Title of Invention

A TOPICAL COMPOSITION FOR THE TREATMENT OF HSV-RELATED PATHOLOGIES

Abstract A composition for treatment of HSV-related pathologies including an expression vector for altering expression of one or more target sequence(s) in an HSV-infected cell by production of single-stranded cDNA (ssDNA) in the cell in vivo suspended for topical application to an affected site in a suitable delivery vehicle. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3' to the inverted tandem repeat, and a reverse transcrip- tase/RNAse H coding gene, and is transfected into the infected cells for inhibition of HSV replication or assimilation into a target cell. The resulting ssDNA binds to the target sequence to alter expression of the target sequence for such purposes as gene activation or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding to specific cellular proteins, or interfering with RNA splicing functions.
Full Text The present invention relates to the treatment of HSV-related pathologies using a
composition that produces ssDNA in vivo for altering the expression of genes involved in
HSV replication. More particularly, the present invention relates to a composition and
method for delivering the information required for production of ssDNA in vivo for
altering the function of a gene or genes involved in replication and self-protection of HSV
against the host immune system by topical application to an affected site.
Herpes simplex virus types I and II (HSV-I and HSV-II) are the etiological agents
of several clinically significant pathologies including oropharyngeal infections, skin
infections, ocular infection, and central nervous system disorders such as meningitis and
encephalitis (see, for instance, U.S. Patent No. 5,795,721). HSV seropositive individuals
are found in virtually all human populations. It is estimated that more than 60% of the
population is seropositive for HSV-I and more than 16% of the population is seropositive
forHSV-II.
Primary HSV is often asymptomatic. Whether asymptomatic or not, HSV
infection invariably leads to establishment of latent viral infection in nerve cells. No
known treatment is available for latent infection and the persistent and frequent outbreaks
it provokes. The consequences of latent infection cover a spectrum from no symptoms to
repeated and/or severe episodes of the active form of the disease.
As used herein, the term "HSV" is intended to refer to all known and yet to be
discovered and/or identified strains of herpes simplex virus, including known and yet to
be discovered and/or identified strains of herpes simplex and/or other viruses, as well as
human papilloma (HPV) and varicella zoster (VZ) viruses, that share some of the same
genome and/or modes of infection and replication. The term "HSV-related pathology" is
intended to refer to any pathological condition in which HSV or other related viruses are
a causative agent and/or in which such viruses have been and/or will at some time in the
future be implicated, even if HSV or these other viruses are not the cause of the pathology
(for instance, if HSV infection is a symptom and/or consequence of the pathological
condition).
Most HSV-related pathologies are not life-threatening, although they do represent
a significant proportion of doctor visits for sexually transmitted diseases. Affected
individuals often seek ameliorative treatment to counteract the discomfort caused by HSV
induced lesions. More serious, and even life-threatening, HSV-related pathologies do
occur. For instance, over 50% of neonates are afflicted by HSV-related CNS disorders,
and without appropriate treatment, CNS infection in neonates leads to greater than 60%
mortality. Even with treatment, there is significant mortality in affected neonates.
Sestimates of neonatal herpes infection are approximately 1 in 4000 live births. Two-
thirds of neonatal herpes is attributable to HSV-II Due to the increasing percentage of
HSV-II infected women (20% in the U.S.), neonatal herpes presents a serious public
health problem and, so far as is known, there is no effective treatment for this pathology.
There is, therefore, a long-felt and unfulfilled need for effective treatment against
these, and other HSV-related pathologies. Known treatments simply are not addressing
this need, and it is a primary object of the present invention to provide an effective
treatment against HSV-related pathologies for topical application to an affected site, and
specifically, to provide a treatment that alters the expression of the genes implicated in
the replication and self-protection of HSV against the host immune system.
The HSV genome is a double-stranded, closed, circular DNA molecule consisting
of 1.5 x 105 base pairs, encoding more than 75 polypeptides (B. Roizman and A.E. Sears,
Herpes simplex viruses and their replication, in B. N. Fields and D. M. Knipe (Eds.),
Fundamental Virology, 2nd Ed., pp. 849-895, Raven Press (1991)). The infective HSV
life cycle is governed by three stages of gene expression, referred to as immediate-early
(IE), middle and late (also referred to as a, p, and y). Each of these stages results in
expression of genes whose products are required for subsequent event in the HSV life
cycle, culminating in production and release of infective HSV virions.
Following entry of the HSV virion into the cell, its protein coat is shed, releasing
a capsid structure that transports the viral genome to the nuclear pores and into the
nucleus. Entry into the nucleus marks the beginning of the immediate-early stage. Five
IE genes, a4--ICP4, a0--ICP0, a27--ICP27/UL 54, a22--ICP22/US1, and a47--
ICP47/US12, are expressed and function in the earliest stages of the productive infection
cycle. This immediate-early stage of infection is termed the "a" phase of gene
expression, and is mediated by the action of a-TIF through its interaction with cellular
transcription factors at specific enhancer elements associated with the individual a-
transcript promoters. At least three of the protein products of these genes, ICP0, 1CP4,
and ICP27, are factors involved in regulation of HSV gene expression (ICP, infected-cell
polypeptide). These regulatory factors are expressed throughout the viral life cycle, and
serve to control the complex patterns of gene activation, repression, and de-repression
that determine the course of HSV proliferation. Middle (b) genes primarily encode
proteins involved in DNA replication and late (g) genes encode structural proteins
involved in packaging and assembling mature virions.
The ICP4 protein is the major transcriptional regulator of HSV gene expression.
(Roizman and Sears, supra) The ICP4 gene (also known as the a4 gene) encodes a
polypeptide of 1298 amino acids with a predicted molecular weight of 133,000 daltons.
The mature ICP4 protein exists as a homodimer of 350,000 daltons; the large molecular
weight reflects post-translational modifications. The ICP4 homodimer is a double-
stranded DNA binding protein which functions both as a transcriptional repressor and
activator of HSV gene expression (Beard, P., S. Faber, K. Wilcox, and L. Pizer, Herpes
simplex virus immediate-early infected-cell polypeptide 4 binds to DNA and promotes
transcription, Proc. Natl. Acad. Sci. USA. 83:4016-4020 (1986); Pizer, L., D. Tedder, J.
Betz, and K. Wilcox, Regulation of transcription in vitro from herpes simplex virus
genes, J. Virol. 60:950-959 (1996)). The actual mechanisms by which ICP4 affects
transcription are not well understood. The most well-characterized DNA binding sites are
those where ICP4 acts as a repressor.
ICP4 is an essential gene product whose function is required for HSV
proliferation. HSV-1 mutants which lack both copies of the ICP4 gene (there are two in
different loci in the genome) fail to replicate DNA and produce new virions (DeLuca, N.
A., A. M. McCarthy, and P. A. Schaffer, Isolation and characterization of deletion
mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory
protein ICP4, J. Virol. 56:558-570 (1995)). In addition to ICP4, other essential proteins
are glycoproteins B and D (gB and gD), ICP27, and several proteins involved in DNA
replication (Matthews, J. T., B. J. Terry, and A. K. Field, The structure and function of
the HSV DNA replication proteins: defining novel antiviral targets, Antiviral Res. 20:89-
114 (1993); Olivo, P. D., N. L. Nelson, and M. D. Challberg, Herpes simplex virus type 1
gene products required for DNA replication: identification and overexpression, J. Virol.
63:196-204 (1989); Roizman, B., and A. E. Sears, supra). As mentioned above, the
action of ICP4 is required throughout the HSV life cycle because of its essential role in
viral gene expression.
The biochemical and molecular genetic literature on ICP4 has mostly focused on
HSV-1. Although HSV-1 molecular biology has received more attention than HSV-2, it
is generally agreed that the two perform most critical functions in the same manner, with
closely related proteins. Not surprisingly, the ICP4 proteins from HSV-1 and HSV-2 are
functionally interchangeable, based on genetic recombination experiments between the
two viruses (Smith, C. A., and P. A. Schaffer, Intertypic recombinants of herpes simplex
virus types 1 and 2 infected cell polypeptide 4, Virology. 160:176-182 (1987)). These
experiments demonstrated that virtually any region of the two genes can be interchanged
with no alteration in function. Consistent with these data, HSV-1 and -2 have nearly
identical ICP4 amino acid sequences based on analysis of their respective gene sequences
(McGeoch, D. J., and S. Cook, Molecular phylogeny of the alphaherpesvirinae subfamily
and a proposed evolutionary timescale, J. Mol. Biol. 238:9-22 (1994)).
ICP47 is a cytosolic protein that appears to interfere with transport from cytosol
into the ER of peptides designated for presentation by class I MHC mediated by TAP
heterodimer. Studies have shown that ICP47 blocks TAP transportation and interacts
directly with the TAP-class I complex. The action of ICP47 enables the HSV viruses to
"cloak" themselves and elude the host's immune system by producing surface peptides
