U.S. patent application number 13/384020 was filed with the patent office on 2012-07-26 for method of providing agents to the cochlea.
This patent application is currently assigned to NewSouth Innovations Pty. Limited. Invention is credited to Gary David Housley.
Application Number | 20120191032 13/384020 |
Document ID | / |
Family ID | 43448802 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120191032 |
Kind Code |
A1 |
Housley; Gary David |
July 26, 2012 |
METHOD OF PROVIDING AGENTS TO THE COCHLEA
Abstract
Provided is a method of transfecting cells of the cochlea with
an agent by electroporation, and in certain embodiments using a
cochlear implant to provide at least one electroporation
electrode.
Inventors: |
Housley; Gary David;
(Connells Point, AU) |
Assignee: |
NewSouth Innovations Pty.
Limited
New South Wales
AU
|
Family ID: |
43448802 |
Appl. No.: |
13/384020 |
Filed: |
July 15, 2010 |
PCT Filed: |
July 15, 2010 |
PCT NO: |
PCT/AU2010/000899 |
371 Date: |
April 9, 2012 |
Current U.S.
Class: |
604/20 ;
607/57 |
Current CPC
Class: |
A61K 41/0047 20130101;
A61F 11/00 20130101; A61N 1/327 20130101; A61K 48/0075 20130101;
A61N 1/0541 20130101; A61N 1/0412 20130101; A61M 2037/0007
20130101 |
Class at
Publication: |
604/20 ;
607/57 |
International
Class: |
A61N 1/30 20060101
A61N001/30; A61F 11/04 20060101 A61F011/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 15, 2009 |
AU |
2009903320 |
Claims
1. A method of transfecting cells with an agent by electroporation
within a cochlea of a subject, the method comprising introducing a
cochlear implant which comprises at least one electrode into the
cochlea, providing the agent to the cochlea, and providing at least
one second electrode, and then providing an electroporation
electric field between the at least one electrode and the at least
one second electrode and thereby transfecting cells with the
agent.
2. The method of claim 1, wherein the agent comprises a nucleic
acid molecule.
3. The method of claim 2, wherein the nucleic acid molecule encodes
a neurotrophic factor for spiral ganglion cells.
4. The method of claim 2, wherein the nucleic acid molecule encodes
a transcription factor which modulates the expression of a
neurotrophic factor for spiral ganglion cells by mesenchymal cells
of the scala tympani.
5. The method of claim 2, wherein the nucleic acid molecule
decreases the expression of a transcription factor within
mesenchymal cells of the scala tympani or scala vestibuli, wherein
the transcription factor modulates the expression of a neurotrophic
factor for spiral ganglion cells by the mesenchymal cell.
6. The method of claim 3, wherein the neurotrophic factor is
selected from any one of Neurotrophin-3, Neurotrophin-3 precursor
molecule, Neurotrophin 4/5, Nerve growth factor, Brain-derived
neurotrophic factor, glial cell line-derived neurotrophic factor,
ciliary neurotrophic factor and Activity dependent neurotrophic
factor.
7. The method of claim 1, wherein the at least one electrode or the
cochlear implant which comprises at least one electrode is
introduced into the scala tympani or the scala vestibuli of the
cochlea.
8. The method of claim 1, wherein the subject is a human.
9. The method of claim 1, wherein the cochlear implant comprises
the at least one electrode and the second electrode.
10. The method of claim 1, wherein the agent is provided to the
cochlea before introducing the cochlear implant into the
cochlea.
11. The method of claim 1, wherein providing the agent to the
cochlea substantially displaces the media lying within the cochlea
chamber.
12. The method of claim 1, wherein the at least one electroporation
electrode of the cochlear implant and the at least one second
electrode also act as auditory nerve stimulator electrodes.
13. A cochlear implant, comprising: electroporation-driving
circuitry and auditory nerve stimulator circuitry such that the
chochlear implant can generate both art electroporation mode
electrical pulse profile or an auditory nerve stimulator electrical
pulse profile, and at least one electrode coupled to the
electroporation-driving circuitry and auditory nerve stimulator
circuitry for providing electroporation and auditory nerve
stimulator electrical pulse profiles to the cochlea.
14. The cochlear implant of claim 13, wherein the implant comprises
a lumen or channel for delivery of an agent to the cochlea.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of providing an
agent to cells of the cochlea using electroporation. The method has
particular application in supporting the survival or neurite growth
of spiral ganglion cells in subjects having a cochlear implant or
intending to undergo a cochlear implant procedure.
BACKGROUND ART
[0002] Sensori-neural deafness arises from the loss of hair cells
found within the cochlea, the sensory receptors of the auditory
system. This loss of hair cells can lead to degeneration of
peripheral processes and death of spiral ganglion neurons which
rely on hair cells for stimulus and trophic support. The spiral
ganglion neurons reside within Rosenthal's canal in the cochlea,
with peripheral neurite processes connecting to the sensory hair
cells, and central axons to the cochlear nucleus in the brainstem.
Thus the spiral ganglion neurons form the cochlear or auditory
nerve which receives auditory stimuli from the auditory receptors
and transmit sound information to the brain. The auditory output
carried by the cochlear nerve is intrinsically tonotopically
mapped, with the basal region of the cochlea being more sensitive
to the highest frequency sounds and progressively lower frequencies
being encoded by more apical regions of the cochlea.
[0003] The cochlear implant is a highly successful application of
bionic engineering which can restore a degree of hearing to many
deaf subjects. Implants are also being provided to patients with
residual low frequency hearing, where the implant provides access
to the higher frequency sounds. Cochlear implants act by bypassing
the cochlear hair cells and directly electrically stimulating
remnant spiral ganglion nerve cell bodies of the cochlear nerve.
Accordingly, there are advantages where a cochlear implant is used,
for the subject to retain as many cochlear nerve fibres as possible
to facilitate the neural transmission of auditory signals to the
brain, and to ensure that nerve fibres are present in proximity to
the series of electrodes of the cochlear implant.
[0004] The implantation procedure may damage a proportion of
remnant nerve fibres, nerve cell bodies, and sensory hair cells in
the subject's cochlea, and so in subjects with only marginal
remnant innervation it is important to maintain as many functional
neurons as possible following implantation. Cochlear implants which
provide therapeutic support to cochlear tissues following
implantation are being investigated. Some strategies for
therapeutic support of cochlear tissues involve the use of
continuous (tonic) electrical stimulation of spiral ganglion cells
to promote the survival of remnant spiral ganglion cells. Other
experimental strategies being considered involve direct injection
of neurotrophic molecules into the cochlea at the time of
implantation. The use of cochlear implants which comprise a drug
delivery channel or an ensheathing matrix which provides passive
diffusion of agents to surrounding cochlear tissue has also been
proposed. These approaches, however, only provide a short-acting
delivery of agent to cochlea, thus limiting the degree to which the
agents may provide support to the cochlear tissue. The use of
encapsulated cells which can be placed within the cochlea to
secrete neuroactive molecules is also under investigation as a
means of providing longer term therapeutic support to cochlear
nerve cells.
[0005] Other proposed therapeutic approaches to supporting cochlear
neural function utilize gene therapy. These approaches focus on the
use of viral-vector based transformation of cochlear tissues, as
these techniques are generally considered efficient delivery
systems for cell transformation. Viral-based delivery systems are
however limited by the lack of site-specific control of the gene
delivery, resulting in a dispersed and largely uncontrolled
transfection of cells throughout the tissue exposed to the virus,
such as cells lining the scala tympani. In addition, the potential
for causing undesirable immunological reactions to the viral vector
constructs inhibits the immediate translation of this research into
clinical use in human subjects.
[0006] There is therefore a need for techniques which allow the
introduction of genetic constructs to the cochlea and which
substantially overcome or ameliorate at least one or more of the
above disadvantages.
SUMMARY
[0007] Provided herein is a method of transfecting cells with an
agent by electroporation within a cochlea of a subject to receive a
cochlear implant, the method comprising introducing at least one
electrode into the cochlea, providing the agent to the cochlea, and
providing at least one second electrode, and then providing an
electroporation electric field between the at least one electrode
and the at least one second electrode and thereby transfecting
cells with the agent. Certain embodiments of the present invention
are based on the recognition that a cochlear implant may be
modified to deliver an electrical signal of sufficient strength and
amplitude to result in the transduction of an agent, such as a
nucleic acid molecule into cochlear tissues, such as mesenchymal
cells of the scala tympani, by electroporation.
[0008] Described herein are methods for directed gene delivery by
transfecting cells close to the cochlear implant electrode array
using electroporation. This provides, for example, nucleic acid
molecule delivery to cells close to the cochlear implant within the
cochlea, which potentially offers an enhancement to the subsequent
performance of the cochlear implant neural prosthesis.
[0009] Accordingly, in one aspect there is provided a method of
transfecting cells with an agent by electroporation within a
cochlea of a subject, the method comprising introducing a cochlear
implant which comprises at least one electrode into the cochlea,
providing the agent to the cochlea, and providing at least one
second electrode, and then providing an electroporation electric
field between the at least one electrode and the at least one
second electrode and thereby transfecting cells with the agent.
[0010] In particular embodiments the agent comprises or consists of
a nucleic acid molecule. The nucleic acid molecule may encode a
neurotrophic factor for peripheral sensory neurons, and in
particular embodiments the nucleic acid molecule encodes a
neurotrophic factor for spiral ganglion cells. In particular
embodiments the nucleic acid molecule encodes a transcription
factor which promotes the expression of a neurotrophic factor. In
particular embodiments the nucleic acid molecule encodes one or
more therapeutic molecules for spiral ganglion cells. In particular
embodiments the nucleic acid molecule decreases the expression of a
transcription factor within mesenchymal cells of the scala tympani
or scala vestibuli, wherein expression of the transcription factor
inhibits the expression of a neurotrophic factor for spiral
ganglion cells by the mesenchymal cell.
[0011] Also provided is the use of a nucleic acid molecule which
encodes a neurotrophic factor for spiral ganglion cells or which
encodes a transcription factor which promotes the expression of a
neurotrophic factor for spiral ganglion cells for transfecting a
cell of a cochlea of a subject in situ by electroporation.
[0012] The neurotrophic factor may be selected from any one of
Neurotrophin-3, Neurotrophin-3 precursor molecule, Neurotrophin
4/5, Nerve growth factor, Brain-derived neurotrophic factor, glial
cell line-derived neurotrophic factor, ciliary neurotrophic factor
and Activity dependent neurotrophic factor.
[0013] In particular embodiments the cells are cells of the scala
tympani or scala vestibuli, such as mesenchymal cells.
