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DESCRIPTION
A.AV VECTOR-BASED COMPOSITIONS
AND THERAPEUTIC USES THEREOF
1.0 BACKGROUND OF THE INVENTION
The present application claims priority to United States serial number
09/138,226, filed
June 9, 1999, the entire contents of which is specifically incorporated herein
by reference in its
entirety. The United States government has certain rights in the present
invention pursuant to
Grant Numbers NS32727, DK37273 and GM53252 from the National Institutes of
Health.
1.1 FIELD OF THE INVENTION
The present invention relates generally to the fields of molecular biology and
virology,
and in particular, to methods for using recombinant adeno-associated virus
(rAAV)
compositions that express nucleic acid segments encoding therapeutic gene
products in the
treatment of complex human disorders. In certain embodiments, the invention
concerns the use
of rAAV in a variety of investigative, diagnostic and therapeutic regimens,
including the
treatment of diseases of the nervous and musculoskeletal systems, blindness,
age-related
macular degeneration, obesity, weight gain, and various eating disorders.
Methods and
compositions are provided for preparing rAAV-based vector constructs that
express one or
more cytokine or cytokine receptor genes) for use in viral-based gene
therapies.
1Z DESCRIPTION OF THE RELATED ART
An estimated 54 percent of adults in the United States are overweight,
according to
the National Institutes of Health. Treating obesity and its related
conditions, including high
blood pressure, heart disease and diabetes, is estimated to cost more than $45
billion
annually, according to an August, 1996 scientific American a~icle. With the
incidence of
obesity on the rise in the United States, there is a need to develop therapies
that will
alleviate the symptoms associated with obesity.
Two appetite-controlling compounds have been previously identified in mammals-
leptin and ciliary neurotrophic factor (CNTF). Leptin is a naturally occurring
protein produced
by fat cells that inhibits appetite and increases energy expenditure. It
signals the brain,
affecting the brain's secretion of appetite-regulating signals. Such signals
include neuropeptide
Y, a chemical that has been found to stimulate appetite. This process is
thought to be faulty in
most obese people so that even high levels of leptin fail to turn off the
hunger signal.
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LIF is a secreted polyfunctional cytokine that elicits a diversity of
biological effects on
many cell types. LIF's action is mediated following binding to specific
cellular receptors that
trigger differentiation-induction, differentiation-suppression, proliferation,
anal activation
depending on the cell type. The distribution of LIF receptor (LIFR) mRNA in
the adult brain
and spinal cord suggests that LIF or LIF related cytokine exert important
actions on the
neuronal cells of the central nervous system in the adult as well as during
development. LIFR~
mRNA expression is largely restricted to specific brain regions relevant to
the motor and
sensory systems, and in the spinal cord expression is largely found in the
motor neurons of the
ventral horn and in the sensory ganglia.
L3 DEFICIENCIES IN THE PRIOR ART
Currently, there are limited pharmacological approaches to treating eating
disorders,
obesity, neurological dysfunction, and musculoskeletal disorders and diseases
in an affected
animal. Many such methods introduce undesirable side-effects, and do not
overcome the
problems associated with traditional modalities and treatment regimens for
conditions such as
retinitis pigmentosa, age-related macular degeneration, age-related muscle
weakness,
amyotrophie lateral sclerosis, and the like. Thus, the need exists for an
effective treatment that
circumvents the adverse effects and provides more desirable results, with
longer acting effects,
and improved patient compliance. In addition, methods for delivery of
polynucleotides to a
host cell that express a cytokine or cytokine receptor polypeptide useful in
the amelioration of
such conditions, and in particular, administration of specific rAAV-based
polynucleotide
constructs to a marninal are particularly desirable.
2.0 SUMMARY OF THE INVENTION
The present invention overcomes these and other limitations inherent in the
prior art by
providing new rAAV-based genetic constructs that encode one or more mammalian
cytokines
or cytokine receptor polypeptides for the treatment or amelioration of various
disorders
resulting from a cytokine or cytokine receptor polypeptide deficiency. In
particular, the
invention provides genetic constructs encoding one or more mammalian cytokine
or cytokine
receptor polypeptides, for use in the treatment of such conditions as
blindness, retinitis
pigmintosa, age-related macular degeneration, obesity, anorexia, weight gain,
and a variety of
eating disorders. Likewise, the invention provides genetic constructs that
encode one or more
cytokines or cytokine receptor polypeptides useful in the treatment or
amelioration of various
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neuromuscular disorders, including for example, amyotrophic lateral sclerosis,
and related
conditions that manifest from a deficiency or absence of physiologically
normal levels of
cytokines or cytokine receptor polypeptides.
The invention provides compositions and methods for treating or ameliorating
such a
cytokine-or a cytokine receptor polypeptide deficiency in a mammal, and
particularly for
treating or reducing the severity or extent of deficiency in a human
manifesting one or more of
the disorders linked to a deficiency in such polypeptides. In a general sense,
the method
involves administration of an rAAV-based genetic construct that encodes one or
more
anorectic cytokines or cytokine receptor polypeptides in a pharmaceutically-
acceptable vehicle
to the animal in an amount and for a period of time sufficient to treat or
ameliorate the
deficiency in the animal suspected of suffering from such a disorder.
Exemplary cytokines
useful in the practice of include, but are not limited to those described
herein in Table 4, and
include polypeptides such as leptin (Lep), BDNF, LIF, BDNF receptor, LIF
receptor, leptin
receptor, ciliary neurotrophic factor (CNTF), and CNTF receptor polypeptide.
In one embodiment, the cytokine or cytokine receptor polypeptide deficiency
manifests itself in musculoskeletal dynfunction. In such instances, the
invention provides a
method for inhibiting, reducing, or ameliorating age-related muscle weakness
or muscle
fatigue in a mammal, and preferably a human. The method generally involves
administering to the mammal an effective amount of an rAAV composition that
comprises a
selected polynucleotide sequence encoding a mammalian cytokine or cytokine
receptor
polypeptide to inhibit, reduce, or ameliorate the muscle dysfunction, weakness
or muscle
fatigue in the mammal.
In a second embodiment, the cytokine or cytokine receptor polypeptide
deficiency
manifests itself in a neuromuscular disease or disorder. In such instances,
the invention
provides a method for treating or reducing the severity of symptoms of the
neuromuscular
disease in an animal. This method generally involves administering to an
animal suspected
of having one or more neuromuscular diseases or disorders a therapeutically-
effective
amount of an rAAV vector construct that comprises a DNA segment that encodes
at least a
first mammalian cytokine or cytokine receptor polypeptide in an amount and for
a time
effective to treat or ameliorate such a neuromuscular disease.
In a third embodiment, the cytokine or cytokine receptor polypeptide
deficiency
manifests itself in an age-related muscle deterioration. In such instances,
the invention
provides a method for treating, ameliorating, or reducing the severity of
symptoms of one or
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more types of age-related muscle deterioration in a mammal. This method
generally
involves administering to the mammal affected with such a condition an amount
of an
rAAV vector construct that comprises a selected polynucleotide sequence
encoding at least
a first mammalian cytokine or cytolcine receptor polypeptide effective to
treat, ameliorate or
reduce the severity of symptoms of such muscle deterioration or dysfunction in
the
mammal.
In a fourth embodiment, the cytokine or cytokine receptor polypeptide
deficiency
manifests itself in the form of visual impairment, blindness, retinitis
pigmentos, or age-
realted macular degeneration. In such instances, the invention provides a
method for
treating, ameliorating, or reducing the severity of symptoms of one or more
defects in
vision. The administration of one or more such cytokine or cytokine receptor
polypeptide
genetic constructs is preferred in treating such ophthalmic conditions that
develop or are
worsened by a deficiency of cytokine or cytokine receptor polypeptides.
3.O BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to
further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to the following description taken in conjunction with
the
accompanying drawings, in which like reference numerals identify like
elements, and in
which:
FIG. 1A shows that body weight was significantly (p<0.05) reduced by a single
50
~,1 intravenous injection of rAAV-leptin in obese male oblob mice (50-60 g) in
a dose
dependent manner, the highest dose being the most effective;
FIG. 1B shows that food intake in ob/ob mice following rAAV-leptin was also
decreased by the highest dose (p<0.05 ys. control);
FIG. 1C shows that serum leptin levels in oblob mice were elevated
significantly
after intravenous injection of 1011 particles of rAAV-leptin;
FIG. 2 shows representative oblob mice control ys, rAAV-Leptin treated;
FIG. 3A shows that body weight was significantly reduced by 2 weeks post
injection in rAAV-leptin-treated SD rats as compared with control rats; this
reduction was
maintained for the duration of the study (*= p <0.05);
FIG. 3B shows that food intake in rAAV-leptin VS. control SD rats. There was
no
change in overall food intake between the control and experimental groups;
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FIG. 3C shows that serum leptin levels were significantly reduced in rAAV-
Leptin
treated SD rats (0.08 ~ 0.01 ng/ml) ys, controls (2.2 ~ 0.42 ng/ml);
FIG. 4 shows the effects of icr rAVV-CNTF in SD rats (p<0.05 Vs. rAAV-GFP
contr ols);
FIG. 5 shows the effects of icr rAW-CNTF in SD rats (p<0.05 v,~. rAAV-GFP
controls);
FIG. 6A shows the effect of icv rAAV-Leptin in 24 day old SD rats (p<p.05 vS_
rAAV-
GFP injected controls at corresponding time points);
FIG. 6B shows the effect of icv rAAV-Leptin in 24 day old SD rats (p<0.05 Vs,
rAAV-
GFP injected controls at corresponding time points);
FIG. 7 shows the effect of rAAV-Leptin on UCP-1 mRNA levels in interscapular
Brown adipose tissue. UCP-1 mRNA levels were significantly upregulated in rAAV-
leptin
treated rats (9) VS. control (9)at 6 weeks post injection;
FIG. 8 shows the effect of rAAV-Leptin on body fat composition. There was
significant reduction in body fat (g) levels (p<.OS) in rAAV-leptin treated
animals (n=3) Vs.
control (n=3) at 6 weeks post injection;
FIG. 9 shows the effect of rAAV-Leptin on protein composition. There was no
change
in protein levels with <.0S in rAAV-leptin treated animals (n=3) 1,5. control
(n=3) at 6 weeks
post injection (p<0.5 p<.OS);
FIG.10 shows the effect of icv injections of leptin and CNTF on STAT-3 and
STAT-1
phosphorylation in hypothalamus of rats. Recombinant rat leptin (R&D Systems;
S~g in 5~1 of
PBS) and CNTF (Amgen; S~g in 5~1 of PBS) were injected icv to SD male rats
(250-300g).
Animals were sacrificed 15 and 45 min. post-injection. Protein extracts of
hypothalamus were
Western blotted with aSTAT-3, aP(Tyr-705)STAT-3, aP(Ser701)STAT-3, aSTAT-1, or
aP(Tyr701)STAT-1 after SDS-PAGE (7.5%). The representative immunolots are
shown. The
intensities of bands of phosphorylated STATs obtained by image analysis were
normalized to
corresponding total STATs levels and expressed as arbitrary units; each bar is
mean + SEM for
3-5 animals for each time point; *, P<0.01 (t-test) VS. corresponding control;
FIG.11A shows a diagram of rAAV-CNTF;
FIG. 11B shows a nucleotide sequence coding for hGH signal peptide. The codons
of
the hGH are shown in italic, the first two codons of the human CNTF cDNA are
shown in bold
uppercase, the consensus Kozak sequence is underlined;
FIG.12 shows a diagram of the rAAV-Ob vector;
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FIG. 13 shows the diagram of rAAV vectors designed and constructed for the
current
project. IRES is a poliovirus type 1 internal ribosome entry site that
mediates a coordinate
expression of a transgene and GFP reporter gene from dicistronic transcription
unit;
FIG. 14 shows the effect of CNTF gene therapy on ONL thickness in the rds +/-
Tg
P216L mouse using various rAAV constructs. ONL thickness was determined at 21
regular
intervals along a full vertical meridian through the optic nerve head (ONH) 75
days
postinjection (P90). The left half is inferior retina; the right half is
superior retina. The
number of eyes averaged for each treatment is indicated; and
FIG. 15 shows a schematic that indicates pathways affected by hypothalamus
appetite
controlling signals and the relation of various inhibiting and stimulatory
signals to gut response
and leptin release.
4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Illustrative embodiments of the invention are described below. In the interest
of clarity,
not all features of an actual implementation are described in this
specification. It will of course
be appreciated that in the development of any such actual embodiment, numerous
implementation-specific decisions must be made to achieve the developers'
specific goals,
such as compliance with system-related and business-related constraints, which
will vary from
one implementation to another. Moreover, it will be appreciated that such a
development effort
might be complex and time-consuming, but would nevertheless be a routine
undertaking for
those of ordinary skill in the art having the benefit of this disclosure.
Using a mammalian animal model, the inventors have demonstrated the beneficial
effects of these constructs and their efFcacy of anorexigenic and body weight
(BW) reducing
effects. As an illustrative example, an rAAV construct comprising a gene
encoding leptin
(rAAV-leptin) delivered either intramuscularly (lj~) or intravenously (ZV)
reduced BW in
leptin-deficient obese oglob ice. rAAV-leptin injected iv produced a dose-
dependent
decrease in BW concurrent with reduction in food intake (FI). In lean Sprague-
Dawley (SD)
rats, rAAV-leptin delivered intracerebroventricularly (icv) also suppressed
the normal BW
gain.
In a second illustrative embodiment, the efficacy of rAAV-introduced CNTF-
encoding
polynucleotides was demonstrated in leptin-resistant genetically obese Zucker
(fa/fa) rats. An
rAAV-based genetic construct encoding a secreted form of CNTF was administered
(100
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physical particles) icv to falfa rats. The results showed that rAAV-CNTF
decreased the rate of
BW gain accompanied by a significant reduction in cumulative FI.
In a third illustrative embodiment, the relative efficacy of other related
cytokines to
regulate BW after peripheral or central administration using rAAV vectors have
been
demonstrated.
These results demonstrate that cytokine and cytokine receptor-encoding gene
therapy
using an xAAV delivery system is a viable alternative to pharmacologic
approaches for
reducing body weight for extended periods of time.
OBESITY
Obesity is a complex disorder and often leads to hyperinsulinemia,
hyperglycemia and
insulin resistance. Obesity is also a major risk factor for hypertension and
cardiovascular
disease. There are multiple pathways controlling the complex balance of energy
intake and
expenditure. The major afferent factor in a negative feedback loop regulating
daily food intake
and body weight is hormone leptin synthesized in adipocytes. Although leptin
administration
has been shown to reduce food intake and body weight in rodents its
effectiveness is only
transient and requires repeated injections. In humans, plasma levels of leptin
increase in direct
correlation with increase in body weight and adiposity; this tolerance to
leptin (leptin
resistance) is believed to be an underlying factor in the loss of leptin
control on energy balance.
In addition, leptin-resistance due to environmental and genetic factors
contributes substantially
to human obesity. The cellular and molecule mechanisms of leptin resistance
are not known.
It is believed that leptin resistance is heterogeneous and multiple factors,
including defective
transport to brain and defective influence on the activity of the neural
circuits that regulate
body weight, are major players. Consequently, there is a clear need to develop
innovative
approaches to control appetite and body weight and correct obesity.
Leptin belongs to a cytokine family of peptides. Several members of cytokines,
e.g
ciliary neurotrophic factor (CNTF), are potent suppressors of appetite and
body weight gain,
and their efficacy has been demonstrated in normal and genetically obese
rodent models. .
These cytokines are the naturally occurring appetite suppressing molecules
(anorexigenic),
potentially useful for circumventing leptin-resistance and to serve as
therapeutic agents not
only to correct obesity but also to either prevent or maintain body weight at
clinically
appropriate ranges in normal and obese patient populations.
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4.2 AMYOTROPHIC LATERAL SCLEROSIS (ALS)
ALS is a chronic progressive disease of unknown disease of unknown etiology
that
results in the selective destruction of spinal cord, brainstem and cortical
motor neurons, with
patients typically dying within 3 years of disease onset. At present there are
no effective
therapies to halt or even slow the progression of the disease. The cause of
ALS remains
unknown although there is a general consensus that its multifactorial with
factors and cytokines
such as LIF or CNTF may prevent or reduce motor neuron cell death and slow
down disease
progression. Unlike the prior art, in which phase II-III clinical trials using
subcutaneously
administered recombinant human CNTF failed to demonstrate slowiilg disease
progression in
ALS patients, the present invention provides tonic expression of a cytokine
that is delivered by
an rAAV expression system. This system overcomes the limitations in the prior
art by
reducing motor neuron cell death and slowing the progression of this
degenerative disease.
4.3 AGE-RELATED MUSCLE WEAKNESS
It has been demonstrated that subcutaneous administration of CNTF in aged 24-
month
old rats increased the muscle strength 2.5-fold. Consistent with this finding
LIF administered
by single im injection to reinnervated muscle in adult rats resulted in an
increase in muscle
fiber diameter. The present invention provides improved methods for delivering
cytokines,
such as CNTF and LIF, using rAAV vector system that are effective therapeutic
factors in
treatment of age-related muscle weakness.