similar to the host's peptides, so that the host does not recognize them as foreign and
destroy them. Other viruses seem to produce substances that block part of the host's
immune reaction (Hill, A. et al, Nature, 375(6530): 41, 1-5 (1995)).
The composition of the present invention includes a plasmid or other expression
vector in a delivery vehicle that includes the information for delivery and subsequent
expression in vivo of (a) one or more sequences of interest (SOI) that code for production
of a ssDNA sequence pr sequences in vivo that target(s) either a target sequence(s)
involved in HSV replication or assimilation, one or more transcriptional produces) of
such a target sequences), or one or more translational product(s) of such a target
sequence(s), and (b) the signaling instructions and enzymatic function(s) for producing
that ssDNA sequence(s) in vivo. Delivery is accomplished by incorporating the SOI, and
the signaling instructions and enzymatic functions, into a viral vector such as an
adenoviral vector, a non-viral vector such as a liposome, or a plasmid, and delivering that
vector in a creme, ointment, lotion, gel, aerosol spray, or similar substance for use as a
vehicle for topical application to an affected site. Regardless of whether the information
is delivered and/or expressed by a viral vector, by a plasmid and delivery vehicle, or by
other mechanism, the phrase "expression vector" is utilized for the purpose of referring to
the system for delivering and expressing the information that causes a change in gene
function in the host cell. The particular expression vector described herein that is
incorporated into a vehicle for topical application to an affected site is a plasmid,
designated pssXE and pssXV, into which any sequence of interest that targets one or
more genes involved in HSV replication or assimilation of HSV into a host cell.
Examples of such sequences of interest that may be incorporated into the plasmid or other
expression vector of the composition of the present invention include:
Sequences that act as antisense oligonucleotides to bind to one or more
target sequences, or to the mRNA molecules transcribed from the target
sequences, of HSV other than ICP4 and ICP47 such as ICPO, ICP22, and ICP27;
Sequences that inhibit expression of transcription regulatory factors such
as described in U.S. Patent No. 5,795,721;
Oligonucleotides for controlling expression of viral structural proteins;
Sequences that act as triplex forming oligonucleotides (TFOs) at one or
more target sequences of the host cell after integration of the HSV genome, for
instance, to down regulate the sites from which the host cell produces ICP4,
ICP47, ICPO, ICP22, and ICP27 or other proteins involved in viral proliferation
and/or protection of the virus from the host immune system;
Oligonucleotides specifically hybridizable with a translation initiation
site, coding region or 5' untranslated region of HSV. The oligonucleotides are
designed to be specifically hybridizable with DNA, or preferably, RNA from one
of the species herpes simplex virus type 1 (HSV-1), herpes simplex virus type
(HSV-2), human herpes virus 6, Epstein Barr virus (EBV), or varicella zoster
virus (VZV). Such oligonucleotides are conveniently and desirably presented as a
pharmaceutical composition in a pharmaceutically acceptable carrier as described
in U.S. Patent No. 5,514,577. Persons skilled in the art who have the benefit of
this disclosure will recognize that the particular genes described for HSV find
counterparts in other viruses. Thus, each of herpes simplex virus type 2, human
herpes virus type 6, EBV, and VZV have genes that code for proteins having
similar functions. Accordingly, the present invention contemplates ODN-
mediated therapy in which the ODNs are directed to any of the foregoing viruses;
and/or
Protein-binding oligonacleotides (aptamers) mat specifically bind target
molecules such as the proteins comprising the HSV protein coat, thereby
preventing assembly of the protein coat.
This list is not intended to be all-inclusive. It is envisioned that expression vectors can be
constructed in accordance with the present invention that are capable of transfection into
any HSV-infected cell for producing a sequence that can target any, or at least a wide
variety, of genes and/or the control functions for the genes involved in HSV replication
and/or assimilation to alter the function of the target gene(s).
More specifically, the composition of the present invention includes an expression
vector that comprises a cassette into which one or more nucleic acid sequences is/are
incorporated for use as a template for production of that sequence in a host cell, and
subsequent expression within an HSV infected cell, as a single-stranded DNA (ssDNA)
sequence(s) without (or with minima!) flanking sequences that binds to or otherwise
interacts with one or more target gene(s) to alter expression of the target gene(s). The
ssDNA that is produced in the infected aril is designed to be complimentary to and/or to
otherwise bind to one or more nucleic acid sequence(s) comprising a portion of a target
gene or genes involved in the replication or assimilation of HSV, thereby interfering with,
or altering, expression of the target geoefs), and for that reason, is sometimes referred to
herein as an oligonucleotide, or ODN. As used in this specification, the term "ODN" is
intended to refer to: 1) DNA-based oligonucleotides such as triplex-forming
oligonucleotides (TFO), antisense ODNs, DNA enzymes, and aptamers and 2) RNA-
based oligonucleotides such as ribozymes and antisense RNA. All of these molecules
modulate gene expression by interacting with DNA or mRNA in sequence-specific
manner. Also, the term "affected site" is intended to refer to any site at which an HSV
infection manifests itself that can be treated by topical application of the composition of
the present invention, regardless of whether the site of the infection is, for instance, on the
skin, in a body cavity or an opening of the body, or even internal, and regardless of the
nature of the tissue that is infected
It is therefore another object of the present invention to provide a method for
treating HSV-related pathologies comprising administering an expression vector for
producing ssDNA of any nucleotide sequence intracellularly in vivo that functions as (but
is not limited to) an inhibitory nucleic acid to down regulate one or more gene product(s)
and/or viral gene(s) involved in HSV replication or assimilation, for instance, by binding
to proteins that recognize a nucleic acid sequence, to an affected site.
Another object of the present invention is to provide a composition for treatment
of HSV-related pathologies that comprises an expression vector for producing ssDNA of
any nucleotide sequence in vivo that functions as (but is not limited to) an excitatory
nucleic acid for, for instance, binding to one or more target chromosomal sequences to
increase production of one or more gene(s) or to "switch on" one or more target gene(s)
for inhibiting replication or assimilation of HSV in a vehicle for topical application to an
affected site.
Another object of the present invention is to provide a composition for treatment
of HSV-related pathologies that comprises an expression vector for producing ssDNA in
vivo that is designed to favor binding to duplex, either endogenous (native DNA) or the
double-stranded DNA comprising the viral genome, to form triplex structures that
interfere with normal gene transcription and regulation of one or more target gene(s)
involved in the replication or assimilation of HSV in a vehicle for topical application to
an affected site.
Another object of the present invention is to produce ssDNA within one or more
target cell populations for the purpose of disrupting and/or altering one or more of the cell
functions required for HSV replication or assimilation.
Yet another object of the present invention is to provide a composition and
method in which an expression vector for producing ssDNA into which secondary
structures are designed so that the ODN(s) produced by the vector in one or more target
cell populations bind to and/or otherwise inhibit or activate various cellular functions in
HSV proliferation or assimilation that rely on the catalytic action of a protein or on
nucleic acid protein interaction such as transcription, translation, or DNA replication.
Another object of the present invention is to provide a composition for treatment
of HSV-related pathologies that comprises an expression vector for in vivo production of
ssDNA including a sequence exhibiting catalytic activity against mRNA targets of the
gene(s) involved in viral replication or assimilation for transfection into HSV-infected
cells that overcomes the obstacles to delivery of direct administration of ssDNA by
lipofection, direct cellular uptake, and/or microinjection when delivered in an appropriate
vehicle for topical application to an affected site.
Another object of the present invention is to provide a composition for treatment
of HSV-related pathologies that comprises an expression system for in vivo production of
ssDNA including a sequence exhibiting catalytic activity against one or more mRNA
targets produced by a specific gene or genes involved in viral proliferation, assimilation,
or other viral functions for transfection into HSV-infected cells that overcomes the well-
known obstacles to delivery of direct administration of ssDNA by lipofection, direct
cellular uptake, and/or microinjection when suspended in a medium for topical
application to an affected site.
Another object of the present invention is to provide a composition for treatment
of HSV-related pathologies that comprises an expression vector for in vivo production of
ssDNA including a sequence exhibiting catalytic activity against mRNA targets of the
gene(s) involved in viral replication or assimilation for transfection into HSV-infected
cells in a vehicle that can be applied topically to HSV-infected cells.