[0014] In particular embodiments, the subject is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Certain embodiments of the present invention will now be
described, by way of example only, with reference to the
accompanying drawings wherein:
[0016] FIG. 1 shows a photograph of an isolated cochlea from an
adult guinea-pig demonstrating the electrode configuration used for
electroporation gene delivery. The image shows placement of a
shaped electroporation electrode through the round window membrane
and into the basal turn scala media compartment (simulating the
cochlear implant electrode prosthesis), and an apical fenestration
with a reference second electrode.
[0017] FIG. 2 shows a group of photomicrographs illustrating the
successful electroporation of guinea-pig cochlea mesenchymal cells
using the method of Example 1. The expression of DsRed2 reporter is
evident in cochlear mesenchymal cells from the basal region of
scala media 24 hours after electroporation of the guinea-pig
cochlea with the CMVp-TRPC3-IRES-dsRed2 cDNA gene construct
(Experiment ID Nos. GP13b and GP8/4b).
[0018] FIG. 3 shows a low-power confocal microscopy image which
demonstrates expression of the CMVp-BDNF-FLAGtag-IRES-eGFPn gene
cassette over approximately 750 .mu.m in the basal turn of scala
tympani, in close proximity to the location of the electroporation
electrode. The fluorescent signal represents eGFP-labelled nuclei
of cochlear mesenchymal cells in the scala tympani region of the
guinea-pig cochlea, on the lateral wall of the spiral limbus, close
to the location of the electroporation electrode (Experiment ID No.
GP26a, after two days in culture).
[0019] FIG. 4 shows a high-power confocal microscopy image
confirming expression of recombinant brain-derived neurotrophic
factor (BDNF) by transformed mesenchymal cells within the scala
tympani, and represents a detailed image of some of the cells
identified by nuclear labelling in FIG. 3. TRITC fluorescence
signal in the cytoplasm of cells demonstrates the expression of
BDNF polypeptide which is identified by the Flag tag. Fluorescence
signal in the nucleus arises from expression of the eGFPn reporter.
CMVp-BDNF-FLAGtag-IRES-eGFPn gene cassette (Experiment ID No.
GP26a).
[0020] FIG. 5 provides a series of diagrams which illustrate
possible cochlear implant electrode "node" configurations which may
be suitable for electroporation. The positions of nodes of the
cochlear implant electrode are set out as grey-shaded boxes along
the length of the electrode, which is shaped to fit the scala
tympani of the cochlea. FIG. 5A illustrates an "alternative
polarity" configuration in which neighbouring nodes are of opposite
polarity. In this illustration the electrode is shown for DC
stimulation, but this and other configurations illustrated in this
figure would be compatible with other electroporation waveforms.
FIG. 5B illustrates a "distributed current return electrodes"
configuration for the electroporation mode of a cochlear implant,
with a preponderance of current return electrode nodes and a
smaller number of current delivery electrode nodes located, for
example, at the proximal and distal ends of the electrode. FIGS. 5C
and 5C illustrate two configurations in which electrodes labelled
in black are dedicated for electroporation mode only and are used
for charge delivery. In FIG. 5C the dedicated electroporation
current delivery electrodes are used in conjunction with the
conventional implant electrode nodes for current return. In FIG. 5D
dedicated electroporation current delivery and return electrodes
are provided. FIG. 5E illustrates a configuration utilising the
integrated electrode array of the cochlear prosthesis and a
separate "return" electrode. The external return electrode may be
provided to complete the circuit for electroporation using the
prosthetic electrode array for delivery of the electroporation
therapeutic molecules to the cochlea. The return electrode may be
inserted into the cochlear scala vestibuli, if the primary array is
placed into scala tympani, or vice versa. Alternatively the return
electrode may be placed into the cochlear modiolus, or external to
the cochlea. The polarity of the cochlear electrode and the
separate electrode may be swapped from the polarity illustrated
herein, or the polarity may be reversed one or more times during
electroporation. A separate return electrode may also be used in
conjunction with a transient electrode array for
electroporation-based delivery of an agent, prior to implantation
of the cochlear prosthetic electrode array.
[0021] FIG. 6 provides a diagram illustrating one possible
configuration of a cochlear implant for delivery of the agent to
sites within the cochlea as well as the delivery of an
electroporation current. In this embodiment the cochlear implant
comprises the "distal current return" electrode configuration for
electroporation and an integrated catheter for agent delivery into
the cochlear fluid compartment prior to electroporation. In this
embodiment the black electrodes are specifically dedicated for
electroporation mode only and are sufficient for efficient gene
delivery. The solid black line is symbolic of an internal lumen,
such as a catheter, which lies within the cochlear implant and
which allows delivery of the therapeutic molecule to one or more
sites within the cochlea in situ, prior to electroporation of the
cochlear tissue.
DESCRIPTION OF EMBODIMENTS
[0022] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0023] Throughout this specification, reference to "a" or "one"
element does not exclude the plural, unless context determines
otherwise. For instance, reference to "a nucleic acid molecule"
should not be read as excluding the possibility of multiple nucleic
acid molecules.
[0024] As described herein there is provided a method of
transfection by electroporation using a cochlear implant for
delivery of an agent, such as a nucleic acid molecule, to cells of
the cochlea. By relying on electroporation techniques rather than
viral mediated transfection it is possible to deliver a wide range
of agents, such as naked nucleic acid molecules, to cochlear cells.
In addition, by utilising one of the perceived limitations of in
vivo electroporation techniques, that is that the efficiency of
transfection drops off rapidly as the distance from the electrodes
increases, the methods described herein offer a previously
unrecognised advantage in the cochlear implant application, as the
use of electroporation mediated transfection constrains
transfection to the tissue immediately adjacent to the electrode,
allowing relatively precise localisation of delivery of the agent
only to cochlear cells in the immediate vicinity of the cochlear
implant. Typically, only cells within 300 .mu.m of the electrode
will be transfected.
[0025] One limitation of the operation of cochlear implants in
present use is the minimum distance which is employed between the
implant electrodes. The cochlea implant is located within the scala
tympani fluid compartment and the spiral ganglion cell auditory
neurons lie within Rosenthal's canal, the auditory neuron processes
having largely retracted following loss of the sensory hair cells.
The distance between the implant and the closest processes of
auditory neurons is sufficient that the number of electrodes
present in an implant, and hence the number of different
frequency/intensity bands provided by the implant to the subject's
auditory system, is limited by the current spread.
[0026] The methods described herein allow the use of gene therapy
which may, for example, enhance the expression of spiral ganglion
cell trophic molecules, such as brain-derived neurotrophic factor,
in cochlear cells which lie close to the cochlear implant. This is
proposed to be beneficial to the function of the cochlear implant
by promoting spiral ganglion cell neurite outgrowth specifically
towards the transformed cells, which in turn bring the spiral
ganglion neuron processes in close proximity to the cochlear
implant electrodes. By providing spiral ganglion neurite outgrowth
close to the position of the cochlear implant, the functionality of
the cochlear implant may be improved by lowering the required
stimulation currents. The use of lowered stimulation currents may
enable the electrode design of the cochlear implant to include a
higher density of electrodes when working as a bionic ear, thus
also improving the auditory information density that can be
delivered to the cochlear nerve.
[0027] Subject
[0028] The present invention contemplates the transfection of cells
of the cochlea with an agent by electroporation which utilises an
electrode forming part of a cochlear implant.
[0029] Where the methods described herein are intended for
therapeutic purposes the cochlea may be present in situ within a
live subject. Where the methods described herein are intended for
experimental or research purposes the cochlea may be present in
situ within a live subject or may be substantially intact but
isolated from the subject. A "subject" refers to any animal with a
cochlea (e.g., a mammal), including, but not limited to any one of
humans, non-human primates, rodents, and the like, which is or is
to be the recipient of a cochlear implant. Typically, the terms
"subject" and "patient" are used interchangeably herein. Typically
the subject is a human subject.
[0030] As used herein, the term "non-human animals" refers to
non-human vertebrate animals including, but not necessarily limited
to, rodents, non-human primates, ovines, bovines, ruminants,
lagomorphs, porcines, caprines, equines, canines, felines, ayes,
etc.
[0031] The cells which are transfected by the methods described
herein will be cells lying within 300 .mu.m to an electrode of the
cochlear implant. Typically, but not necessarily exclusively, cells
transfected by the methods described herein will be cochlear
mesenchymal cells.
[0032] In particular embodiments the subject's cochlea is a mature
cochlea, which as used herein refers to a developmental stage of
the cochlea within which air-borne sound transduction takes place.
In rodents such as rats, mice and guinea pigs, this typically
occurs around one week after birth. In humans air-borne sound
transduction commences at birth.
[0033] Agents
[0034] Nucleic Acid Molecules
[0035] In certain embodiments, the agent may consist of a nucleic
acid molecule. In certain embodiments the agent may comprise a
nucleic acid molecule, in which case the agent may, for example,
also comprise molecules which enhance the stability or transfection
efficiency of the nucleic acid molecule, such as chitosan, and/or
may also comprise non nucleic acid molecules such as small
neuroprotective peptides.
[0036] As used herein, the term "nucleic acid molecule" refers to
any nucleic acid containing molecule, including but not limited to
DNA or RNA molecules. The term encompasses sequences that include
any of the known base analogs of DNA and RNA including, but not
limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinylcytosine, pseudoisocytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil-, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonyl-methyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine,
provided that the analog is not toxic to cochlear cells. A nucleic
acid molecule may include modifications designed to improve
delivery into cells, stability once inside a cell, and/or binding
to the appropriate intracellular target.
[0037] The nucleic acid molecule may be an oligonucleotide or a
polynucleotide. In particular embodiments the nucleic acid molecule
is naked DNA, such as cDNA, or naked RNA. In particular embodiments
the use of encapsulated DNA or RNA, such as polynucleotides present
in liposomes or microspheres is also contemplated, particularly
where such encapsulation improves the stability of the agent or the
efficiency of transfection by electroporation. The use of
encapsulated DNA or RNA may offer a less advantageous embodiment if
encapsulation allows transfection of cells which are distant to the
electrode to take place. It will be understood by a person skilled
in the art that particles may be mixed with the nucleic acid
molecule to condense the nucleic acid molecule molecule, which may
provide increased transduction efficiency.
[0038] (a) Nucleic Acid Molecules which Encode a Polypeptide
Product
[0039] The agent may be a nucleic acid molecule which encodes a
polypeptide product. The expression of the encoded polypeptide
product promotes a desired outcome, such as promoting or
maintaining spiral ganglion cell neurite outgrowth and/or spiral
ganglion cell survival.
[0040] The encoded polypeptide product may be an endogenous
polypeptide to the subject. In certain embodiments the encoded
polypeptide product is an exogenous polypeptide.
