in articles such as Zhang et at
2000, and
in manufacturer's instructions which are included in NGF-ELISA detection kits
that can be
purchased from commercial sources.
As briefly mentioned above, monitoring of a vector-encoded human polypeptide,
in
lab animals such as mice or rats, can be rendered simpler and more certain if
monoclonal
antibodies are used that recognize and bind to the vector-expressed
(presumably human)
polypeptide, but which do not bind to the mouse or rat version of the same
polypeptide.
Hybridoma cell lines which express monoclonal antibodies that selectively bind
to human
NGF but not mice or rat NGF have been created., and such monoclonal antibody
preparations are available. Similar hybridoma cell lines which express
monoclonal
antibodies that selectively bind to human but not rodent forms of other CNS-
active
polypeptides have also been created, or can be developed using methods known
to those
skilled in the art.
Alternately or additionally, as noted above, it is possible to incorporate an
epitopic
tag (such as c-myc) in a neurotrophic gene construct sequence, to facilitate
immunological
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letection of polypeptides expressed by that gene construct in transfected
neurons, using
methods described in articles such as Moller et al 1998. If such an approach
is used, it is
important to confirm that the polypeptide expressed by the vector construct is
able to
undergo all steps that are necessary for proper delivery to the neurons that
are being
targeted for treatment by that polypeptide.
For example, if the goal of a particular procedure is to deliver therapeutic
polypeptides into the main cell bodies of basal forebrain cholinergic neurons,
and an
"epitopic tag" sequence is used to facilitate monitoring of the polypeptide,
the targeted basal
forebrain cholinergic neurons can be evaluated to ensure that they carried out
uptake (such
as receptor-mediated endocytosis) and retrograde transport of the vector-
encoded, epitope-
tagged polypeptides that were secreted by the transfected BBB-straddling
neurons. These
types of confirmatory tests can be carried out by using methods described in
articles such as
Altar & Bakhit 1991, Ferguson et al 1991, DiStephano et al 1992, and von
Bartheld 2000,
using reagents such as monoclonal antibodies that will bind specifically to
the epitope tag
sequence (and/or to a domain in the fusion polypeptide which contains part of
the native
amino acid sequence along with the tag sequence), to demonstrate that the
vector-encoded
polypeptides were taken up by the targeted basal forebrain cholinergic
neurons.
If a vector-derived NGF carries an antigenic or epitopic tag, monoclonal
antibodies
which bind selectively to the tagged form may be creating using well-known
methods, and
can be used for any subsequent immunohistochemistry or ELISA measurements. If
desired,
confirmation that the delivery of NGF or some other CNS-active polypeptide
(and its
subsequent neurological and physiological effects on tissue inside the BBB)
was indeed
mediated by genetic vector administration to olfactory receptor neurons can be
achieved by
ablating these neurons in control animals, by treatment using a compound such
as zinc
sulfate solution, as described by Horowitz et al 1999.
Based on trans-synaptic tracer studies (e.g., Lafay et al 1991, Barnett et al
1993), it
is generally anticipated that a vector-expressed polypeptide such as NGF will
be detectable
in the basal forebrain cholinergic neurons after allowing sufficient time for
gene expression
in the olfactory receptor neurons, which is likely to require a range of
roughly 24 to 72
hours. This time period is anticipated to include anterograde transport of the
polypeptide
(after it has been expressed) to the receptor neuron's synaptic terminals in
the olfactory
glomerufi, secretion of the polypeptides within the glomeruli (roughly 8 to 24
hours), and
transport of the polypeptides to and into basal forebrain cholinergic neurons
(roughly 8 to
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24 hours).
As discussed in Example 8, certain types of "trans-neuronal" vectors are also
disclosed herein. That class of vectors may be able to be transmitted by
sensory neurons to
other types of neurons inside the BBB, in a manner which will transfect the
recipient
neuron with the foreign gene, causing one or more classes of "downstream"
neurons lying
wholly within the BBB to begin expressing the foreign therapeutic polypeptide.
However, unless specific steps are taken to provide "trans-neuronal" transport
between neurons, it is assumed that: (i) transfection of initial or "primary"
neurons (BBB-
straddling neurons) by a genetic vector as disclosed herein will not lead to
transmission of
the vector to other non-primary neurons which are inside the BBB; and, (ii)
transient gene
expression will result, rather than permanent genetic transformation. If
sensory neurons
such as olfactory receptor neurons are used as the transfection targets,
expression of the
vector-borne gene(s) in such cells is expected to decrease substantially, and
may stop
entirely, during a period of several days to several weeks following the
highest peak or
plateau levels of polypeptide expression. If desired, reapplication of the
same or a similar
genetic vector to the olfactory receptor neurons can deliver an additional
supply of NGF
into BBB-protection CNS tissue.
EXAMPLE 4: MONITORING PHYSIOLOGICAL EFFECTS OF NGF DELIVERY
TO CHOLINERGIC NEURONS IN THE BASAL FOREBRAIN
To enable and improve analysis of the actual physiological, behavioral, and
other
effects of CNS-active polypeptide delivery into BBB-protected CNS tissue using
the genetic
vector methods disclosed herein, experimental animals (or humans who have
volunteered
for clinical trials of this type of therapy) may be divided into two groups:
(i) a test group
which will receive a genetic vector which encodes NGF or some other selected
neurotrophic
or CNS-active polypeptide; and, (ii) a control group which receives a placebo
treatment.
The placebo treatment should use identical treatment of the instillation site
(such as identical
treatment with a decongestant, rinsing, swabbing, scraping, and/or other
irritation of the
nasal linings), followed by either: (a) nasal instillation of a plain saline
solution with no
genetic vector; or, (b) nasal instillation of a solution containing a genetic
vector which
might carry, for example, an innocuous and/or non-functional gene, a nonsense
DNA
sequence which does not encode any polypeptide, or a marker gene which encodes
a
polypeptide that can be easily detected if expressed in mammalian cells, but
which has no
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significant physiological effect.
When animals are tested, introducing a CNS-active polypeptide (such as NGF or
some other neurotrophic factor) through the BBB and into protected brain
tissue may lead to
either or both of at least two categories of observable differences: (i)
effects on the
neuroanatomy of the brain, which can be evaluated by histological,
immunological, or other
biochemical analysis of sections of brain tissue removed from animals that
have been
sacrificed; and, (ii) observable and measurable effects on the behavior of the
animal.
A number of tests have been developed for assessing what appears to be
happening
inside a laboratory animal's CNS system, based on observable and measurable
forms of
behavior (such as the ability of treated mice or rats to remember what they
encountered in
prior challenges involving mazes, water mazes, etc.). In addition, other such
tests are being
developed, and any such test which is currently known or hereafter discovered
can be used,
provided that it is appropriate for assessing the physiological effects of the
polypeptide on
targeted neurons lying wholly within the BBB.
It should be noted that many of these tests involve a surgical or drug
intervention
which inflicts some type of damage on the animal's CNS, to model an injury,
stroke,
neurodegenerative disease, or other CNS disorder. Subsequent tests then seek
to evaluate
whether a certain treatment can help such animals recover from the inflicted
injury or
disorder.
One example of such a test is the "limbria forth lesion model", a commonly
used
model in which the basal forebrain cholinergic neuron axons projecting to the
hippocampus
are "axotomized" (i.e., the main axon of a neuron is surgically severed; if
untreated, this
typically will cause the neuron to atrophy and die over a period of about two
weeks or less,
in most species). This induces atrophy and degeneration of the basal forebrain
cholinergic
neurons (e.g., Hefti 1994). However, that may not be a preferred injury model
for
evaluating the invention disclosed herein, because the basal forebrain
cholinergic neurons
which project through the flmbria fomix do not also project to the olfactory
bulb.
Instead, preferred injury models for use herein should disrupt one of the
neuronal
pathways that is likely to be directly involved in, or affected by, the type
of treatment
disclosed herein. For example, surgically severing the cortical projections of
neurons in the
hindlimb of Broca (which project to both the olfactory bulb and the cortex)
while leaving
intact the olfactory bulb projection (so that retrograde axonal flow of NGF
from the
olfactory bulb is not blocked) would likely offer a better form of challenge
to allow
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esearchers to evaluation the effects of the treatments disclosed herein.
An alternative animal model approach to severing an axonal tract is to ablate
a
elected type of appropriate target tissue, in a manner which will deprive
basal forebrain
tholinergic neurons of endogenous NGF, thereby inducing atrophy and
degeneration of
)asal forebrain cholinergic neurons. This approach can generally model the
effect of
mcitotoxic injury associated with stroke. Published examples include Sofroniew
et al 1993,
which describes ablation of hippocampal tissue. This procedure can be modified
to ablate
ntorhinal cortex tissue, which receives innervation from the hind limb of
Broca (Wenk et
11 1980), to induce atrophy of basal forebrain cholinergic neurons in the
hindlimb of Broca. ,
The general methods described in Sofroniew et al 1993 may be used to monitor
the
?,ffect of NGF delivery (using genetic vectors as disclosed herein, such as by
transfection of
olfactory receptor neurons) on the basal forebrain cholinergic neuron cell
body size, and/or
by evaluating levels of one or more enzymes or other polypeptides that will be
directly
affected by the presence and quantity of NGF, which can therefore be used as
an indicator
polypeptide (one example of a candidate enzyme that might be useful for such
measurements is choline acetyl transferase, or ChAT; this enzyme is required
for
synthesizing acetylcholine). Another neuroanatomical effect that can be
observed and
measured when NGF is administered into the CNS (such as by ventricular
injection or
infusion) to test animals which have had selected regions of cortical CNS
tissue ablated,
axotomized, or otherwise challenged, is shown by the ability of the NGF
administration to
prevent or reduce injury-induced reductions in the density of ChAT-
immunoreactive fibers
in the surviving cortex material (e.g., Garofalo et at 1992). Accordingly,
methods described
in articles such as Garofalo et al 1992 can be used to monitor the effects of
NGF delivery
(arising from genetic vectors as disclosed herein), on the density of ChAT-
immunoreactive
fibers in the surviving cortex, after a surgical or other intervention which
ablates or
otherwise damages one or more selected regions of cortical material.
In addition to neuroanatomical changes, cortical ablation results in reduction
in
certain measurable behaviors that can distinguish between normal and impaired
performance
on various tests that require learning and memory in test animals. Examples
include the
"Morris water maze", passive avoidance tests, responsive tests, and various
other animal
models, as described in articles such as Garofalo and Cuello 1994. It has been
shown that
administration of exogenous NGF into CNS tissue (such as by ventricular
injection or
infusion) in such test animals can significantly attenuate injury-induced
deficits in behavioral
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)erformance by such animals. Accordingly, such behavioral, memory, and/or
learning tests
nay be adapted and used to monitor the effects of NGF delivery into the
forebrain region
via genetic vector treatment of neurons which straddle the BBB.
Aging also results in a reduction in memory and learning performance, as
measured
Dy tests such as the Morris water maze, (Fischer et al 1987 and 1991), and age-
related
declines in memory and learning performance have been used in efforts to
measure and
quantify, for example, the loss of memory and learning that occurs in
Alzheimer's disease
(both in human patients, and in various animal models of Alzheimer's disease).
It has been
shown that administration of exogenous NGF into CNS tissue (such as by
ventricular
injection or infusion) can significantly attenuate at least some types of age-
related declines
in memory and learning (Fischer et al 1987 and 1991). Accordingly, the types
of learning
and memory tests used in such animal models can be used to monitor the effects
of NGF
delivery into the CNS by the methods and genetic vectors disclosed herein.
The monitoring and measuring methods mentioned in Examples 3 and 4 herein are
not exhaustive or exclusive; they can be supplemented by other monitoring and
measuring
methods known to those skilled in the art, or hereafter discovered.
EXAMPLE 5: CONSTRUCTION AND USE OF VECTORS DERIVED FROM
HERPES SIMPLEX VIRUS FOR NON-INVASIVE DELIVERY OF NGF
The Examples above describe the use and evaluation of adenovirus-derived
vectors,
to genetically transfect BBB-straddling olfactory receptor neurons, as a route
and method
for non-invasive delivery of NGF into BBB-protected CNS tissue. However, it
should be
recognized that adenoviral vectors are not the only types of genetic vectors
that can be used
for non-invasive delivery of polypeptides into BBB-protected CNS tissue.
Accordingly,
=
Examples 5-8 describe the use of several other classes of genetic vectors.
To construct genetic vectors which are derived from herpes viruses, and which
carry
one or more "passenger" genes which encode CNS-active polypeptides as
disclosed herein,
methods described in articles such as Goins et al 1999 or Federoff et al 1992
can be used.
HSV-derived vectors are capable of transfecting a wide variety of human cells,
including
olfactory receptor neurons, and they can induce transfected BBB-straddling
neurons to
express passenger genes, and to secrete significant levels of such
polypeptides.
Accordingly, HSV-derived vectors may be used, if desired, in a manner directly
comparable to the use of adenovirus-derived vectors as described in Examples 1-
4. The
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methods described in Examples 2-4 (supplemented when appropriate by other or
additional
methods known to those skilled in the art) may be used for nasal instillation
of HSV-
derived vectors, and for post-transfection monitoring and evaluation.
EXAMPLE 6: CONSTRUCTION AND USE OF LIPID-BASED VECTORS FOR NON-
INVASIVE DELIVERY OF NGF
Methods for preparing DNA plasmid-lipid complexes designed for transfecting
mammalian cells are described in numerous publications, such as the chapter by
Nabel (pp.
127-133) in Methods in Molecular Medicine: Gene Therapy Protocols (P. Robbins,
ed.,
1997). A range of liposome preparation kits designed for use in gene transfer
are available
commercially, or can be designed and created by any skilled technician using
published
methods. The preferred choice of lipid formulation and preparation parameters
(including
lipid and DNA concentrations and ratios in the preparation mixture,
temperature, etc.) for
use with a particular size, type, or concentration of plasmid DNA or other DNA
preparation can be determined by routine testing of various preparative
mixtures, using
parameter ranges and confirmatory tests that are discussed in numerous
articles and in the
instructions that accompany most commercially available kits.
Administration of lipid-based genetic vectors to olfactory epithelium, via
nasal
instillation of a lipid-gene complex suspended in an aqueous saline solution
or other carrier
liquid, can use the same general procedures described in Example 2, adapted
for such use
by means known to those skilled in the art. The methods described in Examples
3 and 4
(supplemented when appropriate by other or additional methods known to those
skilled in
the art) may be used for post-transfection monitoring and evaluation.
EXAMPLE 7: CONSTRUCTION AND USE OF DNA VECTORS THAT TARGET
ENDOCYTOTIC RECEPTORS; CYCLIC LIGAND SELECTION PROCESS
The general principals and procedures that can be used to prepare "receptor-
targeting gene vectors" are described in various publications, such as the
chapter by Findeis
(pp. 135-152) in Methods in Molecular Medicine: Gene Therapy Protocols (P.
