Note: Descriptions are shown in the official language in which they were submitted.
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SYSTEM FOR GENETICALLY TREATING
CARDIAC CONDUCTION DISTURBANCES
FIELD OF THE INVENTION
The present invention relates to systems for
delivering conduction protein genetic material to cardiac
cells in localized areas of the heart to improve the
conductance therein.
BACKGROUND OF THE INVENTION
The conduction system of the human heart is
normally automatic, resulting in the contraction of the
atria and ventricles by means of electrical impulses that
originate in cardiac tissue. The cardiac cycle is separated
into the contraction phase (systole) and relaxation phase
(diastole). Although the rhythm of the cardiac cycle is
intrinsic, the rate of this rhythm is modified by autonomic
nerves and hormones such as epinephrine. The autonomic
nervous system is comprised of parasympathetic and
sympathetic nerves which release neurotransmitters such as
acetylcholine and norepinephrine, respectively.
The natural pacemaker of the human heart is
located in the posterior wall of the right atrium in a small
area, approximately 2 by 5 by 15 mm, referred to as the
sinoatrial node (SA node). The SA node initiates the
cardiac cycle of systole and diastole phases by generating
an electrical impulse that spreads over the right and left
atria, causing them to contract almost simultaneously. This
electrical impulse, referred to as the pacemaker potential,
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is created by the depolarization of the myocardial cells of
the SA node, which results from changes in membrane
permeability to cations. When the cell membrane is
depolarized to about -30 mV, an action potential is
produced. This impulse then passes to the atrioventricular
node (AV node), which is located on the inferior portion of
the interatrial septum. The impulse then continues through
the atrioventricular bundle, referred to as the bundle of
His, which is located at the top of the interventricular
septum. The bundle of His divides into right and left
branches which lead to the right and left ventricles
respectively. Continuous with both branches of the bundle
of His are the Purkinje fibers, which terminate within the
ventricular walls. Stimulation of these fibers causes the
ventricles to contract almost simultaneously and discharge
blood into the pulmonary and systemic circulatory systems.
Abnormal patterns of electrical conduction in the
heart can produce abnormalities of the cardiac cycle and
seriously compromise the function of the heart, sometimes
being fatal. For example, patients having such cardiac
conduction disturbances may suffer from sick sinus syndrome
(SSS), "brady-tachy syndrome," bradycardia, tachycardia, and
heart block. Artificial pacemakers are often used in
patients which suffer from these cardiac conduction
disturbances.
In SSS, the conduction problem relates to, inter
alia, intermittent reentry of the electrical impulse within
the nodal tissue, sometimes resulting in rapid heart beats.
A dual chamber pacemaker is often used to sense atrial
activity and control the ventricle at the appropriate rate.
In some congenital diseases such as "brady-tachy
syndrome," bradycardia, a slow rate of impulse, and
tachycardia, a rapid rate of impulse, occur intermittently.
The disease can be fatal where long pauses allow premature
ventricular contractions (PVCs) to occur in multiples,
initiating tachycardia. A pacemaker and/or cardioverter can
be used to control episodes of tachycardia, and conventional
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demand type pacemakers have long been effective in treating
bradycardia.
Excessive delay or failure of impulse transmission
in abnormally slow impulse conduction is known as heart
block. Heart block is often caused by scar tissue
disrupting the conduction system. The cardiac impulse is
believed to normally spread from the SA node along
internodal pathways to the AV node and ventricles within
0.20 seconds. Heart block occurs in three progressively
more serious stages. In first-degree heart block, although
all impulses are conducted through the AV junction,
conduction time to the ventricles is abnormally prolonged.
In second-degree heart block, some impulses are conducted to
the ventricles, whereas some are not. In third-degree heart
block, no impulses from the natural pacemaker are conducted
to the ventricles. This results in a slower ventricular
contraction rate. The rate of contraction in this case is
usually determined by the rate of the fastest depolarizing
His-Purkinje cell distal from the block site. Typically,
heart rates in third-degree block are in the 20 to 60 bpm
range, but can also be as low as zero in some cases.
Arrhythmias resulting from cardiac conduction
disturbances can be treated with a variety of drugs that
inhibit specific aspects of the cardiac action potentials
and inhibit the production or conduction of impulses along
abnormal pathways. Drugs used to treat these arrhythmias
block the fast Na+ channels (quinidine, procainamide,
lidocaine), block the slow Ca" channel (verapamil), or block
S-adrenergic receptors (propranolol).
The cardiac conduction system, or electrical
activation of the heart, involves the transfer of current,
in the form of chemical ion gradients, from one myocardial
cell to another. Conductive proteins in cardiac cells
facilitate this transfer of current. Individual cardiac
cells express a plurality of gap junction channel proteins,
through which ions traverse. The intercellular channels of
gap junctions are assembled from,individual membrane-
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spanning connexin proteins, several of which have been
cloned and sequenced in mammals. These proteins facilitate
the transfer of ions from cell to cell and are responsible
for electronic coupling of cells. Saffitz, et al., J. Card.
Electrophys., 1995, 6, 498-510.
Connexin proteins comprise a family of related
proteins and include, for example, Cx43 (Fishman, et al., J.
Cell Biol., 1990, 111, 589-598), and Cx40 and Cx45 (Kanter,
et al., J. Mol. Cell Cardiol., 1994, 26, 861-868). Cx43
appears to be the most abundant connexin in the heart, with
expression in the ventricle and atrial myocardium, and
distal bundle of His and Purkinje fibers, while being absent
from the SA node, AV node, and proximal bundle of His.
Gourdie, et al., J. Cell Sci., 1993, 105, 985-991, and
Davis, et al., J. Am. Coll. Cardiol., 1994, 24, 1124-1132.
Cx40 is most abundantly expressed in the atrial myocardium,
and in the distal bundle of His and Purkinje fibers, while
present at reduced levels in the ventricular myocardium, and
the nodes. Gourdie, et al., J. Cell Sci., 1993, 105, 985-
991, and Davis, et al., J. Am. Coll. Cardiol., 1994, 24,
1124-1132. Cx45 is moderately expressed in the ventricle
and atrial myocardium, and distal bundle of His and Purkinje
fibers, while present at lower levels in the SA node, AV
node, and proximal bundle of His. Gourdie, et al., J. Cell
Sci., 1993, 105, 985-991, and Davis, et al., J. Am. Coll.
Cardiol., 1994, 24, 1124-1132. Cx43 and Cx40 connexins are
relatively fast conductive proteins, whereas Cx45 is a
relatively slow conductive protein.
Gene therapy has recently emerged as a powerful
approach to treating a variety of mammalian diseases.
Direct transfer of genetic material into myocardial tissue
in vivo has recently been demonstrated to be an effective
method of expressing a desired protein. For example, direct
myocardial transfection of plasmid DNA by direct injection
into the heart of rabbits and pigs (Gal, et al., Lab.
Invest., 1993, 68, 18-25), as well as of rats (Ascadi, et
al., The New Biol., 1991, 3, 71-81), has been shown to
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result in expression of particular reporter gene products.
In addition, direct in vivo gene transfer into myocardial
cells has also been accomplished by directly injecting
adenoviral vectors into the myocardium. French, et al.,
Circulation, 1994, 90, 2415-2424, and PCT Publication WO
94/11506.
It has long been desired to effectively treat
conduction pathway abnormalities. To this end, conventional
procedures including drug therapy, pacemaker technology, or
a combination thereof, have been employed. In contrast to
these therapeutic procedures, Applicants' invention is
directed to delivery systems for treating and/or correcting
disturbances in the cardiac conduction pathway by delivering
conduction protein genetic material into myocardial tissue.
In patients with cardiac conduction disturbances, it is
desirable to locate the problematic area within the heart,
and either treat the problematic cells to restore proper
cardiac conduction or enhance the cardiac conduction of
normal cells surrounding the problematic area. For example,
in a patient with heart block, a tract of normal, healthy
cells surrounding the scar in the ventricle, which is
causing the heart block, is identified and treated with
Applicants' delivery system by expressing cardiac conduction
proteins, such as, for example, gap junction proteins to
impart a faster or otherwise enhanced conduction system. In
this case, the block can be effectively bridged, or shunted,
resulting in a QRS of a width intermediate between a
normally conducted beat and a PVC.
SUMMARY OF THE INVENTION
In accordance with the above, the primary purpose
of Applicants' claimed invention is to provide delivery
systems for treating cardiac conduction disturbances. Upon
identifying a problematic area within the heart, conduction
protein genetic material is selected such that expression of
a selected conduction protein corrects or improves the
cardiac conduction of the cells in the problematic area.
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Alternatively, expression of a selected conduction protein
can improve the cardiac conduction of normal, healthy cells
surrounding the problematic cells. Improvement of cardiac
conduction can be manifested by a replacement, a speeding
up, or a slowing down of the existing conduction pathway.
The conduction protein genetic material comprises
recombinant nucleic acid molecules comprising a nucleic acid
molecule encoding the conduction protein inserted into a
delivery vehicle, such as, for example, plasmids or
adenoviral vectors, and the appropriate regulatory elements.
Alternatively, the conduction protein genetic material
comprises the conduction protein itself. Expression of the
desired conduction protein from recombinant nucleic acid
molecules is controlled by promoters, preferably cardiac
tissue-specific promoter-enhancers, operably linked to the
nucleic acid molecule encoding the conduction protein. The
conduction protein is preferably a gap junction protein,
such as, for example, the connexins Cx40, Cx43, and Cx45,
which is used to correct or improve the cardiac conduction
of cells within the problematic area. For example, if the
cardiac conduction pathway disturbance is a heart block or
bradycardia, Cx43 or Cx40 is preferably used. However, if
the cardiac conduction pathway disturbance is tachycardia,
Cx45 is preferably used. The cardiac conduction genetic
material is delivered to specific sites within the heart by
perfusion or injection of a therapeutically effective
amount, which is that amount which corrects or improves the
cardiac conduction of the myocardial cells. The
therapeutically effective amount can be delivered to the
specific site in the heart in a single dose or multiple
doses, as desired.
The present invention provides a delivery system
for delivering a therapeutically effective amount of a
predetermined conduction protein genetic material to an
identified cardiac location, the genetic material being
selected for altering the conductivity of cardiac cells to
which it is delivered. The delivery system includes the
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se'_ected genetic material contained in a reservoir, and a
catheter subsystem for delivering the genetic material from
the reservoir to the identified cardiac location so as to
contact a plurality of cells in the proximity of the
selected cardiac location.