[0041] The nucleic acid molecule which encodes a polypeptide
product comprises a coding sequence for a polypeptide product or a
precursor polypeptide of the polypeptide product. The polypeptide
product may be a full-length polypeptide which occurs in nature, or
it may be a portion of a polypeptide which occurs in nature,
provided that the desired activity or functional properties (such
as receptor binding and/or the ability to induce signal
transduction) of the polypeptide product are retained. The nucleic
acid molecule may also comprise a sequence located adjacent to the
coding sequence on the 5' or 3' end of the coding sequence or on
both the 5' and 3' ends for a distance of about 1 kb or more on
either end such that the sequence corresponds to the length of a
full-length mRNA sequence. Sequences located 5' of the coding
region and present on the mRNA are referred to as 5' non-translated
sequences. Sequences located 3' or downstream of the coding region
and present on the mRNA are referred to as 3' non-translated
sequences.
[0042] The nucleic acid molecule may comprise a cDNA sequence
encoding a polypeptide product. The nucleic acid molecule may
comprise a genomic sequence encoding a polypeptide product. A
genomic sequence contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (nRNA); introns may contain regulatory
elements such as enhancers. Introns are removed or "spliced out"
from the nuclear or primary transcript; introns therefore are
absent in the messenger RNA (mRNA) transcript. The mRNA functions
during translation to specify the sequence or order of amino acids
in a nascent polypeptide.
[0043] In certain embodiments, the expression of a nucleic acid
molecule refers to the process of converting genetic information
encoded in the nucleic acid molecule into RNA (including for
example mRNA or snRNA) through "transcription" of the nucleic acid
molecule (i.e., via the enzymatic action of an RNA polymerase), and
for nucleic acid molecules encoding a polypeptide, into a
polypeptide product through translation of mRNA. Expression can be
regulated at many stages in the process. "Up regulation" refers to
regulation that increases the production of expression products
(i.e., RNA or protein), while "down regulation" refers to
regulation that decreases production. Molecules (e.g.,
transcription factors) that are involved in up regulation or down
regulation are often called "activators" and "repressors"
respectively.
[0044] In addition to containing introns, genomic forms of a
nucleic acid molecule may also include sequences located on both
the 5' and 3' end of the sequences that are present on an RNA
transcript. These sequences are referred to as "flanking" sequences
or regions (these flanking sequences are located 5' or 3' to the
non-translated sequences present on the mRNA transcript). The 5'
flanking region may contain regulatory sequences such as promoters
and enhancers that control or influence the transcription of the
gene. The 3' flanking region may contain sequences that direct the
termination of transcription, post-transcriptional cleavage and
polyadenylation.
[0045] A nucleic acid molecule may be present in a cDNA, genomic
DNA or RNA form. When present in a DNA form, the nucleic acid
molecule may be single-stranded (i.e., the sense strand) or
double-stranded. Suitable control elements such as
enhancers/promoters, splice junctions, polyadenylation signals,
etc. may be placed in close proximity to the coding region of the
nucleic acid molecule if needed to permit proper initiation of
transcription and/or correct processing of the primary RNA
transcript. Alternatively, the coding region utilized in expression
vectors may contain endogenous enhancers/promoters, splice
junctions, intervening sequences, polyadenylation signals, etc. or
a combination of both endogenous and exogenous control
elements.
[0046] A nucleic acid molecule may produce multiple RNA species
that are generated by differential splicing of the primary RNA
transcript. cDNAs that are splice variants of the same gene will
contain regions of sequence identity or complete homology
(representing the presence of the same exon or portion of the same
exon on both cDNAs) and regions of complete non-identity (for
example, representing the presence of exon "A" on cDNA 1 wherein
cDNA 2 contains exon "B" instead). Because the two cDNAs contain
regions of sequence identity they will both hybridize to a probe
derived from the entire nucleic acid molecule or portions of the
nucleic acid molecule containing sequences found on both cDNAs; the
two splice variants are therefore substantially homologous to such
a probe and to each other.
[0047] The terms "in operable combination," "in operable order,"
and "operably linked" as used herein refer to the linkage of
nucleic acid molecule sequences in such a manner that a nucleic
acid molecule capable of directing the transcription of a given
sequence and/or the synthesis of a desired polypeptide product is
produced. The term also refers to the linkage of amino acid
sequences in such a manner so that a functional protein is
produced.
[0048] In certain embodiments the nucleic acid molecule is provided
in the form of "expression vector" or "expression cassette", which
as used herein refers to a recombinant DNA molecule containing at
least one desired coding sequence and appropriate nucleic acid
sequences necessary for the expression of the operably linked
coding sequence in a particular host organism. Nucleic acid
sequences necessary for expression in eukaryotic cells are known to
utilize promoters, enhancers, and termination and polyadenylation
signals.
[0049] In certain embodiments the nucleic acid molecule may encode
a polypeptide product which is expressed episomally, for example
when the nucleic acid is presented in the form of a vector, such as
a plasmid. The use of a suitable episomal vector provides a means
of maintaining a polynucleotide sequence in target cells in high
copy number extra-chromosomally, thereby eliminating potential
effects of chromosomal integration. In these embodiments, the
sequence which encodes the polypeptide product may be associated
with a promoter which facilitates transcription of the sequence
which encodes the polypeptide product. The promoter may be a
constitutive promoter or an inducible promoter. Suitable promoters
may include, but are not necessarily limited to a cytomegalovirus
(CMV) promoter (see, for example Chen, X., Frisina, R. D., Bowers,
W. J., Frisina, D. R., Federoff, H. J. (2001) HSV amplicon-mediated
neurotrophin-3 expression protects murine spiral ganglion neurons
from cisplatin-induced damage. Molecular Therapy 3 (6): 958-63 or
Kawamoto, K., Sha, S. H., Minoda, R., Izumikawa, M., Kuriyama, H.,
Schacht, J., Raphael, Y. (2004) Antioxidant gene therapy can
protect hearing and hair cells from ototoxicity. Molecular Therapy
9 (2): 173-181, the entire contents of which are incorporated
herein by reference). The promoter may comprise a Myosin VIIa (Myo)
or neuron specific enolase (NSE) promoter, as these are cell
specific transcription regulators for hair cells (Myo) and spiral
ganglion neurons (NSE). The promoter may comprise a cytomegalovirus
immediate early (IE) enhancer (CMV-IE), or a chicken beta-actin
promoter (CAG), or an elongation factor 1alpha promoter (EF-1alpha)
or a Rous sarcoma virus promoter (RSV) (see, for example, Liu, Y.,
Okada, T., Nomoto, T., Ke, X., Kume, A., Ozawa, K., Xiao, S. (2007)
Promoter effects of adeno-associated viral vector for transgene
expression in the cochlea in vivo. Exp Mol. Med. 39 (2) 170-175,
the entire contents of which are incorporated herein by reference).
The promoter may comprise a glial fibrillary acidic protein (GFAP)
promoter. The promoter may comprise a CBA promoter, which is a
hybrid promoter comprising a sequence from the chicken .beta.-actin
promoter and from the CMV promoter.
[0050] In certain embodiments the nucleic acid molecule comprises a
post-transcriptional regulatory element, for example to enhance
expression of the agent, such as the woodchuck hepatitis virus
posttranscriptional regulatory element (WPRE) (see, for example
Stone, I. M., Lurie, D. I., Kelley, M. W., Poulsen, D. J. (2005)
Adeno-associated virus-mediated gene transfer to hair cells and
support cells of the murine cochlea. Mol Ther 11(6): 843-84 and Xu,
L., et al. (2001) CMV-beta-actin promoter directs higher expression
from an adeno-associated viral vector in the liver than the
cytomegalovirus or elongation factor 1 alpha promoter and results
in therapeutic levels of human factor X in mice. Hum. Gene Ther.
12: 563-573, the entire contents of which are incorporated herein
by reference.)
[0051] In certain embodiments the Nrg-ICD enhancer and/or PSD-95
promoter is used for targeting of the expression of agent in spiral
ganglion neurons (see for example Bao, J., Lin, H., Ouyang, Y.,
Lei, D., Osman, A., Kim, T. W., Mei, L., Dai, P., Ohlemiller, K.
K., Ambron, R. T. (2004) Activity-dependent transcription
regulation of PSD-95 by neuregulin-1 and Eos. Nature Neuroscience 7
(11): 1250-8, the entire contents of which is incorporated herein
by reference)
[0052] In certain embodiments the nucleic acid molecule may be
incorporated into the genome of cells, such as mesenchymal cells of
the scala tympani or scala vestibuli.
[0053] The polypeptide product which is encoded by the nucleic acid
molecule may be a neurotrophic factor for spiral ganglion cells or
cochlear hair cells. As used herein a "neurotrophic factor" is a
polypeptide possessing at least one activity selected from
promoting or maintaining neurite outgrowth from spiral ganglion
cells and/or directing neurite outgrowth from spiral ganglion cells
towards the source of the neurotrophic factor, and/or supporting
the survival of spiral ganglion cells, and/or supporting the
survival of cochlear hair cells. In a particular embodiment the
neurotrophic factor possesses activity in promoting or maintaining
neurite outgrowth from spiral ganglion cells and/or directing
neurite outgrowth from spiral ganglion cells towards the source of
the neurotrophic factor, and/or supporting the survival of spiral
ganglion cells. The neurotrophic factor may be encoded as a
preprotein, and optionally subsequently processed to a mature form.
In certain embodiments, the neurotrophic factor may be selected
from Neurotrophin-3 (NT-3) or Neurotrophin-3 precursor molecule or
a preprotein form thereof (NCBI Accession No. HGNC:8023, Swiss Prot
Accession Number P20783) or homologs or human variants thereof;
Neurotrophin 4/5 (NT-4/5, NT-4, NT-5, NTF4, NTF5) or a preprotein
form thereof ((HGNC:8024), Ensembl accession No. ENSP00000328738,
GenBank accession No. AAA60154, version GI:190265) or homologs or
human variants thereof; Nerve growth factor (NGF) (NCBI Accession
No. NP.sub.--002497.2 version GI:70995319) (HGNC:7808), Ensembl
assession no. ENSP00000358525), preprotein forms thereof or
homologs or human variants thereof; Brain derived neurotrophic
factor (BDNF, Brain-derived neurotrophic factor Precursor,
Abrineurin) (the preprotein form of which is described in NCBI
Accession No. NP.sub.--001137277.1 version GI:219842288,
UniProtKB/Swiss-Prot Accession No P23560) or homologs or human
variants thereof; glial cell line-derived neurotrophic factor
(GDNF, glial cell derived neurotrophic factor precursor)(NCBI
Accession No. HGNC:4232 (gene) or the preprotein forms as described
in NCBI Accession Numbers NP.sub.--000505 and NP.sub.--000505.1) or
homologs or human variants thereof; ciliary neurotrophic factor
(CNTF)(NCBI Accession No. NP.sub.--000605 version GI:4758020
(HGNC:2169), UniProtKB/Swiss-Prot Accession No. P26441) or homologs
or human variants thereof; Activity dependent neurotrophic factor
(ADNF, ADNP1; KIAA0784; ADNP, or Activity-dependent neuroprotector
homeobox protein) (Ensembl Accession Numbers: ENSP00000360662,
ENSP00000379346, ENSP00000342905, and ENSP00000379349) or homologs
or human variants thereof, or fragments thereof, or the ADNF
homologues ADNF-9 or the active peptide region with an amino acid
sequence of SALLRSIPA, or ADNF-14 (neuropeptide) or the
neuroprotective NAP peptide of ADNF with an amino acid sequence of
NAPVSIPQ; the cytokine trophic factor leukaemia inhibitory factor
(LIF) (NCBI accession No. HGNC:6596 (genomic sequence),
UniProtKB/Swiss-Prot Accession No. NP.sub.--002300.1 version
GI:4504991 (polypeptide), typically in conjunction with a
neurotrophin such as NT-3; and Fibroblast growth factor (FGF 1, FGF
2) (NCBI accession No NM.sub.--002006 (genomic sequence),
UniProtKB/Swiss-Prot Accession No. P09038 (polypeptide)) or
homologs or human variants thereof, which is believed to interact
with NT3 and to support neurite outgrowth and growth cone
development, or in a combination of FGF-2 & GDNF.