Robbins, ed.,
1997). As described previously, these vectors contain ligands that will bind
to certain types
of receptors that are exposed on the surfaces of neurons.
As mentioned in the Background section, a number of known types of neuronal
surface receptors undergo a process called "endocytosis", after a ligand
molecule becomes
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bound to the receptor. As suggested by the name, an endocytotic receptor will
be taken
inside the cell, after its ligand molecule becomes bound to it. This type of
activity can be
shown by tests using radiolabelled ligands.
One example of an endocytotic receptor is the "p75" receptor, which is
accessible
on the surfaces of various types of sensory neurons (including olfactory
receptor neurons).
It has been shown to exist in both humans and rats, making it highly useful
for various
types of research. Also known as the p75NGFR receptor (where NGFR indicates
"nerve
growth factor receptor"), it is of special interest for use as described
herein, because it has
been shown to be "up-regulated" (i.e., the expression of mRNA encoding the
receptor is
increased, and the number of receptors that appear on the surfaces of the
neurons is
increased) in various types of neurons that are subjected to crisis or stress
conditions. As
examples, p75 expression increased in the motor neurons of rats following a
peripheral
nerve injury (Yan et .al 1988), and p75 expression also increased in the motor
neurons of
human patients suffering amyotrophic lateral sclerosis (Seeburger et al 1993).
Selection and/or creation of an appropriate ligand that will bind to an
endocytotic
receptor on the type of neuron being targeted (ideally, in the species being
tested or treated)
is key to construction of an effective receptor-targeting gene vector of this
type. A variety
of such ligands are already known, and others can be created, using methods
such as briefly
summarized above.
Since endocytotic receptors are proteins, it is usually possible to create a
complementary polypeptide that will bind to any such receptor, by using a
polypeptide
sequence from the receptor as an antigen, during the creation of monoclonal
antibodies.
Using well-known techniques, the antigenic sequence derived from the receptor
is injected
into animals such as mice, rats, or rabbits; the resulting antibody-producing
cells are then
fused with an immortalized cell line; and, the resulting "hybridoma" cells are
screened, to
identify and isolate a clonal cell line which secretes monoclonal antibodies
that will bind
with high affinity to the receptor of interest. Once the desired monoclonal
antibody line has
been created and identified, a smaller domain or fragment usually can be
isolated from the
variable binding portion of that monoclonal antibody (often referred to by
acronyms such as
the "scFv" fragment, where "Sc" refers to ::single chain" and "Fv" refers to
the variable
fragment, which comprises the binding domain). A gene sequence which encodes
that
binding fragment can then be incorporated into a plasmid or other vector, to
allow
unlimited quantities of the receptor ligand fragment to be synthesized by
fermentation of
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host cells.
Using that general type of approach, monoclonal antibodies can be created
which
will bind to essentially any type of known endocytotic receptor, on any type
of sensory
neuron having one or more peripheral projections.
To continue the example of the p75 receptor, a monoclonal antibody known as
192-
IgG (described in Chandler et al 1984), has been shown to bind with high
affinity to p75
receptors in rats. The 192-IgG monoclonal antibody (as well as various
fragments derived
from it) has been shown to undergo endocytosis, and retrograde transport, in
peripherally-
projecting neurons which express the p75 receptor (Yan et al 1988).
Once a i3olypeptide sequence that can serve as a receptor-binding ligand has
been
identified, various reagents can be used to temporarily couple a DNA segment
to the ligand
polypeptide, thereby creating a polypeptide-DNA complex. In one preferred
method, a
polypeptide can be created having "polylysine" domain, with a series of lysine
residues (or
other amino acid residues having a positive charge at physiological pH)
coupled to the
ligand polypeptide. This can be done by any of several methods, such as adding
a string of
lysine codons to the polypeptide-encoding gene which is used to express the
polypeptide in
a host cell, or chemically conjugated polylysine to a polypeptide by using
bifunctional
crosslinldng reagents. Because of chemical attraction (a polylysine strand has
a positive
charge, and a DNA strand has a negative charge), DNA segments will adhere in a
non-
covalent manner to polypeptides that have polylysine "tails". When the ligand
portion of the
polypeptide binds to an endocytotic receptor, it commences a process that will
draw the
entire polypeptide-polylysine-DNA complex (plus the receptor protein as well)
into the cell.
After that complex has been taken inside the cell, the DNA molecule will be
released, and
can be retrogradely transported inside the cell.
Alternately, a normal ligand for a neurotrophin receptor (such as NGF, which
binds
to NGF receptors) may be used. Radiolabelled NGF has been shown to undergo
receptor-
mediated internalization and retrograde transport, in sensory neurons which
express a
receptor called "trkA" (e.g., Barbacid 1995); accordingly, the NGF polypeptide
itself can
be used as a ligand, for cells having that receptor. It should be noted that
the NGF
polypeptide itself (and, it is believed, various other neuronal receptor
ligands) has a net
positive charge, similar to the positive charge found on histones, a class of
DNA-binding
proteins that are associated with chromosomes; accordingly, the need to first
attach a
polylysine or other similar positively-charged domain or conjugate to an NGF
or other
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positively-charged polyp eptide might be avoided entirely, or minimized, and
the polypeptide
itself may be able to function as both a receptor targeting ligand and a DNA
carrier.
Various other known compounds also provide good candidates which can be
evaluated for potential use as ligands which can bind to endocytotic receptors
on one or
more types of BBB-straddling neurons, and/or as peptide sequences which can
promote
retrograde transport or some other function after a genetic vector or some
portion thereof
has been taken inside a neuron. As just one example, Wiley and Lappi 1993
describes a
conjugate formed by coupling (i) monoclonal antibody 192-IgG, which binds to
the p75
neuronal receptor, to (ii) a plant-derived toxin called saporin, which is
believed to promote
intracellular transport to a neuronal cell body, where it inactivates
ribosomes. This
conjugate was reported by Wiley and Lappi to be useful for causing selective
and targeted
neuronal lesions, for research purposes; accordingly, it can be regarded
herein as a "probe"
compound. It may be possible, using methods known to those skilled in the art,
to modify
and adapt that or similar types of probe compounds, to render them nonlethal
to neurons, in
a manner that will allow a segment of DNA to be (i) non-covalently coupled to
the "probe"
compound; (ii) transported to the neuronal cell body, with the aid of the
probe compound,
and then (iii) released from the probe compound, into the cell body, after the
internalization
and transport steps have been completed.
In addition; numerous types of neurotoxins have been derived from the venoms
of
spiders, wasps, snakes, marine snails, and other venomous organisms. One of
the more
common features of neurotoxins is that they bind (often with extremely high
levels of
binding affinity) to one or more types of receptors and/or ion channels on the
surfaces of
neuronal projections; this is one of the principle mechanisms that organisms
in the wild use
to paralyze or otherwise incapacitate their prey, and to defend against
attackers. A
substantial number of such neurotoxins have been modified by research
biochemists, to
form analogs, conjugates, and other variants and derivatives which have lower,
non-toxic
levels of binding affinity for neuronal receptors or ion channels, in the
search for
therapeutic agents that can be medically useful without inflicting pain at the
levels normally
caused by a bite or sting from a highly venomous animal.
In addition, various types of toxins from pathogenic microbes (including
tetanus
toxin, cholera toxin, etc.), and various compounds derived from plants
(including
compounds that fall within a category known as lectins or agglutinins) offer a
number of
candidates for use as described herein, which can be evaluated to determine
whether that
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an provide selective ligands that will help a genetic vector bind to and
transfect one or
more types of BBB-straddling neurons.
Accordingly, any such known or hereafter-discovered neurotoxin, microbial
toxin,
plant lectin, or other functionally similar compound which can bind
selectively to one or
more receptors, ion channels, proteins, glycoconjugates, or other molecules
that are found
on the surfaces of BBB-straddling neurons, and any analog, conjugate, or other
variant or
derivative of any such neuron-binding compound, can be evaluated for potential
use as
disclosed herein, as a ligand that may help genetic vectors selectively bind
to and transfect
one or more types of BBB-straddling neurons.
As a third alternative approach, a "combinatorial chemistry" approach which
uses
phage display libraries can be used, as described in the Preferred Embodiments
section, and
as illustrated in FIG. 7. Briefly, this approach uses phage display libraries
to generate an
array of potential ligand polypeptides, wherein each phage expresses a single
polypeptide
fragment. The entire array of phages is screened, using a process of receptor-
mediated
endocytosis carried out by neurons in vivo, such as in rats that have had
ligature-type
constrictions placed on nerve bundles such as a sciatic nerve. Intact phage
particles (which
can infect host bacteria) are subsequently harvested from a segment of
extracted nerve cells,
immediately adjacent and "distal" to the point where the ligature was placed.
The harvested
phage particles are then inoculated into E. coil cells, which are used to grow
a subsequent
generation of phages. This new generation of phages will contain those
particular phages
which were present inside the nerve cells, at the location adjacent to the
ligature. As
described in more detail in the Background section, because of how these
phages were
inoculated into and subsequently harvested from a treated lab animal, the
particular phages
which are selected by this screening process presumably must express ligand
polypeptide
sequences which caused the selected phages to be: (i) taken inside the
peripheral projections
of neurons, by a process of endocytosis; and, (ii) retrogradely transported,
inside the
neuronal axon, to the site adjacent to the ligature.
By repeating that type of screening and selection process several times (and
by
subjecting phage lines which performed well to random or targeted mutagenesis,
if desired),
clonal phage lines can be identified which will have incorporated genes that
encode
polypeptide sequences which are highly effective as ligands that can bind to
endocytotic
receptors and trigger endocytosis. Those phages can be analyzed, and the gene
and amino
acid sequences of those ligands can be determined, for subsequent use in
creating highly
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affective receptor-targeting transfection vectors.
If desired, this type of cyclic screening process can be further enhanced by
the use
of various types of assays, such as "fluorescent activated cell sorting"
(FACS), which can
be carried out using known types of machines that are usually called "flow
cytometers". As
an example of how this process can be used, plasmid, phage, or "phagemid" DNA
can be
fluorescently labelled, using a compound such as rhodamine. Cells grown in
tissue culture,
which are known to have p75 receptors on their surfaces, are incubated with
various
polypeptide-DNA complexes, and the cells are then passed through a cell
sorting machine
equipped with a fluorescent detector. If the fluorescent light emitted by a
particular cell is
relatively strong, that will indicate that a certain cell took in large
quantities of labelled
polypeptide-DNA complexes, and a synchronized very brief jet of gas or fluid
can cause the
fluid stream carrying that particular cell to be diverted into a separate
collection device. In
this manner, cells which contain large quantities of "uptaken" DNA can be
isolated rapidly
and easily, using high-speed automated equipment, and can subsequently be
reproduced,
analyzed. or otherwise processed to evaluate or replicate the types of
polypeptides which
caused high levels of polypeptide-DNA uptake into the cells. This is just one
example of
how automated analysis and manipulation can be used to simplify and enhance
this
invention, and other methods will also become apparent to those skilled in the
art.
As yet another alternative, DNA-protein complexes may be prepared by coupling
a
DNA segment to a known type of capsid protein derived from adenovirus (or from
various
other viruses which act in a similar manner). This adenoviral capsid protein
is known to
help promote efficient release of the vector-carried DNA from the endocytotic
vesicle, after
the complex has entered the target cell. This approach is described in the
chapter by Curiel
(pp. 25-40) in Methods in Molecular Medicine: Gene Therapy Protocols (P.
Robbins, ed.,
1997). If desired, the adenoviral capsid protein sequence and the receptor-
binding ligand
sequence may be combined, in a fusion protein.
Nasal instillation of these types of receptor-targeting transfection vectors
can use the
same general procedures described in Examples 2 and 6; those general
procedures can be
adapted specifically for use with receptor-targeting ligand vectors, by means
known to those
skilled in the art. The methods described in Examples 3 and 4, and other
methods known to
those skilled in the art, can be used for post-transfection monitoring and
evaluation.
EXAMPLE 8: CONSTRUCTION AND USE OF TRANS-NEURONAL VECTORS TO
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DELIVER NGF GENE TO SECOND-ORDER NEURONS IN 'THE BRAIN
This example describes a method of developing "transneuronal" gene vectors,
which
may be able to transport foreign genes (rather than just polypeptides
expressed by foreign
genes) into certain classes of neurons that are located entirely within the
BBB. In other
words, the goal of such transneuronal gene vectors is to transfect, and
genetically
transform, not just neurons which straddle the BBB, but also other "secondary"
or "second
order" neurons which share synaptic junctions with transfected BBB-straddling
"primary"
neurons (and, potentially, tertiary neurons as well, which share synaptic
junctions with
secondary neurons). Transneuronal vectors may be able to greatly increase both
the quantity
and the distribution of new and/or supplementary polypeptides, which can be
secreted by
neurons that reside wholly within the BBB. Such treatment may become an
effective way to
treat certain neurodegenerative diseases characterized by widespread and
disseminated brain
damage, such as Parkinson's disease and Alzheimer's disease.
It should be recognized that these types of transneuronal vectors are not
expected to
replicate and produce multiple copies of themselves, after they enter
secondary (or tertiary,
or subsequent) neurons. Instead, the goal of such transneuronal vectors is
simply to place
these vectors in "downstream" neurons which are fully within the BBB, rather
than limiting
their placement to "primary" neurons which straddle the BBB. However, it is
recognized
that in some cases (which will become more probable and more frequent if
additional steps
are taken to promote integration of the gene sequences into chromosomes within
the
downstream neurons, by steps such as making use of terminal repeats derived
from adeno-
associated virus to bracket the transcription unit of the plasmid), this
approach may lead to
non-invasive methods of genetic therapy on CNS neurons that are entirely
within, and
protected by, the blood-brain barrier.
The currently anticipated approach to constructing transneuronal vectors
arises out of
various facts known about transneuronal transport of certain pathogens and
polypeptides,
including: (i) the "nontoxic fragment C" of tetanus toxin (e.g., Knight et al
1999); (ii)
barley lectin (Horowitz et al 1999); and, (iii) wheat germ agglutinin
(Yoshihara et al 1999).
Barley lectin and wheat germ agglutinin have been shown to undergo active
transneuronal
transport from olfactory receptor neurons, into basal forebrain cholinergic
neurons, and into
other classes of neurons as well, including neurons in the locus ceroleus and
raphe nuclei.
Other polypeptide sequences with transneuronal transport capability can also
be discovered
and created, using the same type of combinatorial selection strategy described
in Example
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, above; this can be done by repeated cyclic testing of phage display
libraries, to identify
,hages which have gene sequences that encode polypeptides which drive
transneuronal
ransport of phage into secondary (or tertiary, or subsequent) neurons.