Another object of the present invention is to provide an improved method for
treating HSV-related pathologies involving the application of a topical ointment to an
affected site wherein the improvement comprises contacting the affected site with an
expression vector including the information required for expression of one or more
ssDNA sequences) in vivo that target(s) a dsDNA sequence that is involved in the
proliferation or assimilation of HSV by inclusion of the vector in the topical ointment.
Another object of the present invention is to provide pharmacologically
acceptable compositions for delivering an inhibitory or excitatory nucleic acid sequence,
including a sequence with enzymetic activity, to HSV-infected cells in a manner that
produces a therapeutic effect.
This listing of the objects of the present invention is not intended to be a list of all
the objects of this invention. There are a number of other cellular functions that are used
to advantage by replicating and/or latent HSV virus that are mediated by the cellular
genome which can be altered in accordance with the teachings of the present invention
and which are amenable to regulation by in vivo production of ssDNA. In the interest of
brevity and practicality, those many other functions are not mentioned here. Instead, this
list of some of the objects of the present invention is provided for exemplification and is
not intended to limit the scope of the invention.
These objects are provided by a composition for treating HSV-related pathologies
comprising a vector including the information required for expression of a ssDNA
sequence in vivo that targets or otherwise interacts with one or more nucleic acid target
sequences) involved in the replication or assimilation of HSV in a suitable vehicle for
topical application to an affected site. In one embodiment, the vector comprising the
present composition includes a cassette comprised of a sequence of interest flanked by an
inverted tandem repeat, a 3' primer binding site (PBS), and a gene encoding a reverse
transcriptase for transcribing the mRNA transcript of the cassette from the PBS to release
a single-stranded cDNA transcript in cell into which the expression vector has been
incorporated at the affected site. The sequence of interest is comprised of a nucleic acid
sequence that produces a sequence of nucleic acids mat targets, binds to, or otherwise
interacts with one or more target nucleic acid sequence(s) that is/are involved in
proliferation or assimilation of HSV when reverse transcribed to alter expression of the
target sequence(s).
Several embodiments of the invention are illustrated in the figures, in which
Figure 1 is a schematic illustration of the production of ssDNA in a host cell in
accordance with the teachings of the present invention.
Figure 2 is a schematic illustration of the stem-loop intermediate formed by the
method illustrated in Fig. 1.
Figures 3A and 3B represent schematic illustrations of two embodiments of the
pssXE plasmid comprising one embodiment of an expression vector constructed in
accordance with the teachings of the present invention.
Figures 4A and 4B represent schematic illustrations of two embodiments of the
pssXV plasmid comprising a second embodiment of an expression vector constructed in
accordance with the teachings of the present invention.
Figures 5 and 6 are bar graphs showing the reduction of HSV1 viral infection in
African green monkey (Vero) cells transfected with the pssXE(59) plasmid (Fig. 5) and
the lack of any significant reduction in infection in cells transfected with the control
pssXE(59)S plasmid (Fig. 6).
Figures 7 and 8 are bar graphs showing the reduction of HSV2 viral infection in
African green monkey (Vero) cells transfected with the pssXE(59) plasmid (Fig. 7) and
the lack of any significant reduction in infection in cells transfected with the control
pssXE(59)S plasmid (Fig. 8).
In this description of the present invention, an expression vector is described for
use in producing single-stranded deoxyribonucleic acid (ssDNA) oligonucleotides
(ODNs) of virtually any predefined or desired nucleotide base composition intracellularly
in vivo in HSV-infected cells, with or without flanking nucleotide sequences, that is either
substantially homologous or complementary to the target sequence for binding to the
target sequence to alter the expression of that sequence and/or inactivate or destroy the
product of that sequence. The expression vector is delivered to the infected cells at an
affected site by topical application in a creme, lotion, ointment, spray, or gel.
In one embodiment, the expression vector of the present invention is designed to
produce an ssDNA molecule, preferably free of most contiguous vector sequences, within
an HSV-infected cell. The vector contains all the necessary enzymatic functions and
signaling instructions for producing ssDNA in the infected cell. As shown in Fig. 1
(illustrating the use of a plasmid such as the pssXE or pssXV plasmids described herein
as an expression vector in accordance with the teachings of the present invention), the
infected cell to which the vector is delivered produces one or more RNA transcript(s),
driven by an eukaryotic promoter, that is used as a template to direct the synthesis of the
desired single-stranded DNA sequence(s) for altering the function of one or more gene(s)
involved in replication or assimilation of HSV.
The ssDNA produced in vivo may be any ODN, including ODNs that function as
inhibitory nucleic acids or excitatory nucleic acids. Inhibitory nucleic acids may be
ssDNA synthesized from the mRNA template, or the mRNA template itself, that
specifically bind to complementary nucleic acid sequence(s) in the infected cell. By
binding to the appropriate target nucleic acid sequencers), an RNA--KNA, a DNA--DNA,
or RNA--DNA duplex or triplex is formed More commonly, these nucleic acid
sequences are termed "antisense" sequences because they are usually complementary to
the sense or coding strand of the gene, but the "sense" sequence is also utilized in the cell
for therapeutic purposes. The term "inhibitory nucleic acids" as used herein, therefore,
includes both "sense" and "antisense" nucleic acids, but as set out below, is not limited to
sense and/or antisense nucleic acids.
By binding to a target sequence (and by the phrase "target sequence," it is
intended to refer not only to the target sequence itself, regardless of whether the target
sequence is a sequence that is endogenous to the host cell and/or a part of the genome of
the virus, but also to the transcriptional and translational products of that target sequence),
an inhibitory nucleic acid alters the function of a gene involved in viral replicatioa This
alteration (usually an inhibitory effect) results from, for example, blocking DNA
transcription, processing or poly(A) addition to mRNA, DNA replication, translation, or
promoting inhibitory mechanisms of the cells (such as promoting RNA degradation).
Inhibitory nucleic acid methods therefore encompass a number of different approaches,
functioning in several different ways, for altering gene expression in an HSV-infected
cell. Because of the many ways in which they function to alter gene function, broad
reference is made herein to binding, or otherwise interacting with, the target sequence.
The different types of inhibitory nucleic acid technologies are described in Helene, C. and
J. Toulme (1049 Biochim. Biophys. Acta. 99-125 (1990)), hereinafter referred to as
"Helene and Toulme," which is incorporated herein in its entirety by this specific
reference thereto.
Focusing further on the expression vector comprising the composition of the
present invention, the vector comprises a set of genetic elements adapted for delivery into
a cell to produce ssDNA in vitro or in vivo for altering expression of one or more target
sequence(s) involved in HSV proliferation or assimilation that includes:
(A) an RNA dependent DNA polymerase (reverse transcriptase) gene,
and
(B) a cassette including (1) an inverted tandem repeat (IR), (2) one or
more sequences of interest (SOIs) located (a) between the inverted repeat (IR), (b)
3' to the IR, or (c) both between the IR and 3' to the IR and (3) a primer binding
site (PBS) for the reverse transcriptase that is located 3' to the IR as shown in Fig.
2.
Although not required, the expression vector also preferably includes the functions and
signaling instructions for transcription of these components in vivo and the functions and
signaling instructions for translation of the reverse transcriptase (RT) gene. Additional
elements that are optionally included in the expression vector comprising the composition
of the present invention may include one or more of an RNAse gene, usually associated
with the RT gene, a restriction endonuclease (RE) gene (for a purpose described below), a
downstream polyadenylation signal sequence for expression in eukaryotic cells so mat the
mRNA produced by the sequence of interest includes a poly(A) tail (see Fig. 1), and a
DNA sequence having enzymatic activity when the linearized ssDNA folds into the
appropriate secondary configuration. Although the present invention is not so limited, in
one embodiment of the expression vector, the DNA enzymatic sequence is located within
a sequence of interest, regardless of whether the sequence of interest is located between
the inverted repeat (IR) or between the 3' aspect of the IR and the PBS.
As noted above, in a first embodiment of the composition of the present
invention, the expression vector comprises a plasmid, designated pssXE (for cell culture
testing) and pssXV (for use in animals, including humans), including the three above-
listed elements of the cassette, namely, a primer binding sequence (PBS) matched to the
reverse transcriptase (RT), a sequence of interest (SOI), and an inverted repeat (IR). As
shown in Figs. 3 and 4, the SOI is located either between the inverted tandem repeats or
in a 5' position (with respect to the mRNA transcript) to the PBS, the PBS being located