4.4 BLINDNESS
In the human disease retinitis pigmentosa (RP) and age-related macular
degeneration
(AMD) blindness results from photorecptors degenerating via apoptic pathways
due to. a
wide variety of genetic defects in a wide variety of genes expressed either in
photoreceptors
themselves or in neighboring cells supporting photoreceptor function. Many
cytokines,
particularly the neurotrophins, have been shown to slow this process of cell
death, but
singular intracular injections of cytokine proteins have not proven effective
at the long-term
protection required for RP and AMD. The present invention utilizes an rAAV
system to
deliver specific cytokine-encoding genes or cytokine receptor-encoding genes
under
regulation of either general promoter elements or photoreceptor specific
promoter elements
to ocular tissues to provide gene expression in the retina. This leads to a
long-term
continuous release of the passenger cytokine and prolonged photorecptor
rescue. Animal
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model studies of RP have shown that gene transfer of ciliary neurotrophic
factor (CNTF) or
brain-derive neurotrophic factor (BDNF) using either the CMV or a proximal
murine rod
opsin (MOPS) promoter leads to rescue of vision for at least 3 months. Because
cytokine
receptor density may be low on many retinal cell types, analogous cytokine
receptor gene
delivery, either in isolation or combined with the cytokine gene itself,
further enhances this
rescue, and provides a superior method for the treatment of such disorders in
mammals, and
in particular, humans.
4.5 ADENO-ASSOCIATED VIRUS
Adeno-associated virus-2 (AAV) is a human parvovirus that can be propagated
both as
a lytic virus and as a provirus (Cukor et al. ~ 1984; Hoggan et al. ~ 1972).
The viral genome
consists of linear single-stranded DNA (Rose et al., 1969), 4679 bases long
(Srivastava etal.~
1983), flanked by inverted terminal repeats of 145 bases (Lusby et al. ~
1982). For lytic growth
AAV requires co-infection with a helper virus. Either adenovirus (Atchinson et
al. ~ 1965;
Hoggan, 1965; Parks et al. ~ 1967) or herpes simplex (Buller ~t al. ~ 1981)
can supply helper
function. Without helper, there is no evidence of AAV-specific replication or
gene expression
(Rose and Koczot, 1972; Carter et al. ~ 1983). When no helper is available,
AAV can persist as
an integrated provirus (Hoggan, 1965; Berns et al. ~ 1975; Handa et al. ~
1977; Cheung et al.
1980; Berns et al. ~ 1982).
Integration apparently involves recombination between AAV termini and host
sequences and most of the AAV sequences remain intact in the provirus. The
ability of AAV
to integrate into host DNA is apparently an inherent strategy for insuring the
survival of AAV
sequences in the absence of the helper virus. When cells carrying an AAV
provirus are
subsequently superinfected with a helper, the integrated AAV genome is rescued
and a
productive lytic cycle occurs (Hoggan, 1965).
AAV sequences cloned into prokaryotic plasmids are infectious (Samulski et al.
~ 1982).
For example, when the wild type AAV/pBR322 plasmid, pSM620, is transfected
into human
cells in the presence of adenovirus, the AAV sequences are rescued from the
plasmid and a
normal AAV lytic cycle ensues (Samulski et al. ~ 1982). This renders it
possible to modify the
AAV sequences in the recombinant plasmid and, then, to grow a viral stock of
the mutant by
transfecting the plasmid into human cells (Samulski et al. ~ 1983; Hermonat et
al. ~ 1984). AAV
contains at least three phenotypically distinct regions (Hermonat Et al. ~
1984). The y.ep region
codes for one or more proteins that are required for DNA replication and for
rescue from the
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recombinant plasmid, while the yap and lip regions appear to code for AAV
capsid proteins
and mutants within these regions are capable of DNA replication (Hermonat et
al. ~ 1984). It
has been shown that the AAV termini are required for DNA replication (Samulski
et al. ~ 1983).
Laughlin et .al. (1983) have described the construction of two E. coli hYb~d
plasmids,
each of which contains the entire DNA genome of AAV, and the transfection of
the
recombinant DNAs into human cell lines in the presence of helper adenovirus to
successfully
rescue and replicate the AAV genome (See also Tratschin et al. ~ 1984a;
1984b).
Adeno-associated virus (AAV) is particularly attractive for gene transfer
because it
does not induce any pathogenic response and can integrate into the host
cellular chromosome
(Kotin et al. ~ 1990). The AAV terminal repeats (TRs) are the only essential
His-components for
the chromosomal integration (Muzyczka and McLaughin, 1988). These TRs are
reported to
have promoter activity (Flotte et al~~ 1993). They may promote efficient gene
transfer from the
cytoplasm to the nucleus or increase the stability of plasmid DNA and enable
longer-lasting
gene expression (Bartlett and Samulski, 1998). Studies using recombinant
plasmid DNAs
containng AAV TRs have attracted considerable interest. AAV-based plasmids
have been
shown to drive higher and longer transgene expression than the identical
plasmids lacking 'the
TRs of AAV in most cell types (Philip et al. ~ 1994; Shafron et al. ~ 1998;
Wang et al. ~ 1998). .
There are several factors that prompted researchers to study the possibility
of using
rAAV as an expression vector. One is that the requirements fox delivering a
gene to integrate
into the host chromosome are surprisingly few. It is necessary to have the 145-
by ITRs, which
are only 6% of the AAV genome. This leaves room in the vector to assemble a
4.5-kb DNA
insertion. While this carrying capacity may prevent the AAV from delivering
large genes, it is
amply suited for delivering the antisense constructs of the present invention.
AAV is also a good choice of delivery vehicles due to its safety. There is a
relatively complicated rescue mechanism: not only wild type adenovirus but
also AAV
genes are required to mobilize rAAV. Likewise, AAV is not pathogenic and not
associated
with any disease. The removal of viral coding sequences minimizes immune
reactions to
viral gene expression, and therefore, rAAV does not evoke an inflammatory
response.
AAV therefore, represents an ideal candidate for delivery of the
polynucleotides or
hammerhead ribozyiue constructs of the present invention.
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4.6 PROMOTERS AND ENHANCERS
Recombinant vectors form important aspects of the present invention. The term
"expression vector or construct" means any type of genetic construct
containing a nucleic
acid in which part or all of the nucleic acid encoding sequence is capable of
being
transcribed. In preferred embodiments, expression only includes transcription
of the nucleic
acid, for example, to generate a cytokine polypeptide product from a
transcribed gene.
Particularly useful vectors are contemplated to be those vectors in which the
nucleic
acid segment to be transcribed is positioned under the transcriptional control
of a promoter.
A "promoter" refers to a DNA sequence recognized by the synthetic machinery of
the cell,
or introduced synthetic machinery, required to initiate the specific
transcription of a gene.
The phrases "operatively positioned," "under control" or "under
transcriptional control"
means that the promoter is in the correct location and orientation in relation
to the nucleic
acid to control RNA polymerase initiation and expression of the gene.
In preferred embodiments, it is contemplated that certain advantages will be
gained by
positioning the coding DNA segment under the control of a recombinant, or
heterologous,
promoter. As used herein, a recombinant or heterologous promoter.is intended
to refer to a
promoter that is not normally associated with a cytokine-encoding gene in its
natural
environment. Such promoters may include promoters normally associated with
other genes,
and/or promoters isolated from any other bacterial, viral, eukaryotic, or
mammalian cell.
Naturally, it will be important to employ a promoter that effectively directs
the
expression of the cytokine-encoding DNA segment in the cell type, organism, or
even animal,
chosen for expression. The use of promoter and cell type combinations for
protein expression
is generally known to those of skill in the art of molecular biology, for
example, see Sambrook
et al. (1989), incorporated herein by reference. The promoters employed may be
constitutive,
or inducible, and can be used under the appropriate conditions to direct high-
level expression
of the introduced DNA segment. ,
At least one module in a promoter functions to position the start site for RNA
synthesis. The best-known example of this is the TATA box, but in some
promoters lacking
a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl
transferase gene and the promoter for the SV40 late genes, a discrete element
overlying the
start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional
initiation.
Typically, these are located in the region 30-110 by upstream of the start
site; although a
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number of promoters have been shown to contain functional elements downstream
of the
start site as well. The spacing between promoter elements frequently is
flexible, so that
promoter function is preserved when elements are inverted or moved relative to
one another.
In the tk promoter, the spacing between promoter elements can be increased to
SO by apart
before activity begins to decline. Depending on the promoter, it appears that
individual
elements can function either co-operatively or independently to activate
transcription.
The particular promoter that is employed to control the expression of a
nucleic acid
is not believed to be critical, so long as it is capable of expressing the
nucleic acid in the
targeted cell. Thus, where a human cell is targeted, it is preferable to
position the nucleic
acid coding region adjacent to and under the control of a promoter that is
capable of being
expressed in a human cell. Generally speaking, such a promoter might include
either a
human or viral promoter, such as a CMV or an HSV promoter. In certain aspects
of the
invention, tetracycline controlled promoters are contemplated.
In various other embodiments, the human cytomegalovirus (CMV) immediate early
gene promoter, the SV40 early promoter and the Rous sarcoma virus long
terminal repeat
can be used to obtain high-level expression of transgenes. The use of other
viral or
mammalian cellular or bacterial phage promoters that are well known in the art
to achieve
expression of a transgene is contemplated as well, provided that the levels of
expression are
sufficient for a given purpose. Tables 1 and 2 below list several
elements/promoters that
may be employed, in the context of the present invention, to regulate the
expression of the
present cytokine-encoding constructs. This list is not intended to be
exhaustive of all the
possible elements involved in the promotion of transgene expression but,
merely, to be
exemplary thereof.
Enhancers were originally detected as genetic elements that increased
transcription
from a promoter located at a distant position on the same molecule of DNA.
This ability to
act over a large distance had little precedent in classic studies of
prokaryotic transcriptional
regulation. Subsequent work showed that regions of DNA with enhancer activity
are
organized much like promoters. That is, they are composed of many individual
elements,
each of which binds to one or more transcriptional proteins.
The basic distinction between enhancers and promoters is operational. An
enhancer
region as a whole must be able to stimulate transcription at a distance; this
need not be true
of a promoter region or its component elements. On the other hand, a promoter
must have
one or more elements that direct initiation of RNA synthesis at a particular
site and in a
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13
particular orientation, whereas enhancers lack these specif cities. Promoters
and enhancers
are often overlapping and contiguous, often seeming to have a very similar
modular
organization.
Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter
Data Base EPDB) could also be used to drive expression. Use of a T3, T7 or SP6
cytoplasmic expression system is another possible embodiment. Eukaryotic cells
can
support cytoplasmic transcription from certain bacterial promoters if the
appropriate
bacterial polymerase is provided, either as part of the delivery complex or as
an additional
genetic expression construct.
TABLE 1
PROMOTER AND ENHANCER ELEMENTS
PROMOTE NHANCER EFERENCES
mmunog o a m eavy C am -l~anery et al., ; 1 es et al. ~ ~ ~ rossc a an
Baltimore, 1985; Atchinson and Perry, 1986, 1987; Imler
et al. ~ 1987; Weinberger et al. ~ 1984; I~iledjian et al.
1988; Porton et al. ~ 1990
Immunoglobulin Light Chain Queen and Baltimore, 1983; Picard and Schaffner,
1984
T-Cell Receptor Luria ~t al,, 1987; Winoto and Baltimore, 1989; Redondo
et al. ~ 1990
HLA DQ a and DQ ~ Sullivan and Peterlin, 1987
~-Interferon Goodbourn et al. ~ 1986; Fujita et al.
~ 1987; Goodbourn
and Maniatis, 1988
Interleukin-2 Greene et al. ~ 1989
Interleukin-2 ReceptorGreene et al.~ 1989; Lin et .al., 1990
MHC Class II 5 Loch et al. ~ 1989
MHC Class II HLA-DraSherman et al. ~ 1989
~-Actin I~awamoto et al~~ 1988; Ng et al. ~ 1989
Muscle Creatine KinaseJaynes ~t al.~ 1988; Horlick and Benfield,
1989; Johnson
et al. ~ 1989
Prealbumin (Transthyretin) Costa et al~~ 1988
Elastase I Omitz et al. ~ 1987
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PROMOTE NHANCER hEFERENCES
eta of ionem ~'ln et al. ~ ; a o a an amer,
Collagenase Pinlcert et al. ~ 1987; Angel et al. ~ 1987
Albumin Gene Pinkert et al. ~ 1987; Tronche et al. ~ 1989, 1990
a-Fetoprotein Godbout et al.~ 1988; Campere and Tilghman, 1989
t-Globin Bodine and Ley, 1987; Perez-Stable and
Constantini, 1990
~-Globin Trudel and Constantini, 1987
e-fos Cohen et al. ~ 1987
c-HA-ras Triesman, 1986; Deschamps et al. ~ 1985
Insulin Edlund et al. ~ 1985
Neural Cell Adhesion Hirsh et al., 1990
Molecule
(NCAM)
a>-antic Latimer et al. ~ 1990
rypain
H2B (TH2B) Histone Hw~g et al. ~ 1990
Mouse or Type I Collagenape et al. ~ 1989
Glucose-Regulated ProteinsChug et al. ~ 1989
(GRP94 and GRP78)
Rat Growth Hormone Larsen et al. ~ 1986
Human Serum Amyloid Edbrooke et al. ~ 1989
A (SAA)
Troponin I (TN I) Yutzey et al. ~ 1989
Platelet-Derived GrowthPech et al. ~ 1989
Factor
Duchenne Muscular DystrophyKlamut et al. ~ 1990
SV40 Banerji et al.~ 198I; Moreau et al.~
198I; Sleigh and
Lockett, 1985; Firak and Subramanian,
1986; Herr and
Clarke, 1986; Imbra and Karin, 1986;
Kadesch and Berg,
1986; Wang and Calame, 1986; Qndek et
al. ~ 1987; Kuhl
et al. ~ 1987; Schaffner et al. ~ 1988
Polyoma Swartzendruber and Lehman, 1975; Vasseur
et al. ~ 1980;
Katinka et al.~ 1980, 1981; Tyndell et
al.~ 1981; Dandolo
et al. ~ 1983; de Villiers et al. ~ 1984;
Hen et al. ~ 1986;
Satake et al.~ 1988; Campbell and Villarreal,
1988
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PROMOTE NHANCER EFERENCES
Retroviruses ieg er an o c an, , ; evmson et al. ~ ;
Kriegler et al. ~ 1983, 1984a, b, 1988; Bosze et al. ~ 1986;
Miksicek et al. ~ 1986; Celander and Haseltine, 1987;
Thiesen et al. ~ 1988; Celander et al. ~ 1988; Chol et al.
1988; Reisman and Rotter, 1989
Papilloma Virus C~po et al. ~ 1983; Lusky et al. ~ 1983; Spandidos and
Wilkie, 1983; Spalholz et al.~ 1985; Lusky and Botchan,
1986; Cripe et al. ~ 1987; Gloss et al. ~ 1987; Hirochilca
et al. ~ 1987; Stephens and Hentschel, 1987
Hepatitis B Virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986;
Shaul and Ben-Levy, 1987; Spandau and Lee, 1988;
Vannice and Levinson, 1988
Human Immunodeficiency Virus Muesing et al.~ 1987; Hauber and Cullan, 1988;
Jakobovits et al., 1988; Feng and Holland, 1988; Takebe
et al. ~ 1988; Rosen et al. ~ 1988; Berkhout et al. ~ 1989;
Laspia et al. ~ 1989; Sharp and Marciniak, 1989; Braddock
et al. ~ 1989
Cytomegalovirus Weber et al. ~ 1984; Boshart et al. ~ 1985; Foecking and
Hofstetter, 1986
Gibbon Ape Leukemia Virus Holbrook et al. ~ 1987; Quinn ~t al., 1989
TABLE 2
INDUCIBLE ELEMENTS
LEMENT NDUCER REFERENCES
or o ster a mi er et al. ~ ; as roger
Heavy metals and Karin, 1985; Searle et al.
1985; Stuart et al. ~ 1985;
Imagawa et al. ~ 1987, I~arin et al.
1987; Angel et al. ~ 1987b;
McNeall et al. ~ 1989
MMTV (mouse mammary Glucocorticoids Hung et al. ~ 1981; Lee et al.
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16
LEMENT NDUCER 1ZEFERENCES
tumor virus ; IVTa~ors an Varmus,
;
Chandler et al. ~ 1983;
Lee et al.