Known methods can be used to construct a gene vector using a polypeptide which
is
mown to have transneuronal transport capability. For example, polylysine can
be covalently
)onded to a transport polypeptide, using a chemical reaction, or using a
genetically
nodified gene which encodes for numerous lysine (or other positively charged)
residues at
me end of the polypeptide. Since polylysine is positively charged, it will
attract DNA
;egments (which are negatively charged) when the polypeptide is mixed with DNA
3egments in solution. This leads to moderately strong but noncovalent binding
of the DNA
to the polypeptide. This type of preparation is described in Knight et al
1999.
Administration of these genetic vectors to olfactory epithelium, via nasal
instillation
of vectors suspended in an aqueous saline solution or other carrier liquid,
can use the same
general procedures described in Example 2, adapted for such use by means known
to those
skilled in the art.
The methods described in Examples 3 and 4 (supplemented when appropriate by
other or additional methods known to those skilled in the art) may be used for
post-
transfection monitoring and evaluation. Demonstration of expression of
transneuronally
transported NGF gene constructs within neurons inside the BBB (such as basal
forebrain
cholinergic neurons) can be done with sensitive procedures such as in situ
hybridization
and/or polymerase chain reaction (PCR). As described in Example 3, detection
of
expression will be facilitated by making use of NGF gene) sequences which
express
polypeptides that are distinguishably different from the corresponding
polypeptides in the
host species.
Additional monitoring of the physiological and behavioral effects caused by
such
transneuronal vectors can be done by procedures such as described in Example
4.
EXAMPLE 9: CONSTRUCTION AND USE OF VIRAL VECTORS TO DELIVER
ANTI-NGF POLYPEPTIDES INTO DORSAL HORN REGIONS IN THE SPINAL
CORD
While Examples 1-8, above, describe delivery of neurotrophic polypeptides such
as
nerve growth factor (NGF) to help stimulate neuronal growth or repair, this
Example 9 and
several following examples describe a completely different type of
polypeptide, referred to
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Lerein as an "anti-NGF" polypeptide. Such polypeptides, if they are
transported through the
3BB and delivered into properly targeted locations, can help treat and reduce
various
;onditions, such as neuropathic pain or autonomic dysreflexia. Such
polypeptides can work
)3T at least two known mechanisms: (i) by binding to NGF molecules in
cerebrospinal fluid
:CSF) or synaptic fluid, thereby inactivating the NGF molecules by rendering
them unable
,o bind to NGF receptors; and/or, (ii) by binding to NGF receptor proteins on
neurons,
hereby occupying those receptors and rendering them inaccessible to NGF
molecules, in a
manner which does not trigger the cellular reactions that occur when free NGF
molecules
oind to the receptors.
Anti-NGF polypeptides which bind to free NGF molecules in CSF or synaptic
fluid
are known, and are described in articles such as Ruberti et al 1993 and 1994.
Gene
sequences which encode these anti-NGF polypeptides are also known, and can be
incorporated into complete gene constructs having suitable gene promoters
which will drive
gene expression in mammalian cells, as disclosed in Example 1 above.
If such a gene construct is intended for expression in a nocioceptive neuron,
rather
than an olfactory receptor or other type of sensory neuron, attention must be
given to the
gene promoter that will be used to drive expression of the gene. A range of
potential gene
promoters that function in nocioceptive neurons are known, and suitable
promoters which
can drive desired levels of gene expression in nocioceptive neurons can be
selected from
those candidate promoters by routine experimentation. Preferred promoter
selection also can
be influenced by expression levels that occur naturally in different types of
neurons.
For example, by using a gene promoter which drives expression of the CGRP gene
(described in Watson et al 1995), expression of a vector-carried anti-NGF gene
can be
restricted to transfected nocioceptive neurons which express the neuropeptide
CGRP. The
CGRP promoter may be especially useful if one objective of a treatment is to
reduce the
level of NGF in the spinal cord of a patient suffering from neuropathic or
other chronic
pain, because the activity of this promoter is enhanced in the presence of
NGF. Thus, when
NGF levels in the spinal cord increase, this promoter may be able to increase
synthesis and
secretion of anti-NGF in response, thereby creating a self-limiting, self-
regulating gene
expression system.
A gene construct having an anti-NGF coding sequence and a selected promoter
can
be placed in a gene vector derived from replication-deficient adenoviruses,
using methods
such as described and cited in Example 1 and Ruberti et al 1993 and 1994, or
in a gene
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ector derived from replication-deficient herpes viruses, using methods such as
described in
)(ample 5 and the articles cited therein.
Since this example relates to methods of reducing and controlling NGF-
aggravated
Leuropathic pain, the anti-NGF vectors disclosed herein will not be inserted
into sensory
eceptor neurons such as olfactory neurons. Instead, these vectors will be
injected into
egions that contain high concentrations of nocioceptive nerves, including
subcutaneous
issues or muscle tissue in regions that are (i) plagued by neuropathic pain,
such as
tllodynia, and/or (ii) innervated by BBB-straddling nocioceptive neurons,
using methods
;uch as described by Sahenk et al 1993, or as determined to be optimally
effective during
31inical trials or during a treatment procedure involving a specific patient.
After injection into regions with nocioceptive nerve projections, at least
some of the
anti-NGF gene vectors will contact nocioceptive neuron projections outside the
BBB. Using
the same mechanisms that viruses normally use to inject DNA into cells during
infection,
the viral vectors (or analogous mechanisms used by non-viral vectors, as
described in
Examples 6-8) will inject the genetically-engineered vector DNA into the outer
projections
of the BBB-straddling nocioceptive neurons that innervate those tissues. Using
retrograde
transport, the vector DNA will be carried through the cell cytoplasm into
neuronal regions
called dorsal root ganglions, which flank the spinal cord. The gene which
encodes the anti-
NGF polypeptide will be expressed in the nocioceptive neurons residing within
the
ganglions (the plural form is also spelled "ganglia"), thereby creating anti-
NGF polypeptide
molecules within BBB-straddling neurons.
At least some of these anti-NGF polypeptides will be transported, using normal
anterograde transport, through the axons of BBB-straddling neurons, until they
reach
synaptic terminals or similar exit points which are located in "dorsal horn"
regions (and
possibly other regions) of the spinal cord, in tissue that is enclosed within
and protected by
the BBB. The efficiency of this anterograde transport step may be enhanced by
including a
leader or signal peptide, at the start of the secreted or mature polypeptide,
that will instruct
the transfected cell to package the polypeptide in vesicles containing
neurotransmitter
peptides destined for anterograde transport and release at synapses lying
within the BBB.
An example of such a leader sequence suitable for use in nocioceptive neurons
is the pre-
pro-BDNF sequence, which directs nocioceptive neurons to anterogradely
transport mature
BDNF (which these neurons normally synthesize) into dorsal horn regions within
the spinal
cord.
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When released by nocioceptive neuron terminals at these locations, the anti-
NGF
molecules will bind to NGF molecules, thereby reducing the amount of free NGF
in the
spinal tissue at that location. Reduced access to NGF does not kill mature
nocioceptive
neurons; however, it does lead to a reversible form of suppression (often
called "down
regulation") of nocioceptive functions and activity, as demonstrated by direct
administration
of anti-NGF into laboratory animals, as described in articles such as
Christensen et al 1996.
Anti-NGF may be detected in the spinal cord after allowing sufficient time for
gene
expression in the dorsal root ganglion (anticipated to be about 24 to 72
hours) and
anterograde transport and release of the anti-NGF into the dorsal horn regions
of the spinal
cord (anticipated to be another 8 to 24 hours).
In laboratory animals that have been sacrificed, the release of anti-NGF
polypeptides
in the spinal cord can be monitored directly with appropriate methods, such as
electron
microscopy, immunological staining, and various methods of labelling the
vector-derived
anti-NGF, such as inclusion of an appropriate antigenic tag in the amino acid
sequence of
the polypeptides encoded by the vector gene. It also may be possible to
monitor the
concentration and/or effects of anti-NGF by measuring the concentration of
free NGF.
When anti-NGF is released, it will bind to and neutrali 7e endogenous NGF in
the spinal
cord. Accordingly, changes in the level of NGF in the spinal cord can be
monitored by
making use of various immunological procedures, such as those outlined in
Example 3, and
applying these procedures to examination of the spinal cord tissue receiving
innervation
from the transfected sensory neurons. Changes in the level of NGF in the
spinal cord can
also be inferred from characteristic neuroanatomical and behavioral changes,
as outlined in
more detail in Example 10.
EXAMPLE 10: MONITORING PHYSIOLOGICAL AND BEHAVIORAL EFFECTS
OF ANTI-NGF DELIVERY INTO DORSAL HORN REGIONS IN THE SPINAL
CORD
To allow for statistical comparison of test results, experimental animals may
be
divided into two groups. One group (the test animals) will receive the anti-
NGF gene
vector, as described above, while the other group (the control group) will
receive a vector
containing noncoding DNA, an innocuous gene, or a marker gene that has no
known
neuroactive effect on CNS tissue. The physiological effects of administering
anti-NGF into
the spinal cord include effects on the neuroanatomy of the brain and effects
on the behavior
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)f the animal, as discussed in articles such as Christensen et al 1996 and
Christensen et al
,997.
Appropriate choice of animal model(s) is important in evaluating the
physiological
.ffects of anti-NGF delivery into the spinal cord. In addition to animal
models of
nflammatory pain, various models of hyperalgesia and "allodynia" (chronic
and/or
leuropathic pain, in which moderate stimuli are interpreted by the CNS as
severe pain) can
)e generated by injuring or challenging a peripheral nerve.
As just one example of a common method for modelling neuropathic pain, and for
testing potential treatments for neuropathic pain, a loop ("ligature") of
suture thread can be
surgically placed and then tightened around half to two-thirds of the sciatic
nerve, in one of
the hind legs of an animal such as a rat. Within several days to a week, the
leg will become
hyper-sensitized and susceptible to serious pain in response to mild stimulus.
Rats that have
been treated in that manner can then be placed in a special box with automated
sensors,
which can measure and record how many seconds pass after the floor plate
begins to warm
up, until the rat lifts the hyper-sensitive paw to get it off the warmed
surface. If a candidate
treatment can extend the average number of seconds that pass before nerve-
ligated paws are
raised, until those values approach the comparable times for unoperated
control animals,
that indicates that the treatment may be effective in reducing neuropathic
pain, and may
deserve more elaborate testing on larger animals, and/or clinical trials on
human patients.
It should be recognized, however, that peripheral nerve ligation or other
challenges
or injuries may interfere with retrograde transport of vector DNA from an
injection site to
the dorsal root ganglion, and may complicate the design and interpretation of
such tests for
use to evaluate the therapies disclosed herein. Accordingly, challenges which
directly affect
and involve the spinal cord may be preferred in at least some situations.
Chronic or
neuropathic pain due to spinal injury is frequently observed in clinics, and
over-production
of NGF within the spinal cord has been implicated in a condition known as
"primary
afferent sprouting", which often follows spinal cord injury (e.g., Krenz et at
2000).
Accordingly, spinal cord injury models which reproducibly causes hyperalgesia
or chronic
pain can be used to monitor the effects of anti-NGF delivery into the spinal
cord on
hyperalgesia or chronic pain. Examples include the spinal cord injury model
described in
Krenz et at 1999, which is believed to be suitable for evaluating the
physiological effects of
anti-NGF delivery into the spinal cord using the methods disclosed herein.
Neuroanatomical changes in the pattern of sensory innervation of the dorsal
horn
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.ssociated with hyperalgesia or chronic pain includes abnormal sprouting of
"A" fibers and
ocioceptive CGRP-containing fibers into lamina II of the dorsal horn (e.g.,
Bennet et al
.996). The effect of administration of an anti-NGF gene vector on such
neuroanatomical
lunges can be monitored by using the methods described by Bennet et al 1996 or
-2hristensen et al 1997, while using an agent such as cholera toxin B subunit
to
ransganglionically label the A fibers, and by using CGRP immunohistochemistry
to
iisualize the nocioceptive (NGF-responsive) fibers in the dorsal horn.
Alternately, the effect of delivery of anti-NGF on the number and extent of
synaptic
onnections between peripherally-projecting sensory neurons and second-order
neurons in
the spinal cord may be evaluated by transneuronal tracing methods, using
procedures such
as described in Blessing et al 1994.
Established methods for functional monitoring of pain responses in animal
models
include: (i) measuring foot withdrawal latency in response to thermal
stimulus, as briefly
summarized above and described in more detail in articles such as Romero et al
2000; (i)
measuring responses to application of von Frey hair stimulus, as described in
articles such
as Deng et al 2000.
Another condition that may be susceptible to treatment by anti-NGF or
comparable
polypeptides is often referred to as "autonomic dysreflexia". This condition
occurs most
commonly in patients with spinal cord injuries in the upper thoracic or
cervical region,
which disrupt the normal set of connections in the preganglionic sympathetic
neurons in the
upper spinal cord. As a result, the normal feedback control mechanisms in the
autonomic
nervous system can be disrupted, in ways that can cause a normal stimulus
(such as colon
distension, indicating fulybowels) to trigger other reflex results, some of
which can be
potentially life-threatening (such as increases in heart rate and blood
pressure, to levels
which can pose major risks of severe cardiovascular consequences, such as a
heart attack or
stroke).
As shown in animal model studies (e.g., Krenz et al 1999), direct infusion of
anti-
NGF into the spinal cord in animal models of this condition can prevent or
help control this
type of unwanted reflex response, by helping suppress abnormal and unwanted
innervation
of the dorsal horn by nocioceptive neurons, as can occur after that type of
spinal cord
injury.
Therefore, the use of this invention to introduce anti-NGF polypeptides into
spinal
regions that are protected by the BBB, by using genetic vectors to transfect
exposed and
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accessible projections of nocioceptive neurons (and possibly other spinal
neurons, including
motor neurons), holds substantial promise in treating autonomic dysreflexia.
EXAMPLE 11: CONSTRUCTION AND USE OF LIPID-BASED GENE VECTORS
TO DELIVER ANTI-NGF POLYPEPTIDES INTO DORSAL HORN REGIONS IN
THE SPINAL CORD
The procedures described in Example 9 and articles cited therein may be used
to
create an anti-NGF gene construct that will be expressed in one or more
targeted classes of
nocioceptive neurons. The resulting gene construct copies can be placed inside
a DNA
vector such as a plasmid or other stable form, using known methods. The DNA
vectors can
then be placed inside lipid Vesicles (liposomes), using methods such as
described in
Example 6 and in the chapter by Nabel (pp. 127-133) in Methods in Molecular
Medicine:
Gene Therapy Protocols (P. Robbins ed., 1997).
