Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DESCRIPTION
A Gene Thera~,~ystem and Method Using Alpha-MSH and Its Derivatives
Field of the Invention
The present invention relates to the field of gene therapy.
Background of the Invention
Various diseases originate from defective genes that are either inherited or
modified during life by environmental agents. Examples of these diseases
include
different forms of cancer, hemophilia, or LDL receptor deficiency. Gene
therapy or gene
replacement therapy promises a fundamental cure for these diseases by
replacing,
augmenting, or inhibiting these defective genes.
Common vectors for introducing the therapeutic gene or nucleic acid include
viral
and non-viral vectors. Although viral delivery systems have been considered to
be most
efficient in delivering genes to cells, it may be limited because of a risk of
triggering
inflammatory or ilnmunogenic responses. Forbes, S.J., Reviet-v Article: Gene
Therapy in
Gastroenterology an c1 Hepatology, Aliment Pharmacol. Ther. 11:823-826 (1997).
Recently, the death of Jesse Gelsinger, a volunteer who died on September 17,
1999 while participating in a gene therapy clinical trial at the Institute for
Human Gene
Therapy, University of Pennsylvania, has fueled the controversy over the use
and safety of
gene therapy. The trial was directed to treat omithine transcarbmylase (OTC)
using a
modified adenoviral vector. The administration, however, of the vector to
Gelsinger
"initiated an unusual and deadly immune-system response that led to multiple
organ
failure and death." Preliminary Finclirags, The Institute of Hurnan Gene
Therapy,
University of Pennsylvania Health System, December 2, 1999,
<http://www.med.upenn.edu/ihgt/findings.html>. Although adenoviral vectors
offer
several advantages over other viral vectors in that they can infect a wide
range of cells and
are not limited to replicating cells, as are retroviral vectors, adenoviral
vectors may
activate the immune system, as seen in the Gelsinger's case, such that the
initial dose or
repeated introduction may become less effective, if not life threatening. See
also Forbes,
S.J., supra.
Moreover, the potential immune response to gene therapy is not limited to the
vector used. Since the vector introduces a genetic sequence that encodes a
protein,
polypeptide, enzyme that may be seen as "foreign" to the host, immune
responses toward
the cells expressing that sequence and the products of that expression limit
the
effectiveness of the therapy. For example, in hemophilia experiments using the
Factor
VIII or IX gene as the therapeutic gene, antibodies generated against the
newly expressed
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proteins may limit the effectiveness of the therapy. Forbes, S.J., supra; see
also Herzog,
R., Problems ahd Prospects in Gehe Therapy for Hefnophilia, Current Opinions
in
Hematolo~y, 5:321-326 (1998). Cytotoxic T cells such as neutrophils may also
attack
these cells that expressed the "foreign" genes or viral vector genes, again
limiting the
effectiveness of gene therapy over a sustained period of time.
Thus, there exists a need to enhance the safety and efficiency of gene therapy
by
alleviating some of the detrimental effects of 'the immune system.
Surnlnary of the Invention
The present invention involves the use of alpha-MSH and/or its derivatives as
an
adjunct to gene therapy. In one aspect of the invention, the gene therapy
vector includes
nucleic-acid sequences that express alpha-MSH and/or its derivatives, and
inflammatory
or immune-response gene promoters may control their expression. The sequences
may
also be expressed together with a therapeutic gene using an internal ribosomal
entry site
sequence. In another aspect of the invention, pharmacologically effective
amount of
alpha-MSH and/or its derivatives may be administered before, after, and/or
concurrently
with the gene therapy vector carrying the appropriate gene.
General Description of the Invention
The references cited below are hereby incorporated by reference as if fully
set forth
herein. The present invention involves a method and system for gene therapy
using alpha-
melanocyte stimulating hormone ("a-MSH") and/or its derivatives as an adjunct
therapy.
Because of its anti-inflammatory and anti-pyretic activities, a-MSH and/or its
derivatives
may supplement gene therapy applications by limiting the inflammatory response
of the
patients to the gene therapy vector or the expressed protein. Unlike other
immunosuppressants, a-MSH and/or its derivatives also possess antimicrobial
properties
that may simultaneously protect the body against infection and limit the
negative effects of
the immune system.
In one aspect of the invention, a pharmacologically effective amount of a-MSH
and/or its derivatives may be administered before or after the gene therapy
vector carrying
the appropriate therapeutic gene or nucleic acid is administered.