1984; Ponta et al. ~ 1985;
Sakai
etal.~ 1988
~-Interferon poly(rI)x Tavernier et al. ~ 1983
poly(rc)
Adenovirus 5 E2 EIa Imperiale and Nevins,
1984
Collagenase Phorbol Ester (TPA) gel et al. ~ 1987a
Stromelysin Phorbol Ester (TPA) gel et al. ~ 1987b
SV40 Phorbol Ester (TPA) Angel et al.~ 1987b
Murine MX Gene Interferon, Newcastle
Disease Virus
GRP78 Gene A23187 Resendez et al.~ 1988
~,-2-Macroglobulin IL-6 Kunz et al. ~ 1989
Vimentin Serum Rittlingetal.~ 1989
MHC Class I Gene Interferon Blanar et al. ~ 1989
H-2~b
HSP70 Ela, SV40 Large T Taylor et al. ~ 1989;
Antigen Taylor and
Kingston, 1990a, b
Proliferin Phorbol Ester-TPA Mordacq and Linzer, 1989
Tumor Necrosis FactorFMA Hensel et al. ~ 1989
Thyroid StimulatingThyroid Hormone Chatterjee et al.~ 1989
Hormone a Gene
As used herein, the terms "engineered" and "recombinant" cells are intended to
refer to
a cell into which an exogenous DNA segment, such as DNA segment that leads to
the
transcription of a cytokine, cytokine receptor or a ribozyme specific for such
a polypeptide
product, has been introduced. Therefore, engineered cells are distinguishable
from naturally
occurring cells, which do not contain a recombinantly introduced exogenous DNA
segment.
Engineered cells are thus cells having DNA segment introduced through the hand
of man.
To express a cytokine gene in accordance with the present invention one would
prepare
an rAAV expression vector that comprises a cytokine-encoding nucleic acid
segment under the
control of one or more promoters. To bring a sequence "under the control of a
promoter, one
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17
positions the 5' end of the transcription initiation site of the
transcriptional reading frame
generally between about 1 and about 50 nucleotides "downstream" of (i.e., 3'
of) the chosen
promoter. The "upstream" promoter stimulates transcription of the DNA and
promotes
expression of the encoded polypeptide. This is the meaning of "recombinant
expression" in
this context. Particularly preferred recombinant vector constucts are those
that comprise an
rAAV vector. Such vectors are described in detail herein.
4.7 PHARMACEUTICAL COMPOSITIONS
In certain embodiments, the present invention concerns formulation of one or
more of
the rAAV compositions disclosed herein in pharmaceutically acceptable
solutions for
administration to a cell or an animal, either alone, or in combination with
one or more other
modalities of therapy.
It will also be understood that, if desired, the nucleic acid segment, RNA,
DNA ox PNA
compositions that express a therapeutic gene product as disclosed herein may
be administered
in combination with other agents as well, such as, e.g., proteins or
polypeptides or various
pharmaceutically-active agents, including one or more systemic or topical
administrations of
cytokine or cytokine receptor polypeptides. In fact, there is virtually no
limit to other
components that may also be included, given that the additional agents do not
cause a
significant adverse effect upon contact with the target cells or host tissues.
The rAAV
compositions may thus be delivered along with various other agents as required
in the
particular instance. Such compositions may be purified from host cells or
other biological
sources, or alternatively may be chemically synthesized as described herein.
Likewise, such
compositions may further comprise substituted or derivatized RNA, DNA, or PNA
compositions.
Formulation of pharmaceutically-acceptable excipients and carrier solutions is
well-
known to those of skill in the art, as is the development of suitable dosing
and treatment
regimens for using the particular compositions described herein in a variety
of treatment
regimens, including e.g oral, parenteral, intravenous, intranasal, and
intramuscular
administration and formulation.
Typically, these formulations may contain at least about 0.1 % of the active
compound
or more, although the percentage of the active ingredients) may, of course, be
varied and may
conveniently be between about 1 or 2% and about 60% or 70% or more of the
weight or
volume of the total formulation. Naturally, the amount of active compounds) in
each
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18
therapeutically useful composition may be prepared is such a way that a
suitable dosage will be
obtained in any given unit dose of the compound. Factors such as solubility,
bioavailability,
biological half life, route of administration, product shelf life, as well as
other pharmacological
considerations will be contemplated by one skilled in the art of preparing
such pharmaceutical
formulations, and as such, a variety of dosages and treatment regimens may be
desirable.
In certain circumstances it will be desirable to deliver the pharmaceutical
compositions
disclosed herein parenterally, intravenously, intramuscularly, or even
intraperitoneally as
described in U. S. Patent 5,543,158; U. S. Patent 5,641,515 and U. S. Patent
5,399,363 (each
specifically incorporated herein by reference in its entirety). Solutions of
the active
compounds as freebase or pharmacologically acceptable salts may be prepared in
water
suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions
may also be
prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under
ordinary conditions of storage and use, these preparations contain a
preservative to prevent the
growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions
or dispersions and sterile powders for the extemporaneous preparation of
sterile injectable
solutions or dispersions (U. S. Patent 5,466,468, specifically incorporated
herein by reference
in its entirety). In all cases the form must be sterile and must be fluid to
the extent that easy
syringability exists. It must be stable under the conditions of manufacture
and storage and
must be preserved against the contaminating action of microorganisms, such as
bacteria and
fungi. The can-ier can be a solvent or dispersion medium containing, for
example, water,
ethanol, polyol (e.g glycerol, propylene glycol, and liquid polyethylene
glycol, and the like),
suitable mixtures thereof, andlor vegetable oils. Proper fluidity may be
maintained, for
example, by the use of a coating, such as lecithin, by the maintenance of the
required particle
size in the case of dispersion and by the use of surfactants. The prevention
of the action of
microorganisms can be brought about by various antibacterial ad antifungal
agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the
like. In many cases,
it will be preferable to include isotonic agents, for example, sugars or
sodium chloride.
Prolonged absorption of the injectable compositions can be brought about by
the use in the
compositions of agents delaying absorption, for example, aluminum monostearate
and gelatin.
For parenteral administration in an aqueous solution, for example, the
solution should
be suitably buffered if necessary and the liquid diluent first rendered
isotonic with sufficient
saline or glucose. These particular aqueous solutions are especially suitable
for intravenous,
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19
intramuscular, subcutaneous and intraperitoneal administration. In this
connection, a sterile
aqueous medium that can be employed will be known to those of skill in the art
in light of the
present disclosure. For example, one dosage may be dissolved in 1 ml of
isotonic NaCI
solution and either added to 1000 ml of hypodermoclysis fluid or injected at
the proposed site
of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages
1035-1038 and 1570-1580). Some variation in dosage will necessarily occur
depending on the
condition of the subject being treated. The person responsible for
administration will, in any
event, determine the appropriate dose for the individual subject. Moreover,
for human
administration, preparations should meet sterility, pyrogenicity, and the
general safety and
purity standards as required by FDA Office. of Biologics standards.
Sterile injectable solutions are prepared by incorporating the active
compounds in .the
required amount in the appropriate solvent with various of the other
ingredients enumerated
above, as required, followed by filtered sterilization. Generally, dispersions
are prepared by
incorporating the various sterilized active ingredients into a sterile vehicle
which contains the
basic dispersion medium and the required other ingredients from those
enumerated above. In
the case of sterile powders for the preparation of sterile injectable
solutions, the preferred
methods of preparation are vacuum-drying and freeze-drying techniques which
yield a powder
of the active ingredient plus any additional desired ingredient from a
previously sterile-filtered
solution thereof.
The compositions disclosed herein may be fornmlated in a neutral or salt form.
Pharmaceutically-acceptable salts, include the acid addition salts (formed
with the free amino
groups of the protein) and which are formed with inorganic acids such as, for
example,
hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and
the like. Salts formed with the free carboxyl groups can also be derived from
inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such
organic bases as isopropylamine, trimethylamine, histidine, procaine and the
like. Upon
formulation, solutions will be administered in a manner compatible with the
dosage
formulation and in such amount as is therapeutically effective. The
formulations are easily
administered in a variety of dosage forms such as injectable solutions, drug-
release capsules,
and the like.
As used herein, "carrier" includes any and all solvents, dispersion media,
vehicles,
coatings, diluents, antibacterial and antifungal agents, isotonic and
absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like. The use of
such media and agents
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for pharmaceutical active substances is well known in the art. Except insofar
as any
conventional media or agent is incompatible with the active ingredient, its
use in the
therapeutic compositions is contemplated. Supplementary active ingredients can
also be
incoyorated into the compositions.
The phrase "pharmaceutically-acceptable" refers to molecular entities and
compositions
that do not produce an allergic or similar untoward reaction when administered
to a human.
The preparation of an aqueous composition that contains a protein as an
active. ingredient is
well understood in the art. Typically, such compositions are prepared as
injectables, either as
liquid solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid
prior to injection can also be prepared. The preparation can also be
emulsified.
4.8 LIPOSOME-, NANOCAPSULE-, AND MICROPARTICLE-MEDIATED DELIVERY
In certain embodiments, the inventors contemplate the use of liposomes,
nanocapsules,
microparticles, microspheres, lipid particles, vesicles, and the like, for the
introduction of the
compositions of the present invention into suitable host cells. In particular,
the rAAV vector
compositions of the present iilvention may be formulated for delivery either
encapsulated in a
lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the
like.
Such formulations may be preferred for the introduction of pharmaceutically
acceptable
formulations of the nucleic acids or the rAAV-cytokine constructs disclosed
herein. The
formation and use of liposomes is generally known to those of skill in the art
(see for example,
Couvreur et al. ~ 1977; Couvreur, 1988; Lasic, 1998; which describes the use
of liposomes and
nanocapsules in the targeted antibiotic therapy for intracellular bacterial
infections and
diseases). Recently, liposomes were developed with improved serum stability
and circulation
half times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U. S.
Patent
5,741,516, specifically incorporated herein by reference in its entirety).
Further, various
methods of Iiposome and liposome like preparations as potential drug carriers
have been
reviewed (Takakura, 1998; Chandran et al~~ 1997; Margalit, 1995; U. S. Patent
5,567,434; U.
S. Patent 5,552,157; U. S. Patent 5,565,213; U. S. Patent 5,738,868 and U. S.
Patent 5,795,587,
each specifically incorporated herein by reference in its entirety).
Liposomes have been used successfully with a number of cell types that are
normally
resistant to transfection by other procedures including T cell suspensions,
primary hepatocyte
cultures and PC 12 cells (Renneisen et al., 1990; Muller et al. ~ 1990). In
addition, liposomes
are free of the DNA length constraints that are typical of viral-based
delivery systems.
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21
Liposomes have been used effectively to introduce genes, drugs (Heath and
Martin, 1986;
Heath ~t al.~ 1986; Balazsovits et al.~ 1989; Fresta and Puglisi, 1996),
radiotherapeutic agents
(Pilcul et al. ~ I 987), enzymes (Imaizumi ~t al. ~ 1990a; Imaizumi et al. ~ I
990b), viruses (Faller
and Baltimore, 1984), transcription factors and allosteric effectors (Nicolau
and Gersonde,
1979) into a variety of cultured cell lines and animals. In addition, several
successful clinical
trails examining the effectiveness of liposome-mediated drug delivery have
been completed
(Lopez-Berestein et al., I985a; 1985b; Coune, 1988; Sculier et al., 1988).
Furthermore,
several studies suggest that the use of liposomes is not associated with
autoimmune responses,
toxicity or gonadal localization after systemic delivery (Mori and Fukatsu,
1992).
Liposomes are formed from phospholipids that are dispersed in an aqueous
medium
and spontaneously form multilarnellar concentric bilayer vesicles (also termed
multilamellar
vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 p,m.
Sonication of
MLVs results in the formation of small unilamellar vesicles (SLTVs) with'
diameters in the
range of 200 to 500 ~, containing an aqueous solution in the core.
Liposomes bear resemblance to cellular membranes and are contemplated for use
in
connection with the present invention as carriers for the peptide
compositions. They are
widely suitable as both water- and lipid-soluble substances can be entrapped,
i~ e. in the aqueous
spaces and within the bilayer itself, respectively. It is possible that the
drug-bearing liposomes
may even be employed for site-specific delivery of active agents by
selectively modifying the
liposomal formulation.
In addition to the teachings of Couvreur et al. (I977; 1988), the following
information
may be utilized in generating liposomal formulations. Phospholipids can form a
variety of
structures other than liposomes when dispersed in water, depending on the
molar ratio of lipid
to water. At low ratios the liposome is the preferred structure. The physical
characteristics of
liposomes depend on pH, ionic strength and the presence of divalent canons.
Liposomes can
show low permeability to ionic and polar substances, but at elevated
temperatures undergo a
phase transition which markedly alters their permeability. The phase
transition involves a
change from a closely packed, ordered structure, known as the gel state, to a
loosely packed,
less-ordered structure, known as the fluid state. This occurs at a
characteristic phase-transition
temperature and results in an increase in permeability to ions, sugars and
drugs.
In addition to temperature, exposure to proteins can alter the permeability of
liposomes.
Certain soluble proteins, such as cytochrome c, bind, deform and penetrate the
bilayer, thereby
causing changes in permeability. Cholesterol inhibits this penetration of
proteins, apparently
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22
by packing the phospholipids more tightly. It is contemplated that the most
useful liposome
formations for antibiotic and inhibitor delivery will contain cholesterol.
The ability to trap solutes varies between different types of liposomes. Fox
example,
MLVs are moderately efficient at trapping solutes, but SUVs are extremely
inefficient. SUVs
offer the advantage of homogeneity and reproducibility in size distribution,
however, and a
compromise between size and trapping efficiency is offered by large
unilamellar vesicles
(LUVs). These are prepared by ether evaporation and are three to four times
more efficient at
solute entrapment than MLVs.
In addition to liposome characteristics, an important determinant in
entrapping
compounds is the physicochemical properties of the compound itself. Polar
compounds are
trapped in the aqueous spaces and nonpolar compounds bind to the lipid bilayer
of the vesicle.
Polar compounds are released through permeation or when the bilayer is broken,
but nonpolar
compounds remain affiliated with the bilayer unless it is disrupted by
temperature or exposure
to lipoproteins. Both types show maximum efflux rates at the phase transition
temperature.
Liposomes interact with cells via four different mechanisms: Endocytosis by
phagocytic cells of the reticuloendothelial system such as macrophages and
neutrophils;
adsorption to the cell surface, either by nonspecific weak hydrophobic or
electrostatic forces, or
by specific interactions with cell-surface components; fusion with the plasma
cell membrane
by insertion of the lipid bilayer of the liposorne into the plasma membrane,
with simultaneous
release of liposomal contents into the cytoplasm; and by transfer of liposomal
lipids to cellular
or subcellular membranes, or vice versa, without any association of the
liposome contents. It
often is difficult to determine which mechanism is operative and more than one
may operate at
the same time.
The fate and disposition of intravenously injected liposomes depend on their
physical
properties, such as size, fluidity, and surface charge. They may persist in
tissues for h or days,
depending on their composition, and half lives in the blood range from min to
several h.
Larger liposomes, such as MLVs and LLTVs, are taken up rapidly by phagocytic
cells of the
reticuloendothelial system, but physiology of the circulatory system restrains
the exit of such
large species at most sites. They can exit only in places where large openings
or pores exist in
the capillary endothelium, such as the sinusoids of the liver or spleen. Thus,
these organs are
the predominate site of uptake. On the other hand, SWs show a broader tissue
distribution but
still are~sequestered highly in the liver and spleen. In general, this i~ vivo
behavior limits the
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23
potential targeting of liposomes to only those organs and tissues accessible
to their large size.
These include the blood, liver, spleen, bone marrow, and lymphoid organs.
Targeting is generally not a limitation in terms of the present invention.
However,
should specific targeting be desired, methods are available for this to be
accomplished.
Antibodies may be used to bind to the liposome surface and to direct the
antibody and its drug
contents to specific antigenic receptors located on a particular cell-type
surface. Carbohydrate
determinants (glycoprotein or glycolipid cell-surface components that play a
role in cell-cell
recognition, interaction and adhesion) may also be used as recognition sites
as they have
potential in directing liposomes to particular cell types. Mostly, it is
contemplated that
intravenous iizjection of liposomal preparations would be used, but other
routes of
administration are also conceivable.
Alternatively, the invention provides for pharmaceutically acceptable
nanocapsule
formulations of the compositions of the present invention. Nanocapsules can
generally entrap
compounds in a stable and reproducible way (Henry-Michelland et al. ~ 1987;
Quintanar-
Guerrero et al-~ 1998; Douglas et al-~ 1987). To avoid side effects due to
intracellular
polymeric overloading, such ultrafine particles (sized around 0.1 Vim) should
be designed using
polymers able to be degraded i~ vivo- Biodegradable polyalkyl-cyanoacrylate
nanoparticles
that meet these requirements are contemplated for use in the present
invention. Such particles
may be are easily made, as described (Couvreur et al., 1980; Couvreur, 1988;
zur Muhlen et
al-~ 1998; Zambaux et al- 1998; Pinto-Alphandry ~t al., 1995 and U. S. Patent
5,145,684,
specifically incorporated herein by reference in its entirety).
4.J ADDITIONAL MODES OF DELIVERY
In addition to the methods of delivery described above, the following
techniques are also
contemplated as alternative methods of delivering the rAAV vector compositions
to a target cell
or animal. Sonophoresis (i. e., ultrasound) has been used and described in U.