The resulting liposome vectors may be administered to peripheral projections
of
nocioceptive neurons by means such as injecting an aqueous suspension of
liposomes in
saline solution into regions that contain high concentrations of nocioceptive
nerves,
including subcutaneous tissues or muscle tissue in regions suffering from
neuropathic or
other chronic pain, using methods such as described by Sahenk et al 1993, or
as determined
to be optimally effective during clinical trials or during a treatment
procedure involving a
specific patient. At least some of the liposome vectors will adhere to and
merge with
nocioceptive neuron projections which are located outside the BBB, in the
muscle tissue,
and that reaction will deliver the anti-NGF encoding DNA which was carried by
the
liposomes into the neuronal projections. Retrograde transport of the DNA
segments will
carry at least some of the anti-NGF DNA to the neuronal cell body, where the
anti-NGF
genes will be expressed into anti-NGF polypeptides.
Subsequent delivery of the anti-NGF polypeptides into the spinal cord, and the
physiological and behavioral effects of the anti-NGF polypeptides in spinal
tissue, can be
measured and monitored using procedures such as described in Examples 9 and
10, above.
EXAMPLE 12: CONSTRUCTION AND USE OF DNA VECTORS THAT TARGET
ENDOCYTOTIC RECEPTORS ON NOCIOCEPTIVE NEURONS, TO DELIVER
ANTI-NGF POLYPEPTIDES INTO DORSAL HORN REGIONS IN THE SPINAL
CORD
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Various known methods, such as those described in Example 9, can be used to
create anti-NGF gene constructs that will be expressed in nocioceptive
neurons. Other
known methods, such as those described in Example 11, can be used to place the
anti-NGF
gene constructs into plasmid form or other stable forms that can be
transported into
nocioceptive neurons by non-viral vectors such as liposomes.
These stable DNA forms can then be used to form protein-DNA complexes, which
incorporate polypeptide segments that will bind in a specific manner, as
ligands, to
nocioceptive neuron receptors which undergo endocytosis, using procedures such
as
described in Example 7. Various such receptor-specific polypeptide segments
are already
known, and others can be identified and developed using the phage library
approach
described in Example 7.
Accordingly, these steps, when compiled together in proper sequence, will
create
genetic vectors that can specifically target endocytotic receptors on
peripheral projections of
nocioceptive neurons, in regions that are not enclosed within the BBB and
which therefore
provide relatively simple access to the peripheral projections. Such receptor-
specific
endocytotic gene vectors can be used to transfect such neurons with anti-NGF
genes that
will be expressed in those neurons, and the neurons themselves will then
deliver and secrete
the anti-NGF polypeptides in dorsal horn regions of the spinal cord, in a
manner which can
help control and reduce neuropathic pain and possibly other pain disorders.
Delivery of such anti-NGF polypeptides into spinal cord tissue can be
monitored
using procedures such as described in Example 9, and the physiological and
behavioral
effects of anti-NGF polypeptides in such spinal cord tissue can be evaluated
using
procedures such as described in Example 10.
EXAMPLE 13: TRANS-NEURONAL ANTI-NGF VECTORS THAT WILL
TRANSPORT ANTI-NGF GENE TO SECOND-ORDER NEURONS IN THE SPINAL
CORD
Gene constructs (including suitable gene promoters) that can express anti-NGF
polypeptides in nocioceptive neurons can be created as described in Example 9.
These gene
constructs can be placed in plasmids or other stable forms, using known
methods.
Such anti-NGF plasmids or other DNA vectors can then be coupled to
"transneuronal polypeptides" that can help enable and promote the transfer of
a protein-
DNA complex from one neuron, to another. Various such "transneuronal
polypeptides" are
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known, and include, for example, the "nontoxic fragment C" of tetanus toxin
(e.g., Knight
et al 1999), barley lectin (Horowitz et al 1999), wheat germ agglutinin
(Yoshihara et al
1999) (also listed in Example 8, above).
By binding reversibly to proteins that are (i) exposed at synaptic terminals
in the
periphery, and (ii) internally transported by the BBB-straddling neuron to
synapses within
the BBB, such "transneuronal polypeptides" can enable the transport of protein-
DNA
complexes through the cytoplasm of transfecteci "primary" BBB-straddling
nocioceptive
neurons, through the BBB, and into neuronal terminals located in the dorsal
horn, and
possibly elsewhere in spinal tissue. The transneuronal proteins will then help
the protein-
DNA complexes exit the synaptic terminals of the BBB-straddling nocioceptive
neurons,
and enter into adjacent spinal cord neurons that are located entirely within
the BBB, thereby
transfecting "secondary" spinal cord neurons protected by the BBB.
Delivery of such anti-NGF gene vectors and encoding sequences into BBB-
protected
"secondary" spinal neurons can be monitored by methods such as in situ DNA
probe
hybridization and PCR analysis, using spinal cord cells and tissue from
sacrificed lab
animals. Expression of anti-NGF polypeptides by transfected "secondary" spinal
neurons
can be monitored using procedures such as described in Example 9, and the
physiological
and behavioral effects of anti-NGF genes and polypeptides in such spinal cord
cells can be
evaluated using procedures such as described in Example 10.
EXAMPLE 14: USE OF ADENOVIRAL VECTORS FOR TRANSFECTING SPINAL
MOTOR NEURONS THAT WILL TRANSPORT NEUROTROPHIC POLYPEPTIDES
TO UPPER MOTOR NEURONS
Procedures for preparing non-pathogenic adenoviral vectors that cannot
replicate in
normal cells have been published in articles such as Graham and Prevec 1995.
Articles that
describe examples of such vectors which contain genes that encode various
neurotrophic
factor include Dijkhuizen et al 1997, Gravel et al 1997, Baumgartner et al
1998, and
Romero et al 2000.
Gene sequences which encode "glial cell line derived neurotrophic factor"
(GDNF),
and which have been isolated and put into conveniently-handled forms such as
plasmids or
adenoviral vectors, are described in articles such as Lindsay et al 1996, Choi-
Lundberg et
al 1997, and Baumgartner et al 1997, and in various other articles cited
therein.
Gene sequences which encode "neurotrophin-3" (NT-3) and which have been
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solated and put into conveniently-handled forms such as plasmids or adenoviral
vectors, are
lescribed in articles such as Snider 1994 and Bothwell 1995, and in various
other articles
ited therein.
GDNF and NT-3 are regarded as preferred candidates for initial testing for use
in
mating motor neurons as disclosed herein, since they appear to have relatively
strong
activity and effects (compared to other known neurotrophic factors) when
administered to
motor neurons in particular, as indicated by tests done in the prior art.
Numerous gene promoters that drive gene expression in motor neurons are known
and available, and can be used in tests such as disclosed herein. One class of
promoters that
may deserve particular attention for use to transfect spinal motor neurons
include the
promoters that drive expression of the so-called alpha-1 subunits of glycine
receptors, in
spinal motor neurons. Since glycine receptors are not present at high
concentrations in
nocioceptive or other sensory neurons, it is anticipated that use of one or
more types of
promoters derived from genes which express one or more subunits of such
glycine receptors
(or other genes that are expressed more actively in spinal motor neurons than
in sensory
neurons) may help increase selective expression of vector-borne neurotrophic
genes in the
desired target neurons, while minimizing potential adverse side effects that
might be caused
by unwanted expression in untargeted cells.
Another class of promoter that might be useful in some situations involving
spinal
motor neuron transfection drives the expression of a protein known as the
polio virus
receptor; this receptor protein is not present at substantial levels in
sensory neurons, so the
promoter is presumed to be silent in sensory neurons. However, it should be
noted that
many types of lab animals (including mice and rats) do not have polio virus
receptor genes
or promoters; therefore, it likely would be somewhat more difficult to carry
out various
types of research in animal models, if polio virus promoters are used.
Alternately, various types of viral and other promoters which are known to be
unusually strong promoters in mammalian cells can be used if desired, for
purposes such as
inducing the highest possible levels of expression of NGF (or other CNS-active
polypeptides) in transfected cells. Examples of such strong promoters include
the early gene
promoter from cytomegalovirus, and the late gene promoter from simian virus-
40. Inducible
gene promoters can also be used if desired, so long as the inducing factor
which activates
the selected promoter can be administered in a way which ensures that it will
be transported
into transfected neurons in adequate quantities.
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Accordingly, adenoviral vectors carrying neurotrophic genes that function in
motor
neurons, such as GDNF or NT-3, can be assembled, using various components and
methods as disclosed in the articles cited above.
If desired, an "epitopic tag" sequence can be incorporated into the coding
region of
the vector gene, to facilitate detection and monitoring of the polypeptide
encoded by the
vector gene in various tissues of test animals. This approach is discussed in
more detail in
Example 1, and in articles such as Moller et al 1998. If such an approach is
used, it is
important to confirm that the polypeptide expressed by the vector construct is
able to
undergo all steps that are necessary for proper delivery to the neurons that
are being
targeted for treatment by that polypeptide, using methods described in
articles such as Altar
& Bakhit 1991, Ferguson et al 1991, DiStephano et al 1992, and von Bartheld
2000.
Adenovirus vectors carrying the desired gene construct can be administered to
spinal
motor neurons via intramuscular injection, into a lower limb, of adenovirus
vector
suspended in a volume of solution compatible with adenovirus and tissue vigor
(such as
physiological saline solution). Methods for propagating, purifying,
concentrating, and
titering solutions containing adenoviral vectors can be found in publications
such as the
chapter by Engelhardt (pp. 169-184) in Methods in Molecular Medicine: Gene
Therapy
Protocols (P. Robbins, ed., 1997), and Haase et al 1998 provides information
on dosages
and administration techniques for efficient administration of adenovirus
vectors via
intramuscular injection, for transfer of genes into spinal motor neurons in
laboratory rats. If
desired, electromyographic injection procedures can be used to help ensure
that the fluid is
injected into the exact desired location.
EXAMPLE 15: MONITORING VECTOR GENES AND POLYPEPTIDES IN
TRANSFECTED MOTOR NEURONS, UPPER MOTOR NEURONS, AND BRAIN
TISSUE
Tests that use laboratory animals should be designed in a way that will
simplify the
tasks involved in, (i) measuring and monitoring the movement, locations, and
concentrations
of the genetic vectors and the vector-encoded polypeptides in various cells
and spinal cord
regions that are likely to be contacted and affected by the polypeptides; and
(ii) obtaining
reliable and useful statistical data which accurately reflect such results.
The necessary tasks
can be simplified and rendered more reliable by administering the gene vector
to the
muscles on one side of a test animal (such as injecting the vector solution
into a hind leg),
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and treating the other side of the same animal as a control, by using a
control vector which
carries, for example, an innocuous and/or non-functional gene, a nonsense DNA
sequence
which does not encode any polypeptide, or a marker gene which encodes a
polypeptide that
can be easily detected if expressed in mammalian cells, but which has no
significant
physiological effect. Upon subsequent histological processing, the left and
right regions of
the spinal cord (and the brain, if desired) can be compared against each
other, to assess the
movement, concentrations, and effects of the vector DNA and/or the
polypeptide(s) encoded
by the vector gene(s).
After allowing sufficient time for gene expression (24 to 72 hours), the
effectiveness
of gene vector delivery can be assessed by removing the lumbar spinal cord
from some
experimental animals and processing the tissue to monitor for expression of
the
neurotrophic factor gene within the spinal motor neurons. These types of
analyses can use
methods such as: (i) hybridization of cellular mRNA with DNA probes that are
complementary to the gene vector mRNA, but not to endogenous mRNA sequences,
using
procedures as described in articles such as Xian and Zhou 2000; (ii)
techniques which use
"polymerase chain reaction" (PCR) reagents and methods to detect DNA or mRNA
sequences from the viral vector, as described in articles such as Chie et al
2000; and, (iii)
immunostaining, ELISA, or similar methods which use antibodies that
selectively bind to
the vector polypeptide but not to the endogenous polypeptide in the test
species. Many such
antibody preparations are commercially available; alternately, if desired
(such as to detect
polypeptides having a specific "epitopic tag"), they can be prepared using
methods
disclosed in articles such as Conner 2000, Rush et al 2000, and Zhang et al
2000.
The goal of the procedures outlined in Examples 14 and 15 is to use
transfected
spinal motor neurons which straddle the blood-brain barrier to deliver
therapeutic
polypeptides to the upper motor neurons that lie protected wholly within the
BBB. This is
achieved by: (i) creating vector-encoded neurotrophic polypeptides, by
expressing the
vector-borne neurotrophin genes, and then, (ii) transporting and delivering
those
polypeptides to locations in the spinal cord which are protected from foreign
polypeptides
by the BBB, but which are accessible to axonal processes of upper motor
neurons.
To facilitate the process of identifying the exact regions within the spinal
cord where
such polypeptides are most likely to be delivered by transfected spinal motor
neurons,
"transneuronal labelling" studies can be carried out, using appropriate tracer
procedures and
reagents. Such studies which made use of the pseudorabies virus are described
in articles
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;uch as Card et al 1990. =
Transneuronal labelling can also be used to obtain evidence of delivery of
?olypeptides such as NT-3 or GNDF to the upper motor neurons resulting in
upper motor
aeuron sprouting and establishment of synaptic connections with the
transfected BBB
straddling lower motor neuron. The number of transneuronally labelled upper
motor
neurons in the brain and brain stern will be greater by virtue of an increased
strength and
number of synaptic contacts between upper and lower motor neurons.
Where a control and test vector are administered to opposite sides of the
experimental animal, appropriate differences between the left and right sides
of the spinal
cord will be observed. Severing the descending axonal tracts in some animals
can be used
to confirm that the NT-3 or GDNF detected in the brain in other experimental
animals was
derived from retrograde transport from transfected lower motor neurons.
EXAMPLE 16: MONITORING PHYSIOLOGICAL AND MUSCULAR EFFECTS
CAUSED BY DELIVERING NEUROTROPHIC FACTORS TO UPPER MOTOR
NEURONS
The physiological effects of administering neurotrophic factors such as NT-3
or
GDNF into the spinal cord or brain, using genetic vectors as disclosed herein,
include
effects on the neuroanatomy of the spinal cord and brain, and effects on the
physiology and
behavior of the animal. In particular, improvements and benefits in muscle
strength, muscle
control, and muscle tone that can be provided by using the genetic vectors
disclosed herein
can be evaluated using various methods.
These methods require a basic understanding of what has previously been seen
in
various prior tests using animal models. It must also be recognized that most
such tests are
necessarily carried out by first inflicting some sort of neuronal damage or
injury upon the
spinal cord or motor neuron system of an animal, then waiting for some period
of time for
the injury to be more fully manifested, then administering some sort of test
treatment (such
as direct infusion of a neurotrophic factor into the spinal fluid of the
animal, using a
hypodermic needle), and finally, by subsequent testing of the animal to
determine whether
the test treatment was able to reduce the extent of damage that was inflicted
by that same
type of injury in control animals or limbs. As summarized in Example 4,
comparative tests
usually involve either or both of the following: (i) different control
animals, or (ii)
treatment of the two different sides of the same animal in different manners.