The delivery system may utilize an external
reservoir for providing the genetic material, or alt*rnately
may utilize an implantable reservoir. In either embodiment,
a controllable pump mechanism is provided for transferring
therapeutic-doses of the genetic material from the
reservoir, through a'catheter, and to the selected cardiac
location. The catheter subsystem may be of a type for
direct introduction into the-myocardium, as with a
transthoracic procedure, or, more preferably, a endocardial
catheter having a distal tip portion adapted for positioning
and injecting the genetic material into the-myocardium from
within a heart chamber. In a preferred embodiment, the
catheter distal tip has a normally withdrawn helical needle,
which is extendable when positioned in the vicinity of the
selected site so as to be screwed into the heart. The
needle is hollow and connects with the catheter lumen so as
to receive the pumped genetic material;.it has one or more
ports located so as to effectively release the genetic
material for transduction into the mapped area. in another
preferred embodiment of the 'invention, the delivery system
is combined with the mapping catheter such that once the
selected site is identified, the delivery system, which is
within the mapping catheter, is engaged without removing the
mapping catheter. The delivery system can be used for one
treatment and then removed, or can be implanted for
subsequent treatments, in which 1_atter case it is
controllable by an external programmer type device.
According to one aspect of the present invention, '
there is provided a delivery system for delivering genetic
material to cardiac tissue, comprising: a supply of
conduction protein genetic material; a reservoir for holding
the supply of conduction protein genetic material; a mapping
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catheter for mapping said cardiac tissue for locating a site
for delivering said conduction protein genetic material in
said cardiac tissue; and a delivery means for delivering
said conduction protein genetic material from said reservoir
to said site in said cardiac tissue so as to contact a
plurality of cells in said cardiac tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a flow diagram presenting the primary
steps involved in the practice of this invention, including
mapping the patient's conductive system to determine the
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location of the problem, choosing an appropriate genetic
material, and expressing the genetic material in an
appropriate dose into the determined location.
Figure 2 is a schematic representation of a
delivery system in accordance with this invention,
illustrating delivery of genetic material into a patient's
heart at the chosen location.
Figure 3 is a schematic drawing of the distal
portion of a catheter, which can be extendable and
retractable, used for injecting a solution carrying chosen
genetic material into a patient's heart.
Figure 4 illustrates the distal end of a catheter,
having a distal portion which encloses an osmotic pump.
Figure 5 illustrates a delivery system in which
the delivery means comprises a mapping catheter combined
with a delivery system for injecting a solution carrying
chosen genetic material into a patient's heart'.
Figure 6A is a schematic representation of a
delivery system in accordance with this invention, having a
combined catheter and pacing lead, with a separate pump;
Figure 6B is another embodiment of a combined pacing lead
and delivery catheter having a reservoir located at the
distal end of the catheter.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Applicants' invention provides delivery systems
for treating cardiac conduction pathway disturbances. A
problematic area exhibiting, for example, SSS, "brady-tachy
syndrome," bradycardia, tachycardia, or heart block, within
the heart is identified by routine and conventional
techniques known to the skilled artisan. Once the specific
problem has been identified, conduction protein genetic
material is selected such that expression of a selected
conduction protein corrects or improves the cardiac
conduction of the problematic cells or improves the cardiac
conduction of normal cells surrounding the problematic
cells. The conduction protein genetic material comprises
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either the conduction protein itself or recombinant nucleic
acid molecules comprising a nucleic acid molecule encoding
the conduction protein inserted into a delivery vehicle,
such as, for example, plasmid, cosmid, YAC vector, viral
vectors, and the like, and the appropriate regulatory,
elements. In preferred embodiments of the present
invention, the nucleic acid molecule encoding the conduction
protein is the full length coding sequence cDNA of a
conduction protein, and is inserted into a plasmid or
adenoviral vector, such as, for example, pGEM3 or pBR322,
and Ad5, respectively. The regulatory elements are capable
of directing expression in mammalian cells, specifically
human cells. The regulatory elements include a promoter and
a polyadenylation signal. Expression of the desired
conduction protein is preferably controlled by cardiac
tissue-specific promoter-enhancers, operably linked to the
nucleic acid molecule encoding the conduction protein. The
conduction protein is preferably a gap junction protein,
such as, for example, the connexins Cx40, Cx43, and Cx45,
which is used to correct or improve the cardiac conduction
of cells within the problematic area. The specific gap
junction protein chosen is dependent upon the nature of the
identified problem. For example, where the conduction is
slow or non-existent, such as in heart block or bradycardia,
introduction of Cx40 or Cx43 would enhance conduction. In
contrast, introduction of the slower conducting Cx45 into
the AV node and His tissues would result in the prevention
of brady-tachy syndrome and tachycardia. The conduction
protein genetic material is preferably delivered in a
pharmaceutical composition comprising, for example, the
conduction protein genetic material in a volume of
phosphate-buffered saline with 5% sucrose. The cardiac
conduction genetic material is delivered to specific sites
within the heart by perfusion or injection of a
therapeutically effective amount, which is that amount which
corrects or improves the cardiac conduction of the
myocardial cells. The therapeutically effective amount can
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be delivered to the specific site in the heart in single or
multiple doses, as desired, using the delivery systems of
the invention.
The present invention comprises a delivery system
for delivering a therapeutically effective amount of
conduction protein genetic material to a mapped cardiac
location in such a way as to enhance the effective
conduction of the myocardial cells around the area of
disturbance. In a first embodiment, the delivery system
basically comprises a reservoir subsystem for holding the
genetic material, and a catheter subsystem in communication
with the reservoir subsystem for placement of the genetic
material in and around the identified cardiac location. As
seen in the following discussion of several preferred
embodiments, the reservoir subsystem and catheter subsystem
may be separate, or they may be combined. Preferably the
reservoir contains up to 25 ml of a genetic material for
delivery to the myocardium. In some applications, only a
bolus of about 0.1-10 ml, or more preferably 1-5 ml, is
delivered to the targeted areas. In other applications,
such as where conduction protein is being delivered in
repeated doses, 25 ml or more may be used. Also, the
genetic material may be diluted in a saline solution, such
as, for example, phosphate-buffered saline (PBS), the
reservoir holding the diluted solution for controlled
delivery. Additionally, it is to be understood that the
reservoir and associated control apparatus may be either
implantable or external to the body, depending upon the
circumstances, e.g., whether metered doses are to be
administered to the patient over a period of time, or
whether the delivery of the genetic material is essentially
a one time treatment.
Referring now to Fig. 1, the primary steps
involved in the practice of this invention are shown in the
flow diagram. As illustrated in 30, the first step is to
determine the nature of the cardiac conduction disturbance,
which can manifest itself in ineffective or harmful
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conductive properties. This step can constitute diagnosis
of SSS, "brady-tachy syndrome," bradycardia, tachycardia,
heart block, etc. The next step, illustrated in 32, is
mapping the patient's heart to determine the location, size
and extent of the disturbance of problematic area.
Intracardiac electrocardiographic techniques, or
electrophysiology (EP) studies, permit a detailed analysis
of the mechanisms of cardiac impulse formation and
conduction. The testing and mapping protocol utilized and
the sites selected for recording depend upon the symptoms
manifested in the individual. One skilled in the art is
readily familiar with cardiac mapping techniques, such as,
for example, those described in U.S. Patent 4,699,147, U.S.
Patent 5,297,549, and U.S. Patent 5,397,339.
The mapping techniques known
to those skilled in the art will readily identify those
cardiac locations encompassing cardiac cells with abnormal
conduction properties. As shown in 33, the next step is to
select the appropriate conduction protein genetic material.
This selection, which yields the "preselected genetic
material," is dependent upon the nature of the cardiac
conduction disturbance, as discussed below. The conduction
protein genetic material is next prepared, as illustrated in
34, by either inserting the nucleic acid molecules encoding
the appropriate conduction protein into a delivery vehicle
with the appropriate regulatory elements, in the case of a
recombinant nucleic acid molecule, or expressing the
conduction protein from an expression vector, in the case of
the conduction protein itself. As shown in 35, the next
step is to prepare and load the delivery system with a
therapeutically effective amount of the conduction protein
genetic material. As illustrated in 37, the next step
comprises administering the therapeutically effective amount
to the patient by contacting the appropriate location in the
heart, as determined earlier, using the delivery system
described herein. An alternative method of administering
the therapeutically effective amount of the conduction
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protein genetic material is to directly inject the heart of
the patient. The final step, shown in 38, is to evaluate
the response of the patient to the treatment.
Referring now to Fig. 2, there is shown an
illustrative embodiment of a delivery system useful for
certain applications of this invention, e.g., where larger
amounts of genetic material alone or in solution are
employed. A catheter 36, preferably a transvenous catheter,
includes an elongated catheter body 40, suitably an
insulative outer sheath which may be made of polyurethane,
Teflon, silicone, or any other acceptable biocompatible
plastic. The catheter has a standard lumen (illustrated in
Fig. 3) extending therethrough for the length thereof, which
communicates through to a hollow helical needle element 44,
which is adapted for screwing into the patient's myocardium.
The outer distal end of helical element 44 is open,
permitting genetic material in fluid form to be dispensed
out of the end, as is discussed in more detail below in
connection with Fig. 3. At the proximal end of the
catheter, a fitting 46 is located, to which a Luer lock 48
is coupled. Luer lock 48 is coupled to the proximal end of
elongated catheter body 40 and receives the lumen. A swivel
mount 50 is mounted to Luer lock 48, allowing rotation of
the catheter relative to Luer lock 52. Luer lock 52 in turn
is coupled through control element 54 to a tube 58 which
communicates with reservoir 55, suitably through flow
control 57 and filter 56. Reservoir 55 holds a supply of
the selected genetic material. Control elements 57 and 54
are used for adjustment of the pressure and flow rate, and
may be mechanically or electronically controlled. Thus,
unit 54 or 57 may be used to control either rate of
delivery, or dosage size, or both. Control unit 54 may be
programmed to automatically release predetermined doses on a
timed basis. Further, for an implanted system, control unit
54 may be activated from an external programmer as
illustrated at 51. Reference is made to international
application published under the PCT, International
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Publication No. WO 95/05781,
for a more detailed description of such a
reservoir and catheter combination. It*is to be understood
that such a system is useful for this invention only for
applications where larger fluid amounts are to be expressed,
e.g., where a diluted saline solution is used to wash or
perfuse a selected area.