S. Patent 5,656,016
(specifically incorporated herein by reference in its entirety) as a device
for enhancing the rate
and e~cacy of drug permeation into and through the circulatory system. Other
drug delivery
alternatives contemplated are intraosseous injection (U. S. Patent 5,779,708),
microchip devices
(LT. S. Patent 5,797,898), ophthalmic formulations (Bourlais et al-~ 1998),
transdermal matrices
(U. S. Patent 5,770,219 and U. S. Patent 5,783,208) and feedback-controlled
delivery (U. S.
Patent 5,697,899), each specifically incorporated herein by reference in its
entirety.
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24
4.10 THERAPEUTIC AND DIAGNOSTIC KITS
The invention also encompasses one or more compositions together with one or
more;
pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and/or
other components,
as may be employed in the formulation of particular rAAV-polynucleotide
delivery
formulations, and in the preparation of therapeutic agents for administration
to a mammal, and
in particularly, to a human, for one or more of the cytol~ine-deficient
conditions described
herein. In particular, such kits may comprise one or more rAAV-cytokine
composition in
combination with instructions for using the viral vector in the treatment of
such disorders in a
mammal, and may typically further include containers prepared for convenient
commercial
packaging.
As such, preferred animals for administration of the pharmaceutical
compositions
disclosed herein include mammals, and particularly humans. Other preferred
animals include
marines, bovines, equines, porcines, canines, and felines. The composition may
include
partially or significantly purified rAAV-cytokine compositions, either alone,
or in combination
with one or more additional active ingredients, which may be obtained from
natural or
recombinant sources, or which may be obtainable naturally or either chemically
synthesized, or
alternatively produced i~ vitro h'om recombinant host cells expressing DNA
segments
encoding such additional active ingredients.
Therapeutic kits may also be prepared that comprise at least one of the
compositions
disclosed herein and instructions for using the composition as a therapeutic
agent. The
container means for such kits may typically comprise at least one vial, test
tube, flask, bottle,
syringe or other container means, into which the disclosed rAAV compositions)
may be
placed, and preferably suitably aliquoted. Where a second cytokine or cytokine
receptor
composition is also provided, the kit may also contain a second distinct
container means into
which this second composition may be placed. Alternatively, the plurality of
cytokine or
cytokine receptor compositions may be prepared in a single pharmaceutical
composition, and
may be packaged in a single container means, such as a vial, flask, syringe,
bottle, or other
suitable single container means. The kits of the present invention will also
typically include a
means for containing the vials) in close confinement for commercial sale, such
as, e.g.
inj ection or blow-molded plastic containers into which the desired vials) are
retained.
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4.1.1 METHODS OF NUCLEIC ACID DELIVERY AND DNA TRANSFECTION
In certain embodiments, it is contemplated that one or more of the rAAV-
delivered
cytokine-encoding RNA, DNA, PNAs and/or substituted polynucleotide
compositions
disclosed herein will be used to transfect an appropriate host cell.
Technology for introduction
of rAAV's comprising one or more PNAs, RNAs, and DNAs into target host cells
is well
known to those of skill in the art.
Several non-viral methods for the transfer of expression constructs into
cultured
mammalian cells also are contemplated by the present invention for use in
certain i~ vita°o
embodiments, and under conditions where the use of rAAV-mediated delivery is
less desirable.
These include calcium phosphate precipitation (Graham and Van Der Eb, 1973;
Chen and
Okayama, 1987; Rippe ~t al.~ 1990) DEAE-dextran (copal, 1985), electroporation
(along and
Neumann, 1982; Fromm et al. ~ 1985; Tur-Kaspa et al. ~ 1986; Potter et al. ~
1984; Suzuki et al.,
1998; Vanbever ~t al., 1998), direct microinjection (Capecchi, 1980; Harland
and Weintraub,
1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al. ~ 1979;
Takakura, 1998)
and lipofectamine-DNA complexes, cell sonication (Fechheimer ~t al. ~ 1987),
gene
bombardment using high velocity microprojectiles (Yang ~t al., 1990; Klein et
al., 1992), and
receptor-mediated transfection (Curiel et al. ~ 1991; Wagner ~t al. ~ 1992; Wu
and Wu, 1987;
Wu and Wu, 1988). Some of these techniques may be successfully adapted for iy~
vivo or ex
vzvo use.
4.12 EXPRESSION IN ANIMAL CELLS
The inventors contemplate that a polynucleotide comprising a contiguous
nucleic acid
sequence that encodes a therapeutic cytokine polypeptide of the present
invention may be
utilized to treat one or more cellular defects in a transformed host cell.
Such cells are
preferably animal cells, including mammalian cells such as those obtained from
a human or
other primate, marine, canine, bovine, equine, epine, or porcine species. In
particular, the use
of such constructs for the treatment and/or amelioration of eating disorders
or neurological
dysfunction in a human subject suspected of suffering from such a disorder is
highly
contemplated. The cells may be transformed with one or more rAAV vectors
comprising one
or more therapeutic cytokine genes of interest, such that the genetic
construct introduced into
and expressed in the host cells of the animal is sufficient to alter, reduce,
ameliorate ox prevent
the deleterious or disease conditions either i~ vitro ~~or ire vivo~
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4.13 TRANSGENIC ANIMALS
It is contemplated that in some instances the genome of a transgenic non-human
animal
of the present invention will have been altered through the stable
introduction of one or more
of the rAAV-delivered polynucleotide compositions described herein, either
native,
synthetically modified, or mutated. As used herein, the term "transgenic
animal" is intended to
refer to an animal that has incorporated exogenous DNA sequences into its
genome. In
designing a heterologous gene for expression in animals, sequences which
interfere with the
efficacy of gene expression, such as polyadenylation signals, polymerise II
termination
sequences, hairpins, consensus splice sites and the like, are eliminated.
Current advances in
transgenic approaches and techniques have permitted the manipulation of a
variety of animal
genomes via gene addition, gene deletion, or gene modifications (Franz et al~~
1997). For
example, mosquitos (Fallon, 1996), trout (Ono et al., 1997), zebrafish
(Caldovic and Hackett,
1995), pigs (Van Cott et al~~ 1997) and cows (Haskell and Bowen, 1995), are
just a few of the
many animals being studied by txansgenics. The creation of transgenic animals
that express
hmnan proteins such as a-1-antitrypsin, in sheep (Carver et al~~ 1993); decay
accelerating
factor, in pigs (Cozzi et al~~ 1997), and plasminogen activator, in goats
(Ebert et al., 1991) has
previously been demonstrated. The transgenic synthesis of human hemoglobin (U.
S. Patent
5,602,306) and fibrinogen (LJ. S. Patent 5,639,940) in non-human animals have
also been
disclosed, each specifically incorporated herein by reference in ifs entirety.
Further, transgenic
mice and rat models have recently been described as new directions to study
and treat
cardiovascular diseases such as hypertension in humans (Franz et al~~ 1997;
Pinto-Siestma and
Paul, 1997). The construction of a transgenic mouse model has recently been
used to assay
potential treatments for Alzheimer's disease (U. S. Patent 5,720,936,
specifically incorporated
herein by reference in its entirety). It is contemplated in the present
invention that transgenic
animals contribute valuable information as models for studying the effects of
cytokine and
cytokine receptor compositions on correcting genetic defects and treating a
variety of disorders
in an animal.
4.14 SELECTION AND CHARACTERIZATION OF CYTOKINE GENETIC CONSTRUCTS
The enzyme luciferase is useful as a screenable marker in the context of the
present
invention (Kung et al~~ 1998). In the presence of the substrate luciferin,
cells expressing
luciferase emit light that can be detected on photographic or x-ray film, in a
luminometer (or
liquid scintillation counter), by devices that enhance night vision, or by a
highly light sensitive
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video camera, such as a photon counting camera. All of these assays are
nondestructive and
transformed cells may be cultured further following identification. The photon
counting
camera is especially valuable as it allows one to identify specific cells or
groups of cells that
are expressing luciferase and manipulate those in real time. The above
techniques also could
be utilized if the screenable marker is a protein such as green fluorescent
protein (gfp). '
To confirm the presence of the exogenous DNA or "transgene(s)" in the
transformed
cells, and in particular, a transgene delivered by an rAAV vector composition,
a variety of
assays may be performed. Such assays include, for example, "molecular
biological" assays,
such as Southern and Northern blotting, RT-PCRTM and PCRTM; "biochemical"
assays, such as
detecting the presence of a protein product, e.g by immunological means
(ELISAs and
Western blots) or by enzymatic function assay.
While Southern blotting and PCRTM may be used to detect the transgene(s) in
question,
they do not provide information as to whether the gene is being expressed.
Expression may be
evaluated by RT-PCRTM for mRNA and/or specifically identifying the protein
products of the
introduced genes or evaluating the phenotypic changes brought about by their
expression.
Assays for the production and identification of specific proteins may make use
of
physical-chemical, structural, functional, or other properties of the
proteins. Unique physical-
chemical or structural properties allow the proteins to be separated and
identified by
electrophoretic procedures, such as native or denaturing gel electrophoresis
or. isoelectric
focusing, or by chromatographic techniques such as ion exchange or gel
exclusion
chromatography. The unique str~.ictures of individual proteins offer
opportunities for use of
specific antibodies to detect their presence in formats such as an ELISA
assay. Transgenic
animals are described that synthesize epitope tagged prion proteins as a
method of detecting the
expressed proteins) (U. S. Patent 5,789,655, specifically incorporated herein
by reference iiz its
entirety). Combinations of approaches may be employed with even greater
specificity such as
western blotting in which antibodies are used to locate individual gene
products that have been
separated by electrophoretic techniques. Additional techniques may be employed
to absolutely
confirm the identity of the product of interest such as evaluation by amino
acid sequencing
following purification. Although these are among the most commonly employed,
other
procedures may be additionally used.
Assay procedures may also be used to identify the expression of proteins by
their
functionality, especially the ability of enzymes to catalyze specific chemical
reactions
involving specific substrates and products. These reactions may be followed by
providing and
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28
quantifying the loss of substrates or the generation of products of the
reactions by physical or
chemical procedures. Examples are as varied as the enzyme to be analyzed and
may include
assays fox PAT enzymatic activity by following production of radiolabeled
acetylated
phosphinothricin from phosphinothricin and 14C-acetyl CoA or for anthranilate
synthase
activity by following loss of fluorescence of anthranilate, to name two.
Very frequently the expression of a gene product is determined by evaluating
the
phenotypic results of its expression. These assays also may take many forms
including but not
limited to analyzing changes in the chemical composition, morphology, or
physiological
properties of the cells of the animal or human.
4.15 DNA INTEGRATION, RNA EXPRESSION AND INHERITANCE
Genomic DNA may be isolated from animal cell Lines or any animal parts to
determine
the presence of the exogenously introduced cytokine-encoding genenetic
construct through the
use of one or more readily-available techniques that are well known to those
skilled in the art.
The presence of DNA elements introduced through the methods of this iilvention
may be
determined by polymerise chain reaction (PCRTM). Using this technique,
discreet fragments of
DNA axe amplified and detected by gel electrophoresis. This type of analysis
permits one to
determine whether a gene is present in a stable transformant, but does not
prove integration of
the introduced gene into the host cell genome. In addition, it is not possible
using PCRTM
techniques to determine whether transformants have exogenous genes introduced
into different
sites in the genome, i~ e, , . whether iTansformants axe of independent
origin. It is contemplated
that using PCRTM techniques it would be possible to clone fragments of the
host genomic DNA
adjacent to an introduced gene.
Positive proof of DNA integration into the host genome and the independent
identities
of transformants may be determined using the technique of Southern
hybridization. Using this
technique specific DNA sequences that were introduced into the host genome and
flanking
host DNA sequences can be identified. Hence the Southern hybridization pattern
of a given
transformant serves as an identifying characteristic of that transformant. In
addition it is
possible through Southern hybridization to demonstrate the presence of
introduced genes in
high molecular weight DNA, i.e., confirm that the introduced gene has been
integrated into the
host cell genome. The technique of Southern hybridization provides information
that is
obtained using PCRTM e.g. the presence of a gene, but also demonstrates
integration into the
genome and characterizes each individual transformant.
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It is contemplated that using the techniques of dot or slot blot hybridization
which are
modifications of Southern hybridization techniques one could obtain the same
information that
is derived from PCRTM, e_g the presence of a gene.
Whereas DNA analysis techniques may be conducted using DNA isolated from any
part of an animal, RNA will only be expressed in particular cells or tissue
types and hence it
will be necessary to prepare RNA for analysis from these tissues. PCRTM
techniques may also
be used for detection and quantitation of RNA produced from introduced genes.
In tlus
application of PCRTM it is first necessary to reverse transcribe RNA into DNA,
using enzymes
such as reverse transcriptase, and then through the use of conventional PCRTM
techniques
amplify the DNA. In most instances PCRTM techniques, while useful, will not
demonstrate
integrity of the RNA product. Further information about the nature of the RNA
product may
be obtained by Northern blotting. This technique will demonstrate the presence
of an RNA
species and give information about the integrity of that RNA. The presence or
absence of an
RNA species can also be determined using dot or slot blot Northern
hybridization. These
techniques are modifications of Northern blotting and will only demonstrate
the presence or:
absence of an RNA species.
4.16 SELECTABLE MARKERS
In certain embodiments of the invention, the delivery of a nucleic acid in a
cell, and in
particular, an rAAV construct that expresses one or more therapeutic cytokine
or cytokine
receptor compositions may be identified l~z vitro or in vivo bY ~cluding a
marker in the
expression construct. The marker would result in an identifiable change to the
transfected cell
permitting ready identification of expression. Usually the inclusion of a drug
selection marker
aids in cloning and in the selection of transformants, fox example, neomycin,
puromycin,
hygromycin, DHFR, GPT, zeocin and histidinol. Alternatively, enzymes such as
herpes
simplex virus thymidine kinase (tk) (eukaryotic) or chloramphenicol
acetyltransferase (CAT)
(prokaryotic) may be employed, as well as markers such as green fluorescent
protein,
luciferase, and the like. Immunologic markers also can be employed. The
selectable marker
employed is not believed to be important, as long as it is capable of being
expressed
simultaneously with the nucleic acid encoding a gene product. Further examples
of selectable
markers are well known to one of skill in the art.
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4.17 SITE-SPECIFIC MUTAGENESIS
Site-specific mutagenesis is a technique useful in the preparation of
individual peptides,
or biologically functional equivalent polypeptides, through specific
mutagenesis of the
underlying polynucleotides that encode them. The teclnuque, well-known to
those of skill in
the art, further provides a ready ability to prepare and test sequence
variants, for example,
incorporating one or more of the foregoing considerations, by introducing one
or more
nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the
production of
mutants through the use of specific oligonucleotide sequences which encode the
DNA
sequence of the desired mutation, as well as a sufficient number of adjacent
nucleotides, to
provide a primer sequence of sufficient size and sequence complexity to form a
stable duplex
on both sides of the deletion junction being traversed. Mutations may be
employed in a
selected polynucleotide sequence to improve, alter, decrease, modify, or
change the properties
of the polynucleotide itself, and/or alter the properties, activity,
composition, stability, or
primary sequence of the encoded polypeptide.
In certain embodiments of the present invention, the inventors contemplate the
mutagenesis of the contemplated cytokine or cytokine receptor-encoding
polynucleotide
sequences to alter the activity or effectiveness of such constructs in
increasing or altering their
therapeutic activity in a transformed host cell. Likewise in certain
embodiments, the inventors
contemplate the mutagenesis of such genes themselves, or of the rAAV delivery
vehicle to
facilitate improved regulation of the particular cytokine or cytokine receptor
polypeptide's
activity, solubility, stability, expression, or efficacy i~ vitro ~~or i~
vivo.
The techniques of site-specific mutagenesis axe well known in the art, and are
widely
used to create variants of both polypeptides and polynucleotides. For example,
site-specific
mutagenesis is often used to alter a specific portion of a DNA molecule. In
such embodiments,
a primer comprising typically about 14 to about 25 nucleotides or so in length
is employed,
with about S to about 10 residues on both sides of the junction of the
sequence being altered.
As will be appreciated by those of skill in the art, site-specific mutagenesis
techniques
have often employed a phage vector that exists in both a single stranded and
double stranded
form. Typical vectors useful in site-directed mutagenesis include vectors such
as the M 13
phage. These phage are readily commercially-available and their use is
generally well-known
to those skilled in the art. Double-stranded plasmids are also routinely
employed in site
directed mutagenesis that eliminates the step of transferring the gene of
interest from a plasmid
to a phage.
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In general, site-directed mutagenesis in accordance herewith is performed by
first
obtaining a single-stranded vector or melting apart of two strands of a double-
stranded vector
that includes within its sequence a DNA sequence that encodes the desired
peptide. An
oligonucleotide primer bearing the desired mutated sequence is prepared,
generally
synthetically. This primer is then annealed with the single-stranded vector,
and subjected to
DNA polymerizing enzymes such as E, coli polymerase I Klenow fragment, in
order to
complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is
formed wherein
one strand encodes the original non-mutated sequence and the second strand
bears the desired
mutation. This heteroduplex vector is then used to transform appropriate
cells, such as E, coli
cells, and clones are selected which include recombinant vectors bearing the
mutated sequence
arrangement.