[0054] The polypeptide product which is encoded may be an Apyrase
(IUBMB Enzyme Nomenclature Enzyme classification EC 3.6.1.5), an
enzyme which hydrolyses ATP extracellularly and therefore prevents
P2X receptor activation which can inhibit neurotrophin-dependent
neurite outgrowth. The polypeptide product may be an
ectonucleotidase, an enzyme which promotes the production of
adenosine, which is neuroprotective via adenosine receptors on
spiral ganglion neurons. An example of a suitable ectonucleotidase
in human subjects is ectonucleoside triphosphate diphosphohydrolase
2 (ENTPD2) (NCBI Accession no. NM 001246).
[0055] The polypeptide product which is encoded by the nucleic acid
molecule may be involved in the melanocortin signalling pathway,
for example alpha-melanocyte stimulating hormone (alpha-MSH). The
melanocortins make up a family of endogenous peptides derived from
pro-opiomelanocortin, which bind to five melanocortin receptors
(MCRs). Other examples of melanocortins which may be encoded by the
nucleic acid molecule include proopiomelanocortin (POMC) or its
cleavage products
(adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulating
hormone/beta-melanocyte stimulating hormone/beta-endorphin) or
precursors of active peptides (for gene NCBI Accession No.
HGNC:9201; for precursor NCBI accession No AAA60140 version
GI:190188).
[0056] The polypeptide product which is encoded by the nucleic acid
molecule may be a morphogen, for example a bone morphogenic protein
(BMP), Sonic hedgehog, or Wingless/Wnts.
[0057] The polypeptide product which is encoded by the nucleic acid
molecule may be an axon guidance factor which is capable of
attracting spiral ganglion cell neurites, such as laminin,
Netrin-1, an Ephrin, a semaphorin, a Slit such as a 3 slit homolog,
slit2 or slit3 or a cytokine such as Leukemia inhibitory
factor.
[0058] (b) Nucleic Acid Molecules which Modulate Endogenous Gene
Expression
[0059] In certain embodiments the agent may comprise or consist of
a nucleic acid molecule which modulates the expression of an
endogenous gene sequence. The modulation of expression of the
endogenous gene expression may be a direct regulation of
expression, for example by directly modulating transcription or
translation of an endogenous gene. The modulation of expression of
the endogenous gene by the nucleic acid molecule may be an indirect
regulation of expression, for example by expression of a
polypeptide by the nucleic acid molecule which in turn modulates
the expression of the endogenous gene. An example may be be
targeted inhibition of adenosine kinase (NCBI accession No HGNC:257
(genomic sequence); NP.sub.--001114.2 (polypeptide)), leading to
increased extracellular adenosine levels which are
neuroprotective.
[0060] The modulation of expression of an endogenous gene sequence
may be an up regulation of an endogenous gene sequence. For
example, the nucleic acid molecule may encode a transcription
factor which promotes the expression of an endogenous gene, such as
a gene which encodes a neurotrophic factor. The Sox2, Brn-3.1 and
Gata 3 transcription factors are examples of this class of cell
signalling molecule associated with determination of sensory hair
cell, supporting cell and spiral ganglion neuron development and
survival in the cochlea (Keithley, E M, Erkman, L., Bennett, T.,
Lou, L. Ryan, A.F. (1999) Effects of a hair cell transcription
factor Brn-3.1, gene deletion on homozygous and heterozygous mouse
cochleas in adulthood and aging. Hearing Research 134:71-76; Van
der Wees, J., Van Looij, M.A., de Ruiter, MM, Elias, H., van der
Burg, H., Liem, S.S., Kurek, D., Engle, J.D., Karis, A., van
Zanten, B.G., de Zeeuw, C.I., Grosveld, F.G., van Doornick, J.H.
(2004) Hearing loss following Gata3 haploinsufficiency is caused by
cochlear disorder. Neurobiol Dis 16:169-78; Dabdoub, A., Puligilla,
C., Jones, J.M., Fritzsch, B., Cheah, K.S., Pevney, L.H. Kelley,
M.W. (2008) Sox2 signaling in prosensory domain specification and
subsequent hair cell differentiation in the developing cochlea.
Proc Natl Acad Sci USA 105:18396-401; and Appler, J., Koundakjian,
E., Lu, C, Goodrich, L. (2009) Gata3 regulates neurite extension
and targeting of spiral ganglion neurons. Proc, 7th Molecular
Biology of Hearing and Deafness conference. June 20-23, Harvard
Medical School, Boston, Mass., USA).
[0061] The Adenovirus-mediated transduction of the transcription
factor atonal homologue 1 (Atoh1, also known as Math1, ATH1, HATH1
or bHLHa14) (NCBI accession No. BC069578 version GI:47480322
(HGNC:797), Uniprot accession Nos. Q92858; Q14CT9) into mouse or
guinea pig cochlear cells has been described to lead to the
generation of a cochlear hair cell-like phenotype from supporting
cells, which led to secretion of trophic factors, the attraction of
neurite outgrowth and the formation of functional synapses between
the transformed cells and auditory spiral ganglion cells.
Accordingly, the methods described herein contemplate the
transfection of the transcription factor atonal homologue 1 into
cochlear cells using electroporation, without the need for viral
vector-mediated delivery.
[0062] Other transcription factors which modulate the expression of
neurotrophic factors in cells are known in the art. For example,
the transcription factor SRY-box-containing gene 10 (Sox10) induces
a greater than 100 fold up regulation of the expression of ciliary
neurotrophic factor in Schwann cells in the mouse, whilst knockdown
of Sox10 results in a reduction of expression ciliary neurotrophic
factor by more than 80%. Accordingly, the methods described herein
contemplate the transfection of cochlear cells by electroporation
of a nucleic acid molecule encoding a transcription factor, wherein
the transcription factor modulates the expression of an endogenous
neurotrophic factor for spiral ganglion cell neurons, or wherein
the transcription factor modulates the expression of an exogenous
polynucleotide sequence which encodes a neurotrophic factor for
spiral ganglion cells and which is transfected with the
transcription factor.
[0063] The up regulation of a gene sequence may be achieved by the
use of gene targeting constructs to generate transcription units
formed by homologous recombination between an endogenous target
gene and the targeting construct. In such methods, expression of a
desired targeted gene in a cell (i.e., a desired endogenous
cellular gene) is altered by the introduction by homologous
recombination into the cellular genome at a preselected site, of
DNA which includes at least a regulatory sequence, an exon and a
splice donor site. These components are introduced into the
chromosomal (genomic) DNA in such a manner that this, in effect,
results in production of a new transcription unit (in which the
regulatory sequence, the exon and the splice donor site present in
the DNA construct are operatively linked to the endogenous gene).
As a result of introduction of these components into the
chromosomal DNA, the expression of the desired endogenous gene is
altered.
[0064] The production and use of targeting constructs are
described, for example, in U.S. Pat. No. 5,641,670 (Treco et al.),
the entire contents of which are incorporated herein by reference.
Endogenous genes which may be up regulated by these techniques
include neurotrophic factor genes selected from Neurotrophin-3
(NT-3) or Neurotrophin-3 precursor molecule or a preprotein form
thereof (NCBI Accession No. HGNC:8023, Swiss Prot Accession Number
P20783) or homologs or human variants thereof; Neurotrophin 4/5
(NT-4/5, NT-4, NT-5, NTF4, NTF5) or a preprotein form thereof
((HGNC:8024), Ensembl accession No. ENSP00000328738, GenBank
accession No. AAA60154, version GI:190265) or homologs or human
variants thereof; Nerve growth factor (NGF) (NCBI Accession No.
NP.sub.--002497.2 version GI:70995319) (HGNC:7808), Ensembl
accession no. ENSP00000358525), preprotein forms thereof or
homologs or human variants thereof; Brain derived neurotrophic
factor (BDNF, Brain-derived neurotrophic factor Precursor,
Abrineurin) (the preprotein form of which is described in NCBI
Accession No. NP.sub.--001137277.1 version GI:219842288,
UniProtKB/Swiss-Prot Accession No P23560) or homologs or human
variants thereof; glial cell line-derived neurotrophic factor
(GDNF, glial cell derived neurotrophic factor precursor)(NCBI
Accession No. HGNC:4232 (gene) or the preprotein forms as described
in NCBI Accession Numbers NP.sub.--000505 and NP.sub.--000505.1) or
homologs or human variants thereof; ciliary neurotrophic factor
(CNTF)(NCBI Accession No. NP.sub.--000605 version GI:4758020
(HGNC:2169), UniProtKB/Swiss-Prot Accession No. P26441) or homologs
or human variants thereof; Activity dependent neurotrophic factor
(ADNF, ADNP1; KIAA0784; ADNP, or Activity-dependent neuroprotector
homeobox protein) (Ensembl Accession Numbers: ENSP00000360662,
ENSP00000379346, ENSP00000342905, and ENSP00000379349) or homologs
or human variants thereof, or fragments thereof, or the ADNF
homologues ADNF-9 or the active peptide region with an amino acid
sequence of SALLRSIPA, or ADNF-14 (neuropeptide) or the
neuroprotective NAP peptide of ADNF with an amino acid sequence of
NAPVSIPQ; the cytokine trophic factor leukaemia inhibitory factor
(LIF) (NCBI accession No. HGNC:6596 (genomic sequence),
UniProtKB/Swiss-Prot Accession No. NP.sub.--002300.1 version
GI:4504991 (polypeptide), typically in conjunction with a
neurotrophin such as NT-3; and Fibroblast growth factor (FGF 1, FGF
2) (NCBI accession No NM.sub.--002006 (genomic sequence),
UniProtKB/Swiss-Prot Accession No. P09038 (polypeptide)) or
homologs or human variants thereof, which is believed to interact
with NT3 and to support neurite outgrowth and growth cone
development, or in a combination of FGF-2 & GDNF; Apyrase; or
ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) (NCBI
Accession no. NM 001246); a gene encoding a polypeptide involved in
the melanocortin signalling pathway, for example alpha-melanocyte
stimulating hormone (alpha-MSH), proopiomelanocortin (POMC) (HGNC
Accession No. HGNC:9201), or a gene encoding a morphogen, for
example a bone morphogenetic protein (BMP), Sonic hedgehog, or
Wingless/Wnts, a gene encoding an axon guidance factor, such as
laminin, Netrin-1, an Ephrin, a semaphorin, a Slit such as a 3 slit
homolog, slit2 or slit3 or a cytokine such as Leukaemia inhibitory
factor.