Referring now to Fig. 3, there is shown in
expanded detail a schematic of the distal end of the
catheter of Fig. 2, illustrating the interconnection of the
helical element 44 with the interior of the catheter. As
illustrated, the helical needle 44 is provided with an
internal lumen 59 which is in communication with the
internal lumen 63L of the lead formed by tube 63.. In this
embodiment, helical element 44 may also be a paci-ng
electrode, in which case it is formed of conductive material
and welded, crimped, swaged, or connected by other means so
as not to prevent fluid flow, to tip element 61. Tip
element 61 in turn is electrically connected to a conductor
coil or coils 64, 65, which extend the length of the lead
and are connected to a pacemaker. An outer membrane 60
forms the outer wall of elongated catheter body 40, shown in
Fig. 2. Further referring to Fig. 3, element 44 has an
outlet 75 where the genetic material may be expressed, and
holes or ports 76, 77, and 78 may also be utilized for
providing exits for the genetic material which is. supplied
through lumen 59 under a pressure of up to about one
atmosphere from reservoir 55 and the control elements.
In practice, a catheter 36 of the form illustrated
in Figs. 2 and 3 is advanced to the desired site:for
treatment, which site or location has been previously
identified by means of cardiac mapping, as discussed above.
The catheter may be guided to the indicated location by
being passed down a steerable or guidable catheter having an
accommodating lumen, for example as disclosed in U.S. Patent
No. 5,030,204; or by means of a fixed configuration guide
catheter such as illustrated in U.S. Patent No. 5,104,393.
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Alternately, the catheter may be advanced to the desired
location within the heart by means of a deflectable stylet,
as disclosed in PCT Patent Application WO 93/04724,
published March 18, 1993, or by a deflectable guide wire as
disclosed in U.S. Patent No. 5,060,660. In yet another
embodiment, the helical element 44 may be ordinarily
retracted within a sheath at the time of guiding the
catheter into the patient's heart, and extended for screwing
into the heart by use of a stylet. Such extensible helical
arrangements are commercially available and well known in
the pacing art.
It is to be understood that other forms of the
reservoir subsystems and catheter subsystems are within the
scope of this invention. Reservoir embodiments include, for
example, drug dispensing irrigatable electrodes, *uch as
those described in U.S. Patent 4,360,031; electrically
controllable, non-occluding, body implanting drug delivery
system; such as those described in U.S. Patent No.
5,=041,107; implantable drug infusion reservoir such as those
described in U.S. Patent No. 5,176,641; medication delivery
devices such as those described in U.S. Patent 5,443,450;
and infusion pumps, such as SYNCHROMEO made by Medtronic,
Inc.; and osmotic pumps such as those made by Alza.
Referring.now to Fig. 4, there is shown, by way of
illustration, another embodiment of a delivery system having
a combined catheter and reservoir, useful for applications
involving delivery of a relatively small bolus of genetic
material, e.g., 1-5 ml. Fig. 4 illustrates the distal end
of a catheter, having a distal portion 70 which encloses an
osmotic pump. See U.S. Patent 4,711,251, assigned to
Medtronic, Inc.. The pump
includes an inner chamber 68 and an outer chamber 66, which
chambers are separated by an impermeable membrane 67. A
semi-permeable outer membrane 72 forms the outer wall of
chamber 66. The tubular portion 74 of the helical member
connects to lumen 74L within inner chamber 68. A conductor
80, which runs the length of the catheter, extends into t:~.e
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inner chamber 68 and connects with extension 74E as shown at
74C to provide electrical contact through to element 44, in
an application which the element 44 is used as a pacing
electrode. A seal 79 is provided at the point where the
conductor passes through outer membrane 72 and inner
membrane 67. An insulating cover 86 encompasses the
conductor 80 from the point of contact with seal 79. An end
cap 73, which may be integral with outer membrane 72 closes
the chamber. Alternately, end cap 73 may be constructed to
elute a predetermined medication, such as, for example,
steroids. Steroids, such as dexamethasone sodium phosphate,
beclamethasone, and the like, are used to control
inflammatory processes.
In this arrangement, prior to inserting the
catheter distal end into the patient's heart, the inner
chamber 68 is charged with the genetic material which is to
be dispensed into the myocardium. This may be done, for
example, by simply inserting a micro needle through end cap
73, and inserting the desired bolus of genetic material into
chamber 68. After the chamber 68 is filled and the
catheter is implanted, body fluids will enter chamber 66
through membrane 72 to impart a pressure on the inner
chamber 68 via the impermeable membrane 67. This results in
a dispensing of the genetic material stored within chamber
68 through the lumen 74L of extension 74E and the helical
element 44. Although the preferred needle or element 44 is
helical, additional configurations of needles or elements
can also be used as known to those skilled in the art.
Still referring now to Fig. 4, there is
illustrated another embodiment of a catheter tip useful for
delivering a small bolus of the selected genetic material.
In this embodiment, the bolus of material is stored within
the hollow interior of helical element 44, i.e., the
interior is the reservoir. The interior reservoir is
maintained sealed by use of a soluble material which is
normally solid, but which dissolves when subjected to body
fluids for a period of time. An.example of such material is
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mannitol, which can be used when the delivery system is not
preloaded with the conduction protein genetic material.
Plugs or globules 81-85 of mannitol are illustrated (by
dashed lines) in place to block the two ends of element 44,
as well as the ports 76, 77, 78. In instances where the
conduction protein genetic material is preloaded into the
delivery system, a shape memory metal can be used in place
of the mannitol. Such metals are well known to the skilled
artisan. Either of these features can be combined with an
osmotic pump, as described in connection with Fig. 3, where
the outer chamber is filled with a saline solution which
forces the genetic material out of the ports of element 44.
Alternatively, the outer chamber can be filled with the
genetic material, which is then forced out of the ports of
element 44. Another alternate embodiment, not shown, is to
use a stylet which inserted through to the distal end of the
catheter, to push a piston which aids in expressing the
genetic material into the myocardial cells.
Although a transvenous form of delivery system is
preferred, it is to be understood that the invention can
employ other methods and devices. For example, a small
bolus of selected genetic material can be loaded into a
micro-syringe, e.g., a 100 l Hamilton syringe, and applied
directly from the outside of the heart.
Referring now to Fig. 5, there is shown, by way of
illustration, another embodiment of a delivery system having
a combined mapping catheter and delivery means. The
delivery system of this embodiment comprises a catheter 90
with a distal end 91 having an opening at the distal end.
The catheter 90 further comprises mapping electrode means 92
at the distal end 91. The mapping electrode means carries
out the mapping of the patient's heart. Conductor means 93
electrically connects the mapping electrode means 92 to the
proximal end 94 of the catheter 90. The delivery system
further comprises a delivery means within the catheter. The
embodiment of the delivery means illustrated in Fig. 5 is
the delivery means shown in Fig. 3. However, any of the
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delivery means described herein can be used in combination
with the mapping catheter shown in Fig. S. The catheter 90
is inserted into the patient's heart and the site located by
routine mapping procedures. Once a site is identified in
the heart, the mapping catheter 90 remains in place and the
delivery means is then extended through the distal end 91 of
the catheter 90, and the heart tissue or cells is contacted
with the conduction protein genetic material. In another
embodiment of the invention, the catheter 90 is a peelable
introducer sheath, with two conductor means 93 electrically
connecting the introducer sheath, which serves to map the
heart, to electrode means 92. Once the cardiac site is
mapped, the delivery means is contacted with the heart
tissue, and the introducer is removed and peeled away.
Referring now to Fig. 6A, there is shown, by way
of illustration, another embodiment of an implantable
delivery system comprising a combined pacing lead and
delivery catheter, hereinafter referred to simply as a
catheter. In this embodiment, the catheter 90 is combined
with a pacemaker or pulse generator (not shown) and a source
of genetic material such as illustrated by pump 100 which is
suitably implanted near the pacemaker. The proximal end 101
of the catheter is connected to the pacemaker in the
standard fashion. The genetic material is delivered through
connecting tube 102 to a proximal section 88 of the
catheter, communicating with lengthwise catheter lumen
illustrated at 89. Alternately, the pacemaker head may
contain a reservoir and micropump, for providing delivery of
the genetic material directly to the lumen 89. The main
length of the catheter has an outside sheath of
biocompatible insulating material 96, and at least one
conductor coil 95 which communicates electrically from the
pacemaker to electrode 97 at the distal tip of the catheter.
The catheter further comprises an axially positioned
polymeric cannula 103, having lumen 87, through at least a
portion of the catheter length and positioned within coil
95, which provides an inner surface for the catheter lumen.
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The cannula terminates at the distal end of the catheter,
just proximal to the tip portion of electrode 97, which is
illustrated as having an outer porous surface. Electrode 97
has a central opening, shown covered with the porous
electrode material, through which genetic material can pass
when the catheter is positioned in the patient. As shown,
conductor coil 95 is electrically connected to electrode 97,
and connects pace pulses and sensed cardiac signals between
the pacemaker and the electrode. Of course, for a bipolar
embodiment, the lead/catheter 90 carries a second electrode
(not shown), suitably a ring electrode just proximal to
electrode 97. Also, as illustrated, a fixation mechanism
such as tines 98 are employed for fixing or anchoring the
distal tip to the heart wall of the patient.
In one embodiment, pump 100 is suitably an osmotic
minipump, which pumps fluid contained within through tube
102, into catheter portion 88 and through lumens 89, 87 to
the tip electrode 97. As mentioned previously, the
reservoir and pump may alternately be mounted in the
pacemaker device itself. In either instance, the genetic
material is delivered under very minimal pressure from the
reservoir through the lumen of the catheter to the
electrode, where it is passed through the electrode central
channel to contact myocardial cells. In yet another
embodiment, the lumen portion 87 provided by the cannula is
utilized as the reservoir. In this embodiment, delivery may
either be passive, or with the aid of a micropump (not
shown). The genetic material can be preloaded into the
cannula, or it can be inserted by a needle just before the
catheter is introduced and positioned with the patient.