at the most 3' aspect of the mRNA transcript, or in both locations. In other words, the
SOI is located (1) between the IR, (2) between the IR and the PBS, and/or (3) both
between the IR and between the IR and the PBS. The PBS, SOI, and IR reside in the
untranslated 3' portion of the RT polyprotein in the E plasmid shown in Fig. 3. When the
RT-RNAse H component of the plasmid is transcribed under control of an appropriate
promoter (in the embodiments described herein, the CMV promoter was utilized), the
resulting mRNA transcript contains the coding region for the RT-RNAse H polyprotein
and, at the end of translation at the stop signals, the additional mRNA transcript contains
(3' to this translated protein) these three elements with further 3' downstream signaling
events for polyadenylation signals, which remain intact from the RT-RNAse H
component.
The pssXE and pssXV plasmids shown in Figs. 3 and 4 comprising one
embodiment of the expression vector of the present invention include a multiple
subcloning site that facilitates subcloning of the SOI. As known in the art, a multiple
cloning site (MCS) containing a number of restriction enzyme (usually 4-10) recognition
sequences, is designed to make a vector more flexible for the insertion of different DNA
sequences. As will be apparent to those skilled in the art, however, only restriction
enzymes that do not cut the vector can be chosen. Although many are known, the
following is a list of restriction enzymes that can be selected for use in connection with
pssXE, pssXV, or any other plasmid constructed in accordance with the teachings of the
present invention:
AfIII Ascl, BsiWl, BsmBI BspMI, BsrGI, BsBI, ClaI, E1047111, Hpal, Narl
PF1MI, PshAI, SfiI,, SgfI, SrfI, Sse838871, SwaI, XcmI,
as well as the EcoRI, PacI, PstI, and SacII sites that were selected for inclusion in the
pssXE and pssXV plasmids. Those skilled in the art who have the benefit of this
disclosure will recognize that these particular cloning sites were chosen for the particular
systems described herein and that other cloning sites may be equally useful for this same
purpose.
The nucleic acid sequence that is referred to herein as a cassette that includes the
SOI, IR, and PBS is preferably regulated by an appropriate wide spectrum or tissue-
specific promoter/enhancer, such as the CMV promoter, or a combination of
promoters/enhancers, located upstream of the genetic element. The promoter/enhancer
can either be constitutive or inducible promoter. As set out in more detail below, those
skilled in the art who have the benefit of this disclosure will recognize that a number of
other eukaryotic promoters may be used to advantage to control expression of the SOI
including SV-40, RSV (non-cell type specific) or tissue-specific glial fibulary acidic
protein (GFAP).
The primer binding site (PBS) for initiation of priming for cDNA synthesis is
located between the 3' IR and the polyadenylation signal. The PBS is a sequence that is
complementary to a transfer RNA (tRNA) which is resident within the eukaryotic target
cell. In the case of the mouse Maloney reverse transcriptase (MoMULV RT) that is one
RT that may be utilized in conjunction with the present invention, the PBS takes
advantage of the proline tRNA. The PBS utilized in connection with the expression
vector described herein including the RT gene from human immunodeficiency virus was
taken from the nucleotide sequence of HTV. Y. Li, et al, 66 J. Virology 6587-6600
(1992). However, those skilled in the art who have the benefit of this disclosure will
recognize that any PBS that is matched to a particular RT is utilized for this purpose. The
PBS is exclusively recognized by a primer tRNA that is endogenous to the target cells.
Each tRNA has the ability to recognize a unique sequence (i.e., codon) on the mRNA
transcript coding for an amino acid, and has the ability to covalently link to a specific
amino acid (i.e., the tRNA becomes "charged" when bound to a specific amino acid).
However, a primer tRNA, when bound to the mRNA transcript PBS and not covalently
linked with an amino acid (i.e., "uncharged"), may be used to initiate ssDNA synthesis by
the RT. Thus, each PBS incorporated into the expression vector comprising the
composition of the present invention must contain the unique sequence recognized b"y"the
primer tRNA, and the primer tRNA must be a primer tRNA that is recognized by the
particular RT utilized.
Other retroviral RT/RNAse H genes may be used to advantage in connection with
the present invention, it being preferred that the RT/RNase H gene be an RT/RNase H
gene that is regulated by an appropriate upstream eukaryotic promoter/enhancer such as
the CMV or RSV promoter for expression in human cells. RNA-dependent DNA
polymerase/RT genes suitable for use in connection with the present invention include
those from retroviruses, strains of hepatitis B, hepatitis C, bacterial retron elements, and
retrons isolated from various yeast and bacterial species. As found in nature, these RNA-
dependent DNA polymerases usually have an associated RNase H component enzyme
within the same coding transcript. However, the present invention does not require the
naturally-occurring RNase H gene for a particular RT. In other words, those skilled in the
art will recognize from this disclosure that various combinations of RT and RNase H
genes can be spliced together for use in connection with the present invention to fulfill
this function and that modifications and/or hybrid versions of these two enzyme systems
are available and/or known to those skilled in the art which will function in the intended
manner. Those skilled in the art will also recognize that the target cell may itself have
sufficient endogenous RNase H to fulfill this function. Similarly, those skilled in the art
will recognize that the target cell may itself have sufficient endogenous RT activity from,
for instance, prior retroviral infection, to fulfill this function. It will also be recognized by
those skilled in the art who have the benefit of this disclosure that the use of a viral vector
as the expression vector comprising the composition of the present invention makes
possible the use of a number of viral RT genes that are otherwise not well-suited for use
in a plasmid expression vector system.
The RT/RNase H gene also preferably includes a downstream polyadenylation
signal sequence so that the mRNA produced from the RT/RNase H gene includes a 3'
poly(A) tail for mRNA stability. As known to those skilled in the art, multiple poly(A)
tails are available and are routinely used for production of expressed eukaryotic genes.
Those skilled in the art will also recognize that a number of tissue-specific or
wide spectrum promoters/enhancers, or combinations of promoters/enhancers other than
those listed above may also be used to advantage to regulate the RT/RNAse H gene, the
RE gene (if utilized), and the sequence of interest. Although a list of all available
promoters/enhancers is not needed to exemplify the invention, as noted above, the
promoters/enhancers may be constitutive or inducible and may include the CMV or RSV
(non-cell type specific) or GFAP (tissue specific) promoters/enhancers listed here and
many other viral or mammalian promoters. Representative promoters/enhancers that are
appropriate for use in connection with the cassette of the present invention may include,
but are not limited to, HSVtk (S.L. McKnight, et al., 217 Science 316 (1982)), human 15-
globulin promoter (R. Breathnach, et al, 50 Ann. Rev. of Biochem. 349 (1981)), 15-actin
(T. Kawamoto, et al., 8 Mol. Cell Biol. 267 (1988)), rat growth hormone (P.R Larsen, et
al., 83 Proc. Natl. Acad. Sci. U.S.A. 8283 (1986)), MMTV (AL. Huang, et al., 27 Cell
245 (1981)), adenovirus 5 E2 (M.J. Imperiale, et al., 4 Mol. Cell. Biol. 875 (1984)), SV40
(P. Angel, et al., 49 Cell 729 (1987)), a-2-macroglobulin (D. Kunz, et al., 17 Nucl. Acids
Res. 1121 (1989)), MHC class I gene H-2kb (MA. Blanar, et al., 8 EMBO J. 1139
(1989)), and thyroid stimulating hormone (V.K. Chatterjee, et al., 86 Proc. Natl. Acad.
Sci. U.S.A. 9114 (1989)). A list of other promoters that may be suitable for use in
connection with the cassette of the present inventions includes: .
1. SV40 early promoter;
2. Cytomegalovirus (CMV) promoter;
3. Elongation factor-1 a (EF-1 a) promoter;
4. Thyroxine-binding globulin (TBG) promoter;
5. Multidrug resistance gene (mdrl) promoter, drug and heat inducible
6. Heat shock protein (HSP) promoter;
7. Tet-responsive (TRE) promoter, drug inducible;
8. HSV (thimine kinase) TK promoter, heat inducible;
9. Gal4-Elb promoter; drug inducible;
10. Ubiquitin C(UbC) promoter; and
11. Telomerase reverse transcriptase (TERT) promoter, tumor-specific.
Those skilled in the art will recognize that this list of promoters is not intended to be all-
inclusive and mat there are other promoters that will function to advantage when utilized
in the espression vector of the present invention.
The RT produced in the cell synthesizes a complementary DNA (cDNA) using as
the template the cassette including the SOI. The RNase H activity of the RT degrades the
mRNA template component of the RNA/cDNA hybrid to produce a ssDNA in vivo.
The gene encoding the RE (if used, and not a required component of the
invention) may be any of several genes mat encode for REs, and preferably those that are
controlled by one or more constitutive or inducible wide spectrum and/or tissue-specific
promoters/enhancers such as those listed above. Two REs that have been tested are
MboII and FokI, but those skilled in the art who have the benefit of this disclosure will
recognize that any RE (type I, n, US, or HI) site may be included in the IR. These
enzymes "clip," or digest, the stem of the stem-loop intermediate described below to
linearize the SOI as single-stranded DNA. Expression of the RE gene may be regulated
by an appropriate constitutive or inducible promoter/enhancer located upstream from the
restriction endonuclease gene such as the CMV or RSV promoter for expression in
human cells, in plasmid pssXA. The RE gene also preferably includes a downstream