The preparation of sequence variants of the selected peptide-encoding DNA
segments
using site-directed mutagenesis provides a means of producing potentially
useful species and is
not meant to be limiting as there are other ways in which sequence variants of
peptides and the
DNA sequences encoding them may be obtained. For example, recombinant vectors
encoding
the desired peptide sequence may be treated with mutagenic agents, such as
hydroxylamine, to
obtain sequence variants. Specific details regarding these methods and
protocols are found in
the teachings of Maloy etal.~ 1994; Segal, 1976; Prokop and Bajpai, 1991;
Ruby, 1994; and
Maniatis et al. ~ 1982, each incorporated herein by reference, for that
purpose.
As used herein, the term "oligonucleotide directed mutagenesis procedure"
refers to
template-dependent processes and vector-mediated propagation that result in an
increase in the
concentration of a specific nucleic acid molecule relative to its initial
concentration, or in an
increase in the concentration of a detectable signal, such as amplification.
As used herein, the
term "oligonucleotide directed mutagenesis procedure" is intended to refer to
a process that
involves the template-dependent extension of a primer molecule. The term
template dependent
process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein
the sequence of
the newly synthesized strand of nucleic acid is dictated by the well-known
rules of
complementary base pairing. Typically, vector mediated methodologies involve
the
introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal
amplification
of the vector, and the recovery of the amplified nucleic acid fragment.
Examples of such
methodologies are provided by U. S. Patent No. 4,237,224, specifically
incorporated herein by
reference in its entirety.
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32
A number of template dependent processes are available to amplify the target
sequences of interest present in a sample. One of the best lcnown
amplification methods is the
polymerase chain reaction (PCRTM) which is described in detail in U. S. Patent
Nos. 4,683,195,
4,683,202 and 4,800,159, each of which is incorporated herein by reference in
its entirety.
Briefly, in PCRTM, two primer sequences are prepared which are complementary
to regions on
opposite complementary strands of the target sequence. An excess of
deoxynucleoside
triphosphates is added to a reaction mixture along with a DNA polymerase
(e.g., Taq
" polymerase). If the target sequence is present in a sample, the primers will
bind to the target
and the polymerase will cause the primers to be extended along the target
sequence by adding
on nucleotides. By raising and lowering the temperature of the reaction
mixture, the extended
primers will dissociate from the target to form reaction products, excess
primers will bind to
the target and to the reaction product and the process is repeated. Preferably
reverse
transcription and PCRTM amplification procedure may be performed in order to
quantify the
amount of mRNA amplified. Polymerase chain reaction methodologies are well
known in the
art.
Another method for amplification is the ligase chain reaction (referred to as
LCR),
disclosed in Eur. Pat. Appl. Publ. No. 320,308 (specifically incorporated
herein by reference in
its entirety). In LCR, two complementary probe pairs are prepared, and in the
presence of the
target sequence, each pair will bind to opposite complementary strands of the
target such that
they abut. In the presence of a ligase, the two probe pairs will link to form
a single unit. By
temperature cycling, as in PCRTM, bound ligated units dissociate from the
target and then serve
as "target sequences" for ligation of excess probe pairs. U. S. Patent No.
4,883,750,
incorporated herein by reference in its entirety, describes an alternative
method of
amplification similar to LCR for binding probe pairs to a target sequence.
Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880,
incorporated herein by reference in its entirety, may also be used as still
another amplification
method in the present invention. In this method, a replicative sequence of RNA
that has a
region complementary to that of a target is added to a sample in the presence
of an RNA
polymerase. The polymerase will copy the replicative sequence that can then be
detected.
An isothermal amplification method, in which restriction endonucleases and
ligases are
used to achieve the amplification of target molecules that contain nucleotide
5'-[~,-thio]triphosphates in one strand of a restriction site (Walker et al. ~
1992), may also be
useful in the amplification of nucleic acids in the present invention.
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33
Strand Displacement Amplification (SDA) is another method of carrying out
isothermal amplification of nucleic acids that involves multiple rounds of
strand displacement
and synthesis, i~ e, nick translation. A similar method, called Repair Chain
Reaction (RCR) is
another method of amplification which may be useful in the present invention
and is involves
annealing several probes throughout a region targeted for amplification,
followed by a repair
reaction in which only two of the four bases are present. The other two bases
can be added as
biotinylated derivatives for easy detection. A similar approach is used in
SDA.
Sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a
probe
having a 3' and 5' sequences of non-target DNA and an internal or "middle"
sequence of the
target protein specific RNA is hybridized to DNA which is present in a sample.
Upon
hybridization, the reaction is treated with RNaseH, and the products of the
probe are identified
as distinctive products by generating a signal that is released after
digestion. The original
template is annealed to another cycling probe and the reaction is repeated.
Thus, CPR involves
amplifying a signal generated by hybridization of a probe to a target gene
specific expressed
nucleic acid.
Still other amplification methods described in Great Britain Pat. Appl. No. 2
202 328,
and in PCT Intl. Pat. Appl. Publ. No. PCT/LJS89/01025, each of which is
incorporated herein
by reference in its entirety, may be used in accordance with the present
invention. In the
former application, "modified" primers are used in a PCR-like, template and
enzyme
dependent synthesis. The primers may be modified by labeling with a capture
moiety (e.g
biotin) and/or a detector moiety (e.g. enzyme). In the latter application, an
excess of labeled
probes is added to a sample. In the presence of the target sequence, the probe
binds and is
cleaved catalytically. After cleavage, the target sequence is released intact
to be bound by
excess probe. Cleavage of the labeled probe signals the presence of the target
sequence.
Other nucleic acid amplification procedures include transcription-based
amplification
systems (TAS) (Kwoh et al., 1989; PCT Intl. Pat. Appl. Publ. No. WO 88/10315,
incorporated
herein by reference in its entirety), including nucleic acid sequence based
amplification
(NASBA) and 3 SR. In NASBA, the nucleic acids can be prepared for
amplification by
standard phenol/chloroform extraction, heat denaturation of a sample,
treatment with lysis
buffer and minispin columns for isolation of DNA and RNA or guanidinium
chloride
extraction of RNA. These amplification techniques involve annealing a primer
that has
sequences specific to the target sequence. Following polymerization, DNA/RNA
hybrids are
digested with RNase H while double stranded DNA molecules are heat-denatured
again. In
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34
either case the single stranded DNA is made fully double stranded by addition
of second target-
specific primer, followed by polymerization. The double stranded DNA molecules
are then
multiply transcribed by a polymerase such as T7 or SP6. In an isothermal
cyclic reaction, the
RNAs are reverse transcribed into DNA, and transcribed once again with a
polymerase such as
T7 or SP6. The resulting products, whether truncated or complete, indicate
target-specific
sequences.
Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference in its
entirety,
disclose a nucleic acid amplification process involving cyclically
synthesizing single-stranded
RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in
accordance with the present invention. The ssRNA is a first template for a
first primer
oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent
DNA
polymerase). The RNA is then removed from resulting DNA:RNA duplex by the
action of
ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA
or RNA).
The resultant ssDNA is a second template for a second primer, which also
includes the
sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5'
to its
homology to its template. This primer is then extended by DNA polymerase
(exemplified by
the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a
double-stranded
DNA ("dsDNA") molecule, having a sequence identical to that of the original
RNA between
the primers and having additionally, at one end, a promoter sequence. This
promoter sequence
can be used by the appropriate RNA polymerase to make many RNA copies of the
DNA.
These copies can then re-enter the cycle leading to very swift amplification.
With proper
choice of enzymes, this amplification can be done isothermally without
addition of enzymes at
each cycle. Because of the cyclical nature of this process, the starting
sequence can be chosen
to be in the form of either DNA or RNA.
PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein by reference
in its
entirety, disclose a nucleic acid sequence amplification scheme based on the
hybridization of a
promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by
transcription of many RNA copies of the sequence. This scheme is not cyclic;
i.e. new
templates are not produced from the resultant RNA transcripts. Other
amplification methods
include "RACE" (Frohman, 1990), and "one-sided PCR" (Ohara et al., 1989) which
are well-
known to those of skill in the art.
Methods based on ligation of two (or more) oligonucleotides ill the presence
of nucleic
acid having the sequence of the resulting "di-oligonucleotide", thereby
amplifying the
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di-oligonucleotide (Wu and Dean, 1996, incorporated herein by reference in its
entirety), may
also be used in the amplification of DNA sequences of the present invention.
4.1g BIOLOGICAL FUNCTIONAL EQUIVALENTS
Modification and changes may be made in the structure of the rAAV vector-
delivered
cytokine compositions, or the polynucleotides and/or encoded polypeptides of
the present
invention and still obtain a functional molecule that encodes a cytokine or
cytokine receptor
polypeptide with desirable characteristics. As mentioned above, it is often
desirable to
introduce one or more mutations into a specific polynucleotide sequence. In
certain
circumstances, the resulting encoded polypeptide sequence is altered by this
mutation, .or in
other cases, the sequence of the polypeptide is unchanged by one or more
mutations in the
encoding polynucleotide.
When it is desirable to alter the amino acid sequence of a polypeptide to
create an
equivalent, or even an unproved, second-generation molecule, the amino acid
changes may be
achieved by changing one or more of the codons of the encoding DNA sequence,
according to
Table 3.
For example, certain amino acids may be substituted for other amino acids in a
protein
structure without appreciable loss of interactive binding capacity with
structures such as, for
example, antigen-binding regions of antibodies or binding sites on substrate
molecules. Since
it is the interactive capacity and nature of a protein that defines that
protein's biological
functional activity, certain amino acid sequence substitutions can be made in
a protein
sequence, and, of course, its underlying DNA coding sequence, and nevertheless
obtain a
protein with like properties. It is thus contemplated by the inventors that
various changes may
be made in the peptide sequences of the disclosed compositions, or
corresponding DNA
sequences which encode said peptides without appreciable loss of their
biological utility or
activity.
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TABLE 3
AMINO ACIDS I :ODONS
amne a G G
Cysteine Cys C UGC UGU
Aspartic Asp D GAC GAU
acid
Glutamic Glu E GAA GAG
acid
PhenylalaninePhe F UUC UUU
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine ' Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gln Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
In making such changes, the hydropathic index of amino acids may be
considered. The
importance of the hydropathic amino acid index in conferring interactive
biologic function on a
protein is generally understood in the art (Kyte and Doolittle, 1982,
incorporate herein by
reference). It is accepted that the relative hydropathic character of the
amino acid contributes
to the secondary structure of the resultant protein, which in turn defines the
interaction of the
protein with other molecules, for example, enzymes, substrates, receptors,
DNA, antibodies,
antigens, and the like. Each amino acid has been assigned a hydropathic index
on the basis of
its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
These values are:
isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteinelcystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-
0.8); tryptophan (-
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37
0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.S);
glutamine (-3.S);
aspartate (-3.S); asparagine (-3.S); lysine (-3.9); and arginine (-4.S).
It is known in the art that certain amino acids may be substituted by other
amino acids
having a similar hydropathic index or score and still result in a protein with
similar biological
activity, i. e, still obtain a biological functionally equivalent protein. In
making such changes,
the substitution of amino acids whose hydropatluc indices are within -~-2 is
preferred, those
witlun ~1 are particularly preferred, and those within ~0.S are even more
particularly preferred.
It is also understood in the art that the substitution of like amino acids can
be made effectively
on the basis of hydrophilicity. U. S. Patent 4,SS4,101 (specifically
incorporated herein by
reference in its entirety), states that the greatest local average
hydrophilicity of a protein, as
governed by the hydrophilicity of its adjacent amino acids, correlates with a
biological property
of the protein.
As detailed in U. S. Patent 4,SS4,101, the following hydrophilicity values
have been
assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate
(+3.0 -~- .l); glutamate
(+3.0 ~ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);
threonine (-0.4);
proline (-O.S ~ 1); alanine (-O.S); histidine (-O.S); cysteine (-1.0);
methionine (-1.3); valine (-
l.S); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-
2.S); tryptophan (-3.4).
It is understood that an amino acid can be substituted for another having a
similar
hydrophilicity value and still obtain a biologically equivalent, and in
particular, an
immunologically equivalent protein. In such changes, the substitution of amino
acids whose
hydrophilicity values are within +2 is preferred, those within ~1 are
particularly preferred, and
those within ~0.S are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on
the
relative similarity of the amino acid side-chain substituents, for example,
their hydrophobicity,
hydrophilicity, charge, size, and the Like. Exemplary substitutions that take
various of the
foregoing characteristics into consideration are well known to those of skill
in the art and
include: arginine and lysine; glutamate and aspartate; serine and threonine;
glutamine and
asparagine; and valine, leucine and isoleucine.
4.19 RIBOZYMEs
Although proteins traditionally have been used for catalysis of nucleic acids,
another
class of macromolecules has emerged as useful in this endeavor. Ribozymes are
RNA-
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38
protein complexes that cleave nucleic acids in a site-specific fashion.
Ribozymes have
specific catalytic domains that possess endonuclease activity (I~im and Cech,
1987; Gerlach
et al. ~ 1987; Forster and Symons, 1987). Far example, a large number of
ribozymes
accelerate phosphoester transfer reactions with a high degree of specificity,
often cleaving
only one of several phosphoesters in an oligonucleotide substrate (Cech et
al.~ 1981; Michel
and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been
attributed
to the requirement that the substrate bind via specific base-pairing
interactions to the
internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific
cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al.
~ 1981). For
example, U. S. Patent No. 5,354,855 (specifically incorporated herein by
reference) reports that
certain ribozymes can act as endonucleases with a sequence specificity greater
than that of
known ribonucleases and approaching that of the DNA restriction enzymes. Thus,
sequence-
specific ribozyme-mediated inhibition of gene expression may be particularly
suited to
therapeutic applications (Scanlon et al. ~ 1991; Sarver et al. ~ 1990).
Recently, it was reported
that ribozymes elicited genetic changes in some cells lines to which they were
applied; the
altered genes included the oncogenes H-y~as, c fos and genes of HIV. Most of
this work
involved the modification of a target mRNA, based on a specific mutant codon
that is cleaved
by a specific ribozyme.
Six basic varieties of naturally occurring enzymatic RNAs are known presently.
Each
can catalyze the hydrolysis of RNA phosphodiester bonds i~ ty~ans (~d bus can
cleave other
RNA molecules) under physiological conditions. In general, enzymatic nucleic
acids act by
first binding to a target RNA. Such binding occurs through the target binding
portion of a
enzymatic nucleic acid which is held in close proximity to an enzymatic
portion of the
molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid
first recognizes
and then binds a target RNA through complementary base pairing, and once bound
to the
correct site, acts enzymatically to cut the target RNA. Strategic cleavage of
such a target RNA
will destroy its ability to direct synthesis of an encoded protein. After an
enzymatic nucleic
acid has bound and cleaved its RNA target, it is released from that RNA to
search for another
target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over many technologies,
such as
antisense technology (where a nucleic acid molecule simply binds to a nucleic
acid target to
block its translation) since the concentration of ribozyme necessary to affect
a therapeutic
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39
treatment is lower than that of an antisense oligonucleotide. This advantage
reflects the ability
of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able
to cleave many
molecules of target RNA. In addition, the ribozyme is a highly specific
inlubitor, with the
specificity of inhibition depending not only on the base pairing mechanism of
binding to the
target RNA, but also on the mechanism of target RNA cleavage. Single
mismatches, or base-
substitutions, near the site of cleavage can completely eliminate catalytic
activity of a
ribozyme. Similar mismatches in antisense molecules do not prevent their
action (Woolf ~t al.
1992). Thus, the specificity of action of a ribozyme is greater than that of
an antisense
oligonucleotide binding the same RNA site.
The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a
hepatitis g virus, group I intron or RNaseP RNA (iri association with an RNA
guide sequence)
or Neurospora VS RNA motif. Examples of hammerhead motifs are described by
Rossi et al.
(1992). Examples of hairpin motifs are described by Hampel ~t al. (Eur. Pat.
Appl. Publ. No.
EP 0360257), Hampel and Tritz (1989), Hampel ~t al. (1990) and U. S. Patent
5,631,359
(specifically incorporated herein by reference). An example of the hepatitis g
virus motif is
described by Perrotta and Been (1992); an example of the RNaseP motif is
described by
Guerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif is described
by Collins
(Saville and Collins, 1990; Saville and Collins, 1991; Collins and Olive,
1993); and an
example of the Crroup I intron is described in U. S. Patent 4,987,071
(specifically incorporated
herein by reference). All that is important in an enzymatic nucleic acid
molecule of this
invention is that it has a specific substrate binding site which is
complementary to one or more
of the target gene RNA regions, and that it have nucleotide sequences within
or surrounding
that substrate binding site which impart an RNA cleaving activity to the
molecule. Thus the
ribozyme constructs need not be limited to specific motifs mentioned herein.