In another embodiment, as illustrated in Figure
6B, a chamber 99 is provided just proximal from eluting
electrode 97, and serves as the reservoir of the genetic
material. Insulating material 96 is formed from a self-
sealing material such that it may be pierced with a needle,
or the like, and reseal itself, thus allowing introduction
of the genetic material into the chamber prior to
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implantation. Alternately, insulating material 96 can
contain a port (not shown) through which the needle inserts
the genetic material. In this embodiment, delivery of the
material is without a pump, i.e., passive, the material
draining slowly through the microporous portion of electrode
97.
As used herein, the phrase "cardiac conduction
disturbance" refers to disturbances or disruptions of the
normal cardiac conduction system in a mammal. Such
disturbances may be the result of congenital phenomena or
trauma, and can manifest in conditions such as, for example,
sick sinus syndrome, "brady-tachy syndrome," heart block,
bradycardia, tachycardia, and other arrhythmatic conditions.
Manifestations of such cardiac conduction disturbances have
been traditionally treated by drugs, artificial conduction
systems such as pacemakers, ablation therapy, or a
combination thereof.
As used herein, the phrase "conduction protein
genetic material" refers to recombinant nucleic acid
molecules encoding the conduction proteins or,
alternatively, the conduction proteins themselves, which are
used in the methods and delivery systems of the invention.
For chronic treatment, or long term treatment, the
conduction protein genetic material will be in the form of
recombinant nucleic acid molecules encoding the conduction
protein. In contrast, for acute treatment, or short term
treatment, the conduction protein genetic material will be
in the form of the conduction proteins themselves. Once the
conduction protein genetic material has been selected, it is
referred to as "predetermined genetic material."
A "recombinant nucleic acid molecule", as used
herein, is comprised of an isolated conduction protein-
encoding nucleotide sequence inserted into a delivery
vehicle. Regulatory elements, such as the promoter and
polyadenylation signal, are operably linked to the
nucleotide sequence encoding the conduction protein, whereby
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the protein is capable of being produced when the
recombinant nucleic acid molecule is introduced into a cell.
The nucleic acid molecules encoding the conduction
= proteins are prepared synthetically or, preferably,from
isolated nucleic acid molecules, as described below. A
nucleic acid is."isolated" when purified away from other
cellular constituents, such as, for example, other.cellular
nucleic acids or proteins, by standard techniques known to
those of ordinary skill in the art. The coding region of
the nucleic acid molecule encoding the conduction protein
can encode a full length gene product or a subfragment
thereof, or a novel mutated or fusion sequence. The protein
coding sequence can be a sequence endogenous to the target
cell, or exogenous to the target cell. The promoter, with
which the coding sequence is operably associated;-may or may
not be one that normally is associated with the coding
sequence.
The nucleic acid molecule encoding the conduction
protein is inserted into an appropriate delivery vehicle,
such as, for example, an expression plasmid, cosmid, YAC
vector, and the like. Almost any delivery vehicle can be
used for introducing nucleic acids into the cardiovascular
system, including, for example, recombinant vectors, such as
one based on adenovirus serotype 5, AdS, as set forth in
French, et al., Circulation, 1994, 90, 2414-2424.
An additional protocol
for adenovirus-mediated gene transfer to cardiac cells is
set forth in WO 94/11506 and in Barr, et al., Gene Ther.,
1994, 1, 51-58.
Other recombinant vectors include, for example,
plasmid DNA vectors, such as one derived from pGEM3 or
pBR322, as set forth in Acsadi, et al., The New Biol., 1991,
3, 71-81, and Gal, et al., Lab. Invest., 1993, 68, 18-25,
cDNA-containing liposomes, artificial viruses,
nanoparticles, and the like. It is also contemplated that
conduction proteins be injected directly into the
myocardium.
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The regulatory elements of the recombinant nucleic
acid molecules of the invention are capable of directing
expression in mammalian cells, specifically human cells.
The regulatory elements include a promoter and a
polyadenylation signal. In addition, other elements, such
as a Kozak region, may also be included in the recombinant
nucleic acid molecule. Examples of polyadenylation signals
useful to practice the present invention include, but are
not limited to, SV40 polyadenylation signals and LTR
polyadenylation signals. In particular, the SV40
polyadenylation signal which is in pCEP4 plasmid
(Invitrogen, San Diego, CA), referred to as the SV40
polyadenylation signal, can be used.
The promoters useful in constructing the
recombinant nucleic acid molecules of the invention may be
constitutive or inducible. A constitutive promoter is
expressed under all conditions of cell growth. Exemplary
constitutive promoters include the promoters for the
following genes: hypoxanthine phosphoribosyl transferase
(HPRT), adenosine deaminase, pyruvate kinase, 0-actin, human
myosin, human hemoglobin, human muscle creatine, and others.
In addition, many viral promoters function constitutively in
eukaryotic cells, and include, but are not limited to, the
early and late promoters of SV40, the Mouse Mammary Tumor
Virus (MMTV) promoter, the long terminal repeats (LTRs) of
Maloney leukemia virus, Human Immunodeficiency Virus (HIV),
Cytomegalovirus (CMV) immediate early promoter, Epstein Barr
Virus (EBV), Rous Sarcoma Virus (RSV), and other
retroviruses, and the thymidine kinase promoter of herpes
simplex virus. Other promoters are known to those of
ordinary skill in the art.
Inducible promoters are expressed in the presence
of an inducing agent. For example, the metallothionein
promoter is induced to promote (increase) transcription in
the presence of certain metal ions. Other inducible
promoters are known to those of ordinary skill in the art.
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Promoters and polyadenylation signals used must be
functional within the cells of the mammal. In order to
maximize protein production, regulatory sequences may be
selected which are well suited for gene expression in the
cardiac cells into which the recombinant nucleic acid
molecule is administered. For example, the promoter is
preferably a cardiac tissue-specific promoter-enhancer, such
as, for example, cardiac isoform troponin C (cTNC) promoter.
Parmacek, et al., J. Biol. Chem., 1990, 265, 15970-15976,
and Parmacek, et al., Mol. Cell Biol., 1992, 12, 1967-1976.
In addition, codons may be selected which are most
efficiently transcribed in the cell. One having ordinary
skill in the art can produce recombinant nucleic acid
molecules which are functional in the cardiac cells.
Genetic material can be introduced into a cell or
"contacted" by a cell by, for example, transfection or
transduction procedures. Transfection refers to the
acquisition by a cell of new genetic material by
incorporation of added nucleic acid molecules. Transfection
can occur by physical or chemical methods. Many
transfection techniques are known to those of ordinary skill
in the art including: calcium phosphate DNA co-
precipitation; DEAE-dextran DNA transfection;
electroporation; naked plasmid adsorption, and cationic
liposome-mediated transfection. Transduction refers to the
process of transferring nucleic acid into a cell using a DNA
or RNA virus. Suitable viral vectors for use as transducing
agents include, but are not limited to, retroviral vectors,
adeno associated viral vectors, vaccinia viruses, and
Semliki Forest virus vectors.
Treatment of cells, or contacting cells, with
recombinant nucleic acid molecules can take place in vivo or
ex vivo. For ex vivo treatment, cells are isolated from an
animal (preferably a human), transformed (i.e., transduced
or transfected in vitro) with a delivery vehicle containing
a nucleic acid molecule encoding a conduction protein, and
then administered to a recipient. Procedures for removing
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cells from mammals are well known to those of ordinary skill
in the art. In addition to cells, tissue or the whole or
parts of organs may be removed, treated ex vivo and then
returned to the patient. Thus, cells, tissue or organs may
be cultured, bathed, perfused and the like under conditions
for introducing the recombinant nucleic acid molecules of
the invention into the desired cells.
For in vivo treatment, cells of an animal,
preferably a mammal and most preferably a human, are
transformed in vivo with a recombinant nucleic acid molecule
of the invention. The in vivo treatment may involve
systemic intravenous treatment with a recombinant nucleic
acid molecule, local internal treatment with a recombinant
nucleic acid molecule, such as by localized perfusion or
topical treatment, and the like. When performing in vivo
administration of the recombinant nucleic acid molecule, the
preferred delivery vehicles are based on noncytopathic
eukaryotic viruses in which nonessential or complementable
genes have been replaced with the nucleic acid sequence of
interest. Such noncytopathic viruses include retroviruses,
the life cycle of which involves reverse transcription of
genomic viral RNA into DNA with subsequent proviral
integration into host cellular DNA. Retroviruses have
recently been approved for human gene therapy trials. Most
useful are those retroviruses that are replication-deficient
(i.e., capable of directing synthesis of the desired
proteins, but incapable of manufacturing an infectious
particle). Such genetically altered retroviral expression
vectors have general utility for high-efficiency
transduction of genes in vivo. Standard protocols for
producing replication-deficient retroviruses (including the
steps of incorporation of exogenous genetic material into a
plasmid, transfection of a packaging cell line with plasmid,
production of recombinant retroviruses by the packaging cell
line, collection of viral particles from tissue culture
media, and infection of the target cells with viral
particles) are provided in Kriegler, M. "Gene Transfer and
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24
Expression, a Laboratory Manual", W.H. Freeman Co., New York
(1990) and Murry, E.J. e.d. "Methods in Molecular Biology",
Vol. 7, Humana Press, Inc., Cliffton, New Jersey (1991).
A preferred virus for contacting cells in certain
applications, such as in in vivo applications, is the adeno-
associated virus, a double-stranded DNA virus. The adeno-
associated virus can be engineered to be replication
deficient and is capable of infecting a wide range of cell
types and species. It further has advantages such as heat
and lipid solvent.stability, high transduction frequencies
in cells of diverse lineages, including hemopoietic cells,
and lack of superinfection inhibition thus allowing multiple
series of transductions. Recent reports indicate that the
adeno-associated virus can also function in an
extrachromosomal fashion.
In preferred embodiments of the present invention,
the recombinant nucleic acid molecules comprising nucleic
acid molecules encoding the conduction proteins, or, in the
alternative, the conduction proteins, are delivered to the
cardiac.cells of the identified cardiac location, as
determined by mapping procedures set forth above, using the
delivery systems set forth above. Alternatively, the
.conduction protein genetic material is delivered to the
cardiac cells of the identified cardiac location by direct
injection.