polyadenylation signal sequence so that the mRNA transcript from the RE gene will have
a 3' poly(A) tail.
The cassette also comprises an inverted tandem repeat (JR.). After digestion of the
mRNA from the mRNA-cDNA heteroduplex by RNAse H and the release of the ssDNA,
the IR causes the ssDNA to fold back upon itself to form the stem of a stem-loop
structure, the stem structure being comprised of double stranded, anti-parallel DNA, in
the manner described in U.S. Patent No. 6,054,299 and as shown in Fig. 2. The ssDNA
that is produced is transcribed with the encoded 5' and 3' regions flanking the stem (made
up of the IR) and a loop containing the SOI. The stem is then digested or cleaved by any
of the many RE enzymes that recognize the cut site designed into the stem (again, note
that the endonuclease recognition site may be designed into the stem even though the RE
gene is not included in the vector system of the present invention) to release the ssDNA
loop (see Fig. 1). The loop portion of the ssDNA, which does not form any apparent
duplex DNA, is immune to RE activity since REs recognize only double stranded DNA
as a target substrate.
As noted above, those skilled in the art will recognize that the RE site(s) need not
be designed into the IR which forms the stem of the stem-loop intermediate if it is desired
to produce ssDNA from an SOI located between the PBS and the IR, with transcription of
the cassette terminating at the stem formed by the IR. Another option is to design the IR
to contain eukaryotic, prokaryotic, or viral protein DNA binding sites, that act to
competitively titer out selected cellular proteins. Combinations of restriction sites or
other sequence specific elements may be included in the IR depending on the base pair
composition chosen for the IR such that linear or precisely cut stem-loop intermediate
forms of ssDNA are produced. It is generally preferred to use synthetically constructed
sequence specific elements in the IR since it is unlikely that a naturally occurring inverted
repeat would have the properly aligned restriction sites.
As noted above, the cassette that comprises the expression vector of the
composition of the present invention may also include a DNA sequence with catalytic
activity. Because of the inclusion of the so-called "DNA enzyme" in the cassette (and in
the embodiment described herein, the DNA enzyme is located within the sequence of
interest), the present invention is used to particular advantage when the sequence of
interest serves as the template for synthesis of an inhibitory nucleic acid that is an
antisense sequence, a triplex-forming oligonucleotide (TFO), or a DNA enzyme
sequence. The nucleic acid sequence having enzymatic activity utilized in the method of
altering gene expression described herein is the 10-23 DNA enzyme (Santoro and Joyce,
supra (1997)). The enzymatic sequence is inserted into the cassette in either or both of
the two locations, e.g., (a) between the IR and inside the SOI (at the Noil site) or (b)
inside the second SOI that is located 3' to the IR and 5' to the PBS (at the PacI/BamHI
sites). Either way, the resulting ssDNA is specific for the target DNA sequence(s),
mRNA sequence(s), or any other suitable substrate, to inhibit or change DNA or mRNA
splicing mechanisms, or even to directly alter the cellular genome in a specific manner.
Those skilled in the art will recognize from this disclosure that any DNA sequence having
enzymatic activity will function for the intended purpose when inserted into the cassette
of the present invention. A number of nucleic acid sequences with enzymatic activity
have been reported in the literature, including:
sequences having RNAse activity such as the so-called "10-23" and "8-17
enzymes" (Santoro, S.W. and G.F. Joyce, supra (1997)) and other metal-
dependent RNAses (Breaker, R.R. and G.F. Joyce, 1 Biol. Chem. 223-229 (1994)
and Breaker, R.R. and G.F. Joyce, 2 Biol. Chem. 655-660 (1995)) and histidine-
dependent RNAse (Roth, A. and R.R Breaker, 95 Proc. Natl Acad. Sci. USA
6027-6031(1998));
sequences having DNAse activity such as copper-dependent DNAse
(Carmi, N., el al., 3 Chem. Biol. 1039-1046 (1996), Carmi, et ai, supra (1997);
Sen, D. and C.R. Geyer, 2 Curr. Opin. Chem. Biol. 680-687 (1998)) and the
DNAses which required divalent metal ions as cofactors or hydrolyzed the
substrate independently of divalent metal ions reported in Faulhammer, D. and M.
Famulok (269 J. Molec. Bio. 18-203 (1997));
sequences with DNA ligase activity such as copper-dependent DNAse
(Breaker, R.R., 97 Chem. Rev. 371-390 (1997)) and zinc-dependent E47 ligase
(Cuenoud, B. and J.W. Szostak, 375 Nature 611-613 (1995)); and
sequences with DNA kinase activity such as calcium-dependent DNA
kinase (Li, Y. and R.R. Breaker, 96 Proc. Natl. Acad. Sci. USA 2746-2751
(1999)).
Generally, it is those DNA sequences having enzymatic activity that are derived from
physiological conditions that are preferred for use in the cassette comprising the
expression vector that comprises one component of the composition of the present
invention. Those skilled in the art will recognize that as described above, the expression
vector of the present invention is not utilized solely for the purpose of producing
antisense sequences in vivo, that the antisense sequence need not necessarily contain a
nucleic acid sequence having catalytic activity, and that the inhibitory nucleic acid
sequence could also be any of the other types of inhibitory nucleic acid sequences
described above.
It may also be advantageous if the expression vector comprising the composition
of the present invention contains other specialized genetic elements to facilitate
identification of cells that carry the vector and/or to increase the level of expression of the
set of genetic elements comprising the cassette. The specialized genetic elements may
include selectable marker genes such as genes that confer to the bacteria (e.g., E. coli)
resistance to antibiotics such as zeocin (described infra), ampicillin, chloramphenicol,
kanamycin (neomycin), or tetracycline so that the vector can be transformed and
amplified in a prokaryotic system.
Incorporation of the above components into the expression vector of the present
invention makes available at least two convenient methods for removing predetermined
vector sequences after the production of ssDNA as shown in Fig. 1. In the first method,
the cassette that comprises a portion of the expression vector is reverse transcribed in the
infected cell from the PBS so that the SOI between the IR comprises the loop portion of
the ssDNA stem-loop intermediate that is produced when the nucleotides comprising the
IR pair up to form the stem of the stem-loop vector, the stem comprising an RE site.
After digestion with the appropriate RE, the loop is released as linearized, single-stranded
cDNA without (and/or with minimal) flanking sequences. In the second method, the
cassette is reverse transcribed from the PBS and an SOI included in the cassette 3' to the
IR is likewise transcribed, but reverse transcription is terminated at the stem of the stem-
loop structure formed by the pairing of the nucleotides of the IR. Either way, the
resulting ssDNA is produced without (and/or with minimal) flanking sequences. If it is
desired to produce ssDNA utilizing the second method, the cassette is designed so that the
transcript of the cassette includes an IR that forms a stem that is more stable than the stem
produced when ssDNA is produced by digestion of the stem in accordance with the first
aspect of the present invention (for instance, by designing the IR so as not to include an
RE site). By designing the cassette to produce an IR that forms a stem that is easily
denatured in accordance with the first aspect of the invention, reverse transcription
proceeds right on through the second SOI (if it is even designed into the cassette) to the
SOI located between the IR This "premature termination" of the reverse transcriptase
cDNA transcript at the 3' aspect of the stem structure therefore provides a second method
for limiting the intervening vector sequences contained with an in vivo-produced ssDNA.
A stem that is intermediate in stability allows production of both the first and second
SOIs.
It will also be evident to those skilled in the art from this description that the
intact stem-loop ssDNA structure can function similarly in many applications as the
linearized ssDNA form. Consequently, the cassette is also used to advantage without the
restriction endonuclease gene and associated regulatory elements and/or with a sequence
of interest that lacks the corresponding restriction endonuclease site.
It will also be evident to those skilled in the art from this description that a
cassette can be made that encodes a ssDNA mat has a "trimmed" stem-loop structure.
The RE sites encoded in the IR flanking the SOI are designed such that the stem portion
(after duplex formation) is digested with the corresponding RE so as to cut the dsDNA
comprising the stem in a way that removes a portion of the stem and the associated
flanking sequences, yet leaves sufficient duplex DNA that the transcript retains the
above-described stem-loop structure. Such ssDNA structure may be more resistant to
intracellular nucleases by retaining the "ends" of a ssDNA in double stranded form.
The expression vector of the present invention is constructed using known
digestion and ligation techniques to splice the particular SOI into the cassette (between
inverted tandem repeats or between PBS and inverted tandem repeats). Those skilled in
the art who have the benefit of this disclosure will also recognize that the above-described
signals used for expression within eukaryotic cells may be modified in ways known in the
art depending upon the particular host cell and sequence being targeted. For instance, a
likely modification is to change the promoter so as to confer advantageous expression
characteristics on the cassette in the system in which it is desired to express the SOI.