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It should be recognized that many such tests use "axotomy", which refers to
urgically severing the axon. As noted in Example 4, the axon is crucial to a
neuron's
'unctioning, and over a span of time measured in days, if a neuron's axon has
been severed
n a location that is relatively close to the neuron's cell body, the neuron
likely will begin
o atrophy and will eventually die, even though the entire remainder of the
neuron is
mdamaged. The reasons for this are complex, and are generally believed to
involve cellular
Tactors (and especially neurotrophic factors) involved in nervous system
development.
kccording to the so-called "neurotrophic hypothesis", a developing brain in a
fetus initially
generates an oversupply of neurons, then a pruning process begins. During the
pruning
stage, neurons that do not actively continue to receive incoming nerve signals
(and/or are
riot contacted by one or more types of neurotrophic factors) die off, in a
form of
programmed cell death called "apoptosis."
Many animal studies have demonstrated that application of various neurotrophic
factors (usually by injection into spinal fluid or spinal tissue) can prevent
the type of
atrophy, degeneration, and death that can be induced in upper motor neurons by
axotomy
injury. Examples of such animal tests, and the results that arise when
neurotrophic factors
are applied to such neurons, are reported in articles such as Novikova et al
2000, Giehl and
Tetzlaff 1996, and Giehl et al 1997.
However, where the axotomizing injury is distant from lower motor neurons that
have been genetically transfected with foreign genes (as will occur if and
when the genetic
vectors disclosed herein are used in the manner disclosed in Example 14), it
will not be
easy to demonstrate that the vectors and genes of this invention had an effect
on axotomy-
induced atrophy or degeneration, because the axotomy injury will interfere
with the normal
retrograde axonal flow of neurotrophic factor within the transfected
peripheral motor
neuron. If the transfected lower motor neurons lie within a few millimeters of
the severed
end of the upper motor neuron, the upper motor neuron is more likely to have
access to the
neurotrophic polypeptide that is expressed and secreted by transfected motor
neurons.
Accordingly, appropriate experimental designs which use axotomy injuries
preferably
should use an axotomic locations that are within reasonably close distances to
the closest
tips of the motor neurons that can reasonably be transfected by using muscle
injections.
Administration of neurotrophic factors has also been shown, in published
reports, to
stimulate sprouting and regeneration of injured axons (e.g., Schnell et al
1994).
Accordingly, anterograde tract tracing studies, involving injection of tracer
compounds such
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as biotinylated dextran into the motor cortex (as described in Ferguson et al
2001) may be
used to study and demonstrate various effects (such as stimulation of
sprouting and
regeneration) when genetic vectors such as disclosed herein are used to
deliver factors such
as NT-3 or GDNF via lower motor neurons that lie within about 1 to 5 mm of the
severed
end of an injured upper motor neuron.
Use of the invention to deliver NT-3 or GDNF via lower motor neurons is also
expected to establish a chemoattractant gradient, where the highest
concentrations of the
attractant compound are likely to be centered near the transfected lower motor
neurons
(unless such molecules are rapidly cleared or dispersed, such as by active
uptake into other
cells). Regenerating injured motor neurons typically will respond to this type
of
chemoattractant gradient, by growing in the direction of increasing
concentrations of the
chemoattractant. By making use of anterograde tract tracing methods, such as
described in
Ferguson et al 2001, the influence of such chemoattractant gradient on the
directional
growth of the regenerating injured upper motor neurons may be evaluated and
demonstrated.
Establishment of a chemoattractant gradient emerging from transfected lower
motor
neurons is also likely to accelerate the rate at which regenerating injured
upper motor
neurons will form new synaptic connections with the neurons that are releasing
a
chemoattractant compound, such as GDNF or NT-3. This type of acceleration in
the rate of
synaptic connection can be demonstrated by undertaking time-course studies in
animal
models, and monitoring the time course of appearance of a transneuronal tracer
compound
in the upper motor neuron, if the tracer was injected into regions outside the
BBB. Suitable
transneuronal tracer methods are described in articles such as Ugolini 1995,
Travers et al
1995, and Card et al 1990.
Establishment of functional synaptic connections between upper and lower motor
neurons also will alter theelectrical behavior of the lower motor neurons. The
primary
effect of upper motor neuron activity tends to be inhibitory: therefore, when
this form of
inhibition is lost, lower motor neurons tend to enter a hyperactive status.
Accordingly,
restored or regenerated connections with upper motor neurons is expected to
reestablish
more normal levels of activity in the lower motor neurons, which will manifest
as more
normal sensory reflexes.
Electrophysiologica1 methods can be used to monitor the time course of such
changes, and can provide an indicator of whether functional synaptic
reconnections are
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)eing established between upper and lower motor neurons.
Establishment of functional reconnection between upper and lower motor neurons
will also result in observable changes in motor function, including muscle
strength, muscle
Lone, and coordination. The time course of change in muscle strength, and the
ability of an
animal to perform fine motor or coordinated motor tasks (such as by overcoming
challenges
in order to obtain food), can be tested and monitored by methods described in
the literature
and known to those skilled in the art.
EXAMPLE 17: USE OF LENTIVIRUS-DERIVED VECTORS FOR TRANSFECUNG
SPINAL MOTOR NEURONS THAT WILL TRANSPORT NEUROTROPHIC
POLYPEPTIDES TO UPPER MOTOR NEURONS
The methods described in articles such as Hottinger et al 2000 can be used to
construct
a lentivirus vector capable of carrying a gene construct which has (i) a GDNF,
NT-3, or
other neurotrophic polypeptide coding sequence, and (ii) a gene promoter
suited for gene
expression in transfected spinal motor neurons, as described in Example 14.
This type of
lentivirus-derived gene vector may be administered to the accessible
projections of spinal
motor neurons via intramuscular injection, using procedures such as described
in Example
14, above.
After transfection of the spinal motor neurons, expression of the encoded
polypeptide in the transfected neurons, and delivery of the polypeptide into
spinal cord or
brain tissue piotected by the BBB, can be measured and monitored by methods
such as
described in Example .15, above. Muscular and other physiological effects of
the
neurotrophic polypeptide, and the ability of the neurotrophic polypeptide to
prolong
neuronal survival following axotomy or similar challenge, can be measured and
monitored
by methods such as described in Example 16, above.
EXAMPLE 18: PREPARATION AND USE OF LIPOSOME VECTORS FOR
TRANSFECTING SPINAL MOTOR NEURONS THAT WILL TRANSPORT
NEUROTROPHIC POLYPEPTIDES TO UPPER MOTOR NEURONS
As noted in Example 6, methods for preparing DNA plasmid-lipid complexes for
transfecting mammalian cells are described in publications such as the chapter
by Nabel
(pp. 127-133) in Methods in Molecular Medicine: Gene Therapy Protocols P.
Robbins, ed.,
1997), and can be adapted for cell types or specialized uses by routine
testing of various
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preparative mixtures.
Liposomal vectors carrying neurotrophic factor genes may be administered to
the
accessible projections of spinal motor neurons via intramuscular injection,
using procedures
such as described in Example 14, above. After transfection of the spinal motor
neurons,
expression of the encoded polypeptide in the transfected neurons, and delivery
of the
polypeptide into spinal cord or brain tissue protected by the BBB, can be
measured and
monitored by methods such as described in Example 15, above. Muscular and
other
physiological effects of the neurotrophic polypeptide, and the ability of the
neurotrophic
polypeptide to prolong neuronal survival following axotomy or similar
challenge, can be
measured and monitored by methods such as described in Example 16, above.
EXAMPLE 19: CONSTRUCTION AND USE DNA VECTORS THAT TARGET
ENDOCYTOTIC RECEPTORS ON SPINAL MOTOR NEURONS THAT WILL
TRANSPORT NEUROTROPHIC POLYPEPTMES TO UPPER MOTOR NEURONS
Reagents and procedures for preparing genetic vectors using ligands which bind
to
endocytotic receptors on neurons are discussed in Example 7, above. Those
procedures
include methods for using monoclonal antibodies, or repeated selection cycles
involving
phage display libraries, to identify and create high-affinity ligands that
will actively bind to
endocytotic receptors on spinal motor neurons.
As introduced in Example 7, ligands to the p75 receptor can be used to target
gene
delivery to the spinal motor neurons. While spinal motor neurons normally
express only
low levels of p75, these spinal motor neurons upregulate their expression of
p75 in
response to injury or deprivation of neurotrophic factors, and in various
diseases such as
amyotrophic lateral sclerosis. Therefore, a p75 targeting gene vector enables
enhanced or
targeted delivery of genes encoding therapeutic proteins to spinal motor
neurons in need of
therapeutic support.
The efficiency of gene vectors which target spinal motor neurons can be
enhanced
by including, in the vector construct itself or in an injectable solution
which contains the
gene construct, a compound that can concentrate the gene vector at the "motor
endplate" in
an injected muscle. For this purpose, botulinum toxin may be useful;
alternately, a
monoclonal antibody may be generated, using a 17-amino acid peptide sequence
from the 0/1
subunit of the acetylcholine receptor as the antigen (see Yoshikawa et al
1997). In a disease
called myasthenia gravis, a patient develops antibodies against this epitope
of the
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acetylcholine receptor, and these antibodies are localized on the muscle
endplate in the
synaptic cleft.
Accordingly, "re,ceptor-targeting" gene vectors which use such receptor-
binding
ligands and possibly other enhancements can be used to carry gene constructs
that have a
GDNF, NT-3, or other neurotrophic polypeptide coding sequence, and a gene
promoter
suited for gene expression in transfected spinal motor neurons, as described
in Example 14.
Administration to spinal motor neurons via intramuscular injection of ligand-
DNA
complexes can use procedures such as described in Example 14, above. After
transfection
of the spinal motor neurons, expression of the encoded polypeptide in the
transfected
neurons, and delivery of the polypeptide into spinal cord or brain tissue
protected by the
BBB, can be measured and monitored by methods such as described in Example 15,
above.
Muscular and other physiological effects of the neurotrophic polypeptide, and
the ability of
the neurotrophic polypeptide to prolong neuronal survival following axotomy or
similar
challenge, can be measured and monitored by methods such as described in
Example 16,
above.
EXAMPLE 20: TRANSFECTION OF SPINAL MOTOR NEURONS USING
TRANS-NEURONAL VECTORS TO DELIVER NEUROTROPHIC FACTOR GENES
TO CNS NEURONS IN CONTACT WITH THE SPINAL MOTOR NEURONS
Genetic vectors that may have "transneuronal" transport capability are
described in
Example 8, above. Such vectors designed for use in transfecting spinal motor
neurons can
be assembled using methods such as described in Example 8, and can carry gene
constructs
such as described in Example 14, above. Administration to spinal motor neurons
via
intramuscular injection can use procedures such as described in Example 14,
and post-
transfection monitoring can use methods such as described in Examples 15 and
16.
EXAMPLE 21: DELIVERY OF NEUROTROPHIC FACTORS INTO THE
BRAINSTEM, BY INJECTION OF VECTORS INTO THE TONGUE TO
TRANSFECT LOWER MOTOR NEURONS OF THE HYPOGLOSSAL NUCLEUS
Genetic vectors derived from adenoviruses, herpes viruses, or lentiviruses, or
using
cationic liposomes, ligands that bind to endocytotic receptors, and/or trans-
neuronal
polypeptides, can be created as described in the examples above. These types
of vectors can
carry neurotrophic gene constructs (or, if desired, gene constructs that
express recombinant
antibodies or similar polypeptides that inhibit one or more neurite inhibitory
molecules such
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.s 1N1 or No-Go) which will be expressed in transfected motor neurons, as
described in the
bregoing examples.
As illustrated in FIG. 6, such vectors can be used to transfect a certain
class of
ower motor neurons which have projections that are accessible outside the BBB,
in the
ongue. These neurons, known as "motor neurons of the hypoglossal nucleus," are
;ynaptically connected to other neurons in certain regions in the brainstem.
Therefore, the
notor neuron terminals inside the tongue offer a relatively direct passageway
for delivering
leurotrophic polypeptides into neurons of the brainstem that synapse with or
have
projections that lie near these transfected lower motor neurons.
Administration to these motor neurons can be achieved via injection into the
tongue,
using general procedures such as described in Example 14 but adapted for
injection into the
muscles of the tongue. Post-transfection monitoring in test animals can use
general methods
such as described in Examples 15 and 16, appropriately adapted to monitor for
delivery of
exogenous peptides (such as epitope-tagged NGF, NT-3, or GDNF) to neurons in
the brain
stem, as well as other methods known to those skilled in the art. As one
example, for use
in animals, various transneuronal tracer methods, such as described by Ugolini
1995,
Travers et al 1995, or Card et al 1990, can be used to label neurons in
brainstem which
make contact with transfected lower motor neurons in the hypoglossal nucleus.
By counting
the number of second-order neurons which contact the hypoglossal nucleus in
animals
treated with the invention, and comparing such data to numbers seen in control
animals,
evidence can be obtained of the delivery of neurotrophic factor to second-
order neurons in
the brainstem.
As another example of a monitoring method which may be useful in human
patients,
hyperexcitable reflex blinks are a symptom of Parkinson's disease, which
correlates
clinically with the severity of the disease. Tonically active serotonergic
"raphe neurons"
normally inhibit the spinal trigeminal neurons involved in reflex blink
circuits, and
deterioration of these serotonergic raphe neurons leads to the hyperexcitable
blink reflex
(e.g., Basso and Evinger 1996). Accordingly, evidence of the effectiveness of
clinical use
of the invention to reduce the rate of degeneration of serotonergic neurons of
the brainstem
can be obtained by monitoring the hyperexcitable blink reflex in patients
suffering
Parkinson's disease.
Another of method for monitoring preservation of brainstem neurons in human
patients with Parkinson's disease involves taking measurements of a patient's
swallowing
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:eflex, The swallowing reflex involves a coordinated movement of tongue and
other oral
ind respiratory muscles and, in Parkinson's disease, the time between stimulus
and reflex
swallowing is abnormally prolonged. A swallowing reflex test involves delivery
of a small
volume of water into the throat, via intranasal catheter, as the swallowing
reflex stimulus.
The initiation of swallowing reflex can be accurately measured using surface
electrode
electromyographic recording of the muscles in the throat and used to calculate
the time
between stimulus and reflex swallowing (Iwasaki et al 2000). Accordingly,
evidence of the
effectiveness of clinical use of the invention to reduce the rate of
degeneration of neurons of
the brainstem can be obtained by monitoring the swallowing reflex in patients
suffering
Parkinson's disease.