In preferred embodiments of the present invention,
the nucleic acid molecules encoding the conduction proteins
comprise the full length coding sequence cDNA of a
conduction protein. Preferably, the conduction proteins are
the gap junction proteins; more preferably, they are the
connexin proteins. Such nucleic acid molecules are
described in the Fishman, et al., J. Cell. Biol., 1990, 111,
589-598, and Kanter, et al., J. Mol. Cell Cardiol., 1994,
26, 861-868 references, which contain the full length coding
sequence cDNA of the connexin gap junction proteins Cx43,
and Cx40 and Cx45, respectively.
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Introduction of the gap junction-encoding nucleic
acid molecules or the gap junction proteins to normal
cardiac cells surrounding a scar causing heart block will
result in normal or enhanced conduction. Alternatively, it
is proposed that introduction of the gap junction-encoding
nucleic acid molecules or the gap junction proteins to
abnormal cardiac cells, those cells exhibiting cardiac
conduction disturbances, will result in normal or enhanced
conduction properties. Determining the appropriate
conduction protein genetic material, i.e., determining which
connexin protein is appropriate, is dependent upon the
particular cardiac conduction disturbance diagnosed. For
example, if the cardiac conduction pathway disturbance is a
heart block or bradycardia, in which conductance is slowed
or non-existent, Cx43 or Cx40, the faster connexins, is
preferably used. However, if the cardiac conduction pathway
disturbance is tachycardia, in which conductance is too
rapid, Cx45 is preferably used.
Nucleic acid molecules comprising nucleotide
sequences encoding the connexin proteins Cx40, Cx43, and
Cx45 are isolated and purified according to the methods set
forth in Fishman, et al., J. Cell. Biol., 1990, 111, 589-
598, and Kanter, et al., J. Mol. Cell Cardiol., 1994, 26,
861-868. The nucleic acid and protein sequences of Cx40 are
set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively. The
nucleic acid and protein sequences of Cx43 are set forth in
SEQ ID NO:3 and SEQ ID NO:4, respectively. The nucleic acid
and protein sequences of Cx45 are set forth in SEQ ID NO:5
and SEQ ID NO:6, respectively. It is contemplated that
nucleic acid molecules comprising nucleotide sequences that
are preferably at least 70% homologous, more preferably at
least 80o homologous, and most preferably at least 90%
homologous to the connexin nucleotide sequences described in
SEQ ID NOs 1, 3 and 5, can also be used.
It is understood that minor modifications of
nucleotide sequence or the primary amino acid sequence may
result in proteins which have substantially equivalent or
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enhanced activity as compared to the conduction proteins
exemplified herein. These modifications may be deliberate,
as through site-directed mutagenesis, or may be accidental
such as through mutations in hosts which produce the
conduction proteins. A "mutation" in a protein alters its
primary structure (relative to the commonly occurring or
specifically described protein) due to changes in the
nucleotide sequence of the DNA which encodes it. These
mutations specifically include allelic variants. Mutational
changes in the primary structure of a protein can result
from deletions, additions, or substitutions. A"deletion"
is defined as a polypeptide in which one or more internal
amino acid residues are absent as compared to the native
sequence. An "addition" is defined as a polypeptide which
has one or more additional internal amino acid residues as
compared to the wild type protein. A "substitution" results
from the replacement of one or more amino acidresidues by
other residues. A protein "fragment" is a polypeptide
consisting of a primary amino acid sequence which is
identical to a portion of the primary sequence of the
protein to which the polypeptide is related.
Preferred "substitutions" are those which are
conservative, i.e., wherein a residue is replaced by another
of the same general type. As is well understood, naturally-
occurring amino acids can be subclassified as acidic, basic,
neutral and polar, or neutral and nonpolar and/or aromatic.
It is generally preferred that encoded peptides differing
from the native form contain substituted codons for amino
acids which are from the same group as that of the amino
acid replaced. Thus, in general, the basic amino acids Lys,
Arg, and Histidine are interchangeable; the acidic amino
acids Asp and Glu are interchangeable; the neutral polar
amino acids Ser, Thr, Cys, Gin, and Asn are interchangeable;
the nonpolar aliphatic acids Gly, Ala, Val, Ile, and Leu are
conservative with respect to each other (but because of
size, Gly and Ala are more closely related and Val, Ile and
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27
Leu are more closely related), and the aromatic amino acids
Phe, Trp., and Tyr are interchangeable.
While Pro is a nonpolar neutral amino acid, it
represents difficulties because of its effects on
conformation, and substitutions by or for Pro are not
preferred, except when the same or similar conformat,ional
results can be obtained. Polar amino acids which represent
conservative changes include Ser, Thr, Gln, Asn; and to a
lesser extent, Met. In addition, although classified in
different categories, Ala, Gly, and Ser seem to be
interchangeable, and Cys additionally fits into this group,
or may be classified with the polar neutral amino acids.
Some substitutions by codons for amino acids from different
classes may also be useful.
Once the nucleic acid molecules encoding=the
connexin proteins are isolated and purified according to.the
methods described above, recombinant nucleic acid molecules
are prepared in which the desired connexin nucleic acid
molecule is incorporated into a delivery vehicle by methods
known to those skilled in the art, as taught in, for
example, Sambrook et al., Molecular Cloning: A Laboratory.
Manual, Second Ed. Cold Spring Harbor Press (1989).
-Preferred delivery vehicles include, for example, plasmids
(Acsadi, et al., The New Biol., 1991, 3, 71-81, and Gal, et
al., Lab. Invest., 1993,-68, 18-25
and adenovirus (WO 94/11506 and in Barr, et al., Gene Ther.,
1994 1, 51-58). The
nucleic acid molecules encoding connexin proteins, or
connexin proteins produced therefrom, are delivered to the
cardiac cells of the identified cardiac location by the
delivery systems of the present invention. Thus, such
delivery systems of the present invention are used to
contact the cardiac cells of the identified cardiac
location, which comprises cardiac cells having cardiac
conduction disturbances, with recombinant nucleic acid
molecules encoding a connexin protein, or connexin proteins.
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28
Where the conduction protein genetic materia_ is
in the form of conduction proteins, such proteins can be
prepared in large quantities by using various standard
expression,systems known to those skilled in the art.
. Sambrook et al., Molecular Cloning: A Laboratory Manual,
Second Ed.-Cold Spring Harbor Press (1989), pp. 16.1-16.55.
The recombinant nucleic acid molecules or connexin
proteins are preferably delivered in a pharmaceutical
composition. Such pharmaceutical compositions can include,
for example, the recombinant nucleic acid molecule or
protein in a volume of phosphate-buffered saline with 5t
sucrose. In other embodiments of the invention, the
recombinant nucleic acid molecule or protein is delivered
with suitable pharmaceutical carriers, such as those
described in the most recent edi-tion of Remington's
Pharmaceutical Sciences, A. Osol, a standard reference text
in this field. The recombinant nucleic acid molecule or
protein is delivered in a therapeutically effective amount.
Such amount is determined experimentally and is that amount
which either restores normal conduction or improves abnormal
conduction of cardiac cells. The amount of recombinant
nucleic acid molecule or protein is preferably between 0.01
g and 100 mg, more preferably between 0.1 g and 10 mg,
more preferably.between 1 g and 1 mg, and most preferably
between 10 g and 1 mg. A single therapeutically effective
amount is referred to as a bolus. Where adenovirus vectors
are used; the amount of reconibinant nucleic acid molecule is
preferably between 10' plaque forming units (pfu) and 10's
pfu, more preferably between 108 pfu and 1019 pfu, and most
preferably between 109 pfu and 10i2 pfu. A single
therapeutically effective amount of conduction protein
genetic material is referred to as a bolus. In some
embodiments of the present invention, the delivery of the
recombinant nucleic acid molecules or proteins is combined
with steroid elution, such as with dexamethasone sodium
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phosphate, beclamethasone, and the like, to control
inflammatory processes.
The following examples are meant to be exemplary
of the preferred embodiments of the invention and are not
meant to be limiting.
EXAMPLES
Example 1: Isolation and Purification of Nucleic Acid
Molecules Encoding the Connexin Proteins
Nucleic acid molecules encoding Cx43, Cx40, and
Cx45 are isolated and purified according to general methods
well known to those skilled in the art. Briefly, total
cellular RNA is isolated and purified (Chomczynsky, et al.,
Anal. Biochem., 1987, 162, 156-159) from heart tissue,
cardiac transplantation donors, or from salvaged tissue, and
selected for poly(A) RNA (Sambrook et al., Molecular
Cloning: A Laboratory Manual, Second Ed. Cold Spring Harbor
Press (1989), pp. 7.26-7.29). cDNA corresponding to the
connexin proteins is prepared from the poly(A) cardiac RNA
by reverse transcription using a GENEAMPTM PCR kit (Perkin
Elmer Cetus, Norwalk, CT), or the like, using random
hexamers according to the manufacturer's instructions. The
specific connexin nucleic acid molecules are amplified by
the polymerase chain reaction (PCR), also using the GENEAMPTM
PCR kit, or the like, using forward and reverse primers
specific for each of the different connexin proteins,
according to the manufacturer's instructions. For example,
the forward primer for Cx43 can be 5'-
ATGCCTGACTGGACCGCCTTAGGC-3' (SEQ ID NO:7), and the reverse
primer can be 5'-GATCTCGAGGTCATCAGGCCGAGG-3' (SEQ ID NO:8).
For example, the forward primer for Cx45 can be 5'-
ATGAGTTGGAGCTTTCTGACTCGC-3' (SEQ ID NO:9), and the reverse
primer can be 5'-AATCCAGACAGAGTTCTTCCCATC-3' (SEQ ID NO:10).
For example, the forward primer for Cx40 can be 5'-
ATGGGCGATTGGAGCTTCCTGGGA-3' (SEQ ID NO:11), and the reverse
primer can be 5'-CACTGATAGGTCATCTGACCTTGC-3' (SEQ ID NO:12).
It is understood that additional primers can be used for
amplification as determined by those skilled in the art.
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These primers may be preceded at the 5' terminus by
nucleotide sequences containing endonuclease restriction
sites for easy incorporation into vectors. The specific
connexin nucleic acid molecules can also be amplified by PCR
from human genomic DNA (Stratagene, San Diego, CA). After
cutting the PCR products with the appropriate restriction
endonuclease(s), the PCR products are purified by
phenol:chloroform extractions, or using commercial
purification kits, such as, for example, MAGICTM Minipreps
DNA Purification System (Promega, Madison, WI). The
specific nucleotide sequence of the PCR products is
determined by conventional DNA sequencing procedures, and
the identity of the PCR products confirmed by comparison to
the published sequences for the connexin proteins.