The expression vector is delivered to the HSV-infected cell by topical application
to the affected site. Topical application is accomplished by formulating the expression
vector with a suitable vehicle for application to the affected site. The location of the
affected site is an important element in formulation of the vehicle. For instance, many
affected sites occur on the patient's skin, yet the skin poses a particular challenge for
topical application. One example of a delivery vehicle for use in the composition of the
present invention (because it provides transport through the outer layers of the skin) is a
two-part gel of the type described in U.S. Patent No. 5,837,289, the specification of which
is hereby incorporated into this specification in its entirety by this specific reference.
Briefly, this patent describes a composition comprised of two so-called "penetration
enhancers" and a medication capable of being administered transdermally. In a preferred
embodiment described in that patent, an organogel is prepared by blending a fatty acid
phospholipid emulsifying agent such as lecithin with a fatty acid or an ester thereof, such
as isopropyl palmitate or isopropyl myristate, to form an organogel that is then blended
with a polyoxymer such as a polyoxyalkylene polymer, and the medication is solubilized
with a solvent such as water, alcohol, or other suitable solvent and mixed with the
organogel and polymeric component. However, contrary to the teaching of Patent No.
5,837,289, it has been discovered that the expression vector that comprises one
component of the composition of the present invention need not be solubilized before
mixing with the organogel and polymeric component to be delivered effectively to an
affected site in accordance with the method of the present invention. In fact, the
expression vector that comprises the composition of the present invention is quite large
such that solubilization is effectively impossible in any known solvent, yet it has been
discovered that the expression vector can be suspended in the blend of the organogel and
the polymeric component taught by the above-incorporated patent for successful delivery
to an affected site.
Consequently, in one embodiment, the two-part gel that is utilized as a delivery
vehicle for the expression vector is a PLURONIC™ (Wyandotte Chemical Co.) lecithin
oranogel, or PLO, which is a combination of PLURONIC™ gel and soy lecithin.
PLURONIC™ gel is a poloxamer comprised of poly(ethylene oxide)-b-
poly(propyleneoxide)-b-poly(ethylene-oxide). The most common form of PLURONIC7111
gel used in pharmaceutical compounding is PLURONIC™ F-127 (BASF Corporation).
Lecithin is a permeation enhancer that acts as an amphoteric surfactant in the two-part
gel, thus facilitating penetration of the expression vector into the dermal layer, and the
concentration of lecithin in the two-part gel yields control of various bulk properties of
the two-part gel such as thixotrophy, viscosity and gelation temperature.
This composition is generally made up in accordance with the same general
formulas as described in the above-incorporated U.S. Patent No. 5,837,289 with the
following modification. That patent discloses a formula comprised of about parts by weight of a medication and about 40 parts by weight of the first penetration enhancer (the organogel), and 40 to 70 parts by
weight of the second penetration enhancer (the polyoxymer). In the present invention, the
composition is comprised of a mixture of the first and second penetration enhancers in the
same ratios as described in the above-incorporated patent (about 20 to about 40 parts by
weight of the organogel and about 40 to about 70 parts by weight of the polyoxymer) but
the expression vector comprises between about 0.01% and about 20% (by weight), and
preferably between about 0.1% to 5 %, of the composition. The two-part gel is preferably
prepared by adding the two components of the two-part gel in proportions of between
about 10 and about 40 parts by weight of the organogel and between about 20 to about 70
parts by weight of the polyoxymer, in small increments while applying sheer force, for
instance, with syringe techniques, hand-held rotor tool, ELECTRO MOTOR, or ointment
mill, for each part of the expression vector (by weight).
By reference to specific ingredients, one embodiment of the two-part gel will now
be described. Between about 4 and about 8 parts (by weight) of isopropyl palmitate are
placed in a beaker or other suitable container, and between about 4 and about 8 parts of
soya granular lecithin and between about 0.05 and about 0.5 parts of sorbic acid are
dispersed in the beaker without shaking (the lecithin dissolves in time). A vessel is then
calibrated to between about 40 ml and about 60 ml and between about 8 and about 12
parts of a polyoxymer such as CARBOPOL™ or PLURONIC™ (Wyandotte Chemical
Co.) are placed in the calibrated vessel. Cold water is added to bring total volume to
between about 40 and about 60 ml and the vessel is shaken before storing at normal
refrigeration temperatures for long enough for the polyoxymer to dissolve (overnight).
To prepare the two-part gel, about 20% (by volume) of the prepared lecithin and about
80% (by volume) of the polyoxymer gel are withdrawn into syringes and the two
mixtures are transferred from one syringe to another by using a LUER LOK™-to-LUER
LOK™ connector until uniform.
The desired expression vector containing the SOI as the active component is
dissolved or dispersed in sterile water. The active component with a final concentration of
0.1%, not to exceed 10% of the final volume, is then added to 2.2 ml of
Lecithin/lsopropyl Palmitate solution that has been placed in a 10 cc syringe. The
mixture is vortexed. Pluronic F-127, 20% is added to a second syringe to q.s. to 10 ml.
Use of the syringe to syringe technique for mixing encourages micelle formation. In a
second formulation, salicylic acid USP, 2 gm, is mixed with vitamin E acetate (125 U/ml)
2 ml, q.s. to 100 gm with Base PCCA Cosmetic HRT (Professional Compounding
Centers of America, Inc., Houston, TX). The expression vector with the SOI is diluted in
sterile water and then q.s to 10 ml in the Base PCCA Cosmetic HRT to a final
concentration of 0.01% weight of active ingredient to volume and is mixed using the
syringe to syringe technique.
In another embodiment, network poly(amino ester) (n-PAE) and other modified
polyethylenimine (PEI) lipopolymers as described by T.M. Klibanov (Enhancing
polyethylenimine's delivery of plasmid DNA into mammalian cells, 99 Proc. Natl Acad.
Sci. USA 14640-5 (2002)) and W.T. Godbey, et al. (Size matters: molecular weight
affects the efficiency of poly(ethyienimine) as a gene delivery vehicle, 45 J. Biomed
Mater. Res. 268-75 (1999)) are used to advantage as a vehicle for delivery of the
expression vector described herein. Alternatively, graft copolymers of PEI and
PLURONIC gel as described by C.L. Gebhardt, et al. (Design and formulation of
polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer, 1
Bioconjug. Chem 937-44 (2002)) and P. Lemieux, el al. (Block and graft copolymers and
NanoGel copolymer networks for DNA delivery into cell, 8 J. Drug Target 91-105
(2000)) are used to advantage as the delivery vehicle of the composition of the present
invention.
In yet another embodiment, the above-described expression vector is delivered to
an affected site in a cationic block copolymer of the type described in A. Caputo, et al.
(Micellar-type complexes of tailor-made synthetic block copolymers containing the HTV-
1 tat DNA for vaccine application, 20 Vaccine 2303-2317 (2002)), the entirety of which
is hereby incorporated herein by this specific reference thereto. Other types of micelles
such as polyion complex (PIC) micelles as described by M. Harada-Shiba, et al. (Polyion
complex micelles as vectors in gene therapy--pharmacokinetics and in vivo gene transfer,
9 Gene Ther. 407-14 (2002)) are also utilized to advantage as the delivery vehicle in the
composition of the present invention. Similarly, the expression vector is delivered to an
affected site in micelles formed of the water-soluble lipopolymers described in D.A.
Wang, et al (Novel Branched Poly(Ethylenimine)-Cholesterol Water-Soluble
Lipopolymers for Gene Delivery, 3 Biomacromolecules 1197-1207 (2002)). All of the
references cited in these two paragraphs are hereby incorporated herein in their entireties
by this specific reference thereto, and the expression vector is mixed with the vehicle in
the proportions and in accordance with the methods set out in the thus-incorporated
references.
In another embodiment, the delivery vehicle of the present invention also includes
an ingredient such as an antibiotic, analgesic, and/or anesthetic for treatment of symptoms
at the affected site. For instance, formulations of the composition of the present invention
for topical application at the site of a viral eruption might include an anesthetic for relief
of pain such as lidocaine; alternatively, if the affected site is also the site of a bacterial
infection, the composition of the present invention may include an antibiotic such as
kanamycin or any of the many known antibiotics that are advantageously applied
topically.
EXAMPLES
Except where otherwise indicated, standard techniques as described by J.
Sambrook, el al. (Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring
Harbor Press (1989), hereinafter referred to as "Maniatis, et al. (1989)") and F.M.
Ausubel, et al. (Current Protocols in Molecular Biology, New York: John Wiley & Sons
(1987)), both of which are hereby incorporated in their entirety by this specific reference
thereto, were utilized in the examples set out below. It should be understood that other
methods of production of ssDNA, both by natural processes and by designed artificial
methods using different enzyme products or systems, may also be utilized in connection
with the method of the present invention and that the examples set out herein are set out
for purposes of exemplification and are not intended to limit the scope of this disclosure