[0065] The modulation of expression of the endogenous gene may be
the down regulation of expression of an endogenous gene. The down
regulation of expression of the endogenous gene may, for example,
be the down regulation of a transcription factor which
constitutively or inducibly inhibits the expression of a
neurotrophic factor by a cochlear cell, such as a cochlear
mesenchymal cell, which results in the mesenchymal cell expressing
a neurotrophic factor.
[0066] The down regulation of an endogenous gene may utilize the
targeted disruption of the expression of one or more endogenous
genes using any nucleic acid molecules which selectively target and
inhibit the expression of the genes, such as antisense sequences,
varieties of small interfering RNA (siRNA) sequences, shRNA
sequences, ribozyme sequences and the like. The "expression" of
endogenous genes is intended to encompass the transcription and/or
translation of endogenous gene sequences.
[0067] Methods for the design, synthesis, and delivery of antisense
nucleic acid molecules are well known in the art. The antisense
molecules may be DNA or RNA, or partial or complete synthetic
analogues thereof. Antisense polynucleotides of the invention may
be generated which are at least substantially complementary along
their length to the region of the gene in question. Binding of an
antisense polynucleotide to its complementary cellular sequence may
interfere with transcription, RNA processing, transport,
translation and/or mRNA stability.
[0068] Suitable antisense oligonucleotides may be prepared by
methods well known to those of skill in the art. Typically
antisense oligonucleotides will be synthesized on automated
synthesizers. Suitable antisense oligonucleotides may include
modifications designed to improve their delivery into cells, their
stability once inside a cell, and/or their binding to the
appropriate target. For example, the antisense oligonucleotide may
be modified by the addition of one or more phosphorothioate
linkages, or the inclusion of one or more morpholine rings into the
backbone. The antisense oligonucleotide may be 10-30 base pairs in
length.
[0069] As a practical matter, whether any particular nucleic acid
molecule is no more than 90% identical to, for instance, the
nucleotide sequence of other known gene or mRNA or cDNA sequence
can be determined conventionally using known computer programs such
as the Bestfit program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the
local homology algorithm of Smith and Waterman to find the best
segment of homology between two sequences (Advances in Applied
Mathematics 2:482-489 (1981)). When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 90% identical to a reference sequence,
the parameters are set such that the percentage of identity is
calculated over the full length of the reference nucleotide
sequence and that gaps in homology of up to 5% of the total number
of nucleotides in the reference sequence are allowed. A preferred
method for determining the best overall match between a query
sequence and a subject sequence, also referred to as a global
sequence alignment, can be determined using the FASTDB computer
program based on the algorithm of Brutlag and colleagues (Comp.
App. Biosci. 6:237-245 (1990)). In a sequence alignment the query
and subject sequences are both DNA sequences. An RNA sequence can
be compared by converting U's to T's. The result of said global
sequence alignment is in percent identity. Preferred parameters
used in a FASTDB alignment of DNA sequences to calculate percent
identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1,
Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length
of the subject nucleotide sequence, whichever is shorter.
[0070] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
Only bases outside the 5' and 3' bases of the subject sequence, as
displayed by the FASTDB alignment, which are not matched/aligned
with the query sequence, are calculated for the purposes of
manually adjusting the percent identity score.
[0071] Small interfering RNA (siRNA) sequences are small, usually
double-stranded RNA oligonucleotides, for example at 19, 21, 27 or
29 bases in length, with or without overhangs, which specifically
hybridize with RNA sequences of interest and which serve as
substrates for the RNA-induced silencing complex. Double-stranded
RNA molecules may be synthesized in which one strand is identical
to a specific region of the mRNA transcript to be silenced, and
this double stranded RNA may be introduced directly. Alternatively,
corresponding dsDNA can be employed, which, once presented
intracellularly is converted into dsRNA. Methods for the design and
synthesis of suitable siRNA molecules for use in RNA interference
(RNAi) and for achieving post-transcriptional gene silencing are
well known to those of skill in the art. For example, rules for the
rational design of siRNA are available online in "Rules of siRNA
design for RNA interference (RNAi)" at
<http://www.protocol-online.org/prot/Protocols/Rules-of-siRNA-design-f-
or-RNA-interference-RNAi-3210.html>. These rational design
principals are also described in Elbashir S M at al. (2001)
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells. Nature. 411:494-498; Elbahir S M et al. (2001).
Functional anatomy of siRNAs for mediating efficient RNAi in
Drosophila melanogaster embryo lysate. EMBO J. 20:6877-6888;
Elbashir S M et al. (2002). Analysis of gene function in somatic
mammalian cells using small interfering RNAs. Methods. 26:199-213;
Reynolds A, Leake D, Boese Q, Scaringe S, Marshall W S, Khvorova A.
Rational siRNA design for RNA interference. Nat. Biotechnol. 2004
March; 22(3):326-30; and online at
<http://www.basic.northwestern.edu/biotools/oligocalc.html>.
The entire contents of each of these publications is incorporated
herein by reference.
[0072] Examples of two siRNA design tools which implement the
rational siRNA principals discussed above are design algorithms
offered by Dharmacon, Inc and a downloadable Microsoft Excel.TM.
template, written by Maurice Ho at
<http://boz094.ust.hk/RNAUsiRNA>.
[0073] The skilled addressee will appreciate that a range of
suitable siRNA or nucleic acid molecules comprising an siRNA
sequence which is capable of inhibiting the expression of an
endogenous gene can be identified and generated based on knowledge
of the sequence of the genes in question using routine procedures
known to those skilled in the art without undue experimentation.
Those skilled in the art will appreciate that there need not
necessarily be 100% nucleotide sequence match between the target
sequence and the siRNA sequence. The capacity for mismatch is
dependent largely on the location of the mismatch within the
sequences. In some instances, mismatches of 2 or 3 nucleotides may
be acceptable but in other instances a single nucleotide mismatch
is enough to negate the effectiveness of the siRNA. The suitability
of a particular siRNA molecule may be determined using routine
procedures known to those skilled in the art without undue
experimentation.
[0074] Although the maximal effects of antisense nucleic acids and
siRNA on the specific inhibition of RNA or protein expression are
comparable, siRNA generally produces a longer-lasting effect.
Although siRNAs may be introduced into a cell by way of a vector,
for example via a viral-mediated delivery mechanism such as an
adeno-associated virus vector, in the context of the present
invention the siRNA will be delivered exogenously by
electroporation. Techniques for the administration of siRNA
sequences locally or systemically to mice (Yano et al, (2004)
Clinical Cancer Research 10: 7721-7726), to primates (Zimmermann et
al, (2006) Nature 441(7089):111-114) and to humans (Nogawa et al.
(2006) J Clin Invest 115:978-985) have been described, and these
have demonstrated that this class of molecules can be used to
reduce the expression of target genes in vivo. The entire contents
of each of these citations is incorporated herein by reference.
[0075] Ribozymes, such as hammerhead or hairpin ribozymes, are
capable of the targeted catalytic cleavage and splicing of specific
RNA sequences, including mRNA and genomic RNA sequences. The design
and methods for the delivery of ribozymes are reviewed, for
example, in Vaish, Kore and Eckstein (1998) Nucleic Acids Research
26:5237-5242; in Lieber and Strauss (1995) Mol. Cell. Biol.
15:540-551; and in Usman and Blatt (2000) J Clin Invest
106:1197-1202, the entire contents of each of which are
incorporated herein by reference.
[0076] Short hairpin RNA (shRNA) is a sequence of RNA with a tight
hairpin turn structure which is introduced into cells as part of a
vector which comprises a constitutive promoter such as a U6
promoter to allow shRNA to be constitutively expressed. The shRNA
hairpin structure is cleaved by the cellular machinery into siRNA,
which is then bound to the RNA-induced silencing complex (RISC).
This complex binds to and cleaves mRNAs which match the siRNA that
is bound to it. The vector comprising the shRNA sequence is usually
passed on to daughter cells in cell division, allowing the gene
silencing to be inherited. Strategies for designing shRNA sequences
and nucleic acid molecules comprising shRNA for use in mammalian
cells are known, for example McIntyre and Fanning, Design and
cloning strategies for constructing shRNA expression vectors, BMC
Biotechnol. (2006) 6:1.
[0077] The nucleic acid may be microRNA. The term microRNA or
"miRNA" as used herein has the ordinary and plain meaning of a
non-coding RNA molecule found in eukaryotes which is involved in
RNA-based gene regulation. The term can be used to refer to the
single-stranded RNA molecule processed from a precursor or in
certain instances the precursor itself or a mimetic or inhibitor
thereof. The involvement of microRNA in normal cochlear function
and in a familial cochlear disorder has been described in Mencia et
al., Nature Genetics (2009) 41(5):609-613. It is anticipated that
the modulation of microRNA activity in cells of the cochlea, either
by the introduction of microRNA or microRNA mimics into cells or
the silencing of endogenous microRNA with a microRNA inhibitor may
provide an approach for modulating the expression of particular
targeted genes. Strategies for the administration of microRNA into
cells or tissues of subjects have been described in International
patent publication No. WO 2008/073920, which describes methods of
modulating gene expression, or biologic or physiologic pathways in
a cell, a tissue, or a subject comprising administering to the
cell, tissue, or subject an amount of an isolated nucleic acid or
mimetic thereof comprising a specific microRNA or microRNA
inhibitor nucleic acid sequence in an amount sufficient to modulate
the expression of a gene positively or negatively modulated by the
microRNA. Studies have demonstrated therapeutic application of
modulating microRNA in animals, including primates.