Example 2: Insertion of Connexin cDNA into Delivery Vehicles
Preferably, connexin cDNA is inserted into either
plasmid or adenoviral vectors. Plasmid vectors include for
example, pGEM3 or pBR322, as set forth in Acsadi, et al.,
The New Biol., 1991, 3, 71-81, and Gal, et al., Lab.
Invest., 1993, 68, 18-25. Adenoviral vectors include for
example, adenovirus serotype 5, Ad5, as set forth in French,
et al., Circulation, 1994, 90, 2414-2424.
Preferably, the primers used to amplify the
connexin nucleic acid molecules are designed with unique
endonuclease restriction sites located at the 5' terminus.
In the absence of such design, polylinker arms, containing
unique restriction sites, can be ligated thereto. After
cutting the purified PCR products with the appropriate
restriction endonuclease(s), the plasmid vector, comprising
a polylinker, is also cut with the same restriction
endonuclease(s), affording the connexin nucleic acid
molecule a site at which to ligate. In a similar manner,
recombinant adenovirus (Gluzman, et al., in Eukaryotic Viral
Vectors, Gluzman, ed., Cold Spring Harbor Press, 1982,
pp.187-192, and French, et al., Circulation, 1994, 90, 2414-
2424) containing connexin cDNA molecules are prepared in
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accordance with standard techniques well known to those
skilled in the art.
It is contemplated that variations of the above-
described invention may be constructed that are consistent
with the spirit of the invention.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Medtrontic, Inc.
(ii) TITLE OF INVENTION: SYSTEM FOR GENETICALLY TREATING CARDIAC
CONDUCTION DISTURBANCES
(iii) NUMBER OF SEQUENCES: 12
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: 'Van Zant & Associates
(B) STREET: 77 :Bloor Street West, Suite 1407
(C) CITY: Toronto
(D) STATE: ON
(E) COUNTRY: Canada
(F) ZIP: M5S 1M2
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WordPerfect 6.1
(vi) CURRENT APPLICAT:ION DATA:
(A) APPLICATION NUMBER: 2,260,756
(B) FILING DATE: April 4, 1997
(C) CLASSIFICAT:ION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Joan M. Van Zant
(B) REGISTRATION NUMBER: 2992
(C) REFERENCE/DOCKET NUMBER: P184PCA1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 921-6260
(B) TELEFAX: (4:L6) 921-8187
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1074 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: l:Enear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATG GGC GAT TGG AGC TTC CTG GGA AAT TTC CTG GAG GAA GTA CAC 45
Met Gly Asp Trp Ser Phe Le:u Gly Asn Phe Leu Glu Glu Val His
1 5 10 15
AAG CAC TCG ACC GTG GTA GGC AAG GTC TGG CTC ACT GTC CTC TTC 90
Lys His Ser Thr Val Val Gly Lys Val Trp Leu Thr Val Leu Phe
20 25 30
ATA TTC CGT ATG CTC GTG CTG GGC ACA GCT GCT GAG TCT ACC TGG 135
Ile Phe Arg Met Leu Val Leu Gly Thr Ala Ala Glu Ser Thr Trp
35 40 45
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GGG GAT GAG CAG GCT GAT TTC CGG TGT GAT ACG ATT CAG CCT GGC 180
Gly Asp Glu Gln Ala Asp Phe Arg Cys Asp Thr Ile Gln Pro Gly
50 55 60
TGC CAC AAT GTC TGC TAC GAC CAG GCT TTC CCC ATC TCC CAC ATT 225
Cys His Asn Val Cys Tyr Asp Gln Ala Phe Pro Ala Ser His Ile
65 70 75
CGC TAC TGG GTG CTG CAG ATC ATC TTC GTC TCT ACG CCC TCT CTG 270
Arg Tyr Trp Val Leu Gln Ile Ile Phe Val Ser Thr Pro Ser Leu
80 85 90
GTG TAC ATG GGC CAC GCC ATG CAC ACT GTG CGC ATG CAG GAG AAG 315
Val Tyr Met Gly His Ala Met His Thr Val Arg Met Gln Glu Lys
95 100 105
CGC AAG CTA CGG GAG GCC GAG AGG GCC AAA GAG GTC CGG GGC TCT 360
Arg Lys Leu Arg Glu Ala Glu Arg Ala Lys Glu Val Arg Gly Ser
110 115 120
GGC TCT TAC GAG TAC CCG GTG GCA GAG AAG GCA GAA CTG TCC TGC 405
Gly Ser Tyr Glu Tyr Pro Val Ala Glu Lys Ala Glu Leu Ser Cys
125 130 135
TGG GAG GAA GGG AAT GGA AGG ATT GCC CTC CAG GGC ACT CTG CTC 450
Trp Glu Glu Glu Asn Gly Arg Ile Ala Leu Gln Gly Thr Leu Leu
140 145 150
AAC ACC TAT GTG TGC AGC ATC CTG ATC CGC ACC ACC ATG GAG GTG 495
Asn Thr Tyr Val Cys Ser Ile Leu Ile Arg Thr Thr Met Glu Val
155 160 165
GGC TTC ATT GTG GGC CAG TAC TTC ATC TAC GGA ATC TTC CTG ACC 540
Gly Phe Ile Val Gly Gln Tyr Phe Ile Tyr Gly Ile Phe Leu Thr
170 175 180
ACC CTG CAT GTC TGC CGC AGG AGT CCC TGT CCC CAC CCG GTC AAC 585
Thr Leu His Val Cys Arg Arg Ser Pro Cys Pro His Pro Val Asn
185 190 195
TGT TAC GTA TCC CGG CCC ACA GAG AAG AAT GTC TTC ATT GTC TTT 630
Cys Tyr Val Ser Arg Pro Thr Glu Lys Asn Val Phe Ile Val Phe
200 205 210
ATG CTG GCT GTG GCT GCA CTG TCC CTC CTC CTT AGC CTG GCT GAA 675
Met Leu Ala Val Ala Ala Leu Ser Leu Leu Leu Ser Leu Ala Glu
215 220 225
CTC TAC CAC CTG GGC TGG AAG AAG ATC AGA CAG CGA TTT GTC AAA 720
Leu Tyr His Leu Gly Trp Lys Lys Ile Arg Gln Arg Phe Val Lys
230 235 240
CCG CGG CAG TAC ATG GCT AAG TGC CAG CTT TCT GGC CCT CTG TGG 765
Pro Arg Gln Trp Met Ala Lys Cys Gln Leu Ser Gly Pro Leu Trp
245 250 255
GCT ATA GTC CAG AGC TGC ACA CCA CCC CCC GAC TTT AAT CAG TGC 810
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Ala Ile Val Gln Ser Cys Thr Pro Pro Pro Asp Phe Asn Gln Cys
260 265 270
CTG GAG AAT GGT CCT GGG GGA AAA TTC TTC AAT CCC TTC AGC AAT 855
Leu Glu Asn Gly Pro Gly Gly Lys Phe Phe Asn Pro Phe Ser Asn
275 280 285
AAT ATG GCC TCC CAA CAA AAC ACA GAC AAC CTG GTC ACC GAG CAA 900
Asn Met Ala Ser Gln Gln Asn Thr Asp Asn Leu Val Thr Glu Gln
290 295 300
GTA CGA GGT CAG GAG CAG ACT CCT GGG GAA GGT TTC ATC CAG GTT 945
Val Arg Gly Gln Glu Gln Thr Pro Gly Glu Gly Phe Ile Gln Val
305 310 315
CGT TAT GGC CAG AAG CCT GAG GTG CCC AAT GGA GTC TCA CCA GGT 990
Arg Tyr Gly Gln Lys Pro Glu Val Pro Asn Gly Val Ser Pro Gly
320 325 330
CAC CGC CTT CCC CAT GGC TAT CAT AGT GAC AAG CGA CGT CTT AGT 1035
His Arg Leu Pro His Gly Tyr His Ser Asp Lys Arg Arg Leu Ser
335 340 345
AAG GCC AGC AGC AAG GCA AGG TCA GAT GAC CTA TCA GTG 1074
Lys Ala Ser Ser Lys Ala Arg Ser Asp Asp Leu Ser Val
350 355
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 358 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Gly Asp Trp Ser Phe Leu Gly Asn Phe Leu Glu Glu Val His
1 5 10 15
Lys His Ser Thr Val Val Gly Lys Val Trp Leu Thr Val Leu Phe
20 25 30
Ile Phe Arg Met Leu Val Leu Gly Thr Ala Ala Glu Ser Thr Trp
35 40 45
Gly Asp Glu Gln Ala Asp Phe Arg Cys Asp Thr Ile Gln Pro Gly
50 55 60
Cys His Asn Val Cys Tyr Asp Gin Ala Phe Pro Ala Ser His Ile
65 70 75
Arg Tyr Trp Val Leu Gln Ile Ile Phe Val Ser Thr Pro Ser Leu
80 85 90
Val Tyr Met Gly His Ala Met His Thr Val Arg Met Gln Glu Lys
95 100 105
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Arg Lys Leu Arg Glu Ala Glu Arg Ala Lys Glu Val Arg Gly Ser
110 115 120
Gly Ser Tyr Glu Tyr Pro Val Ala Glu Lys Ala Glu Leu Ser Cys
125 130 135
Trp Glu Glu Glu Asn Gly Arg Ile Ala Leu Gln Gly Thr Leu Leu
140 145 150
Asn Thr Tyr Val Cys Ser I:le Leu Ile Arg Thr Thr Met Glu Val