In certain embodiments, it may be important to produce enzymatic cleaving
agents that
exhibit a high degree of specificity for the RNA of a desired target, such as
one of the cytokine
or cytokine receptor sequences disclosed herein. The enzymatic nucleic acid
molecule is
preferably targeted to a highly conserved sequence region of a target mRNA.
Such enzymatic
nucleic acid molecules can be delivered exogenously to specific cells as
required, although in
preferred embodiments the ribozymes are expressed from DNA or RNA vectors that
are
delivered to specific cells.
Small enzymatic nucleic acid motifs (e,g, of the hammerhead or the hairpin
structure)
may also be used for exogenous delivery. The simple structure of these
molecules increases
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the ability of the enzymatic nucleic acid to invade targeted regions of the
mRNA structure.
Alternatively, catalytic RNA molecules can be expressed within cells from
eukaryotic
promoters (e.g., Scanlon e~ al., 1991; I~ashani-Sabet et al.~ 1992; Dropulic
et al.~ 1992;
Weerasinghe et al. ~ 1991; Ojwang et al. ~ 1992; Chen et al. ~ 1992; Sarver et
al. ~ 1990). Those
skilled in the art realize that any ribozyme can be expressed in eukaryotic
cells from the
appropriate DNA vector. The activity of such ribozymes can be augmented by
their release
from the primary transcript by a second ribozyme (Int. Pat. Appl. Publ. No. WO
93/23569, and
Int. Pat. Appl. Publ. No. WO 94/02595, both hereby incozpoxated by reference;
Ohkawa et al.
1992; Taira et al.~ 1991; and Ventura e~ al., 1993).
Ribozymes may be added directly, or can be complexed with cationic lipids,
lipid
complexes, packaged within liposomes, or otherwise delivered to target cells.
The RNA or
RNA complexes can be locally administered to relevant tissues ex vivo~ or i~
vivo ~'ough
injection, aerosol inhalation, infusion pump or stmt, with or without their
incorporation in
biopolymers.
Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO
93/23569
and Int. Pat. Appl. Publ. No. WO 94/02595 (each specifically incorporated
herein by reference)
and synthesized to be tested i~ vitro ~d in vivo~ as described. Such ribozymes
can also be
optimized for delivery. While specific examples are provided, those in the art
will recognize
that equivalent RNA targets in other species can be utilized when necessary.
Hammerhead or hairpin ribozymes may be individually analyzed by computer
folding
(Jaeger et al. ~ 1989) to assess whether the ribozyme sequences fold into the
appropriate
secondary structure, as described herein. Those ribozymes with unfavorable
intramolecular
interactions between the binding arms and the catalytic core are eliminated
from consideration.
Varying binding arm lengths can be chosen to optimize activity. Generally, at
least 5 or so
bases on each arm are able to bind to, or otherwise interact with, the target
RNA.
Ribozymes of the hammerhead or hairpin motif may be designed to anneal to
various
sites in the mRNA message, and can be chemically synthesized. The method of
synthesis used
follows the procedure for normal RNA synthesis as described in Usman et al.
(1987) and in
Scaringe et al. (1990) and makes use of common nucleic acid protecting and
coupling groups,
such as dimethoxylTityl at the 5~-end, and phosphoramidites at the 3~-end.
Average stepwise
coupling yields are typically >98%. Hairpin ribozymes may be synthesized in
two parts and
annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992).
Ribozymes may be
modified extensively to enhance stability by modification with nuclease
resistant groups, for
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41
example, 2~-amino, 2~-C-allyl, 2~-flouro, 2~-o-methyl, 2~-H (for a review see
e.g., Usman and
Cedergren, 1992). Ribozymes may be purified by gel electrophoresis using
general methods or
by high-pressure liquid chromatography and resuspended in water.
Ribozyme activity can be optimized by altering the length of the ribozyme
binding
arms, or chemically synthesizing ribozymes with modifications that prevent
their degradation
by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065;
Perreault et ala 1990;
Pieken etal.~ 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO
93/15187; Int.
Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No.92110298.4; U.S.
Patent
5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which describe various
chemical
modifications that can be made to the sugar moieties of enzymatic RNA
molecules),
modifications which enhance their efficacy in cells, and removal of stem II
bases to shorten
RNA synthesis times and reduce chemical requirements.
A preferred means of accumulating high concentrations of a ribozyme(s) within
cells is
to incorporate the ribozyme-encoding sequences into a DNA expression vector.
Transcription
of the ribozyme sequences are driven from a promoter for eukaryotic RNA
polymerase I (pol
I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts
from pol II or pol
III promoters will be expressed at high levels in all cells; the levels of a
given pol II promoter
in a given cell type will depend on the nature of the gene regulatory
sequences (enhancers,
silencers, etc. ) present nearby. Prokaryotic RNA polymerase promoters may
also be used,
providing that the prokaryotic RNA polymerase enzyme is expressed in the
appropriate cells
(Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al. ~ 1993; Zhou
et al. ~ 1990).
Ribozyrnes expressed from such promoters can function in mammalian cells
(Kashani-Sabet
et al. ~ 1992; Ojwang et al. ~ 1992; Chen et al. ~ 1992; Yu et al. ~ 1993;
L'Huillier et al. ~ 1992;
Lisziewicz ~t al. ~ 1993). Although incorporation of the present ribozyme
constructs into
adeno-associated viral vectors is preferred, such transcription units can be
incorporated into a
variety of vectors for introduction into mammalian cells, including but not
restricted to,
plasmid DNA vectors, other viral DNA vectors (such as adenovirus vectors), or
viral RNA
vectors (such as retroviral, semliki forest virus, sindbis virus vectors).
Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes general
methods for
delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by
a variety
of methods known to those familiar to the art, including, but not restricted
to, encapsulation in
liposomes, by iontophoresis, or by incorporation into other vehicles, such as
hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For
some
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42
indications, ribozymes may be directly delivered ex vivo to cells or tissues
with or without the
aforementioned vehicles. Alternatively, the RNA/vehicle combination may be
locally
delivered by direct inhalation, by direct injection or by use of a catheter,
infusion pump or
stmt. Other routes of delivery include, but are not limited to, intravascular,
intramuscular,
subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill
form), topical, systemic,
ocular, intraperitoneal and/or intrathecal delivery. More detailed
descriptions of ribozyme
delivery and administration are provided in Int. Pat. Appl. Publ. No. WO
94/02595 and Int. Pat.
Appl. Publ. No. WO 93/23569, each specifically incorporated herein by
reference.
Ribozymes of this invention may be used to inhibit gene expression and define
the role
(essentially) of specified gene products in.the progression of disease. In
this manner, other
genetic targets may be defined as important mediators of the disease. These
studies lead to
better treatment of the disease progression by affording the possibility of
combination therapies
(e.g, multiple ribozymes targeted to different genes, ribozymes coupled with
known small
molecule inhibitors, or intermittent treatment with combinations of ribozymes
and/or other
chemical or biological molecules).
5.O EXAMPLES
The following examples are included to demonstrate preferred embodiments of
the
invention. It should be appreciated by those of skill in the ar t that the
techniques disclosed in
the examples which follow represent techniques discovered by the inventor to
function well in
the practice of the invention, and thus can be considered to constitute
preferred modes for its
practice. However, those of skill in the art should, in light of the present
disclosure, appreciate
that many changes can be made in the specific embodiments which are disclosed
and still
obtain a like or similar result without departing from the spirit and scope of
the invention.
S.1 EXAMPLE 1 - LONG-TERM CORRECTION OF OBESITY USING CENTRALLY-
DELIVERED RAAV ENCODING ANOREXIGENIC CYTOKTNES
This example describes the construction of a series of rAAV vectors encoding
naturally-occurring anorexigenic cytokines that have been developed for the
purpose of long-
term body weight (BW and food intake (FI) regulation in obese leptin-resistant
patients.
Using these vectors it has been shown that rAAV constructs encoding the
anorexigenic
hormone leptin was efficient in reducing B W and FI in lean as well as in
genetically obese
animal models after intramuscular (iyn), intravenous (iv) or
intracerebroventricular (icv)
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43
injections. It has also been shown that rAAV encoding cytolcines CNTF and
leukemia factor
(LIF) were efficient in maintaining lower BW in normal rodents for a prolonged
period of
time. Lilcewise, it has been shown that rAAV encoding these cytolcines rAAV
reduced the rate
of BW gain up to 15% for 5 months (the duration of the study) after single icv
llijection of
rAAV in leptin-resistant genetically obese Zuker f~fa rats. No side or ill
effects were
encountered during the course of these studies.
These data demonstrate that tonic expression of naturally occurnng
anorexigenic
cytokine delivered by rAAV is an efficient mechanism for the regulation of BW
and obesity in
mammals, and particularly in humans.
5.2 EXAMPLE Z - SEXUAL DIMORPHISM IN THE RESPONSE TO LEPTIN GENE THERAPY
~rIA AN RAAV VECTOR-BASED DELIVERY SYSTEM
This example describes the efficacy of gene therapy with rAAV carrying rat
leptin
cDNA (rAAV-Leptin) in reducing BW of male and female Sprague Dawley rats. The
rAAV
construct consisted of rat leptin cDNA driven by a hybrid promoter containing
the chicken ~-
actin promoter and CMV enhancer in a rAAV vector.
Adult male and female rats (250-300 g body weight [BW]) were implanted with a
permanent injection cannula in the third cerebroventricle. Following post
surgical recovery, 8
male and 8 female rats were injected with 5 ~l of rAAV-leptin (1013
particles/ml). Control rats
(n=6) each were injected iw with 5 p1 of rAAV control virus. BW and 24-hour
food intake
were monitored weekly. At two weeks post injection there was a significant
decrease in BW in
female rates vs. their initial BS (232.4 ~ 5.8 vs. 250.8 ~ 4.6 g. p< 0.05) and
also vs controls
(264.3 ~ 4.5 g, p < 0.010). This decrease was maintained for the 12-week
duration of the
study. In contrast, whereas control male rats steadily gained BW, rAAV-leptin
injected rats
maintained their initial BW. A significantly lower BW first apparent at 4
weeks (330 ~ 7 g vs.
368 ~ 11 g, p < 0.05) was maintained for 9 weeks. Interestingly, there was no
decrease in the
daily food intake or either males or females vs. control rats. Serum leptin
levels in female rats
were significantly reduced in rAAV-leptin treated vs. control rats (0.8 ~ 0.05
vs. 2.3 ~ 0.3
ng/ml, p < 0.05).
Because leptin was believed to regulate BW in a central fashion, NPY and AGRP
gene
expression was examined in the hypothalami of female rats by i~-situ
hybridization. Results
showed no differences in the relative expression of these orexigenic signals
in rAAV-injected
rats vs. control rats. In summary, central administration of leptin cDNA in a
high titer rAAV
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44
vector has a sex-specific effect on the pattern of decrease of BS concomitant
with normal food
intalce. These studies demonstrate the utility of leptin gene therapy in the
treatment of obesity
and maintenance of lowered body weight over an extended period of time via
administration of
5.3 EXAMPLE 3 -- AN INTERACTING APPETITE REGULATING NETWORK, BODY
WEIGHT REGULATION AND GENE THERAPY
An interactive appetite-regulating network (ARN) composed of orexigenic and
anorexigenic peptides is the primary neural substrate in hypothalamic control
of BW.
Peripheral signals (e.g cytokines) acting through NPY ergic signaling in the
ARN regulate
food intake, energy expenditure and metabolism (Yokosuka et al., 1999).
This example illustrates the utility of rAAV-mediated gene therapy to deliver
naturally-
occurring anorexigenic cytokines (e.g. leptin and ciliary neurotropic factor,
CNTF) in
suppressing BW gain in normal and genetically obese animals. The results
showed that a
single iv injection of adeno-associated virus (rAAV) encoding leptin cDNA
decreased BW and
food intake in leptin-deficient oblob mice. In out-bred Sprague-Dawley rats,
rAAV-leptin
delivered icv curbed the rate of BW gain and decreased plasma leptin levels
without any
appreciable effect on food intake for at least 12 weeks. In leptin receptor
mutant obese Zucker
rats (fa/fa), secretable CNTF in rAAV delivered icv significantly reduced the
rate of BW gain
and decreased average daily food intake over a period of 14 weeks.
In summary, these results indicate that the modulation of ARN by anorectic
cytokines
delivered centrally or peripherally by one or more of the rAAV vectors of the
present invention
provide a means for controlling BW in a mammal.
5.4 EXAMPLE 4 -- CONTROL OF BODY WEIGHT IN LEAN AND OBESE RODENT
MODELS USING CENTRALLY DELIVERED RECOMBINANT ADENO-ASSOCIATED
VIRUS (RAAV~ VECTORS ENCODING CYTOKINES LEPTIN, CNTF AND LIF
This example describes the long-term anorexigenic effect of the hormone leptin
and the .
neurocytokines CNTF and LIF that bind to the IL-6 receptor superfamily. A
series of rAAV
vectors encoding rat leptin hum LIF and sCNTF (an engineered secreted form)
cDNAs were
successfully generated. When administered intracerebroventricularly (icv) each
of these
vectors curtailed BW gain for extended periods of time in normal rats and in
genetically obese
Zucker (fa/fa) rats. In the rAAV-leptin treated lean rats, the reduction in BW
resulted from
loss of white adipose tissue concomitant with a reduction of blood leptin
levels in both males
and females; the lean body mass of rAAV-leptin treated animals was essentially
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indistinguishable from control. Fat mass depletion of rAAV-leptin treated rats
as not related to
food intake. It apparently resulted from increased energy expenditure through
thermogenesis
since there was a marked increase in uncoupling protein I (UCPI) mRNA
expression in the
brown adipose tissue of these animals. To explore the efficacy of putative
leptin substitutes for
the treatment of genetically obese rodent models of leptin resistance rAAV
vectors encoding
the cytokines CNTF or LIF cDNAs were administered icv to falfa tucker rats,
and to lean rats.
These vectors exerted long-term effects resulting in sustained significant
weight loss for up to
20 weeks post injection. The postreceptor signal transduction pathways
activated by rAAV-
leptin, rAAV-CNTF and rAAV-LIF appeared to be similar. These results
demonstrate that
gene therapy to centrally deliver cytokines has long-term therapeutic benefits
for controlling
BW in lean and obese mammals.
5.5 EXAMPLE S -- ACTIVATION OF STAT-DEPENDENT SIGNALING IN RAT
HYPOTHALAMUS BY LEPTIN~ CNTF AND LIF TRANSGENES DELIVERED
CENTRALLY ylA RAAV VECTORS
Studies have shown that related cytokines such an CNTF and LIF that also bind
to the
IL-6 receptor superfamily, are anorexigenic and share common intracellular
signaling
pathways. To investigate whether this cormnonality of biological effects can
be gainfully
employed to regular body weight (BW), the effects of centrally administered
rAAV vectors
encoding leptin, CNTF and LIF cDNAs were examined. This example describes the
results
from a series of studies that showed a single injection of rAAV encoding these
cytokines
resulted in a significant weight loss for extended periods of time. To study
the intracellular
mechanism, the early steps of postreceptor signaling activated in the
hypothalamus were
characterized after a intracerebroventricular (icv) injection of (1)
recombinant leptin, CNTF or
LIF peptides, and (2) recombinant rAAV vectors encoding the cognate genes. The
peptides
induced fast (15 to 45 min) activation of STAT-3 by phosphorylation of Tyr705.
On the other
hand, CNTF and LIF, but not leptin also induced STAT-I phosphorylation on
Tyr701. None of
these cytokines induced STAT-3 . phosphorylation on Ser727. The postreceptor
phosphorylation of STAT-3 was also markedly enhanced after administration of
rAAV vectors
at the time of first indication of significant BW loss. Thus, leptin and the
related cytokines
CNTF and LIF phosphorylate STAT-3 acutely after an icv injection and this
activation is
apparently sustained over extended time periods as seen after centrally
administered rAAV
vectors encoding these cytokines.
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46
5.6 EXAMPLE C -- LEPTIN GENE THERAPY REDUCES FOOD INTAKE AND BODY
WEIGHT WITH AFFECTING ONSET OF PUBERTY AND REPRODUCTIVE CYCLES
The example describes an rAAV genetic construct encoding leptin (rAAV-Ob) that
was injected intracerebroventricularly (6 X 1010 particles in 3 ~,1) into 24
day-old female
Sprague-Dawley rats. The control groups consisted of (a) rats injected icv
with rAAV-UFS
(control vector) and (b) unoperated, untreated rats. Rats were maintained on
ad-libitum rat
chow and water. Food intake and body weight were monitored for 6 months. The
results
showed that during the pubertal period through day 44, rAAV-lep-bOb-treated
rats consumed
26.9% less and 22.8% less during the post-pubertal period through day 180.
Body weight gain
was accordingly reduced in these rats, (rAAV-Ob: 114.3 ~ 9.2 g; rAAV-UFS:
206.0 -~-. 4.1 g).