[0078] Techniques for the design of microRNA and microRNA
inhibitors are readily available. A database of known microRNA
sequences, new microRNA sequences and predicted microRNA targets is
maintained at miRBase <http://microrna.sanger.ac.uk/> and
described in "miRBase: tools for microRNA genomics" Griffiths-Jones
S, Saini H K, van Dongen S, Enright A J. NAR 2008 36(Database
Issue):D154-D158; "miRBase: microRNA sequences, targets and gene
nomenclature" Griffiths-Jones S, Grocock R J, van Dongen S, Bateman
A, Enright A J. NAR 2006 34(Database Issue):D140-D144; and "The
microRNA Registry." Griffiths-Jones S. NAR 2004 32(Database
Issue):D109-D111. Commercial services are available for the design
and synthesis of specific microRNA inhibitors based on a provided
target sequence.
[0079] In general, each of these classes of molecules appears to be
well tolerated, either when administered to human or animal
subjects systemically, such as via intravenous routes, as a bolus
or by sustained release techniques subcutaneously, or when directly
applied to cells.
[0080] Agents Other than Nucleic Acid Molecules
[0081] Also contemplated in the methods described herein is the
electroporation of small neuroprotective polypeptides which may
support the survival of cochlear hair cells and/or spiral ganglion
cells. Examples of such molecules include the neuroprotective NAP
peptide, activity dependent neurotrophic factor, FNK peptide,
ACTH.sub.4-9 analogue Org 2766, or molecules comprising these
peptides which retain neuroprotective activity in the cochlea.
[0082] Electroporation
[0083] The methods described herein use electroporation to deliver
the agent into the cytoplasm and/or nucleus of cochlear cells which
lie close to the electrode of the cochlear implant.
[0084] The use of non-invasive electroporation to successfully
deliver a nucleic acid molecule to cells of an intact non-cochlear
tissue has been previously described, for example, in U.S. Pat. No.
6,972,013 (Zhang and Rabussay), the entire contents of which is
incorporated herein by reference.
[0085] As used herein, the term "transfection" refers to the
transfer of an agent, including but not limited to a nucleic acid
molecule, into the cytoplasm and/or nucleus of a cell by
electroporation. In certain embodiments in which the agent is a
nucleic acid molecule, the nucleic acid molecule encodes a
polypeptide product which is expressed by the cell. In certain
embodiments in which the agent is a nucleic acid molecule, the
nucleic acid molecule acts upon the transcription and translation
machinery of the cell. A transfected cell may carry the nucleic
acid molecule product permanently if the nucleic acid molecule
agent is stably incorporated into the cell's genome. A transfected
cell may carry the nucleic acid molecule product transiently if the
nucleic acid molecule agent is not incorporated into the cell's
genome.
[0086] The agent may be delivered to the cochlea at the time the
electroporation electrode is inserted into the cochlea, or before
the insertion of the electroporation electrode or after the
insertion of the electroporation electrode. The agent may be
provided in a diffusible form, such as incorporated in or
associated with a biodegradable or biocompatible viscous liquid or
gel solution surrounding the electrode array of the cochlear
implant. The viscous liquid or gel solution may comprise
polyacrylic acid (PAA), polyvinyl alcohol (PVA), polylactic acid
(PLA) and/or polyglycolic acid (PGA). Pluronic F127 (BASF) at
<30% solution may also be used to stabilise the media containing
the agent. This approach may provide an advantage in that the agent
is presented to the target cochlear cells with the electrode array
in situ in a stable concentration at the time of
electroporation.
[0087] Where the agent comprises a nucleic acid molecule the agent
may be presented in a diluent solution which comprises ions and
proteins or the agent may be diluted in sterile distilled deionised
water, provided that the diluent solution does not substantially
adversely affect the efficiency of electroporation. Typically water
is used as the diluent, with the nucleic acid molecule provided
typically at a concentration of from 10 ng/.mu.l to 1 .mu.g/.mu.l.
The nucleic acid molecule may be provided within a buffered
solution, such as phosphate-buffered saline (PBS; e.g. pH 7.4), or
a perilymph-like solution (for example as described by Salt, A.N.
Konishi, T. (1986) The cochlear fluids: Perilymph and endolymph.
Eds. Altschuler, R.A., Hoffman, D.W., Bobbin, R.P. In: Neurobiology
of Hearing: The Cochlea. New York, Raven Press, pp. 109-122), and
may be in the presence of divalent cation chelators such as
ethylenediaminetetraacetic acid (EDTA) for stabilization of the
molecules.
[0088] Typically the solution comprising agent is delivered in the
vicinity of the apex of the cochlea, so that the solution
comprising the agent flows back down the cochlea chamber and
displaces the media lying within the cochlea chamber. The volume of
the human scala tympani is in the order of 30 .mu.l, the scala
vestibuli 22 .mu.l, and the scala media 2 .mu.l. In order to ensure
that the chamber contains sufficient agent the nominal volume of
solution comprising the agent to be delivered to the cochlea may be
in the order of 30 .mu.l. Typically, the controlled delivery of
such a volume may be achieved through the use of a mechanical
infusion micropump, a microsyringe, or a mini-osmotic pump (Alza
Corporation, Palo Alto, Calif., USA). Once the agent and electrode
are present within the appropriate chamber(s) electroporation takes
place, and in certain embodiments the electroporation electrode is
then removed. In these embodiments of the invention the
electroporation electrode may be withdrawn from the cochlea as a
separate action to the placement of the cochlear implant, and
typically the electrode is removed before the insertion of the
cochlear implant.
[0089] Electrodes for use in the electroporation methods described
herein deliver the electric field which is required for
electroporation to the cochlear tissue. Typically the electrodes
will comprise electrically conductive, non-toxic or bio-inert
metal, for instance platinum, gold, tungsten, or stainless steel.
The electroporation electrode may be electrically conductive at its
surface along the entire length of the electrode inserted into the
cochlea. The electroporation electrode may be electrically
conductive at its surface along one or more portions of the entire
length of the electrode inserted into the cochlea, and electrically
non-conductive at its surface along other portions of its length.
The non-conductive portions may be insulated at the surface of the
electrode with a coating, such as a resin or plastic coating. The
non-conductive portions may be made of a non-conducting material,
such as a resin or plastic.
[0090] In certain embodiments the electrodes may be inserted and
removed from the cochlea independently of the cochlear implant, for
example where the electroporation electrodes are not part of the
cochlear implant. In these embodiments, at least one electrode
which is used for electroporation is typically inserted into and
along one or more of the scala tympani or the scala vestibuli of
the cochlea before or after the agent is introduced into the same
chamber(s) of the cochlea.
[0091] In other embodiments at least one of the electroporation
electrodes is provided as part of the cochlear implant.
[0092] The electroporation electrode may comprise a lumen, channel
or groove which carries the agent and allows the delivery of the
agent to the chamber(s) of the cochlea, or it may be associated
with a separate catheter or cannula, such as a silicon or polymer
tube which is attached to the electrode. Alternatively, the agent
may be introduced into the cochlea via a cannula which may be
inserted or removed independently to the electroporation
electrode.
[0093] In certain embodiments of the methods described herein, at
least one electrode which is used for electroporation is provided
by the cochlear implant. The term "cochlear implant" or "cochlear
implant device" as used herein refers to a device which is at least
partially implanted in the scala tympani or the scala vestibuli of
the cochlea for the purpose of restoring hearing by direct
electrical stimulation of the cochlear nerve. A cochlear implant is
commonly termed a "bionic ear". Examples of cochlear implants
and/or electrodes for cochlear implants are described in U.S. Pat.
No. 7,451,000 (Cochlear Limited), U.S. Pat. No. 7,406,352 (Cochlear
Limited); U.S. Pat. No. 7,367,992 (Cochlear Limited), U.S. Pat. No.
7,349,744 (Cochlear Limited); U.S. Pat. No. 7,346,397 (Cochlear
Limited), U.S. Pat. No. 7,340,308 (Advanced Cochlear Systems,
Inc.), U.S. Pat. No. 7,319,906 (Advanced Bionics Corporation), U.S.
Pat. No. 7,317,944 (Advanced Bionics Corporation), and U.S. Pat.
No. 7,315,763 (Advanced Bionics Corporation), the entire contents
of each of which is incorporated by reference.
[0094] A cochlear implant possesses at least one electrode and more
typically a series of independently addressable "nodes" along the
length of the region of the implant lying within the cochlea, with
each node electrically insulated from other nodes. During the
normal operation of the cochlear implant, the nodes receive
stimulii originating from an external control unit and provide an
electrical stimulus to the spiral ganglion cells by creating a
voltage between the nodes in a bimodal fashion to prevent
polarization of the nodes.
[0095] For the electroporation methods described herein, in certain
embodiments some or all of the nodes of the cochlear implant may
act as a single electrode. In other embodiments, individual nodes
or groups of nodes may act as an electrode and other individual
nodes or groups of nodes act as a second electrode. In certain
embodiments the cochlear implant may provide a plurality of
electrodes and second electrodes. Examples of configurations of
nodes and/or dedicated electroporation electrodes on a cochlear
implant are illustrated in FIG. 5.
[0096] For the purposes of electroporation a cathode and an anode
is required. When using a cochlear implant all the nodes, or
specific nodes along the implant, may be used to generate a
transient dialectric breakdown of the lipid bilayer of the plasma
membrane of the target cells. In one embodiment, this may be
performed by simultaneously polarizing some or all of the nodes
relative to at least one second electrode which is located distant
to the cochlear implant, for example outside of the cochlea, to
provide a current return path. An example of such a configuration
is illustrated in FIG. 5E. For example, an external electrode may
be located in the fascia tissue which lies along the temporal bone
of the subject. This second electrode is typically the cathode
(negative) terminal relative to the nodes of the electrode array.
In this unipolar delivery mode, the electrical voltage used to
electroporate cochlear cells is typically provided within a voltage
range of from 1 to 100 volts, more typically within a voltage range
of from 10 to 20 volts.
[0097] In another embodiment electroporation utilises bimodal
current delivery in which at least one and more typically several
nodes of the implant are configured as anodes, and with one or more
other nodes interspersed along the electrode array of the cochlear
implant configured as cathode second electrodes for current
delivery. Examples of such embodiments are illustrated in FIGS. 5A
and 5B. In this configuration, the polarity of individual nodes may
be altered over time during electroporation to ensure an even
transfection of target cells along the length of the array, and to
minimise toxic effects which may arise at either the cathode or
anode. Using a bimodal approach, the voltage required for
electroporation may be reduced because of the high field strength
between the closely associated nodes of the electrode.
[0098] In certain embodiments the current for electroporation is
provided by dedicated circuitry in the cochlear implant. In these
embodiments the dedicated circuitry generates the required
electrical pulse profile in an electroporation mode for use during
the implantation procedure and at later stages if additional gene
transfection is performed. The implant stimulator may then be
switched to a conventional stimulus mode for operation as a
cochlear prosthesis/auditory nerve stimulator.