Alternatively, a-MSH
and/or its derivatives may also be administered together with the gene therapy
vector as a
cocktail. The gene therapy vector may include both viral vectors such as
adenoviral,
retroviral, lentiviral, or adenovirus-associated viral (AAV) vectors, and non-
viral vectors
such as liposomes, calcium phosphate, antibodies or receptor based transfer
vectors,
electroporation, or direct injection of nucleic acids.
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In another aspect of the invention, the gene therapy vector carries a DNA
molecule
that includes sequences for expressing a-MSH and/or its derivatives in the
host cells.
Using conventional molecular cloning techniques, gene sequences for a-MSH
and/or its
derivatives may be cloned into viral vectors or expression plasmid vectors.
Constitutive
promoters such as cytomegalovirus (CMV) promoter or inducible promoters may
drive
their expression. Preferably, the inducible promoter employs the use of
inflammatory
gene promoters such as the interleulcins, in particular, the IL-6 promoter, or
the
complement system gene promoters. Other inflammatory gene promoters include
promoters for TNF-a or the NF-oB response element.
The gene therapy vector carrying the a-MSH and/or its derivatives may also
carry
the appropriate therapeutic gene or nucleic acid of interest. This localizes
the expression
of a-MSH and/or its derivatives to the area expressing the therapeutic gene of
interest.
The local effect of a-MSH andlor its derivatives may inlubit a local
inflammatory
response without compromising the systemic immune system of the host. It may
also
protect the cells that have incorporated the gene therapy vectors from the
inflammatory
cytotoxic killings of neutrophils or T-cells. In a preferred embodiment of the
invention,
cloning the gene for a-MSH and/or its derivatives and associated promoter in
the same
DNA vector carrying the therapeutic gene of interest may achieve this co-
localization
effect.
Alternatively, the a-MSH and/or its derivatives can be expressed with an
internal
ribosomal entry site (IRES). The IRES sequence may be placed between the
therapeutic
gene and the gene for a-MSH and/or its derivatives. Thus, the two genes may be
transcribed as a bicistronic mRNA transcript from a single promoter, and the
bicistronic
mRNA, in turn, may be translated simultaneously at the 5' end and at the IRES
sequence.
Because both the protein from the therapeutic gene and a-MSH and/or its
derivatives are
produced from a single transcript, it is more likely that a single cell will
express both
proteins. IRES sequences and vectors can be commercially obtained, for
example, from
Clontech Laboratories, Palo Alto, California (AIRES, cat# 6028-1).
Furthermore, stringing multiple genes for a-MSH and/or its derivatives using
multiple IRES sequences may increase the production of a-MSH and/or its
derivatives. A
secretion signal peptide cloned upstream of the gene for a-MSH and/or its
derivatives may
also transport a-MSH andlor its derivatives to the extracellular environment
where they
are needed. Examples of such secretion peptide signal include the signal
peptides for
epidermal growth factor, basic fibroblast growth factors, or interleul~in-6.
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Example I
This example illustrates the anti-inflammatory, anti-pyretic, and anti-
microbial
activities of a-MSH and its derivatives.
a-MSH is an ancient thirteen amino-acid peptide, SYSMEHFRWGKPV (a-MSH
(1-13)), that is produced by post-translational processing of the larger
precursor molecule
propiomelanocortin. It shares the 1-13 amino acid sequence with
adrenocorticotropic
hormone ("ACTH"), also derived from propiomelanocortin. a-MSH is known to be
secreted by many cell types including pituitary cells, monocytes, melanocytes,
arid
lceratinocytes. It can be found in the skin of rats, in the human epidermis,
or in the
mucosal barrier of the gastrointestinal tract in intact and hypophysectomized
rats. Sea e.~.
Eberie, A. N., The Melanotrophins, Karger, Basel, Switzerland (1998); Lipton,
J.M., et.
al., Anti-inflammatory Influence of the Neuroimmunomodulator a-MSH, Immunol.
Today
18, 140-145 (1997); Thody, A.J., et.al., MSH Peptides are Present in Mammalian
Skin,
Peptides 4, 813-815 (1983); Fox, J. A., et.al., Immunoreactive a-Melanocyte
Stimulating
Hormone Its Distribution in the Gastrointestinal Tract of Intact and
Hypophysectomized
Rats, Life. Sci. 18, 2127-2132 (1981).
a-MSH and its derivatives are known to have potent antipyretic and anti-
inflammatory properties, yet they have extremely low toxicity. They can reduce
production of host cells' proinflammatory mediators in vitro, and can also
reduce
production of local and systemic reactions in animal models for inflammation.