Despite this marked suppression of caloric intake and weight gain, the age at
vaginal opening
of rAAV-Ob-treated rats was not different from that of control rats (rAAV-Ob:
39.9 ~ 0.6
days; rAAV-UFS: 39.0 ~ 0.9 days; untreated control: 38.6 ~ 0.8 days). To
examine the effects
on estrous cycles, vaginal smears were recorded between days 54-70. All groups
exhibited a
similar number of estrous cycles of 4-5 day duration (rAAV-Ob: 3.1 ~ 0.2; rAAV-
UFS: 2.9 ~
0.2; untreated controls: 2.4 ~ 0.2). These results show that leptin gene
therapy admiiustered
prepubertally suppressed food intake and weight gain without altering either
the onset of
puberty or reproductive cycles in adult life. Consequently, centrally
administered leptin gene
therapy is a viable therapy to diminish appetite and maintain a lean phenotype
for long periods
without disrupting the neuroendocrine control of puberty and reproductive
cycles.
5.7 EXAMPLE 7 -- LONG-TERM SUPPRESSION OF WEIGHT GAIN AND OBESITY BY
RAAV-LIF THERAPY
Leptin normally regulates body weight by feedback actions on the hypothalamic
network that regulates appetite and energy expenditure. However, development
of age-related
leptin resistance disrupts the intricate balance thereby leading to excessive
weight gain and
obesity, a major health problem in the United States. A similar age-related
pattern of steady
weight gain in association with leptin resistance is observed in laboratory
rats. This example
demonstrates that by circumventing leptin resistance yia delivery of genes
encoding naturally
occurring anorectic and weight reduced peptides directly into the
hypothalamus, it is possible
to ameliorate the age-related weight gain and obesity. Leukemia inhibitor
factor (LIF), a
potent anorexigenic cytokine, is present in the hypothalamus and LIF receptor
mRNA is
expressed in hypothalamic sites previously implicated in weight regulation.
rAAV vectors
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encoding human LIF injected once intracerebroventricularly (icv, 101 particles
in 5 ~1) into the
adult wild-type female rats completely blocked the weight gain in short-term
(6-week) and
long-term (20-week) studies without affecting caloric consumption. In fact,
weight was
maintained at pre-inj ection range for the duration of the study. This
suppression of weight gain
resulted from blockage of fat deposition as indicated by significantly reduced
blood leptin and
insulin concentrations. Further, administration of rAA.V-LIF twice (l0ln
particles/injections), 2
days apart, was more effective because there was a significant reduction in
daily caloric intake
along with weight loss and drastic reduction in adiposity, as reflected by low
circulating leptin
arid insulin levels in these rats. Therefore, it has been shown that LIF acts
as a novel
hypothalamic anorexigenic signal that can be delivered locally into the
hypothalamus of adult
rats via rAAV vectors to suppress the age-related weight gain and obesity for
extended periods,
and that the two underlying crucial physiological processes in weight
regulation, caloric
consumption and energy expenditure, can be differentially maupulated in a dose-
dependent
manner; low rAAV-LIF titres selectively block weight gain but high rAAV-LIF
titres suppress
both weight gain and daily caloric consumption. Consequently, central delivery
of rAAV-LIF
is an efficient therapy to overcome age-related adverse consequences of leptin-
resistance on
weight and adiposity.
S.8 EXAMPLE S - RAAV-CNTF THERAPY RESCUES RETINITIS PIGMINTOSA
This example describes the ability of CNTF gene therapy to rescue or delay PR
loss
in the rds +/- P216L transgenic mouse. Retinas in this animal degenerate due
to a
combination of a dominant P216L rds transgene and rds +/- haploinsufficiency,
thus
mimicking rds-caused human RP. An improved version of human CNTF (DH-CNTF)
containing 166D and 167H mutations knov~m to enhance its affinity for CNTFRa,
was
created, thus increasing human CNTF's stability and potency. Four rAAV vectors
were
tested, two with the CMV promoter (secreted sDH-CNTF and nonsecreted DH-CNTF,
defined by the presence or lack of a human growth hormone secretory signal)
and two (DH-
CNTF and sDh-CNTF) with the mops500 rod opsin promoter (MOPS) that also
supports
expression efficiently and specifically in rodent rods. The CMV promoter
supports
expression well in RPE cells and moderately in PR's when injected into the
subretinal
space. Rescue was initially identified by retinal morphology, expressed as ONL
thickness
along a full vertical meridian through the optic nerve head (ONH) at P90, 75
days post
inj ection.
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The results clearly demonstrate that reproducible photoreceptor survival
occurred
only when the secretable form of CNTF (sDH-CNTF) was expressed from the CMV
promoter (p<0.000004). More limited data on 4 animals shows that the CMV-sDH-
CNTF
rescue effect persists fox at least 4 months after injection (p<0.0025). Since
both CMV and
MOPS promoters drive expression in rods, but only CMV promoter supports RPE
expression, rod expression of CNTF, whether secretable or not, is ineffective.
This might
suggest that rods do not contain the necessary CNTFRa receptor necessary for
efficient
CNTF interaction. Alternatively (but not excluding the first possibility), rod
survival
mediated by CNTF may require a CNTF-induced RPE trophic factor. Aspects of
both
scenarios are being tested.
S.9 EXAMPLE 9 -- INTRATHECAL ADMINISTRATION OF RAAV-CYTOKINE
CONSTRUCTS
While the rAAV constructs of the present invention are most directly
administered
via central administration, peripheral delivery of the vector constructs may
exploit the
natural properties of cytokines such as LIF to decrease fat storage in
adipocytes locally via
inhibition of lipoprotein lipase (LPL) activity, decreasing lipogenesis and
stimulating
lipolysis. This example describes the intrathecal (it) injection of the rAAV-
cytokine
constructs directly into cerebrospinal fluid (CSF), to provide an efficient
modality of
delivery that by-passes the blood-brain barrier and direct transduction of
brain tissues. It
has been shown that it-administered rAAV encoding ~-glucuronidase (GUS)
resulted in a
complete elimination of the storage granules in the brain of a mouse model of
the
mucopolysaccharidosis type VII (MPS VII). Moreover, an it route of
administration is
relatively benign, and has been routinely used in clinical settings. As such,
the inventors
contemplate the use of it administration routes for delivery of the rAAV-
cytokine constructs
disclosed herein. Alternatively, the use of traditional modes of delivery,
such as those
discussed Supra, may be contemplated for administration of particular
constructs depending
upon the particular therapeutic benefit to be achieved.
S.IO EXAMPLE 10 -- CONSTRUCTION OF RAAV VECTORS
Several rAAV vectors have been constructed for use in the methods described
herein.
pTR-sCNTF encodes a secreted form of human CNTF. To construct this vector a
synthetic
sequence coding for human growth hormone (hGH) signal peptide was fused in-
frame to the
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coding sequence of CNTV cDNA. pTR-CBA-Ob encodes rat leptin cDNA under the
control
of chicken ~-actin promoter linked to CMV enhancer (CBA). hLIF-containing
constructs
encode human LIF cDNA, cloned and sequenced by a PCR-mediated protocol from
LIF-
expressing melanoma cell line G-361. In some vectors two transgenes are linked
within
dicistronic cassettes through an IRES element for coordinate expression. All
vectors contain
AAV terminal repeats at both sides of the cassette to mediate replication and
packaging of the
vector.
5.11 EXAMPLE 11 -- REGULATION OF BODY WEIGHT BY RAAV-OB, RAAV-SCNTF
AND RAAV-HLIF VECTORS
It has been recently shown that a single i~,2 injection of rAAV encoding mouse
leptin in
mice deficient for this hormone (oblob) leads to prevention of obesity and
diabetes. To design
a positive control vector rAAV encoding rat leptin cDNA was constructed (pTR-
CBA-Ob).
Initially, injection of 1011 particles of this vector has been shown to be
more effcient upon iv
injection of rAAV-Ob into oblob mice. The same vector administered iw in leaiz
rats, resulted
in a long-term statistically significant reduction of normal BW gain in both
males and females.
The dramatic reduction of BW resulted from the loss of adipose tissue. In
spite of that,
the rAAV-Ob-treated animals remained fit and healthy for the duration of the
experiment. The
protein content of rAAV-Ob-treated rats was essentially indistinguishable from
the control.
To evaluate CNTF as an anorexigenic agent in the context of rAAV vector rAAV-
sCNTF has been tested in lean aamals. It was shown that a single icv injection
of lOlo
physical particles of this vector resulted in long-term BW maintenance in lean
female rats.
Leptin-resistant f~fa Zucker rats have also shown a statistically significant
reduction iiz BW
upon single icv injection of this vector. Mutant forms of CNTF (DH-CNTF) have
also been
constructed that have a higher affinity towards forms of CNTF receptor, as
well as CNTF-
lamtin W cinn rnnetn~rtc Tn arlraitinn it hac he r n chn~am that tha
r~rtnl~ina T TT'-, a mamhar nf+ha
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showed that hLIF was as potent as leptin in this study. In order to identify
cells transduced
with rAAV-CBA-hLIF, a GFP reporter gene was placed within the same transgene
cassette. In
a separate study, the GFP reporter gene linked to the hLIF through an IRES
element in one
dicistronic transcription unit was also tested. rAAV-CBA-hLIF was injected
into spinal cord
of a SD rat. One month post-injection the spinal cord was processed, and
neurons transduced
with rAAV-CBA-hLIF were easily identifiable proving the functional reliability
of the GFP
reporter gene. The glial cells (oligodendrocytes and astrocytes) have been
transduced with
rAAV-CBA-hL,IF at about 100 times lower rate as compared to neurons.
5.12 EXAMPLE 12 -- LEPTIN, CNTF AND LIF INITIATE SIMILAR DOWNSTREAM SIGNAL
TRANSDUCTION CASCADE IN HYPOTHALAMUS
Intraperitoneally injected recombinant CNTF and Ieptin have been previously
shown to
activate a similar pattern of STAT factors in hypothalamic satiety centers. On
the other hand,
it was shown that recombinant leptin administered iv in rats activates STAT-3
phosphorylation,
but fails to activate either Jak proteins, or STAT-1 and STAT-5, or even
mitogen-activated
protein (MAP) kinase iii the hypothalamus. Therefore, leptin signaling differs
from that of
other ligands that bind members of this class of receptor by failing to
stimulate. measurable
responses in any of the known upstream signal transduction proteins.
rAAV vectors encoding Iipostatic hormone leptin, neurocytokines CNTF and LIF
exert
strong anorexigenic effect if applied peripherally (rAAV-CBA-Ob), or centrally
in genetically
obese (rAAV-CBA-OB in oblob ice and rAAV-CNTF in falfa rats), as well as in
lean SD
rats (all three vectors). Although leptin, CNTF and LIF ligands all interact
with different
specific receptors, they all activate similar downstream of Jak STAT-3
signaling cascases
resulting in loss of BW. Jaks are tyrosine kinases that associate with
cytokine and leptin
receptors. Upon ligand binding, they activate members of the STAT family
through
phosphorylation on a single tyrosine. Activated STATs form dimers, translocate
to the
nucleus, bind to specific response elements in promoters of target genes, and
transcriptionally
activate these genes. In the case of leptin signaling, the early transduction
events preceding
STAT-3 phosphorylation, have not been identified yet.
To characterize the signaling mechanisms and establish their similarity lean
rats were
inj ected icv with recombinant leptin, CNTF and LIF and analyzed the
activation of the pathway
through STAT-3 signaling in hypothalamus and pituitary. Although the levels of
non-
phosphorylated-, or serine-phosphorylated STAT-3 remained unchanged, the
tyrosine-
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phosphorylated STAT-3 increased in leptin-injected rats and even more so in
CNTF-injected
animals. The same observation was also true for pituitary. It is conceivable,
though, that the
difference in leptin-induced activation pattern of STAT-3 vs. those induced
with CNTF, or LIF
is due to the difference in the up-stream signaling and, as a result, to the
timing of STAT-3
phosphorylation. For example, leptin causes tyrosine phosphorylation of STAT-3
in the
hypothalamus as early as 5 min. after injection and reaches maximum at 30 min.
These data
were obtained at 45 min. post administration, by which time the leptin-
iilduced activation
might have subsided. Consistent with this assumption are data obtained by Li
and Friedman,
who isolated an SH2 domain containing protein tyrosine phosphatase 2 (SHP-2)
that binds
Tyr985 of the leptin receptor. Tyrosyl phosphorylation of SHP-2 i~ vitro was
associated with
decreased phosphorylation of Jak-2 but not Ob-Rb or STAT-3. These data suggest
that SHP-2
is a component of the leptin signal transduction pathway and it may terminate
leptin signaling
by decreasing the level of phosphorylation of Jak-2.
Likewise, the i~l, administration of human recombinant LIF activated the STAT-
3
pathway in hypothalamus through the phosphorylation of the tyrosine residue as
early as 15
min. post-injection. Similarly to leptin and CNTF, this activation was
specific since the serine
residue phosphorylation remained unchanged.
These data demonstrate that recombinant leptin, CNTF and LIF activate the STAT-
3
signal transduction cascade, albeit with a different efficiencies (or timing).
5.13 EXAMPLE 13 -- RAAV VECTORS ENCODING TRANSGENES INDUCIBLE WITH DOX
This example describes the use of the tet-inducible system in brain using a
combination
of two rAAV vectors: a) rAAV-tetR-hLIF encoding hLIF under the control of the
tet-inducible
promoter CMVm~ntetO~; b) rAAV-rtTA/tTS encoding transcription transactivator
and
txanscriptional silencer under the control of a strong constitutive CBA
promoter. There are
several lines of evidence indicating that such an approach will be successful.
Recently
Haberman et al. (Habennan et al., 1998) have demonstrated the long-term gene
expression in
rat brain of a GFP reporter gene under the control of tet-responsive system
and the tet-
transactivator. 1~ vivo s~dies, carried out for 6 weeks, demonstrated that
rAAV vector-
mediated expression is sustained until Dox administration upon which reporter
gene expression
is reduced (Habennan et al., 1998). However, the tet construct design used by
the authors is
always characterized by a background leaky transcription from the CMVmintetO~
promoter,
even under conditions of no induction with the effector. On the other hand,
delivering a
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transcription suppressor (silencer) that binds to the tet-responsive promoter
in the absence of
the effector Dox eliminates the leakiness of transcription. Unfortunately, the
packaging size
limitation of rAAV does not allow one to fit all four genes (hLIF, GFP, rtTA
and tTS) into one
vector. Therefore a double vector paradigm that recently was successfully
demonstrated by
Rendahl et al. (1990 may be employed for rAAV vectors.
Precise control over gene expression is an important issue both in gene
therapy
protocols and for the exploration of gene function. One of the most elegant
regulatable
promoter systems in use today, developed by Gossen and co-workers, involves
the E coli
tetracycline (tet)repressor fused with the C-terminal domain eulcaryotic
transcriptional activator
VP16. This tet-regulatable transcription factor (tTA) specifically ~.ahs-
activates artificial
"minimal" promoters carrying multiple tet operator sites (tet07). Additionally
tet to the system
reversibly inhibits binding of tTA to promoter DNA and blocks gene expression.
A variation
of the original system involves a tTA derivative that carries four point
mutations, (reverse tet-
regulatable ~.ahs-activator, rtTA), which does not tya~s activate in the
default state, but
requires a tet-like compound (doxycycline, Dox) for gene activation. However,
the drawback
of these systems is that tTA and rtTA both display basal leaky expression of
the transgene. A
recent improvement of the tet system incorporates a tet-controlled ~.ahs-
repressor to silence the
Ieaky transcriptional activity of the eukaryotic promoters that are stably
integrated into the
chromatin of human cells. By fusing the KRAB domain of human Koxl to the E.
coli tet
repressor, a tet-controlled hybrid protein (TetR-KRAB, or rTS) was generated.
The rTS binds
to the tet07 operator sequence in the absence but not in the presence of tet.
Therefore,
combining rtTA and tTS genes into one system provides mutually exclusive
repression/induction of tet07 promoter-driven gene. In the absence of Dox
complete
transcription shut-off is mediated by the tTS bound to the tet07. Upon adding
Dox at
concentrations higher than lOng/ml, tTS is displaced by the rtTA trans-
activator and
transcription is induced.
In order to~ exploit rtTA/tTS system the vector rAAV-rtTA/tTS encoding two
chimeric
genes rtTA and tTS was constructed. Expression of both genes linked by IRES
element into
one dicistronic cassette, is mediated by a constitutive CBA promoter. The CBA
promoter is
active in all cell types tested, including neurons.
To test this vector a transient transfection study was performed using HEK292
cells,
with a destabilized GFP reporter gene driven by the CMVm~ntetO~ promoter (pTRE-
dEGFP,
Clontech). This study clearly demonstrates that the combination of two rAAV
transfer vectors,
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one carrying transgene under the control of CMVm~ntet0~ promoter, the other a
combination of
transcription activator and repressor driven by the strong housekeeping
promoter, provides
tight control over expression using Dox. This Dox-inducible system is very
useful for a
temporal conditional expression of transgene, whose product exerts a strong
biological effect
potentially leading to side effects. Therefore an rAAV vector expressing hLIF
under control of
the inducible CMVmintet0~ promoter (pTR-tetR-hLIF) may provide particular
advantages upon
icv injection in the methods disclosed herein.