or the invention described herein.
Construction of plasmids was as described in co-pending U.S. application Serial
No. 10/136,218, filed May 1, 2002, which application is hereby incorporated into this
specification in its entirety by this specific reference thereto. Specifically, expression
vectors constructed in accordance with the teachings of the present invention are the
pssXE (for cell culture studies) and pssXV (for animals, including humans) plasmids
shown in Figs. 3 and 4. To make pssXE and pssXV, the pssXB and pssXD plasmids
described in the above-incorporated co-pending application were double-digested with
Nhel and Xhol. The DNA fragment digested from pssXD (containing RT, PBS, and
other component necessary for synthesis of ssDN A) was subcloned into the vector double
digested from pssXB with the same enzymes that contains a CMV promoter. A multiple
cloning site (MCS) was created in order to facilitate subcloning the sequence of interest
into the plasmid by annealing and subcloning the sequences
5'-E/S/P/P-LINKER (5'-TCGAGCGGCCAGGGGTCTCCCGATCCCGGAC-
GAGCCCCCAAAGAATTC-CGCGGCTGCAGTTAAT-3') and [1]
3'-E/S/P/P-LINKER (S'-TAACTGCAGCCGCG-GAATTCTTTGGGGGCTC-
GTCCGGGATCGGGAGACCCCTGGCCGC-3,) [2]
into Pad and Xhol sites of pssXD plasmid to create four new restriction enzyme
recognition sites, EcoRI, SacII, PsfI and PacI.
The following sequences were cloned into the PacI and EcoRI sites of pssXE and
pssXV:
5'-CCCTTATACGATTTTCGCGCATATCGTATCCCGCCG-3' [3]
5'-CGGCGGGATACGATATGCGCGAAAATCGTATAAGGG-3' [4]
5'-TCCGCCAGGCTACGTAC AACGATTCC AGG-3' [5]
5'-CGGCGTAGGCTAGCTACAACGAGTCCTGG^' [6]
5--AGAAAC AGGCTAGCTACAACGAGTCCGTC-3' [7]
5'-ATCGCCAGGCTAGCTACAACGAACGTCCG-3' [8]
Sequence [3] binds to the ICP4 transcription factor that regulates expression of many
HSV genes by binding to their promoter regions and works in competitive fashion to
prevent ICP4 from interacting with gene promoters in both HSV1 and HSV2. Sequences
were confirmed by automatic sequencing using the BGHRev primer.
The expression vector containing sequence [3] was designated as pssXE(59) (Fig.
3A). A control vector containing complementary sequence of [3], listed above as
sequence [4], was also constructed and designated as pssXE(59)S (Fig. 3B). Both
constructs were transfected into African green monkey kidney (Vero) cells according to
Maniatis, el al. (1989). A number of clones were randomly selected and expanded.
These cell lines were seeded in 24-well culture dishes in Dulbecco's modified Eagle's
medium containing heat-inactivated 10% newborn calf serum (Life Technologies,
Gaithersburg, Md) plus 50 mg of penicillin/ml, 50 mg of streptomycin/ml, and 0.15 mg of
Fungizone/ml and incubated overnight. The medium was then aspirated and replace with
0.2 ml of culture medium containing 50 PFU of either HSV1, ATCC strain 17 or HSV2,
ATCC strain 186. After one hour's adsorptions, 1 ml of blocking medium (growth
medium supplemented with 0.3% human gamma globulin) was added to each well.
Three days later, when well-defined plaques were visualized, the cells were fixed in
methanol and stained in 0.8% crystal violet (Sigma, St. Louis, MO), rinsed, and then air
dried. Plaque counts were recorded in triplicate and then averages were calculated. As
shown in Figure 5, various degrees of reduction of HSV 1 viral infection was observed in
cells transfected with pssXE(59). Particularly, at least 500 fold reductions were observed
in clone r4 and r8 compared to untransfected Vero cells. However, there is no significant
reduction of HSV1 viral infection in cells transfected with the control vector, pssXE(59)S
(Figure 6). In separate experiments, these same clones were also infected with HSV2,
stain 186. As shown in Figure 7, reduction of HSV2 viral infection was observed in cells
transfected with pssXE(59). One thousand fold reduction of viral infection was observed
in clone r4 compared to the control Vero cells. Similarly, there is no significant reduction
of HSV2 viral infection in cells transfected with the control vector, pssXE(59)S (Figure
8).
Sequences [5] and [6] were designed to include DNA enzymes that target a
protein, ICP47-1, from HSV-1, sequence [5] binding to the ICP-47 mRNA sequence at
the position between 12nt and 26nt and sequence [6] binding at the position between 56nt
and 70nt. Similarly, sequences [7] and [8] were designed to include DNA enzymes that
target a protein ICP47-2 from HSV-2, sequence [7] binding to the ICP-47 mRNA
sequence at the position between 21nt and 35nt and sequence [8] binding to the mRNA
sequence at the position between 58nt and 72nt. Again, sequences were confirmed using
the BGHRev primer.
*****
The experiments described above demonstrate the use of an expression
vector for production of ssDNA in HSV-infected cells by multiple stepwise reactions
using eukaryotic RT reactions and various cDNA priming reactions that alter gene
function of the genes involved in replication or assimilation of HSV into target cells.
Those skilled in the art will recognize that the present invention is not limited to the
specific embodiments of such expression vectors described herein. It will be
recognized, for instance, that many nucleic acid sequences may be utilized depending
upon the specific target sequence and/or mode of inhibitory action of the SOI.
Similarly, the SOI may be located in either or both of the two positions, e.g., between
the IR and/or between the PBS and the 3' aspect of the IR. Likewise, the SOI may or
may not include a DNA enzyme sequence depending upon the particular target
sequence and/or mode of action of the SOI and/or the DNA enzyme sequence.
Although described with reference to the figures and specific examples set out
herein, those skilled in the art will recognize that certain changes can be made to the
specific elements set out herein without changing the manner in which those elements
function to achieve their intended respective results. For instance, the cassette described
herein is described as comprising three genetic elements, a sequence of interest, a primer
binding sequence, and a tandem inverted repeat, and when transfected into a target cell
with a reverse transcriptase gene under control of a suitable promoter, produces the
inhibitory nucleic acid sequence described herein. However, those skilled in the art will
recognize that, for instance, the reverse transcriptase gene of the cassette can be replaced
with other reverse transcriptase genes (the reverse transcriptase gene from human
immunodeficiency virus was one such gene which was noted above) and that promoters
other than the CMV promoter described herein may be used to advantage. As noted
above, the stem-loop intermediate that is formed may or may not include a restriction
endonuclease site and its susceptibility to denaturation is manipulated to advantage
depending upon the particular sequence of interest that is intended to be produced from
that intermediate. All such changes, and others that will be made clear to those skilled in
the art by this description modifications which do not depart from the spirit of the present
invention, are intended to fall within the scope of the following claims.
WE CLAIM :
1. A topical composition for the treatment of HSV-related pathologies comprising:
(a) a single-stranded DNA (ssDNA) expression vector having a gene encoding for a reverse
transcriptase (RT) and a ssDNA expression cassette comprising in 5' to 3' order:
(i) a cytomegalovirus (CMV) or Rous Sarcoma virus (RSV) promoter sequence,
(ii) an inverted tandem repeat (IR) sequence,
(iii) a sequence of interest comprising SEQ ID NO: 3, or a portion thereof, and
(iv) a mammalian reverse transcriptase primer binding site (PBS) sequence; and
(b) a topical ointment or cream,
wherein the ssDNA so produced has minimal flanking sequence.
2. The composition as claimed in claim 1 in which the RT is mouse Maloney reverse transcriptase
(MoMuLV-RT).
3. The composition as claimed in claim 1 in which the promoter is either a cytomegalovirus
(CMV) or a Rouse Sarcoma virus (RSV) promoter.
4. The composition as claimed in claim 1 in which the IR sequence comprises
5'-GGTCGGCGGCCTTGAAGAGCGGCCGCACT-3'.
5. The composition as claimed in claim 1 in which the PBS site on the transcribed mRNA
promotes binding of the MoMuLV-RT with an appropriate tRNA.
6. The composition as claimed in claim 1 additionally comprising an antibiotic.
7. The composition as claimed in claim 1 additionally comprising either or both of an anaesthetic
or analgesic.
8. The composition as claimed in claim 1 additionally comprising one or more of vitamins A, D,
and E.
9.T he composition as claimed in claim 1 wherein said ointment or cream comprises a two-part
gel, a Base PCCA Cosmetic HRT, or PLO.
10. A pharmaceutical composition for the treatment of HSV-related pathologies involving the
application of a composition comprising an in vivo ssDNA expression vector carrying SEQ ID NO:
3, or a portion thereof, to an affected site.
11. The pharmaceutical composition as claimed in claim 10 wherein the composition includes a
topical ointment, a topical cream, or a topical spray.
12. A ssDNA expression vector comprising a gene encoding for a reverse transcriptase and a
ssDNA expression cassette comprising in 5' to 3' order:
(i) a cytomegalovirus (CMV) or Rous Sarcoma virus (RSV) promoter sequence,
(ii) an inverted tandem repeat (IR) sequence comprising
5'-GGTCGGCGGCCTTGAAGAGC GGCCGCACT-3',
(iii) a convenient cloning site, and
(iv) a mammalian reverse transcriptase primer binding site (PBS),
wherein the ssDNA so produced has minimal flanking sequence.
13. The ssDNA expression vector as claimed in claim 12 having the genetic composition of
plasmid pssXV.
14. The composition as claimed in claim 1 in which the sequence of interest comprises one or more
sequences having SEQ ID NO: 3.
15. The composition as claimed in claim 1 wherein the ssDNA expression vector is either pssXE or
pssXV, each containing a cassette including an SOI, IR and PBS regulated by a promoter/enhancer.
16. The composition as claimed in claim 1 wherein the carrier is designed for topical
administration.