The "core"
a-MSH sequence (4-10), for example, has learning and memory behavioral effects
but
little antipyretic and anti-inflammatory activity. In contrast, the active
message sequence
for the antipyretic and anti-inflammatory activities resides in the C-terminal
amino-acid
sequence of a-MSH, that is, lysine-proline-valine ("Lys-Pro-Val" or "KPV").
This
tripeptide has activities in vitro and in vivo that parallel those of the
parent molecule. The
anti-inflammatory activity of a-MSH and/or its derivatives are disclosed in
the following
two patents and references, which are hereby incorporated by reference as if
fully set forth
the herein: U.S. Patent No. 5,028,592, issued on July 2, 1991 to Lipton, J.M.,
entitled
Antipyretic and Anti-inflammatory Lys Pro Val Compositions and Method of Use;
U.S.
Patent No. 5,157,023, issued on October 20, 1992 to Lipton, J.M., entitled
Antipreytic and
Anti-inflammatory Lys Pro Val Compositions and Method of Use; see also
Catania, A., et.
al., a-Melanocyte Stimulating Hormone in the Modulation of Host Reactions,
Endocr.
Rev. 14, 564-576 (1993); Lipton, J. M., et.al., Anti-inflammatory Influence of
the
Neuroimmunomodulator of -MSH, hyafnunol. Today 18, 140-145 (1997); Rajora, N.,
et.
al., a-MSH Production Receptors and Influence on Neopterin, in a Human
Monocytelmacrophag~e Cell Line, J. Leukoc. Biol. 59, 248-253 (1996); Star,
R.A., et. al.,
Evidence of Autocrine Modulation of Macrophage Nitric Oxide Synthase by a-MSH,
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Proc. Nat'l. Acad. Sci. (USA) 92, 8015-8020 (1995); Lipton, J.M., et.al., Anti-
inflammatory Effects of the Neuropeptide a-MSH in Acute Chronic and Systemic
inflammation. Anh. NY. Acad. Sci. 741, 137-148 (1994); Fajora, N., et.al., a-
MSH
Modulates Local and Circulating_ tumor Necrosis Factor-a in Experimental Brain
5 Inflammation, J. Neu~oosci, 17, 2181-2186 (1995); Richards, D.B., et. al.,
Effect of a-
MSH (11-131 (lysine-proline-valinel on Fever in the Rabbit, Peptides 5, 815-
817 (1984);
Hiltz, M. E., et. al., Anti-inflaimnatory Activity of a COOH-terminal Fragment
of the
Neurope~tide a-MSH, FASEB J. 3, 2282-2284 (1989).
a-MSH derivatives include, but are not limited to, peptides with the amino-
acid
sequence I~PV (a-MSH (11-13)), MEHFRWG (a-MSH (4-10)), or HFRWGKPV (a-
MSH (6-13)). These derivatives may also include homodimers or heterodimers of
the
above peptides, which may be achieved by adding cysteine residues at the N
termini of
any of the above polypeptides and allowing the cysteines of two polypeptides
to form a
disulfide bond. The peptides may also be N-acetylated and/or C-amidated.
Substituting or deleting certain amino acid residues may also create
biologically
functional derivatives without altering the effectiveness of the peptides. For
example, it is
known that stabilization of the a-MSH sequence can greatly increase the
activity of the
peptide and that substitution of D-amino acid forms for L-forms can improve or
decrease
the effectiveness of the peptides. A stable analog of a-MSH, [Nle4,D-Phe7]-a-
MSH,
which is known to have marked biological activity on melanocytes and melanoma
cells, is
approximately ten times more potent than the parent peptide in reducing fever.
Further,
adding amino acids to the C-terminal of an a-MSH(11-13) sequence can reduce or
enhance antipyretic potency. Addition of glycine to form the 10-13 sequence
slightly
decreased potency; the 9-13 sequence was almost devoid of activity, whereas
the potency
of the 8-13 sequence was greater than that of the 11-13 sequence. It is lcnown
that Ac-[D-
Kl l]-a-MSH 11-13-NH2 has the same general potency as the L-form of the
tripeptide a-
MSH (11-13). However, substitution with D-proline in position 12 of the
tripeptide
rendered it inactive. see e.~. . Holdeman, M., et. al., Anti~yretic Activity
of a Potent a-
MSH Analog, Peptides 6, 273-5 (1985). Deeter, L.B., et. al., Antipyretic
Properties of
Centrally Administered a-MSH Fragments in the Rabbit. Peptides 9, 1285-8
(1989).
Hiltz, M.E., Anti-inflammatory Activity of a-MSH (11-13 Analogs: Influences of
Alterations in Stereochemistrv, Peptides 12, 767-71 (1991).