155 160 165
Gly Phe Ile Val Gly Gln Tyr Phe Ile Tyr Gly Ile Phe Leu Thr
170 175 180
Thr Leu His Val Cys Arg Arg Ser Pro Cys Pro His Pro Val Asn
185 190 195
Cys Tyr Val Ser Arg Pro Thr Glu Lys Asn Val Phe Ile Val Phe
200 205 210
Met Leu Ala Val Ala Ala Leu Ser Leu Leu Leu Ser Leu Ala Glu
215 220 225
Leu Tyr His Leu Gly Trp Lys Lys Ile Arg Gln Arg Phe Val Lys
230 235 240
Pro Arg Gln Trp Met Ala Lys Cys Gln Leu Ser Gly Pro Leu Trp
245 250 255
Ala Ile Val Gln Ser Cys Thr Pro Pro Pro Asp Phe Asn Gln Cys
260 265 270
Leu Glu Asn Gly Pro Gly Gly Lys Phe Phe Asn Pro Phe Ser Asn
275 280 285
Asn Met Ala Ser Gln Gln Asn Thr Asp Asn Leu Val Thr Glu Gln
290 295 300
Val Arg Gly Gln Glu Gln Thr Pro Gly Glu Gly Phe Ile Gln Val
305 310 315
Arg Tyr Gly Gln Lys Pro Glu Val Pro Asn Gly Val Ser Pro Gly
320 325 330
His Arg Leu Pro His Gly Tyr His Ser Asp Lys Arg Arg Leu Ser
335 340 345
Lys Ala Ser Ser Lys Ala Arg Ser Asp Asp Leu Ser Val
350 355
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1146 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
CA 02260756 1999-07-16
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATG GGT GAC TGG AGC GCC TTA GGC AAA CTC CTT GAC AAG GTT CAA 45
Met Gly Asp Trp Ser Ala Leu Gly Lys Leu Leu Asp Lys Val Gln
1 5 10 15
GCC TAC TCA ACT GCT GGA GGG AAG GTG TGG CTG TCA GTA CTT TTC 90
Ala Tyr Ser Thr Ala Gly Gly Lys Val Trp Leu Ser Val Leu Phe
20 25 30
ATT TTC CGA ATC CTG CTG CTG GGG ACA GCG GTT GAG TCA GCC TGG 135
Ile Phe Arg Ile Leu Leu Leu Gly Thr Ala Val Glu Ser Ala Trp
35 40 45
GGA GAT GAG CAG TCT GCC TTT CGT TGT AAC ACT CAG CAA CCT GGT 180
Gly Asp Glu Gln Ser Ala Phe Arg Cys Asn Thr Gln Gln Pro Gly
50 55 60
TGT GAA AAT GTC TGC TAT GAC AAG TCT TTC CCA ATC TCT CAT GTG 225
Cys Glu Asn Val Cys Tyr Asp Lys Ser Phe Pro Ile Ser His Val
65 70 75
CGC TTC TGG GTC CTG CAG ATC ATA TTT GTG TCT GTA CCC ACA CTC 270
Arg Phe Trp Val Leu Gln Ile Ile Phe Val Ser Val Pro Thr Leu
80 85 90
TTG TAC CTG GCT CAT GTG TTC TAT GTG ATG CGA AAG GAA GAG AAA 315
Leu Tyr Leu Ala His Val Phe Tyr Val Met Arg Lys Glu Glu Lys
95 100 105
CTG AAC AAG AAA GAG GAA GAA CTC AAG GTT GCC CAA ACT GAT GGT 360
Leu Asn Lys Lys Glu Glu Glu Leu Lys Val Ala Gln Thr Asp Gly
110 115 120
GTC AAT GTG GAC ATG CAC TTG AAG CAG ATT GAG ATA AAG AAG TTC 405
Val Asn Val Asp Met His Leu Lys Gln Ile Glu Ile Lys Lys Phe
125 130 135
AAG TAC GGT ATT GAA GAG CAT GGT AAG GTG AAA ATG CGA GGG GGG 450
Lys Tyr Gly Ile Glu Glu His Gly Lys Val Lys Met Arg Gly Gly
140 145 150
TTG CTG CGA ACC TAC ATC ATC AGT ATC CTC TTC AAG TCT ATC TTT 495
Leu Leu Arg Thr Tyr Ile Ile Ser Ile Leu Phe Lys Ser Ile Phe
155 160 165
GAG GTG GCC TTC TTG CTG ATC CAG TGG TAC ATC TAT GGA TTC AGC 540
Glu Val Ala Phe Leu Leu I].e Gln Trp Tyr Ile Tyr Gly Phe Ser
170 175 180
TTG AGT GCT GTT TAC ACT TGC AAA AGA GAT CCC TGC CCA CAT CAG 585
Leu Ser Ala Val Tyr Thr Cys Lys Arg Asp Pro Cys Pro His Gln
185 P 190 195
GTG GAC TGT TTC CTC TCT CGC CCC ACG GAG AAA ACC ATC TTC ATC 630
Val Asp Cys Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile Phe Ile
200 205 210
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ATC TTC ATG CTG GTG GTG TCC TTG GTG TCC CTG GCC TTG AAT ATC 675
Ile.Phe Met Leu Val Val Ser Leu Val Ser Leu Ala Leu Asn Ile
215 220 225
ATT GAA CTC TTC TAT GTT TTC TTC AAG GGC GTT AAG GAT CGG GTT 720
Ile Glu Leu Phe Tyr Val P:he Phe Lys Gly Val Lys Asp Arg Val
230 235 240
AAG GGA AAG AGC GAC CCT TAC CAT GCG ACC AGT GGT GCG CTG AGC 765
Lys Gly Lys Cys Asp Pro Tyr His Ala Thr Ser Gly Ala Leu Ser
245 250 255
CCT GCC AAA GAC TGT GGG TCT CAA AAA TAT GCT TAT TTC AAT GGC 810
Pro Ala Lys Asp Cys Gly Ser Gln Lys Tyr Ala Tyr Phe Asn Gly
260 265 270
TGC TCC TCA CCA ACC GCT CCC CTC TCG CCT ATG TCT CCT CCT GGG 855
Cys Ser Ser Pro Thr Ala Pro Leu Ser Pro Met Ser Pro Pro Gly
275 280 285
TAC AAG CTG GTT ACT GGC GAC AGA AAC AAT TCT TCT TGC CGC AAT 900
Tyr Lys Leu Val Thr Gly Asp Arg Asn Asn Ser Ser Cys Arg Asn
290 295 300
TAC AAC AAG CAA GCA AGT GAG CAA AAC TGG GCT AAT TAC AGT GCA 945
Tyr Asn Lys Gln Ala Ser Glu Gln Asn Trp Ala Asn Tyr Ser Ala
305 310 315
GAA CAA AAT CGA ATG GGG CAG GCG GGA AGC ACC ATC TCT AAC TCC 990
Glu Gln Asn Arg Met Gly Gly Ala Gly Ser Thr Ile Ser Asn Ser
320 325 330
CAT GCA CAG CCT TTT GAT TTC CCC GAT GAT AAC CAG AAT TCT AAA 1035
His Ala Gln Pro Phe Asp Phe Pro Asp Asp Asn Gln Asn Ser Lys
335 340 345
AAA CTA GCT GCT GGA CAT GAA TTA CAG CCA CTA GCC ATT GTG GAC 1080
Lys Leu Ala Ala Gly His Glu Leu Gln Pro Leu Ala Ile Val Asp
350 355 360
CAG CGA CCT TCA AGC AGA GCC AGC AGT CGT GCC AGC AGC AGA CCT 1125
Gln Arg Pro Ser Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro
365 370 375
CGG CCT GAT GAC CTG GAG ATC 1146
Arg Pro Asp Asp Leu Glu I].e
380
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 382 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Gly Asp Trp Ser Ala Leu Gly Lys Leu Leu Asp Lys Val Gln
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1 5 10 15
Ala Tyr Ser Thr Ala Gly G:1y Lys Val Trp Leu Ser Val Leu Phe
20 25 30
Ile Phe Arg Ile Leu Leu Leu Gly Thr Ala Val Glu Ser Ala Trp
35 40 45
Gly Asp Glu Gln Ser Ala Phe Arg Cys Asn Thr Gln Gln Pro Gly
50 55 60
Cys Glu Asn Val Cys Tyr Asp Lys Ser Phe Pro Ile Ser His Val
65 70 75
Arg Phe Trp Val Leu Gln I:Le Ile Phe Val Ser Val Pro Thr Leu
80 85 90
Leu Tyr Leu Ala His Val Phe Tyr Val Met Arg Lys Glu Glu Lys
95 100 105
Leu Asn Lys Lys Glu Glu Glu Leu Lys Val Ala Gln Thr Asp Gly
110 115 120
Val Asn Val Asp Met His Leu Lys Gln Ile Glu Ile Lys Lys Phe
125 130 135
Lys Tyr Gly Ile Glu Glu His Gly Lys Val Lys Met Arg Gly Gly
140 145 150
Leu Leu Arg Thr Tyr Ile I].e Ser Ile Leu Phe Lys Ser Ile Phe
155 160 165
Glu Val Ala Phe Leu Leu I].e Gln Trp Tyr Ile Tyr Gly Phe Ser
170 175 180
Leu Ser Ala Val Tyr Thr Cys Lys Arg Asp Pro Cys Pro His Gln
185 190 195
Val Asp Cys Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile Phe Ile
200 205 210
Ile Phe Met Leu Val Val Ser Leu Val Ser Leu Ala Leu Asn Ile
215 220 225
Ile Glu Leu Phe Tyr Val Phe Phe Lys Gly Val Lys Asp Arg Val
230 235 240
Lys Gly Lys Cys Asp Pro Tyr His Ala Thr Ser Gly Ala Leu Ser
245 250 255
Pro Ala Lys Asp Cys Gly Ser Gln Lys Tyr Ala Tyr Phe Asn Gly
260 265 270
Cys Ser Ser Pro Thr Ala Pro Leu Ser Pro Met Ser Pro Pro Gly
275 280 285
Tyr Lys Leu Val Thr Gly Asp Arg Asn Asn Ser Ser Cys Arg Asn
- -----=-= - -- -----
CA 02260756 1999-07-16
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290 295 300
Tyr Asn Lys Gln Ala Ser Glu Gln Asn Trp Ala Asn Tyr Ser Ala
305 310 315
Glu Gln Asn Arg Met Gly Gly Ala Gly Ser Thr Ile Ser Asn Ser
320 325 330
His Ala Gln Pro Phe Asp Phe Pro Asp Asp Asn Gln Asn Ser Lys
335 340 345
Lys Leu Ala Ala Gly His Glu Leu Gln Pro Leu Ala Ile Val Asp
350 355 360