[0099] In certain embodiments, cochlear implants are provided in
which one or more dedicated electroporation electrode "nodes" are
built into the implant for the purpose of providing the
electroporation stimulus to the cochlear tissue. These dedicated
nodes may be driven via an external isolated
electroporation-driving circuit, or the one or more dedicated
electroporation electrode nodes could be driven externally by
electroporation circuitry. In certain embodiments the dedicated
electroporation electrode nodes are not used in the normal
operation of the cochlear prosthesis in providing direct electrical
stimulus to the spiral ganglion cells as a result of auditory
stimulation. Examples of such embodiments are illustrated in FIGS.
5 C and 5D.
[0100] In embodiments in which the cochlear implant provides at
least one electroporation electrode, the agent may be provided via
a lumen, channel or groove in the cochlear implant. These forms of
electrode may allow multiple rounds of introduction of agent into
the cochlea following implantation of the cochlear implant and
therefor multiple rounds of electroporation. An example of such an
embodiment is illustrated in FIG. 6. The use of a cochlear implant
with a lumen to deliver agents to the cochlea is described, for
example, in Paasche et al., (2003) Otology & Neurology
24:222-227, the entire contents of which is incorporated herein by
reference.
[0101] The lumen of the cochlear implant may be sealed following
delivery of the agent.
[0102] US patent application publication No. 2008/0214986, the
entire content of which is incorporated herein by reference,
describes the use of gel compositions with a predetermined
conductivity for use in optimising the electroporation of tissues.
In certain embodiments, the electrodes and or cochlear implant may
be coated with gel, as described in this publication, to provide a
degree of control over the conductivity of the volume surrounding
the electrodes.
[0103] In certain embodiments of the methods described herein the
cochlear implant comprises at least one electrode which is used for
electroporation, and the agent is delivered to the desired chamber
of the cochlea using a catheter or cannula which is not associated
with the cochlea implant. In these embodiments typically the
catheter or cannula is inserted into the appropriate chamber(s) of
the cochlea and the solution comprising the agent is delivered. The
catheter or cannula is then withdrawn, the cochlear implant is
introduced and electroporation takes place, as described above.
[0104] The electroporation stimulus is typically provided in a
square wave configuration of from 100 .mu.s to 500 ms pulse
duration, more typically in a 30 ms pulse width being commonly
used. Multiple pulses are commonly used, with typically from 25 to
200 pulses, more typically from 50 to 100 pulses, with an interval
between each pulse of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds.
Ramped waveforms may also be used, as these may provide a faster
onset of electroporation dielectric breakdown of the cell membrane
for agent delivery, while minimizing current delivery. As such
stimuli delivered to the intact cochlea of a subject may be
perceived by the subject as excessively loud noise, typically the
subject will be under general anaesthetic during
electroporation.
[0105] The delivery of the electroporation current during the
electroporation procedure for delivery of an agent may typically
last from 5 to 10 minutes, depending upon the number of pulses and
the delay between pulses.
EXAMPLES
Example 1
Electroporation Transfection of Cochlear Mesenchymal Cells In
Situ
[0106] Experiments were carried out to determine whether pulsed
electrical fields of a specific profile, delivered via electrodes
placed in the cochlea of a guinea-pig, could provide localized
transfection of specific gene constructs in the mesenchymal cells
lining the cochlear scala tympani. One of two different naked cDNA
gene constructs were introduced into each cochlea. The cDNA gene
constructs utilized commercial bicistronic expression vectors where
fluorescence reporter proteins and therapeutic molecules were
jointly expressed via an IRES (internal ribosome entry site)
element, and expression was driven by the cytomegalovirus promoter
(CMVp).
[0107] The two cDNA constructs were:
[0108] 1. CMVp-TRPC3-IRES-DsRed2
[0109] a. The plasmid (pIRES2-DsRed2 (Clontech.TM.), including the
IRES bicystronic expression element and the DsRed2 fluoroescence
reporter.
[0110] b. The TRPC3 gene (Accession no. AB090949.1) element was
cloned from the guinea-pig (Cavia parcellus) by PCR. The complete
open reading frame was subcloned into the vector upstream of the
IRES element.
[0111] 2. CMVp-BDNF-FLAGtag-IRES-eGFPn
[0112] a. The plasmid backbone utilized the
pShuttle-IRES-hrGFP-lvector (Strategene.TM.)
[0113] b. The BDNF construct (Accession No. HM.sub.--183060) was
ligated into the multicloning site 5' to the 3.times.FLAG-tag and
IRES elements. The eGFPn reporter was downstream of the IRES.
[0114] Cochleae from guinea-pig temporal bones were placed in a
humidity chamber with Dulbecco's phosphate buffered saline (PBS).
The vectors were injected into the scala tympani compartment of the
guinea pig cochlea using a polyethelene cannula (10 .mu.l; 0.1 to
1.0 .mu.g/ml, dissolved in distilled water), after perforation of
the round window at the base of the cochlea, scala media
compartment, and placement of a fenestration at the apex of the
cochlea to permit perfusion of the scala tympani compartment.
[0115] After perfusion of the scala media with the cDNA gene
construct, a shaped silver wire electrode, modelling a cochlear
implant prosthesis, was inserted through the round window membrane
perforation, into the basal turn region of scala tympani (see FIG.
1). This approach is a useful model of a cochlear implant, and has
in the past been used in early translational research into
electrical stimulation of the auditory nerve, directed toward
providing a prosthesis for restoring hearing function utilising
unipolar electrodes inserted into the cochlea, including access via
the round window or through the base of the cochlea to directly
stimulate the auditory nerve. In these early studies current return
was provided by a second electrode located at either the base or
apex of the cochlea or in adjacent tissue, as for the current
study. In the present study, a second electrode, inserted into
either the apex of the cochlea, or into the oval window (basal turn
scala vestibuli), completed the current return pathway. The basal
electrode typically acted as an anode pole, with the second
electrode acting as the current source (cathode). In some
experiments the polarity was reversed.
[0116] A range of electroporation profiles were utilized in these
experiments, as set out in Table 1, using an isolated stimulator
(Grass Instruments, USA), gated to produce square wave
voltage-steps via a computer interface (Labview). Controls included
addition of the cDNA gene constructs with and without a liposomal
transfection reagent (lipofectamine 2000; Invitrogen) transfection
media and insertion of the electrodes, but no voltage delivery.
[0117] After electroporation, the cochleae were maintained in PBS
for 30 minutes at room temperature and then the tissues were
dissected and placed into organotypic culture (37.degree. C., 5%
CO.sub.2) in 24 well tissue culture plates. The culture media
included Dulbecco's modified Eagle medium (DMEM; Gibco), 10% fetal
bovine serum (FBS; Gibco), 25 mM Hepes (Sigma-Aldrich); 6 mg/ml
glucose & 300 units/ml penicillin (Sigma-Aldrich).
[0118] The tissue was imaged by removing the tissue from the
culture wells between 24 h and 96 h, after which the tissue was
placed in culture medium on a coverslip for epifluorescence imaging
(Leica, German; Andor emCCD, Ireland). Cells expressing the
CMVp-TRPC3-IRES-DsRed2 gene construct were detected by expression
of the DsRed2 signal in cytoplasm using a red emission filter
(N2.1; Leica, Germany). Expression of the CMVp-BDNF-FLAG
tag-IRES-eGFPn construct was detected using a green emission filter
(GFP, Leica, Germany) for the nuclear GFP signal. Expression of the
therapeutic BDNF molecule by the target cells in the cochlea was
confirmed by detection of the FLAG-tag, using an anti-FLAG antibody
(Sigma, cat. F7425), detected via a secondary antibody
(goat-anti-rabbit IgG; FITC). Expression of BDNF was confirmed
using confocal imaging (Zeiss 710 LSM, Germany).
[0119] The results which were obtained are summarized in Table 1. A
total of 14 experiments were performed to assess the potential for
electroporation-based gene delivery in the adult guinea-pig
cochlea. Gene construct CMVp-TRPC3-IRES-DsRed2 was delivered to 8
cochleae. Two of these experiments were controls in which
electroporation was omitted. In an additional two control
experiments, no cDNA was delivered to the cochlea, but the tissue
was placed into organotypic culture to confirm autofluorescence
levels and check for possible vector carry-over. The control
experiments did not exhibit expression of the reporter gene.
[0120] The electroporation experiments with the
CMVp-TRPC3-IRES-DsRed2 construct showed expression of the reporter
in four out of six experiments. Of these, four experiments used the
basal turn scala tympani electrode as the anode (STb(-)) and
expression of the DsRed2 reporter was imaged in three cases. Two
experiments utilized a cathode (+) electrode placement in the basal
scala vestibuli region, with DsRed2 reporter identified in one of
these. For the STb(-) electrode configuration, reporter gene
expression was primarily localized to the basal half of the
cochlea--in close proximity to the shaped scala tympani electrode.
The majority of transfected cells were mesenchymal cells on the
lateral aspect of the spiral limbus and the medial aspect of the
lateral wall, in the scala tympani compartment (FIG. 2). Limited
expression was detected in supporting cells of the organ of Corti.
Expression was maintained for the duration of the study (96
hours).
[0121] A second set of experiments investigated the delivery and
heterologous expression of BDNF as a putative therapeutic molecule.
Two experiments used the STb(-)-basal anode configuration, and two
experiments, the Stb(+) cathode configuration. Expression of the
CMVp-BDNF-FLAGtag-IRES-eGFPn construct was confirmed in all four
experiments, evident as nuclear GFP labelling, principally within
the basal region (turn1 and turn 2; FIG. 3). This expression was
sustained for the duration of these experiments (72 hours).
Expression of recombinant BDNF by these cells was confirmed by
FITC-positive immunolabelling of the cytoplasm of the GFP-positive
cells, following fixation and processing for immunofluorescence
(FIG. 4).
[0122] Both basal anode and cathode electrode configurations
produced transfection of the target cells with the transgene. The
anodal configuration for the basal scala tympani electrode was
preferred for unipolar electroporation with expression at more
apical regions (Turn 3), closer to the apical anode, when the basal
cathode configuration was used. In summary, 4 out of 6 experiments
using the CMVp-TRPC3-IRES-DsRed2 construct resulted in gene
delivery to the cells lining scala media of the cochlea and 4 out
of 4 experiments using the CMVp-BDNF-FLAGtag-IRES-eGFPn construct
were effective.