EXAMPLE 22: IN VIVO SCREENING: IMAGE TYPES AND LEBRARTES
M13K07 helper phages can be purchased from various commercial suppliers,
such as New England Biolabs and Amersham Biosciences. This strain of helper
phage
contains fully functional genes that encode both the pIII and pVIII coat
proteins. It also
contains an origin of replication (from plasmid pl5a) which is tightly
controlled in a
manner that results in low copy numbers in bacterial cells. It also contains a
mutated
(Met-40-Ile) copy of the phage M13 pII gene, which is essential for phage
replication;
this mutation causes it to be secreted by E. coil cells, as phage particles,
in low copy
numbers. It also carries a kanamycin resistance gene, inserted at the Ava I
site within the
M13 origin of replication. This kanamycin gene functions as a selectable
marker in E.
coil host cells that are not resistant to kanamycin. Additional information on
using and
culturing these helper phages is available from commercial suppliers, and in
various
published articles describing their use.
The ScFv phage library was supplied by Cambridge Antibody Technology. It is
described in various patents (such as US 6,172,197) and published articles. It
was
created by inserting gene sequences obtained from B-lymphocyte cells (which
create
antibodies) into the gene sequences that encode the pIII coat protein (which
is located, in
relatively small numbers, at one end of filamentous M13 phage particles). Each
foreign
gene sequence in the scFv library contains both the "heavy variable" (VH) and
"light
variable" (VI) domains of a single antibody, in a single gene sequence that
will express
the VH and VI, domains in a "single chain" (Sc) polypeptide, having an average
molecular
weight of about 35 kilodaltons. The seFv library
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has an estimated 13 x 109 (i.e., 13 billion) different recombinants. TO ensure
maximal
diversity, it contains "variable fragment" (Fv) antibody domains obtained from
numerous
people of different ancestries. The library is estimated to encode a range and
diversity of
different Fv antibody domains that could be generated by the immune systems of
ten
different people from a varied assortment of racial and ethnic groups.
It should also be noted that the scFv phages are phagernids. They have a
bacterial
origin of replication, which causes them to reproduce in high copy numbers, as
double-
stranded plasmids, in E. coli cells. They also contain a phage origin of
replication, which
can trigger the synthesis of single-stranded DNA for assembly into phage
particles;
however, that ssDNA synthesis requires a phage ssDNA transcribing protein to
be present,
and scFv phages do not encode that protein. That ssDNA transcribing protein
must be
supplied by helper phages, such as the M131(07 helper phages mentioned above.
Therefore, when M131(07 helper phages are used to coinfect E, coli cells that
have
already been infected by scFv pliagernids, the addition of the ssDNA
transcribing protein
(from the helper phages) to cells that already contain large numbers of dsDNA
plasmids
(from the scFv phagemids) will trigger the formation of large numbers of ssDNA
strands,
from the scFV phagemid plasmids. These newly formed ssDNA strands will then be
packaged inside coat proteins (mainly pVIII coat proteins from a particular
scFV clone
which infected that host cell). The newly packaged ssDNA and its coat proteins
will
secreted by the host cell, as filamentous phage particles. Most of these
secreted phage
particles will contain scFv phagemid DNA, rather than M131(07 helper phage
DNA, since
the helper phage DNA sequences will be present in the host cells only in low
copy numbers
(due to the low-copy-number plasmid p1 5a origin of replication in the helper
phage DNA).
The pVIII coat proteins in the phage particles that are secreted by a some
particular
E. coli host cell will contain clonal copies of some particular antibody
"variable fragment"
polypeptide sequence, which was encoded by a DNA sequence that was obtained
from a
human B-lymphocyte. This human antibody DNA sequence was inserted into the
scFv
phagemid DNA at a controlled and targeted site, near the middle of the
phagemid gene that
- encodes the pVill coat protein.
The Ph.D-C7C phage display library was obtained from New England Biolabs.
This library contains an estimated 2 x 109 different recombinants, with
foreign DNA
inserts encoding random sequences of seven amino acid, inserted near the DNA
sequence
that encodes the N-terminus of the pIII coat protein of
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413 phages. This library provided an essentially random repertoire ot peptide
sequences
hat could be tested, to determine whether certain phages would be internalized
and
ransported by neurons in the sciatic nerve bundle.
EXAMPLE 22: CELL TYPES, TRAITS, AND METHODS
Except as otherwise noted, all phage amplification and titering used the TG1
strain
)1. E. coil, from Cambriage Antibody Technology. This strain, which was
specifically
iesigned and developed for working with M13 phages, is also sold by companies
such as
Stratagene (La Jolla, California; www.stratagene.corn). Additional information
describing
culturing and transformation methods for this strain can be downloaded at no
cost from the
web sites of commercial suppliers.
Among ether features, the TG1 strain has a "lacIq" repressor gene which,
together
with catabolite repression by glucose, negatively regulates a "lac" promoter
that has been
placed in control of expression of the M13 gene that encodes the pIII phage
coat protein.
Most types of M13 phages that are used with TG1 cells contain an "amber" stop
codon,
inserted at the start of the pill gene. As described below, this allows
expression of pill
polypeptides (inCluding chimeric pIII polypeptides that contain foreign amino
acid
sequences) in soluble form, in a non-suppressing E. con strain such as HB2151,
without
having to redone the gene.
The "amber" stop codon can be effectively inactivated by transferring TG1
cells into
culture medium that contains no glucose, and that instead contains lactose (a
particular type
of sugar molecule) as the sole source of carbon for the bacteria. A compound
called iso-
propyl-thio-galactopyranoside (IPTG) is also added; this potently induces
expression of the
"lac" operon, which enables the host cells to metabolize lactose molecules as
a nutrient. In
addition to enabling transformed cells to grow in media with lactose as a sole
carbon
source, it also enables transformed cells to convert a chemical called X-Gal
into a bright
blue color, so that transformed colonies can be easily identified and
isolated, on agar plates.
In a typical procedure used to "amplify" (reproduce) a particular assortment
of
phages (such as after an in vitro panning or in vivo selection procedure as
described
herein), a colony of TG1 cells that had been grown on an agar plate was used
to inoculate
liquid culture media which contained 16 g tryptone, 10 g yeast extract, and 5
g NaC1 per
liter (this type of liquid culture media, containing tryptone and yeast
extract, is referred to
as 2TY media). The cells were replicated in a sbaldng incubator to an optical
density
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(0D600, measured at a light wavelength of 600 nanometers) of about 0.5 to 0.8
units (all
incubations were done at 37 C, unless otherwise indicated). A phage
preparation was added
to the E. colt culture, and the mixture was incubated. Initial incubation was
carried out in
stationary conditions, for 30 minutes, to facilitate binding of the phages to
the bacterial
cells. This was followed by 30 minutes in a shaking incubator running at 200
rpm, to
ensure maximal exposure of the cells to fresh nutrients.
These cells were then centrifuged at 3500 rpm for 10 minutes, and the
supernatant
containing old broth and metabolites was discarded. The cell pellet was
resuspended in 500
microliters (AL) of fresh 2TY culture broth, and the mixture was spread across
the surfaces
of four fairly large (24.3 cm x 24.3 cm) square plates containing 2TY agar
media with
ampicillin and glucose. The plates were incubated overnight at 30 C. Because
ampicillin
was present, only E. coli cells that contained scFv phagemids or PhD-C7C
phages gave rise
to colonies on the plates.
The following day, to complete the preparation of standardized phage solutions
that
could be frozen until needed, colonies were scraped from each agar plate into
10 mL of
2TY broth, in a 50 mL tube. A half-volume of sterile 100% glycerol was added,
and the
solution was mixed by placing the tube in an end-over-end rotator for 10
minutes at room
temperature. 1 mL aliquots were frozen at -70 C for storage.
When a batch of phages was needed for a test, a 1 mL aliquot of the glycerol-
containing stock was thawed, and 100 AL of the thawed stock was added to 25 mL
of 2TY
broth containing 2% (w/v) filter-sterilized glucose and 100 mg/mL ampicillin.
The cells
were grown at 37 C in a shaking incubator until they reached an 0D600 density
of about 0.5
to 0.8. M131(07 helper phages were then added, to form a fmal concentration of
5 x 109
"colony forming units" (cfu) per mL. The mixture was incubated for 30 minutes
while
stationary, then for 30 minutes in a shaker tray at 200 rpm. The cells were
then centrifuged
at 3500 rpm for 10 minutes, and the cell pellet was resuspended in 25 mL
prewarmed 2TY
(without glucose) containing kanamycin (50 p,g/mL) and ampicillin (100
lAg/mL). These
were incubated overnight at 25 C, with rapid shaking, to produce phage
particles.
The phage particles were purified from the supernatant by precipitation with
20%
polyethylene glycol (PEG) and 2.5 M NaCl. These particles were then
resuspended in a
final volume of 1.5 mL of sterile phosphate buffered saline (PBS) at about 4
C.
To "titer" a solution that contains phage particles (i.e., to obtain an
estimate of how
many infective phage particles were present in each mL of solution), TG1 cells
were grown
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n 2TY media, in a shaking incubator at 300 rpm for about 4 hrs, until an OD600
density or
%bout 0.5 to 0.8 was reached. A sample of phage supernatant was serially
diluted at 10-fold
iilutions, in 2TY media, by adding 50 pi of each dilution in the series to a
450 ttl.,
suspension of TG1 cells in an EppendorfTm tube. The tube was incubated
stationary for 30
minutes, followed by shaking at 300 rpm for 30 minutes. 100 AL of each
dilution of the
infected TGI cells were streaked onto prewarmed 2TY agar plates (2TY media
containing
100 pg/ml, ampicillin, 2% w/v filter-sterilized glucose, and 1.5% w/v agar).
The plates
were incubated overnight, and the following day, the number of colonies were
counted.
TG1 cells could grow on ampicillin-containing media only if they carried
ampicillin
resistance genes from a phage.
EXAMPLE 24: CROSS-LINKING OF P75 RECEPTOR-BINDING ANTIBODIES
(MC192) TO M13K07 HELPER PHAGES
A monoclonal antibody preparation known as MC192 (and by similar terms, such
as
clone 192; originally described in Chandler et al 1984) is commercially
available from
various suppliers, such as Cell Sciences and Chemicon. The monoclonal
antibodies bind
to "low affinity" (p75) nerve growth factor receptors on rat neurons.
Monoclonal
antibodies that bind to human p75 receptors are also available, from companies
such as
United States Biological.
Unlike various other monoclonal antibodies that also bind to p75 receptors in
rats,
the MC192 antibody can trigger endocytosis of the antibody-receptor complex,
leading to
neuronal uptake of the MC192 antibody. This has been shown by studies using
radiolabelled antibodies (Johnson et al 1987, Yan et al 1988).
To evaluate the ability of the MC192 antibody to drive endocytosis of phages
into
rat neurons, a preparation was made, containing MC192 antibodies that were
chemically
crosslinked to M13K07 helper phages, using a multi-step process. First,
sulfosuccinimidy1-
4-(N-maleimidomethyl) cyclohexane-I-carboxylate (abbreviated as sulfo-SMCC;
purchased
from the Pierce company, Australia) was reacted (through the active sulfo-NHS
ester end)
with primary amine groups on the MC192 antibody. This resulting in the
formation of an
amide bond between the antibody and each cross-linker group, with sulfa-NHS
being
released as a byproduct. To remove unreacted sulfo-SMCC, the activated
antibody was
purified using Microcon Ylv1-100 centrifugal filter (100 kilodalton cut-off;
catalog number
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42412, Millipore Corporation, USA).
In the next phase, M13K07 phages were incubated with 2-iminothiolane (2-IT;
also
called Traut's reagent) to generate free sulfhydryl groups; these groups are
positioned at the
ends of short chains that are bonded to lysine residues, in the pVIII coat
protein of the
phages. The phages were filtered, using YM-100 kDa filters, to remove excess
reagent.
The antibody preparation was then mixed with the phage preparation, at a 1:10
ratio, to form thioester cros slinking bonds, from the maleimide groups on the
activated
antibodies and the sulfhydryl groups on the activated phages. The reaction
product was
filtered to remove excess antibodies, and iodoacetamide (Sigma Chemical) was
added to
block any remaining free reactive sulfhydryl groups. The reaction product was
precipitated
twice in PEG/NaC1 (20% w/v polyethylene glycol, average molecular weight 8000,
in
water with 2.5, molar NaC1) to remove free antibodies.
;
The resulting phage mixture contained various numbers of MC192 antibodies,
located randomly along the length of the phage. Because of the 10:1 ratio of
antibodies to
phages in the reaction mixture, it was presumed and estimated that on average,
from about
2 to about 20 antibodies were bonded to most phage particles.
EXAMPLE 25: SURGICAL TREATMENT AND PHAGE EMPLACEMENT IN RATS
Female Sprague-Dawley rats were used, and surgeries were performed under
halothane anaesthetic (2% in oxygen, administered by nose cone connected by
tubing to
anesthetic machine). Alternatively, longer-acting injectable anesthetics such
as sodium
pentobarbitone may be used if desired.
Certain comments are offered below, about preferred procedures for doing this
type
of surgery on rats, since the use of correct procedures will substantially
increase the
likelihood of success. Many people who are quite familiar with cells and
phages may not be
familiar with small animal surgery, as is necessary to carry out the in vivo
procedures of
this invention.
It should also be noted that whenever someone is doing this type of surgery
for the
first time, a binocular microscope that can provide up to 20x magnification is
almost always
used, during training, to help focus the vision and attention on important
sites and aspects
of the procedure. After conducting a procedure a few times, a technician can
choose
whether or not to use a microscope during subsequent procedures (or during a
delicate or
difficult part of a procedure, such as suturing the two ends of a sciatic
nerve together, after
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)lacing the phage-containing gel foam between the nerve ends). If mice are
used, their
;mailer size might dictate use of a binocular microscope for all procedures,
until a
echnician develops a fairly high level of experience and familiarity with the
procedures.
When the rats were 6 weeks of age, an initial surgery was performed, to
"upregulate" (increase) expression of the p75 cell receptors. If the sciatic
nerve is injured in
this manner, the motor neurons (which have their cell bodies in the spinal
cord, and axonal
fibers projecting through the sciatic nerve) are stimulated to express
increased numbers of
p75 receptors on their cell and axonal surfaces. Tests that were conducted to
compare the
differences between phage uptake by pre-ligated neurons, versus phage uptake
by
non-ligated neurons, indicated that the pre-ligation step increased p75
receptor density and
phage uptake by roughly 13-fold. These tests included phage uptake tests, as
well as
staining (using MC192 antibodies) of tissue sections taken from the lumbar
regions of spinal
cords of rats.