Gln Arg Pro Ser Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro
365 370 375
Arg Pro Asp Asp Leu Glu I.le
380
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1188 bases
(B) TYPE: nucleic acid
(C) STRANDEDNES,3: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATG AGT TGG AGC TTT CTG ACT CGC CTG CTA GAG GAG ATT CAC AAC 45
Met Ser Trp Ser Phe Leu Thr Arg Leu Leu Glu Glu Ile His Asn
1 5 10 15
CAT TCC ACA TTT GTG GGG AAG ATC TGG CTC ACT GTT CTG ATT GTC 90
His Ser Thr Phe Val Gly Lys Ile Trp Leu Thr Val Leu Ile Val
20 25 30
TTC CGG ATC GTC CTT ACA GCT GTA GGA GGA GAA TCC ATC TAT TAC 135
Phe Arg Ile Val Leu Thr A:La Val Gly Gly Glu Ser Ile Tyr Tyr
35 40 45
GAT GAG CAA AGC AAA TTT GTG TGC AAC ACA GAA CAG CCG GGC TGT 180
Asp Glu Gln Ser Lys Phe Val Cys Asn Thr Glu Gln Pro Gly Cys
50 55 60
GAG AAT GTC TGT TAT GAT GCG TTT GCA CCT CTC TCC CAT GTA CGC 225
Glu Asn Val Cys Tyr Asp Ala Phe Ala Pro Leu Ser His Val Arg
65 70 75
TTC TGG GTG TTC CAG ATC ATC CTG GTG GCA ACT CCC TCT GTG ATG 270
Phe Trp Val Phe Gln Ile Ile Leu Val Ala Thr Pro Ser Val Met
80 85 90
TAC CTG GGC TAT GCT ATC CAC AAG ATT GCC AAA ATG GAG CAC GGT 315
Tyr Leu Gly Tyr Ala Ile His Lys Ile Ala Lys Met Glu His Gly
95 100 105
GAA GCA GAC AAG AAG GCA GCT CGG AGC AAG CCC TAT GCA ATG CGC 360
Glu Ala Asp Lys Lys Ala Ala Arg Ser Lys Pro Tyr Ala Met Arg
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110 115 120
TGG AAA CAA CAC CGG GCT CTG GAA GAA ACG GAG GAG GAC AAC GAA 405
Trp Lys Gln His Arg Ala Leu Glu Glu Thr Glu Glu Asp Asn Glu
125 130 135
GAG GAT CCT ATG ATG TAT CCA GAG ATG GAG TTA GAA AGT GAT AAG 450
Glu Asp Pro Met Met Tyr P:ro Glu Met Glu Leu Glu Ser Asp Lys
140 145 150
GAA AAT AAA GAG CAG AGC C:AA CCC AAA CCT AAG CAT GAT GGC CGA 495
Glu Asn Lys Glu Gln Ser G:Ln Pro Lys Pro Lys His Asp Gly Arg
155 160 165
CGA CGG ATT CGG GAA GAT GGG CTC ATG AAA ATC TAT GTG CTG CAG 540
Arg Arg Ile Arg Glu Asp G:Ly Leu Met Lys Ile Tyr Val Leu Gln
170 175 180
TTG CTG GCA AGG ACC GTG TTT GAG GTG GGT TTT CTG ATA GGG CAG 585
Leu Leu Ala Arg Thr Val Phe Glu Val Gly Phe Leu Ile Gly Gln
185 190 195
TAT TTT CTG TAT GGC TTC CAA GTC CAC CCG TTT TAT GTG TGC AGC 630
Tyr Phe Leu Tyr Gly Phe G:Ln Val His Pro Phe Tyr Val Cys Ser
200 205 210
AGA CTT CCT TGT CCT CAT AAG ATA GAC TGC TTT ATT TCT AGA CCC 675
Arg Leu Pro Cys Pro His Lys IleAsp Cys Phe Ile Ser Arg Pro
215 220 225
ACT GAA AAG ACC ATC TTC CTT CTG ATA ATG TAT GGT GTT ACA GGC 720
Thr Glu Lys Thr Ile Phe Leu Leu Ile Met Tyr Gly Val Thr Gly
230 235 240
CTT TGC CTC TTG CTT AAC ATT TGG GAG ATG CTT CAT TTA GGG TTT 765
Leu Cys Leu Leu Leu Asn Ile Trp Glu Met Leu His Leu Gly Phe
245 250 255
GGG ACC ATT CGA GAC TCA CTA AAC AGT AAA AGG AGG GAA CTT GAG 810
Gly Thr Ile Arg Asp Ser Leu Asn Ser Lys Arg Arg Glu Leu Glu
260 265 270
GAT CCG GGT GCT TAT AAT TAT CCT TTC ACT TGG AAT ACA CCA TCT 855
Asp Pro Gly Ala Tyr Asn Tyr Pro Phe Thr Trp Asn Thr Pro Ser
275 280 285
GCT CCC CCT GGC TAT AAC ATT GCT GTC AAA CCA GAT CAA ATC CAG 900
Ala Pro Pro Gly Tyr Asn I]Le Ala Val Lys Pro Asp Gln Ile Gln
290 295 300
TAC ACC GAA CTG TCC AAT GCT AAG ATC GCC TAC AAG CAA AAC AAG 945
Tyr Thr Glu Leu Ser Asn Ala Lys Ile Ala Tyr Lys Gln Asn Lys
305 310 315
GCC AAC ACA GCC CAG GAA CAG CAG TAT GGC AGC CAT GAG GAG AAC 990
Ala Asn Thr Ala Gln Glu Gln Gln Tyr Gly Ser His Glu Glu Asn
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320 325 330
CTC CCA GCT GAC CTG GAG GCT CTG CAG CGG GAG ATC AGG ATG GCT 1035
Leu Pro Ala Asp Leu Glu Ala Leu Gln Arg Glu Ile Arg Met Ala
335 340 345
CAG GAA CGC TTG GAT CTG GCA GTT CAG GCC TAC AGT CAC CAA AAC 1080
Gln Glu Arg Leu Asp Leu Ala Val Gln Ala Tyr Ser His Gln Asn
350 355 360
AAC CCT CAT GGT CCC CGG GAG AAG AAG GCC AAA GTG GGG TCC AAA 1125
Asn Pro His Gly Pro Arg G:lu Lys Lys Ala Lys Val Gly Ser Lys
365 370 375
GCT GGG TCC AAC AAA AGC ACT GCC AGT AGC AAA TCA GGG GAT GGG 1170
Ala Gly Ser Asn Lys Ser Tlar Ala Ser Ser Lys Ser Gly Asp Gly
380 385 390
AAG AAC TCT GTC TGG ATT 1188
Lys Asn Ser Val Trp Ile
395
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 396 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: tuzknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ser Trp Ser Phe Leu Thr Arg Leu Leu Glu Glu Ile His Asn
1 5 10 15
His Ser Thr Phe Val Gly Lys Ile Trp Leu Thr Val Leu Ile Val
20 25 30
Phe Arg Ile Val Leu Thr Ala Val Gly Gly Glu Ser Ile Tyr Tyr
35 40 45
Asp Glu Gln Ser Lys Phe Val Cys Asn Thr Glu Gln Pro Gly Cys
50 55 60
Glu Asn Val Cys Tyr Asp Ala Phe Ala Pro Leu Ser His Val Arg
65 70 75
Phe Trp Val Phe Gin Ile Ile Leu Val Ala Thr Pro Ser Val Met
80 85 90
Tyr Leu Gly Tyr Ala Ile His Lys Ile Ala Lys Met Glu His Giy
95 100 105
Glu Ala Asp Lys Lys Ala Ala Arg Ser Lys Pro Tyr Ala Met Arg
110 115 120
Trp Lys Gln His Arg Ala Leu Glu Glu Thr Glu Glu Asp Asn Glu
125 130 135
Glu Asp Pro Met Met Tyr Pro Glu Met Glu Leu Glu Ser Asp Lys
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140 145 150
Glu Asn Lys Glu Gln Ser Gln Pro Lys Pro Lys His Asp Gly Arg
155 160 165
Arg Arg Ile Arg Glu Asp G:ly Leu Met Lys Ile Tyr Val Leu Gln
170 175 180
Leu Leu Ala Arg Thr Val Phe Glu Val Gly Phe Leu Ile Gly Gln
185 190 195
Tyr Phe Leu Tyr Gly Phe G:Ln Val His Pro Phe Tyr Val Cys Ser
200 205 210
Arg Leu Pro Cys Pro His Lys Ile Asp Cys Phe Ile Ser Arg Pro
215 220 225
Thr Giu Lys Thr Ile Phe Leu Leu Ile Met Tyr Gly Val Thr Gly
230 235 240
Leu Cys Leu Leu Leu Asn Ile Trp Glu Met Leu His Leu Gly Phe
245 250 255
Gly Thr Ile Arg Asp Ser Leu Asn Ser Lys Arg Arg Glu Leu Glu
260 265 270
Asp Pro Gly Ala Tyr Asn Tyr Pro Phe Thr Trp Asn Thr Pro Ser
275 280 285
Ala Pro Pro Gly Tyr Asn Ile Ala Val Lys Pro Asp Gln Ile Gln
290 295 300
Tyr Thr Glu Leu Ser Asn Ala Lys Ile Ala Tyr Lys Gln Asn Lys
305 310 315
Ala Asn Thr Ala Gln Glu Gln Gln Tyr Gly Ser His Glu Glu Asn
320 325 330
Leu Pro Ala Asp Leu Glu Ala Leu Gln Arg Glu Ile Arg Met Ala
335 340 345
Gln Glu Arg Leu Asp Leu Ala Val Gln Ala Tyr Ser His Gln Asn
350 355 360
Asn Pro His Gly Pro Arg Glu Lys Lys Ala Lys Val Gly Ser Lys
365 370 375
Ala Gly Ser Asn Lys Ser Thr Ala Ser Ser Lys Ser Gly Asp Gly
380 385 390
Lys Asn Ser Val Trp Ile
395
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1:Lnear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATGCCTGACT GGACCGCCTT AGGC 24
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GATCTCGAGG TCATCAGGCC GAGG 24
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATGAGTTGGA GCTTTCTGAC TCGC 24
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
AATCCAGACA GAGTTCTTCC CATC: 24
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ATGGGCGATT GGAGCTTCCT GGGA 24
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE : nucleic acid
(C) STRANDEDNESSI: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CACTGATAGG TCATCTGACC TTGC: 24