A composition for treatment of HSV-related pathologies including an expression vector for altering expression of
one or more target sequence(s) in an HSV-infected cell by production of single-stranded cDNA (ssDNA) in the cell in vivo suspended
for topical application to an affected site in a suitable delivery vehicle. The expression vector is comprised of a cassette comprising
a sequence of interest, an inverted tandem repeat, and a primer binding site 3' to the inverted tandem repeat, and a reverse transcrip-
tase/RNAse H coding gene, and is transfected into the infected cells for inhibition of HSV replication or assimilation into a target
cell. The resulting ssDNA binds to the target sequence to alter expression of the target sequence for such purposes as gene activation
or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding
to specific cellular proteins, or interfering with RNA splicing functions.

Documents:

1271-kolnp-2005-abstract.pdf

1271-kolnp-2005-assignment.pdf

1271-kolnp-2005-claims.pdf

1271-kolnp-2005-correspondence.pdf

1271-kolnp-2005-description (complete).pdf

1271-kolnp-2005-drawings.pdf

1271-kolnp-2005-examination report.pdf

1271-kolnp-2005-form 1.pdf

1271-kolnp-2005-form 13.pdf

1271-kolnp-2005-form 18.pdf

1271-kolnp-2005-form 3.pdf

1271-kolnp-2005-form 5.pdf

1271-kolnp-2005-gpa.pdf

1271-kolnp-2005-granted-abstract.pdf

1271-kolnp-2005-granted-assignment.pdf

1271-kolnp-2005-granted-claims.pdf

1271-kolnp-2005-granted-correspondence.pdf

1271-kolnp-2005-granted-description (complete).pdf

1271-kolnp-2005-granted-drawings.pdf

1271-kolnp-2005-granted-examination report.pdf

1271-kolnp-2005-granted-form 1.pdf

1271-kolnp-2005-granted-form 13.pdf

1271-kolnp-2005-granted-form 3.pdf

1271-kolnp-2005-granted-form 5.pdf

1271-kolnp-2005-granted-gpa.pdf

1271-kolnp-2005-granted-reply to examination report.pdf

1271-kolnp-2005-granted-specification.pdf

1271-kolnp-2005-reply to examination report.pdf

1271-kolnp-2005-specification.pdf


Patent Number 235814
Indian Patent Application Number 1271/KOLNP/2005
PG Journal Number 36/2009
Publication Date 04-Sep-2009
Grant Date 02-Sep-2009
Date of Filing 30-Jun-2005
Name of Patentee CYTOGENIX INC.
Applicant Address 3100 WILCREST DRIVE, SUITE 140 HOUSTON, TEXAS
Inventors:
# Inventor's Name Inventor's Address
1 YIN CHEN 2414, LANSING CIRCLE, PEARLAND, TEXAS 77584
PCT International Classification Number A61K
PCT International Application Number PCT/US2003/039033
PCT International Filing date 2003-12-06
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10/313-828 2002-12-06 U.S.A.