TABLE-US-00001 TABLE 1 [cDNA] (.mu.g/.mu.l) & Pulse Pulse
Plasmid Voltage width # interval Electrode ID (a or b) (V) (ms)
pulses (s) config Results GP12.aA3 1, a 0 0 0 0 STb(-); Control,
placement of SVb(+) electrodes but no voltage applied; outcome =
N/E GP12.b 1, a 10 30 100 10 STb(-) +ve GFP, location C2 = T1;
SVb(+) D3 = T2, 48 h GP13.a 1, a 40 50 60 1 STb(+) N/E SVb(-)
GP13.b 1, a 100 50 60 5 STb(-) +ve GFP, location C2 = T4; SVb(+) D3
= T2; D5 = T2; 24 h, 48h GP8/4a 0.1, a 0 0 0 0 STb(-) Control,
Lipofectamine 2000 A(+) in Opti-MEMI media; N/E GP8/4b 0.1, a 100
50 100 5 STb(-) +ve; D2 = T1, many cells; A(+) GP9/4a 0 0 0 0 0 0
Control, no DNA GP9/4b 1, a 100 50 100 5 STb(-) N/E @ 24 h A(+)
GP17a 0 0 0 0 0 -- Control, no DNA; N/E GP17b 1, a 100 50 100 5
Outer +ve; C1 = T1; C4 = T2; spiral C5 = T3; 24 hr & 96 hr
limbus(+) A(-) GP25a 1, b 100 30 100 5 STb(-) +ve; A5 = T1; many
nuclei GFP A(+) +ve; A5 = T1(fixed for immunofluorescence anti-
FLAG = BDNF @ 48 hr); A6 = T1; B2 = T3 lateral wall; B3 = T2
lateral wall; 48 hr; B1 = T1; B3 = T2 lateral wall. GP26a 1, b 100
50 50 5 STb(+) +ve; C1 = T1-T3 lateral wall, A(-) nuclei +ve in
base-apex; C3 = T1, 20 nuclei +ve on target region (processed for
BDNF); C4 = T2; C5 = T2; 72 hrs. GP27a 1, b 100 50 50 5 STb(-) +ve;
B3 = T1, ST-spiral limbus A(+) region, B4 = T2; B5 = T2; 72 hr
GP27b 1, b 100 50 50 5 STb(+) =ve; C1 = T1-T2 ST; C5 = T2, A(-) 7
nuclei; T1 = T1; 72 hr. Key: plasmid a = CMVp-TRPC3-IRES-DsRed2;
plasmid b = CMVp-BDNF-FLAG tag-IRES-eGFPn; Stb(-), anode inserted
into basal turn scala tympani via round window; SVb(+) cathode
inserted into scala vestibule basal turn via oval window; A(+)
cathode into apex of cochlea. An . . . Cn refers to organotypic
culture plate location, T1 . . . T4 refers to the four turns of the
cochlea (base to apex). N/E = no expression; +ve = expression
detected.
[0123] These experiments demonstrated the feasibility of
electroporation-mediated transfer of naked therapeutic
polynucleotides into target tissues in an adult mammalian cochlea.
This was achieved through the use of a shaped electrode placed
within the scala tympani perilymphatic chamber, as a model for
cochlear implant prosthesis. Both unipolar and bipolar electrode
configurations for electroporation-based gene delivery in the adult
cochlear have been validated by the results achieved using
basal-basal and basal-apical electrode placement, as well as both
cathode and anode configurations of the scala tympani
electroporation electrode. The density of transduced cells was
greatest in the tissue immediately adjacent to the basal
electroporation electrode, and the numbers of cells expressing the
reporter genes decreased in more apical regions of scala tympani.
These data indicate the electroporation based gene delivery is
efficient and site-specific.
Example 2
Assessment of Neuroprotection Provided by Cochlear Cell
Transfection
[0124] A guinea-pig model is employed to study the ability of
electroporetic transfection of a polynucleotide construct encoding
a spiral ganglion trophic factor to support the survival of spiral
ganglion neurons.
[0125] Cochlear sensory hair cells are destroyed using a single
co-administration of kanamycin (400 mg/kg provided subcutaneously)
and frusemide (100 mg/kg intravenously, causing a progressive loss
of the cochlear (spiral ganglion) neurons in following weeks.
[0126] After kanamycin/frusemide treatment, a solution comprising
an expression cassette, such as the cassettes described in Example
1, encoding a spiral ganglion cell neurotrophic factor, for example
any one of glial cell line-derived neurotrophic factor,
brain-derived neurotrophic factor, neurotrophin-3,
neurotrophin-4/5, and ciliary neurotrophic factor, is introduced to
the cochleae of the anaesthetised guinea pigs. Electrodes inserted
into the scala tympani are used to create an electroporetic
stimulus to transfect mesenchymal cells of the scala tympani. The
electrodes are then removed, the cochleae closed and the animals
allowed to recover.
[0127] At a series of time points commencing immediately after
surgery and at one weekly intervals up to four months, animals are
euthanazed and tissue sections used to assess spiral ganglion cell
numbers, and the outgrowth of spiral ganglion cell neurites towards
transfected cochlear cells, as detected via reporter gene
expression, by immunolabelling with a florescent anti-neurofilament
antibody and/or by detection of therapeutic molecule expression
using immunofluorescence.
Example 3
Use of a Cochlear Implant to Transfect Cochlear Cells by
Electroporation
[0128] Guinea-pigs are deafened by selective lesion of the cochlea
hair cells using kanamycin/frusemide as described in Example 2, and
undergo chronic cochlear implantation, via the round window for
functional validation of the efficacy of cochlear implant-mediated
electroporation gene delivery. A nucleic acid molecule which
encodes a neurotrophic factor for spiral ganglion neurons, for
instance in the form of an expression cassette as described in
Example 1 which also comprises a reporter molecule, or which
modulates endogenous neurotrophic factor expression is delivered
into scala media immediately prior to, or with the implantation of
a cochlear implant prosthesis and then electroporation occurs by
delivering high field strength current through the cochlear implant
prosthesis electrode array. The array is left in place and the
wound closed. The nucleic acid molecule is selected from nucleic
molecules comprising sequences encoding Neurotrophin-3 (NT-3) or
Neurotrophin-3 precursor molecule (NCBI Accession No. HGNC:8023);
Neurotrophin 4/5 (NT-4/5, NT-4, NT-5, NTF4, NTF5) (NCBI Accession
No HGNC:8024); Nerve growth factor (NGF) (NCBI Accession No.
HGNC:7808); Brain derived neurotrophic factor (BDNF) (NCBI
Accession No. HGNC:1033); glial cell line-derived neurotrophic
factor (GDNF)(NCBI Acession No. HGNC:4232); ciliary neurotrophic
factor (CNTF)(HGNC Accession No. HGNC:2169; Activity dependent
neurotrophic factor gene (ADNF); Apyrase; or ectonucleoside
triphosphate diphosphohydrolase 2 (ENTPD2); alpha-melanocyte
stimulating hormone (alpha-MSH); bone morphogenetic protein (BMP);
Sonic hedgehog; Wnts; Laminin; Netrin-1; Ephrins; slit2/slit3; and
Leukaemia inhibitory factor.
[0129] The efficacy of the delivery is assessed by recording the
electrically evoked potentials arising from direct stimulation of
the cochlear implant array in its prosthesis stimulation mode. The
electrically evoked auditory brainstem responses are recorded using
a differential signal between a subdermal electrode located at the
vertex of the skull and a lateral subdermal electrode located
adjacent to the temporal bone on the side receiving the cochlear
implant. Control experiments omit the nucleic acid molecule with
electroporation stimulation, and also include the nucleic acid
molecule without electroporation stimulation.
[0130] The efficacy of the gene therapy for prevention of spiral
ganglion neuron loss and re-growth of the spiral ganglion neurites
towards the cochlear implant array is assessed over a period of
weeks by determining electrical stimulus threshold strengths and
input/output rate functions for the evoked potentials that arise
from activity in the cochlear nerve and from the down-stream
auditory activity in the brain.
[0131] After four months, the guinea pig cochleae are taken and
neuronal survival and neurite outgrowth assessed by histochemistry
and anti-neurofilament immunofluorescence. Sustained expression of
the nucleic acid molecule is detected by the fluorescence reporter
detection, or immunofluorescence detection of the reporter protein
(anti-GFP antibody).
Example 4
Treatment of Human Subjects
[0132] Human subjects are recruited on the basis of sudden onset
hearing loss attributable to lesion of the cochlear hair cells, as
assessed by loss of distortion product otoacoustic emissions
(DPOAE). This condition provides a differential for analysis of
long-term neuronal survival and outgrowth of the neurites to the
region of the cochlear prosthesis, based on electrically evoked
auditory brainstem response (EABR) measurements undertaken
post-operatively over a 12 month period.
[0133] Cochlear implant surgery will not differ from the standard
practice of implantation of the cochlear electrode array via an
incision through the round window or cochleostomy, except that
immediately prior to insertion of the electrode array, a
therapeutic gene construct (cDNA) is introduced into scala tympani
using, for example, a fine catheter of equivalent length to the
cochlear implant. The cDNA is typically provided at 1 .mu.g/.mu.l
concentration in water or in a buffered artificial perilymph with
reduced osmolarity (total volume delivered 30 .mu.l).
[0134] The catheter is then removed and the cochlear implant's
electrode array is inserted. The cochlear implant is then
immediately driven in electroporation mode, to deliver a field
strength between electrodes of sufficient intensity and duration to
produce dialectric breakdown of the cells in closest proximity to
the electrodes, which are typically the mesenchymal cells lining
scala tympani on the modiolar canal wall. The electroporation
stimulus will result in entry of the naked cDNA agent, transfecting
the cells. The number of electrical pulses required for this
procedure is typically fewer than 250, with a pulse duration of
approximately 50 milliseconds, and typically 1 second per pulse. On
this basis the electroporation will require less than 10 minutes
additional time and could be undertaken while the surgeon continues
with the procedure.
[0135] Dedicated circuitry in the stimulator unit of the cochlear
implant, which is typically surgically implanted in a
post-auricular location, is used to drive the electroporation,
however in other embodiments separate electroporation electrode
nodes on the implant may be used and activated by an external
isolated electroporation circuitry for this procedure, and are
subsequently disconnected at the completion of the procedure.
[0136] The surgical procedure otherwise proceeds as for a
conventional cochlear implant procedure and normal activation of
the cochlear implant occurs as a conventional post-operative
undertaking.
[0137] Efficacy of the gene delivery and expression of the
therapeutic molecules would be detected if reduced stimulus
intensities are required to achieve EABR thresholds or in hearing
discrimination testing (audiometry) and audiological assessment of
hearing function and speech discrimination. A successful reduction
in stimulus intensities required for hearing threshold (compared
with controls) would indicate that spiral ganglion cell neurites
are in closer proximity to the cochlear implant, and this would be
a positive indicator for neural protection and neural repair
(neuritogenesis) arising from the neurotrophic stimulation.
[0138] Refinements of the cochlear implant array for cochlear
implants may be possible in the light of increased neurite
proximity to the implant which may be achieved following the
methods of the invention.
* * * * *
References