Two other factors should also be noted about p75 receptors, in rats. First, it
is
present in substantial numbers on the surfaces of motor neurons that originate
in the spinal
cord, and that send out axons or other neuronal fibers into muscle tissues
that are not
enclosed within the blood-brain barrier. This includes sciatic nerves;
however, the p75
receptor is present in substantial copy numbers, on sciatic nerve surfaces,
for only about
two weeks after a rat is born, while the rat is growing rapidly. After about
one to two
weeks, its copy number on sciatic nerves drops off, and by the time rats are
about 6 weeks
old, it is present on sciatic nerve surfaces only in very low and often
undetectable
quantities.
The second notable factor is this: since p75 receptors are not abundant on
sciatic
nerve surfaces in rats that are 3 weeks old or older, even after a controlled
injury has been
inflicted on a sciatic nerve, the p75 receptor endocytosis system can be
saturated, fairly
easily. It apparently was saturated, on a number of occasions, during the in
vivo screening
tests described herein. However, rather than invalidating any of the results
disclosed herein,
this factor should be regarded more as a "ceiling" value, which cannot be
exceeded.
Accordingly, these saturation limits can be approached and utilized in ways
that appear to
confirm and validate the mechanisms and effects that are believed by the
Applicants to be
active in these types of in vivo screening tests using p75 receptors.
During the initial surgery, there is no need to use any mechanical restraint.
The
animal is laid op its side with the hindlimb uppermost, fur shaved and skin
swabbed. A 1 to
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cm midthigh skin incision in parallel with the femur is made, using surgical
scissors or
calpel. Using the tip of closed surgical scissors (blades 2 to 4 cm long), the
femur is
ocated by palpation, and the point of the scissors is pushed, just caudal to
the femur,
hrough the muscle layers to a depth of 1 to 2 cm, depending on the size of the
animal. The
Icissors are then opened, to separate the muscle with minimal bleeding, and to
create a 1 to
cm window which exposes the sciatic nerve lying beneath the muscle. Retractors
or
artures can be used to hold open the muscle and maximize the window of
operation, but
his may not be required by an experienced technician.
By using this entry procedure, the sciatic nerve can be clearly seen. The
sciatic
ierve is only loosely attached to the surrounding tissues, by membranes that
are easily
separated. The nerve itself is protected by a tough nerve sheath, and an
estimated 50,000
axons may be contained within this nerve bundle, depending on the location.
While the
axons of some sympathetic or other nerve may not be myelinated, each motor
axon (and
most sensory nerve axons) is surrounded by a myelin sheath, contributed by
Schwann cells,
which make up the bulk of nerve tissue mass outside the blood brain bather.
A ligature is emplaced by inserting a pair of curved forceps under the sciatic
nerve,
and used the forceps to gently lift the nerve and free any loosely adhering
membranes, if
present, from a 1 to 2 cm length. The forceps are opened and used to grasp a
length of 6/0
silk suture, which is then pulled under the nerve by withdrawing the forceps.
The suture,
which is placed at a site slightly above the location where the tibial branch
bifurcation
divides the sciatic nerve bundle into two smaller bundles, is then tied
tightly around nerve
to ligate it. It is important to use non-resorbable sutures, such as silk or
nylon. Black silk is
generally preferred, since it is less elastic (making tight ligations easier
to secure), and
because a black ligature is more easily located during a subsequent operation.
The instruments are withdrawn, the separated muscles are allowed to rejoin,
and the
skin incision is closed with 1, 2 or 3 sutures, depending on the length of the
incision. The
animal is then allowed to recover from anesthesia.
Seven days later, the sciatic nerve was exposed again, and a 2 to 3 mm section
of
the sciatic nerve which contained the ligature was excised, using a pair of
surgical scissors.
Roughly 9 cubic millimeters of a collagen matrix gel foam, containing 10 Al of
the
MC192-M131(07 antibody-phage conjugate (with titers ranging from about 3.3 x
106 to
about 2.1 x 109 cfu/mI.) was inserted between the two transected ends of the
nerve bundle.
The free ends of the sciatic nerve were sutured together, flanking the gel
foam that
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contained the phages, using a 10-0 nylon surgical suture. A small flexible
sleeve of silicone
rubber was placed around the nerve ends and the gel foam containing the
antibody-phage
conjugates, to ensure that the antibody-phage conjugates in the gel foam would
remain in
direct contact with the ends of the nerve fibers.
During the same surgery when the transection and phage emplacement were made,
the sciatic nerve bundle was ligated, using a 6-0 silk suture, at a location
about 2 cm above
the transection site, near the rat's hip. This ligature is referred to herein
as the hip ligature,
to distinguish it from the initial ligature that was used to increase p75
receptor expression.
The hip ligature created a constriction point that prevented antibody-phage
conjugates that had been taken into sciatic neurons from being retrogradely
transported all
the way to the spinal cords of the neurons. Therefore, antibody-phage
conjugates
accumulated, inside the nerve fibers, at a location that was just below
(distal to) the hip
ligature.
After a delay of 18 hours, to allow enough time for endocytotic uptake and
retrograde transport, the rat was sacrificed by chloroform inhalation, and a
nerve segment
distal to the hip ligature was harvested, as disclosed in the next example.
EXAMPLE 26: NERVE HARVESTING AND INTERNALISED PHAGE
COLLECTION
As mentioned in the prior example, a rat was sacrificed 18 hours after: (i)
emplacement of the collagen gel containing the phage particles, and (ii)
emplacement of the
hip ligature.
A nerve segment which included the hip ligature and a segment of nerve fibers
just
distal to that ligature was harvested. This was done by emplacing and
tightening an
additional ligature around the sciatic nerve, about 0.5 cm below (distal to)
the hip ligature,
to prevent any loss of the phage particles from either of the cut ends of the
nerve bundle. A
segment of the sciatic nerve bundle, which contained both of the two ligature
loops still tied
tightly around both ends of the segment, was then cut out and removed.
The excised nerve bundle was scrubbed 3 times with sterile PBS, using forceps
with
sterilized tissue paper, until the outer membrane was removed. The neurons
were then
transferred onto a dry glass plate, and the ligatures were removed.
450 L of a lysis buffer (which digested cell membranes but not bacteriophage
particles) was then applied, containing 1% Triton X-100, 10 mM Tris, and 2 mM
EDTA at
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pH 8, and also including 1/100 (by volume) of a protease inhibitor mixture
(containing
4-(2-amino-ethyl)-benzenesulfonyl fluoride, pepstatin A, E64, bestatin,
leupeptin, and
aprotinin in dimethylsulfcodde, purchased from Sigma Chemical, Australia).
While in the
lysis buffer, the nerve fibers were cut into small pieces, using a scalpel,
and the resulting
suspension was transferred to an Eppendorf tube and incubated at room
temperature on
vortex for 1 hour. The tube was then centrifuged at 10,000 rpm at 4 C for 10
minutes, to
pellet the sciatic nerve debris.
The supernatant (which contained phage particles) was collected and stored on
ice,
while the debris pellet was incubated with 300 LL more lysis buffer and
vortexed for 1
more hour at room temperature. The lysed debris was then incubated at room
temperature
for 1 hour, and the sample was transferred into another Eppendorf tube and
centrifuged at
10,000 rpm at 4 C for 10 minutes, to pellet any remaining debris. The
supernatant was
collected and added to the previous supernatant, and a 20% volume of CaC12 was
added, to
inactivate the EDTA in the lysis buffer.
Some of the resulting aliquots of the mixed supernatants were titered, as
described
in Example 25, to determine phage particle concentrations. Other aliquots had
a 50%
volume of glycerol added, and the mixture was frozen and stored at -20 C for
subsequent
use or analysis. During all but the final rounds of in vivo selection, still
other aliquots were
used to infect E. con cells, and the amplified phage preparations that
resulted were used as
reagents in subsequent cycles of in vivo selection, using the same procedures
described
above.
EXAMPLE 27: HISTOLOGIC PHOTOGRAPHS OF NERVE SEGMENTS
A fluorescent staining technique was used to generate photomicrographs that
visually
confirmed the accumulation of internalised and transported MC192-M13K07
antibody-phage conjugates in the sciatic nerve bundle, just below the hip
ligature.
To create these photographic confirmations, the animal was euthanised with an
overdose of anesthetic (sodium pentobarbitone, 80 mg/kg, injected into the
abdomen IP). It
was then perfusion-fixed through the heart, using 400 mL of ice cold 0.1 molar
sodium
phosphate buffer containing 2% paraformaldehyde and 0.2% parabenzoquinone over
30
minutes. The sciatic nerve segment containing the hip ligature was then
dissected out and
placed in the same fixative for an additional hour, before being transferred
to 30% sucrose
in sodium phosphate buffer.
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The still-intact nerve bundle was then embedded in OCT compound (Tissue-TekTm,
Sakura Finetechnical Company Ltd., Tokyo, Japan), and frozen. Longitudinal
cryostat
sections (50 microns thick) were cut from the embedded and frozen nerve
bundles, using a
microtome.
Selected longitudinal tissue sections that had been cut from near the center
of the
nerve bundle were then treated with imrnunoreagents, to reveal the presence
and
concentration of phage particles. One reagent was a rabbit-derived antibody
preparation
(Sigma Chemicals, catalog number 132661) that binds to the pill capsid protein
on
bacteriophage particles, and that also contains a Biotin polypeptide sequence.
These
antibodies were incubated with the tissue slice for 1 hour at room
temperature. Alexa Fluor
488 (Molecular Probes, catalog number S-11223), which contains a streptavidin
sequence
that binds very tightly to the Biotin sequence on the phage-binding rabbit
antibody, was
then added and incubated for 1 hour at room temperature.
Fluorescent photographs were taken of the nerve bundle, covering a portion of
the
nerve bundle which included segments of nerve fibers on both sides of the hip
ligature. One
of those photographs, reproduced in black and white, is provided as FIG. 2 in
the
drawings.
That photograph (and others which showed very similar results) clearly shows
that
antibody-phage conjugates did indeed accumulate on the distal side of the hip
ligature.
These and similar photographs from other tests provide clear confirmation that
the binding,
enclocytosis, and transport mechanisms described herein are indeed working
efficiently, and
in the manner disclosed.
Similar photographs and other analytical tests of a control treatment, which
involved
injecting Ml 3K07 phages without any receptor-specific internalizing
antibodies erosslinked
to them, showed no control phages at the same location, on the distal side of
the ligatures.
The foregoing tests and results, using monoclonal antibodies affixed to phage
particles, confirmed several key aspects of the invention disclosed herein.
Among other
things, these results confirmed that: (i) complete filamentous phage particles
that could bind
to p75 receptors in rat neurons could be internalized and then retrogradely
transported, by
sciatic nerve fibers; and, (ii) the ligation, emplacement, and harvesting
protocols described
above can be used satisfactorily and effectively, to accomplish in vivo
screening and
selection of particular ligands that can enable complete, viable, and
infective phage particles
to be internalised into, and then transported within, neuronal fibers, based
on phage-fiber
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contacts that occur outside the blood-brain barrier.
Based on that confirmation of their general approach to in vivo screening
using
nerve fibers, and after conceiving, developing, optimizing, and confirming a
combination of
methods and reagents that enabled these types of in vivo screening tests to be
carried out
successfully and effectively, the Applicants then extended their approach, by
testing it in
actual in vivo screenings of phage display libraries containing huge numbers
of candidate
ligands.
EXAMPLE 28: IN VITRO BIOPANNING OF scFv PHAGE LIBRARY
As mentioned above, the p75 receptor in rat neurons is known to have
endocytotic
activity. It is also known to have its copy numbers, on neuronal surfaces,
increased by a
factor of roughly 10 to 15 fold, in response to various types of neuronal
injuries. In rats,
this type of injury can be created, in a controlled and reproducible manner,
by emplacement
of a tight ligature loop around the sciatic nerve bundle, in a location
slightly above the site
of the tibial branch bifurcation, where the sciatic nerve divides into two
major branches. A
ligature at that site will provoke a substantial increase in p75 receptor
numbers along the
length of the sciatic nerve fibers between the spinal cord and the ligature
site, and the
affected nerve fibers segments with increased p75 receptor numbers will be
long enough to
enable emplacement of a phage-containing gel at one location, followed by
harvesting of
internalized and transported phages at a separate location.
All of these factors made the p75 receptor a useful and controllable target
for initial
testing and confirmation of the in vivo selection process disclosed herein,
using phage
display libraries with huge numbers of candidate ligands.
Accordingly, the Applicants chose to limit their first tests of the scFv phage
display
library to candidate ligands that could bind specifically to the p75 receptor
in particular.
Although the Applicants realized that other ligands in the display library
(which, as
mentioned in Example 22, contains an estimated 13 billion different variable
fragment
antibody sequences, designed to emulate the immune systems of ten different
humans from
widely varying ancestries) would inevitably be able to bind to other neuronal
surface
receptors that could trigger endocytosis and retrograde transport, their
decision and goal, in
their tests using the scFv library, was to limit their tests to ligands that
would bind to the
p75 receptor,
After an initial set of efforts, which confirmed the general principles but
which also
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led to wide variations in the resulting data (those tests and results are
described in Example
30, and arose when the Applicadts began trying to evaluate cyclic repetition
of the in vivo
screening process, using phages selected in one cycle of tests as the starting
population for
the next cycle), the Applicants decided to focus on p75 receptor endocytosis
by processing
the scFv phage display library using an in vitro procedure called
"biopanning".
This in vitro method used a preparation of recombinant human p75 receptor
polypeptides. These polypeptides, which are commercially available, encode
amino acid
residues 1-250 of the extracellular domain of human nerve growth factor (NGF)
receptors,
fused to a carboxy terminal 6x histidine-tagged Fc region of human IgG1
protein, via a
peptide linker. In order to obtain glycosylated proteins, the chimeric protein
is expressed in
eukaryotic rather than bacterial cells, using an insect cell line known as
Sf21 (from the "fall
armyworm" moth, Spodoptera frugiperda), and a baculovirus expression vector.
The
recombinant mature chimeric protein exists as a disulfide-linked homodimer.
Each
monomer contains 466 amino acids and has a calculated mass of 51 kDa; however,
because
glycosylation increases the size and weight of the protein, each monomer
migrates as a
90-100 IcDa protein, when processed by electrophoresis in sodium dodecyl
sulfate-
polyacrylarnide (SDS-PAGE) gels.
These polypeptides were coated onto the surfaces of immunotubes. This was done
by
using 7 mL MAXISORPTM tubes, with a polystyrene hydrophilic surface (catalog
#444474,
from Nalge Nunc International, Denmark). A concentration of 5 g/mL of the
recombinant
human p75 receptor protein, suspended in 0.5 ml phosphate-buffered saline
(PBs), was
incubated overnight in the immunotubes at 4 C.