5.14 EXAMPLE t4 -- PRODUCTION OF RAAV VECTORS
Txaditional protocols to produce rAAV vectors have generally been based on a
three-
component system. One component of tlus system is a proviral plasmid encoding
the
recombinant DNA to be packaged as rAAV. This recombinant DNA is located
between 145
base pair (bp) AAV-2 inverted terminal repeats (ITRs) that are the minimal His
acting AAV-2
sequences that direct replication and packaging of the vector. A second
component of the
system is a plasmid encoding the AAV-2 genes, j.ep and cad. The AAV-2 y.ep
gene encodes
four Rep proteins (Rep 78, 68, 52 and 40) that act in ty.ay~s to replicate the
rAAV genome,
resolve replicative intermediates, and then package single-stranded rAAV
genomes. The
AAV-2 yap gene encodes the three structural proteins (VPl, VP2, and VP3) that
comprise the
virus capsid. Because AAV-2 does not proficiently replicate on its own, the
third component
of a rAAV packaging system is a set of helper functions from another DNA
virus. These
helper functions create a cellular environment in which rAAV replication and
packaging can
efficiently occur. The helper functions provided by adenovirus (Ad) have
almost exclusively
been used to produce rAAV and are encoded by the genes Ela, Elb, E2a, E4orf6,
and VA
RNA. While the first two components of the system are generally introduced
into cells in
which replication and packaging is to occur by transfection, ad helper
functions are introduced
by superinfection with wild type Ad virus.
The traditional rAAV production techniques are limited in their ability to
produce large
quantities of vector because of inherent inefficiencies in transfection.
Serious difficulties are
also encountered when the scale of transfection is increased. 'The requirement
for wild type Ad
may also reduce the amount of rAAV produced since Ad may compete for cellular
and viral
substrates that are required for viral replication but are present only in
limiting amounts.
Another problem encountered in traditional production protocols is that
superinfection with Ad
requires development of effective procedures for purification of Ad from the
rAAV produced.
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While these purification processes are generally successful at eliminating Ad
contamination of
rAAV preparations, they also reduce rAAV titers. Stringent assays for Ad
contamination of
rAAV are also necessary.
To produce rAAV, a double co-transfection procedure is used to introduce a
rAAV
transfer vector plasmid together with pDG (Grimm et al., 1998) AAV helper
plasmid carrying
the AAV rep and cap genes, as well as Ad helper genes required for rAAV
replication and
packaging at a 1:1 molar ratio. Plasmid DNA used in the transfection is
purified by a
conventional alkaline lysis/CsCI gradient protocol. The transfection is
carried out as follows:
293 cells are split 1:2 the day prior to the experiment, so that, when
transfected, the cell
confluence is about 75-80%. Ten 15-cm plates are transfected as one batch. To
make CaP04
precipitate 0.7 mg of pDG are mixed with 180 ~g of rAAV transfer vector
plasmid in a total
volume of 12.5 ml of 0.25 M CaCl2. The old media is removed from the cells and
the
formation of the CaP04 precipitate is initiated by adding I2/5 ml of 2xHBS pH
7.05 (pre-
warmed at 37°C) to the DNA-CaCl2 solution. The DNA is incubated for 1
min; and
transferring the mixture into pre-warmed 200 ml of DMEM-10% FBS then stops the
formation
of the precipitate. Twenty two ml of the media is immediately dispensed into
each plate and
cells are incubated at 37°C for 48 hrs. The CaP04 precipitate is
allowed to stay on the cells
during the whole incubation period without compromising cell viability. Forty-
eight hr post-
transfection cells are harvested by centrifugation at 1,140 x g for 10 min.
Cells are then lysed
in 15 ml of 0.15 m MaCl, 50 mM tris HCl pH 8.5 by 3 freeze/thaw cycles in dry
ice-ethanol
and 37°C baths. Benzonase (Nycomed Pharma A/S, pure grade) is added to
the mixture (50
U/ml, final concentration) and the lysate is incubated for 30 min at
37°C. The lysate is
clarified by centrifugation at 3,700 g for 20 min and the virus-containing
supernatant is further
purified using a discontinuous density gradient.
The typical discontinuous step gradient is formed by underlayering and
displacing the
less dense cell lysate with Iodixanol, 5,5"[(2-hydroxi-1-3-propanediyl)-
bis(acetylamino)] bis
[N,N'bi ,(2,3dihydroxypropyl-2-4,6-triiodo-1,3-enzenecarboxamide], prepared
using a 60%
(wt./vol.) sterile solution of OptiPrep (Nycomed). Specifically, 15 ml of the
clarified lysate are
transferred into Quick-Seal Ultra-Clear 25 x 89 mm centrifuge tube (Beckman)
using a syxinge
equipped with 1/27 x 89 mm spinal needle. Care is taken to avoid bubbles,
which would
interfere with subsequent filling and sealing of the tube. A variable speed
peristaltic pump,
Model EP-1 (Bio-Rad), is used to underlay in order: 9 ml of I S% iodixanol and
1 M NaCl in
PBS-MIA buffer containing Phenol Red (2.5 q,1 of a 0.5% stock solution per ml
of the iodixanol
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solution); 5 ml of 40% iodixanol in PBS-MK buffer; and finally, 5 ml of 60%
iodixanol in
PBS-MK buffer containing Phenol Red (0.1 ~l/1). Tubes are sealed and
centrifuged in a Type
70 Ti rotor (Beckman) at 350,000 x g for 1 hr at 18°C. Four ml of the
clear 40% step is
aspirated after puncturing the tube on the side with a syringe equipped with
an 18-gauge needle
with the bevel uppermost. The iodixanol fraction is further purified using
conventional
Heparin agarose affinity chromatography.
For chromatography, typically, a pre-packed 2.5 ml Heparin agarose Type I
cohunn
(Sigma) is equilibrated with 20 ml of PBS-MK under gravity. The rAAV iodixanol
fraction is
then applied to the pre-equilibrated column, and the column is washed with 10
ml of PBS-MK.
rAAV is eluted with the same buffer containing 1M NaCI. After applying the
elution buffer,
the first 2 ml of the eluant are discarded, and the virus is collected in the
subsequent 3.5 ml of
elution buffer.
Virus is then concentrated and desalted by centrifugation through the BIOMAX
100 K
filter (Millipore) according to the manufacturer instructions. The high salt
buffer is changed by
repeatedly diluting the concentrated virus with the Lactated Ringer's solution
and repeating the
centrifugation.
To characterize the quality of the virus, two assays are used to titer both
physical and
infectious rAAV particles. A conventional dot-blot assay or quantitative
competitive PCRTM
(QR PCRTM) assay are used to determine physical particle titers. Infectious
titers are
determined by infectious center assay (ICA) and fluorescent cell assay (FCA),
wluch scores for
expression of GFP.
QC PCRTM method is based on competitive co-amplified of a specific target
sequence
with internal standard plasmid of known concentration in on reaction tube. It
provides precise
and fast quantitation of viral particles. The internal standard must hare
primer recognition sites
with the specific template. Both the specific template and the internal
standard must be
PCRTM-amplified with the same efficiency and it must be possible to analyze
the PCRTM-
amplified products separately. The easiest way to distinguish between the
template and the
internal standard is to incorporate a size difference in the two products.
This can be achieved,
for example, by constructing standards having the same sequence as the
specific target but
containing a deletion. Quantitation is then performed by comparing the PCRTM
signal of the
specific template with the PCRrM signal obtained with known concentrations of
the competitor
(the internal standard).
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The purified viral stock is first treated with DNAseI to digest any
contaminating
unpaclcaged DNA. Ten ~,l of a purified virus stock is incubated with 10 U of
DNA se I
(Boehringer) in a 100 ~1 reaction mixture, containing 50 mM Tris HCI, pH 7.5,
10 mM MgCl2
for 1 hr at 37°C. At the end of the reaction, 10 ~1 of l OX Protinase
I~ buffer (10 mM Tris HCI,
pH 8.0, 10 mM EDTA, 1% SDS final concentration) is added, followed by the
addition of 1 ~l
of Proteinase I~ (18.6 mg/ml, Boehringer). The mixture is incubated at
37°C for one hour.
Viral DNA is purified by phenol/chloroform extraction (twice), followed by
chloroform
extraction and ethanol precipitation using 10 ~g of glycogen as a carrier. The
DNA pellet is
dissolved in 100 ~1 of water. QC PCRTM reaction mixtures each contains 1 ~l of
the diluted
viral DNA and two-fold serial dilutions of the internal standard plasmid DNA,
such as pdl-
GFP. The most reliable range of standard DNA was found to be between 1 and 100
pg. And
aliquot of each reaction is the analyzed by 2% agarose gel electrophoresis,
until two PCR-rM
products are resolved. The analog image of the ethidium bromide stained gel is
digitized using
and ImageStore 7500 system (UVP). The densities of the target and competitor
bands in each
lane are measured using the ZERO-Dscan Image Analysis System, version 1.0
(Scanalytics)
and their ratios are plotted as a function of the standard DNA concentration.
A ration of 1, at
which the number of viral DNA molecules equals the number of competitor DNA
molecules is
used to determine the DNA concentration of the virus stock.
In case of rAAV-CBA-Ob, as well as the rAAV-rtTA/tTS vector, for which no
plasmid
DNA carrying a deleted standard template is available yet, the titer of
physical rAAV particles
is determined using conventional dot-blot assay and viral DNA purified as
described above.
Sample of serially diluted respective plasmid of sequence is used as a
hybridization probe.
A modification of the previously published protocol (McLaughlin et al., 1988)
is used
to measure the ability of the virus to infect C12 cells, unpackage, and
replicate. Briefly, C2
cells containing integrated wtAAV rep and cap genes (Clark et al., 1995), are
plated in a 96-
well dish at about 75% confluence and infected with Ad5 at the M.O.I of 20.
One ~1 of serially
diluted rAAV-sCNTF is visually scored using a fluorescence microscope. High
sensitivity
CHROMA filter #41012 High Q FITC LP is used to monitor the fluorescence. To
calculate
the titer by hybridization, cells are harvested and processed essentially ad
described earlier
(McLaughlin et al., 1988).
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57
5.15 EXAMPLE 15 -- RHSV-1 VECTORS FOR THE PRODUCTION OF RAAV
In order to make an rAAV production system which did not possess the
restrictions to
large-scale rAAV production inherent in traditional Ad-based transfection
systems, the use of
another DNA helper virus of AAV, herpes simplex virus type I (HSV-1), was
investigated for
the production of rAAV. Like Ad, HSV-1 is able to fully support AAV
replication and
packaging. While little is known about the nature of the interactions between
HSV01 and
AAV, the minimal set of HSV-1 genes required to replicate and package AAV has
been
determined to be ULS, ULB, UL52 and UL29. These genes encode components of the
HSV-1
core replication machinery and by themselves are able to form nuclear
prereplication centers
that develop into mature replication foci. The goal was to create a single
infectious agent
capable of delivering all AAV-2 genes and HSV-1 helper virus functions
required to produce
xAAV. In this recombinant HSV-1, the AAV-2 j.~p and yap genes are recombined
into tine
viral genome (within the tk gene) to create the recombinant vector d27.1-s~c~
Therefore, use of
this infectious agent along with a rAAV proviral cell line would eliminate
restrictions to large-
scale rAAV production inherent in transfection protocols. In addition, use of
a mutant HSV-1
that was replication defective and not complemented by the rAAV proviral cell
line would
eliminate the HSV-1 helper virus from the rAAV preparation. Thus, the two
significant
problems associated with Ad based systems would not be encountered.
The recombinant virus expressing j.ep and yap, d27.1-r~c~ was constructed by
homologous recombination techniques. The HSV-1 background used to make the
recombinant
vector expressing the AAV-2 yep and yap genes was d27.1 ~ ~nis mutant vector,
d27.1, does
not produce ICP17. This vector was chosen because it is replication defective
and should be
less cytotoxic in a non-permissive cell line that wt HSV-1. The virus 427.1
still does, however,
express the early genes known to be required for rAAV production and thus
should still
provide efficient helper function for rAAV production. Using an ICP27 mutant
as the helper
virus for rAAV production has additional advantages. Host cell splicing of
messenger RNA is
inhibited by ICP27. In addition, d27. I over-expresses for AAV replication and
packaging.
5.16 EXAMPLE 16 -- A RECOMBINANT HSV-1 EXPRESSING AAV-2 ~p AND CAP
SUPPORTS EFFICIENT RAAV PRODUCTION
The ability of the recombinant virus, d27.1-~c, to produce rAAV was analyzed.
The
amount of AAV-GFP produced after 293 cells are transfected with a proviral
plasmid and
superinfected with d27.1-rc has been determined. Efficient rescue of rAAV from
the plasmid
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58
pTR-UFS, a proviral rAAV plasmid that encodes the green fluorescent protein
(GFP), is
observed. Up to 380 expression unit (e.u.) of AAV-GFP are produced per cell
after
superinfection with d27.1-y~c at a MOI of ten.
Recombinant 427.1-f°c is even more effective at producing rAAV from
the rAAV
proviral cell line 293:AAV-GFP. At a M.O.I. of ten, 480 e.u. of AAV-GFP per
cell can be
produced from this cell line. The amount of AAV-GFP produced from the cell
line is a
function of the M.O.I. of d27. l -rc. Increasing the M.O.I. to ten resulted in
an increase in
rAAV production. At M.O.Ls higher than ten, significantly less rAAV is
produced. The
efficient production of rAAV using d27.1-rc has been maintained when the scale
of infection
was increased 100-fold in tissue culture flasks. When 109 293:AAV-GFP cells
are infected
~~ d27.1-f~c~ over 350 e.u. of AAV-GFP are produced per cell. Importantly,
rcAAV has not
been observed in any large preparation of AAV-GFP made using d27.1-s°c
(~')~
5.17 EXAMPLE 17 -- CYTOKINE AND CYTOKINE RECEPTOR POLYPEPTIDE-ENCODING
NUCLEIC ACID SEGMENTS
The inventors contemplate the use of one or more of the cytokine and cytokine
receptor
DNA sequences illustrated in Table 4 in the preparation of rAAV vector-based
constructs in
the practice of the present invention. In illustrative embodiments, the
inventors contemplate
the use of mammalian cytokine and cytokine-receptor encoding polynucleotides,
and in
particular, the use of human cytokine and eytokine-receptor encoding
polynucleotides in
creation of the rAAV vector constructs for therapeutic administration.
TABLE 4
POLYNUCLEOTIDE SEQUENCES ENCODING CYTOKINES AND CYTOKINE RECEPTORS USEFUL
IN THE PRACTICE OF THE PRESENT METHODS
UMBER
Mouse Leptin Y10297
Human Leptin NM000230
Rat Leptin Receptor D84126; U52966
Mouse Leptin Receptor Y10298
Pig Leptin Receptor AJ223163; AF092422
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Horse Leptin Receptor AF139663
Sheep Leptin Receptor U62124
Human Leptin Receptor NM002303; AH003667; U59261;
U59262;
U59263
Pig CNTF U57644
Human CNTF NM000614
Rat CNTF receptor 554212
Human CNTF receptor M73238; NM001842
Human Leukemia Inhibitory FactorNM002309
Pig LIF Receptor U97364
Rat LIF Receptor a D86345
Mouse LIF Receptor X99779; X99778
Human LIF Receptor NM002310; X61615
Human Interleukin-1 (IL-1) E00909; E00619
Human IL-1 Receptor, type I NM000877; NM003856
Human IL-1 Receptor, type II NM004633
Human IL-1 Receptor-like 2 NM003854
(IL1RL2)
Human Interleukin-2 (IL-2) E00573; 577834; 577835
Human IL-2 like E0201 l; E00211
Human IL-2 Receptor XOI057; E00727
Human IL-2 Receptor a (IL2RA) NM000417
Human IL-2 Receptor ~ (IL2RB) NM000878
Human IL-2 Receptor Y (IL2RG) NM000206; L 19546
Human Interleukin-6 (IL-6) 556892
Human IL-6 Receptor (IL6R) NM000565
Human Interleulcin-10 (IL-10) U16720; AF043333
Human IL-10 Receptor a (IL10RA)NM001558
Human IL-10 Receptor ~ (ILlORB)NM000628
Brain-Derived Neurotrophic M61176; NM001709
Factor (BDNF)
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All of the compositions and methods disclosed and claimed herein can be made
and
executed without undue experimentation in light of the present disclosure.
While the
compositions and methods of this invention have been described in terms of
preferred
embodiments, it will be apparent to those of skill in the art that variations
may be applied to
the compositions and methods and in the steps or in the sequence of steps of
the method
described herein without departing from the concept, spirit and scope of the
invention.
More specifically, it will be appaxent that certain agents which are both
chemically and
physiologically related may be substituted for the agents described herein
while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent
CA 02410512 2002-11-27
WO 01/94605 PCT/USO1/40901
to those skilled in the art are deemed to be within the spirit, scope and
concept of the
invention as defined by the appended claims.