The next day, the tubes were poured out, and then rinsed with PBS, filled to
the
brim with 3% w/v skim milk in PBS, and blocked for 2 hours at room temperature
(RT).
Meanwhile, 50 kcl of the scFv phage display library was pre-blocked with 450
1 of 3%
skim milk in PBS in an Eppendorf tube for 1 hour at RT. The immunotubes were
rinsed
with PBS, then 500 i.t1 of pre-blocked phage solution was added to the tubes,
and incubated
for 2 hours at RT.
After 15 washes with PBS containing 0.1% Tween 20, the remaining bound phages
were eluted with 15 minute incubation of 500 jzL fresh triethylamine (TEA) in
a 100 mM,
pH 11 solution at RT. The eluted phage were transferred into an Eppendorf
tube, and
neutralized by adding 250 4, of 1M Tris-HC1 buffer, pH 7.4.
Half of the elutant was used to infect TG1 E. coil host cells, which were
cultured to
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in 0D6 optical density of 0.5-0.8. The other half of the elutant was stored
as a backup.
Fhe biopanned phage preparation was incubated with TG1 cells for 1 hour at 37
C, with 30
minutes under stationary, then 30 minutes of 300 rpm shaking. The infected
cell solutions
were then serially diluted and plated on 2TY agar plates with ampicillin, to
determine
phage titer.
Remaining cells were plated on four 243 mm x 243 mm 2TY agar plates with
ampicillin, for amplification. These plates were incubated at 30 C overnight.
Colonies were
scraped into liquid 2TY broth, and grown to 0D500 levels of 0.5-0.8. M13K07
helper
phages were then added, to form a final concentration of 5 x 109 "colony
forming units"
(cfu) per mL. The mixture was incubated for 30 minutes while stationary, then
for 30
minutes in a shaker tray at 200 rpm. The cells were then centrifuged at 3500
rpm for 10
minutes, and the cell pellet was resuspended in 25 mL prewarmed 2TY (without
glucose)
containing Icanamycin (50 p,g/mL) and ampicillin (100 ,ug/mL). These were
incubated
overnight at 25 C, with rapid shaking, to produce phage particles. The phage
particles were
precipitated, using 20% polyethylene glycol (PEG) and 2.5 M NaCl. These
particles were
then mixed witil'a collagen gel, and a bolus of gel containing roughly 50
billion (5 x 1010)
cfu of phages was emplaced in a rat leg during an in vivo screening operation.
Only a single round of in vitro biopanning was used prior to in vivo
screening,
because the Applicants wanted to preserve maximal diversity of the p75-binding
ligands that
would be tested in vivo. This diversity would have been jeopardized by
successive in vitro
screenings, because it is known that many types of p75-binding antibodies are
not
internalized by cells having p75 receptors. The concern is that tightly-
binding ligands
appear to somehow lock up and/or contort in vivo p75 receptors, in ways that
impede the
ability of the p75 receptors to carry out the normal process of endocytosis.
Since additional
rounds of in vitro biopanning would tend to select for tight-binding ligands
without regard
to their ability to trigger endocytosis, it could lead to elimination of
candidate ligands that
might be substantially more effective in achieving actual endocytotic
transport into cell
interiors, in vivo.
Nevertheless, three successive rounds of biopanning were carried out, using
the scFy
phage library, to evaluate how this library would respond to repeated rounds
of in vitro
screening. To establish comparable results, each solution of phages used in
the second and
third rounds was diluted, by PBS, to match the titer of the solution that had
been used in
the first round.
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While the first round of biopanning resulted in scFv titers of roughly 3000
cfu
(compared to control values of roughly 1200 cfu, when PBS was tested), the
second round
of biopanning resulted in major increases, to about 84 million cfu. A third
round of
biopanning resulted in titers of about 85 million cfu, which was not a
significant increase
over the second round.
EXAMPLE 29: IN VIVO (SCIATIC NERVE) SELECTION OF INTERNALISED
PHAGES FROM THE scFv LIBRARY
As mentioned in the previous example, the "enriched" portion of the scFv phage
display library that was selected by one round of biopanning (using human p75
receptor
polypeptides), as described in Example 29, was used as the starting reagent in
a series of in
vivo screenings in rats. These in vivo screenings used the procedures and
methods that had
been developed, tested, and optimized by using MC192/M12K07 antibody-phage
conjugates as described in Examples 25 and 26.
Briefly, in a first operation, an initial ligature was placed just above the
tibial
branching of the sciatic nerve, to induce increased p75 receptor expression on
the sciatic
nerve fibers above the ligature. A week later, in a second operation, the
sciatic nerve
bundle was cut, and the cut end was packed inside a silicone rubber sleeve
with collagen
gel containing about 50 billion cfu of scFv phages that had been obtained by a
single round
of p75 biopanning. During the second operation, a ligature was also emplaced
and tightened
around the sciatic nerve in the hip region, to create an obstacle that would
cause
internalised and retrogradely transported phage particles to accumulate,
inside the nerve
fibers, just distal to the ligature. Eighteen hours later, in a third
operation, the rat was
sacrificed and a segment of nerve fibers was harvested, including the hip
ligature and
roughly half a centimeter of nerve fibers distal to the ligature. The
harvested nerve fibers
were washed, cut into small pieces, and treated to remove and isolate phage
particles. The
phage particles were amplified, and titers were determined, using E. coli
cells and helper
phages.
While the absolute number of phage recovered from an excised nerve segment
varied between experiments, a standardized measure of uptake and transport was
generated,
by always testing a control phage population, and comparing the results to the
data from the
test phage population.
To illustrate, using absolute numbers that resulted from a representative
experiment
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'n=3 for both control and test selections), 5 x 101 (i.e., 50 billion) cfu
(titered estimate) of
control phages (unmodified M131(07 helper phages) were emplaced into the phage
contact
site. The number of control phages that were recovered from the nerve segment
excised 18
hours later was titered, giving a value of 14,650 (+ 2,975, standard error of
the mean
(SEM), n=3). The same quantity (5 x 1010 cfu) of scFv-phage (biopanned once to
recognize
p75, as described in Example 28) was emplaced in a phage contact site, and the
number of
scFv phages recovered from the nerve segment excised 18 hours later was
titered at 190400
(+ 14,415 SEM, n=3). By comparing those two results, it was calculated that 13-
fold
more scFv phage (biopanned once for p75) were recovered from the excised nerve
segments, than control phage. This experiment was repeated 3 times, with
similar results
each time.
To test whether this marked increase in uptake and transport of scFv phage was
indeed the result of the scFv binding to p'75, the experiment was repeated in
other sets of
animals, in which the sciatic nerve had not been pre-ligated (and, therefore,
the motor
neurons had not upregulated their expression of p75 above the very low and
frequently
undetectable levels that appear in rats that are more than about 2 weeks old).
In these tests,
the amounts of control phage (M13K07) and test phage (scFv) that were applied
were held
the same as before, at 5 x 10' cfu. The number of control phage that were
recovered from
nerve segments excised 18 hours later was 14,815 + 4,481 (n=3). The amount of
scFv
phage (biopanned once for p75 recognition) that were recovered from nerve
segments
excised 18 hours later was 16,413 + 4,541 (n=3). These data clearly showed
that the
efficiency of cellular intake and transport of scFv phage (biopanned once for
p75
recognition) was essentially no different from that of control phage, when
rats were tested
that had very low levels of p75 receptors, as occurs naturally in rats that
are six weeks of
age or older. This confirmed that there was a clear relationship between p75
expression
levels, and efficiency of uptake and transport of scFv phage that had been
biopanned to
recognize p75 receptors. This experiment was repeated 3 times, with similar
results.
EXAMPLE 30: CYCLIC TESTING OF UNPANNED scFv LIBRARY
In a series of tests that were performed before the in vitro biopanning
procedure
(described in Example 28) was settled upon and used, the scFv library was
tested in a series
of cyclic in vivo screening tests, using sciatic nerve fibers as described in
Examples 25 and
26. These tests are referred to as "cyclic", because a screened and selected
phage
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population that was obtained from one round of tests was then used as a
starting reagent, in
the next cycle of tests.
Although these tests provided clear evidence that the phage libraries
contained
particular phage-borne ligands that activated and drove endocytotic
internalisation and
retrograde transport, the results of these cyclic tests were highly variable.
The data
scattering left the Applicants to conclude that the data were consistent with
the following
interpretations:
(i) the ligand-receptor binding and uptake process was saturable, due to the
limited
number of p75 receptors on the surfaces of the nerve fibers;
(ii) the input phage populations were highly diverse, with only one or a few
copies
of any one particular phage present in any initial round(s), and with
subsequent rounds
likely to contain hundreds or even thousands of different phage candidates;
(iii) therefore, the probability was quite low that any one particular phage
would be
selected in two different experiments on different animals (this probability
can be regarded
as being roughly equal to the number of copies of any one particular phage in
a test
population, divided by the number of alternative phages that the nerve bundle
could
effectively sample from).
These factors clearly can account for the very high variability seen between
different
experiments using different animals. Therefore, after encountering and
pondering those high
levels of variability, the Applicants decided to experiment with a pre-
screening step (i.e., in
vitro biopanning) that would reduce the variation within the input population,
and that
would also substantially increase the number of multiple copies of p75-binding
phage
candidates that would be available for the nerve bundle to sample from.
The data obtained from the scFv library tests that were done prior to the
pre-screening step did indicate that the first, the second, and possibly the
third successive
screening cycles all appeared to lead to greater efficiencies, in
internalisation and retrograde
transport by the cells. However, under the particular conditions that were
used, those
efficiencies tended to drop off if still more cycles of in vivo screening were
used. While the
cause for the eventual fall-off was not clear, a significant proportion of
individual colonies
of phage selected from the second or third round selections can reasonably be
anticipated to
display ligands that bind to neuronal receptors and stimulate internalisation
and retrograde
transport (since they were repeatedly selected, the probability that they
might be false
positives is low).
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Cyclic in vivo screening has not yet been evaluated thoroughly, and in
particular, it
las not yet been tested with a phage population that has been pre-screened, by
biopanning
r similar efforts. Nevertheless, the work done to date is believed to clearly
demonstrate
md confirm that:
(1) with at least some types of phage populations, a series of cyclic in vivo
screenings, where a candidate population that has been selected by one round
of screening
is used as a starting reagent in the next round of screening, is indeed
possible, and in at
Least some cases is likely to help identify and isolate candidate ligands that
are exceptionally
effective in triggering and driving endocytotic transport into cells; and,
(2) it is feasible to develop numerical indices that will provide useful
indicators of
when a cyclic piocess should be stopped, to allow careful analysis and
sequencing of
candidate ligands that appear to offer the best performers that have been
identified up until
that point in the screening process.
(3) it is also likely that at least some of the phage ligands stimulated
internalisation
and retrograde transport after binding to surface molecules that were not
previously known
to mediate endocytotic events.
Finally, in considering the implications of these analyses, it should be borne
in mind
that the fundamental and overriding goal of this type of in vivo screening is
not to create a
highly enriched or "elite" phage population, that can offer many thousands of
phage
candidates that will be internalised by nerve fibers. Instead, the goal is to
identify and
isolate (and, in the case of polypeptide ligands, to determine the nucleotide
gene sequence
and/or the amino acid polypeptide sequence of) just one or a small number of
particular
ligands that are highly effective in activating and drive the process of
endocytotic
internalisation. These are the types of ligands that, once they have been
identified and
isolated, can be replicated in mass, and incorporated into molecular complexes
that will
transport useful passenger or payload molecules into specific classes of cells
that have
specific targeted endocytotic receptors or similar molecules on their
surfaces.
It must also be kept in mind that a biopanning step, as described above for
using
p75 polypeptide sequences to pre-screen the scFv phage library, can be carried
out by using
(as the "antigen" molecule that will be affixed to the surfaces of the
immunotubes) any
known polypeptide sequence or fragment, from any type of known or suspected
endocytotic
receptor, or from other surface molecule suspected of having endocytotic
activity. It can
also be carried out by using any glycosylated cell surface molecules that are
suspected of
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having endocytotic activity.
EXAMPLE 31: IN VIVO SELECTION, USING PhD-C7C PHAGE LIBRARY
As briefly mentioned in Example 22, the Ph.D-C7C phage display library
contains
an estimated two billion different phages, with foreign DNA inserts that
encoding random
sequences of seven amino acids, inserted near the DNA sequence that encodes
the N-
terminus of the pill capsid protein of M13 phages. This library provided an
essentially
random repertoire of peptide sequences that could be tested, to determine
whether certain
phages would be internalized and transported by neurons in the sciatic nerve
bundle.
In vivo screening of the PhD-C7C library, using the sciatic nerve procedures
disclosed above, indicated that this library performed just as expected.
Substantial numbers
of phages were internalized by the sciatic nerve fibers, and transported to a
phage
accumulation zone immediately distal to the hip ligature. Selected phages were
removed, in
viable form, from the harvested nerve segments, and those p75-selected viable
phages could
be replicated and manipulated in any way of the ways described above.
If desired, as indicated above, the PhD-C7C library also can be pre-screened,
using
a biopanning technique (as described above for the p75 biopanning of the scFv
library),
using any known and available type of receptor polypeptide sequence as the
biopanning
antigen.
Thus, there has been shown and described a new and useful method for (i) using
in
vivo screening, to identify ligands that can efficiently activate and drive
the process of
cellular endocytosis, via selected endocytotic molecules that are present on
the surfaces of
only limited numbers and types of cells, and (ii) incorporating those ligands
into molecular
complexes that can be used to efficiently transport useful passenger or
payload molecules
into cells having the targeted endocytotic receptors or other surface
molecules. Although
this invention has been exemplified for purposes of illustration and
description by reference
to certain specific embodiments, it will be apparent to those skilled in the
art that various
modifications, alterations, and equivalents of the illustrated examples are
possible. Any
such changes which derive directly from the teachings herein, and which do not
depart from
the spirit and scope of the invention, are deemed to be covered by this
invention.
Thus, there has been shown and described a new and useful means for non-
invasive
transport of therapeutic or other useful polypeptides through the BBB, into
brain and spinal
tissue (and in particular to neurons that lie wholly within the BBB), and for
using in vivo
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screening to identify ligands that can efficiently activate and drive the
process of cellular
endocytosis, via selected endocytotic molecules that are present on the
surfaces of only
limited numbers and types of cells. Although this invention has been
exemplified for
purposes of illustration and description by reference to certain specific
embodiments, it will
be apparent to those skilled in the art that various modifications,
alterations, and equivalents
of the illustrated examples are possible. Any such changes which derive
directly from the
teachings herein, and which do not depart from the spirit and scope of the
invention, are
deemed to be covered by this invention.
1: =
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