Biological fwctional equivalents can also be obtained by substitution of amino
acids having similar hydropathic values. Thus, for example, isoleucine and
leucine, which
have a hydropathic index +4.5 and +3.8, respectively, can be substituted for
valine, which
has a hydropathic index of +4.2, and a protein having like biological activity
can still be
obtained. Alternatively, at the other end of the scale, lysine (-3.9) can be
substituted for
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arginine (-4.5), and so on. In general, it is believed that amino acids can be
successfully
substituted where such amino acid has a hydropathic score of within about +/-
1
hydropathic index unit of the replaced amino acid.
As to the anti-microbial properties of a-MSH and/or its derivatives, they have
been
described in PCT Application Serial No. PCT/LTS00/06917, published as
WO00/59527,
entitled "Anti-microbial Amino Acid Sequences Derived from alpha-Melanoctye
Stimulating Hormone, filed March 17, 2000 by inventors Catania, A. and Lipton,
J.
claiming priority to U.S. Provisional Patent Application Serial No. 60/126,233
entitled
Antimicrobial Amino-Acid Sequences Derived from Alpha-Melanocyte Stimulating
Hormone, filed March 24, 1999. The above PCT publication and the provisional
patent
application are hereby incorporated by reference as if fully set forth herein.
Example II
This example illustrates the use of a-MSH and/or its derivatives in
conjunction
with gene therapy.
Preparation and purification of a-MSH and/or its derivatives may employ
conventional solid-phase peptide synthesis and reversed-phased high-
performance liquid-
chromatography techniques. Patients who will undergo gene therapy may receive
a
pharmacologically effective amount of a-MSH and/or its derivatives either
through
injections or oral administration. The injections, for example, can be
performed
intravenously, intraperitionally, or intradermally depending on the specific
location
targeted by the gene therapy. After adminstration of a-MSH and/or its
derivatives and
under supervision of a physician, the patient can then receive a
pharmacologically
effective amount of the gene therapy vector containing the therapeutic gene or
nucleic acid
of interest according to conventional gene therapy protocols. If needed,
additional
administration of a-MSH and/or its derivatives may be given following the
administration
of the gene therapy vector.
Alternatively, the delivery cocl~tail for the gene therapy vector may include
a
pharmacologically effective amount of a-MSH and/or its derivatives that is
concurrently
or simultaneously administered to the patients.
Example III
Thus example illustrates the construction of gene therapy vector that
expresses a-
MSH and/or its derivatives.
Preparation and purification of gene sequences that express a-MSH and/or its
derivatives may use, among other techniques, conventional oligonucleotide
synthesis
techniques. Complementary oligonucleotides can be made and annealed to form
double
stranded DNA molecules capable of being cloned. Additional sequences
representing
appropriate restriction enzyme sites may be engineered at the ends of each
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oligonucleotide. Preferably, the oligonucleotide sequence downstream of the a-
MSH
sequences includes a stop codon (TAG).
In addition, using polymerase chain reaction, a fragment corresponding to the
signal peptide of IL-6 cDNA, nucleotides 33 to 120 (Genbank Accession No.
J03783),
may be synthesized, cloned into a vector such as pBluescript KS (Stratagene,
San Diego,
CA). Similarly, promoter regions for IL-6 or NF-~cB may also be synthesized
using
oligonucleotides with appropriate matching restriction enzyme sites and cloned
upstream
of the pBluescript carrying signal sequence. Using standard restriction enzyme
digestion
and DNA ligation, a-MSH or its derivatives sequences may be ligated to the
signal
sequence and the promoter.
If an internal ribosomal entry site (IRES) sequence is desired, the
oligonucleotides
sequence above may include such a sequence or it can be incorporated into PCR
primers
and linced by conventional PCR techniques. Alternatively, the a-MSH and/or its
derivatives may be cloned into the pIRES vector from Clontech Laboratories.
Multiple a-
MSH and/or its derivatives may be constructed with multiple IRES sequence if
so desired.
An effective amount of the expression plasmid containing these constructs and
the
therapeutic gene of interest can be directly injected or introduced into
patients using non-
viral vectors such as liposomes, electroporation, or using a gene gun.
Alternatively, the a-MSH and/or its derivatives constructs can be inserted
into
appropriate replication deficient retroviral, lentiviral, adenoviral, or
adenovirus-associated
viral vectors using standard restriction enzyme and ligation techniques, blunt
end cloning,
or PCR techniques. Packaging cell lines using helper viruses may then package
the vector
DNA into viral particles for use in gene therapy.
Titer of the recombinant virus may first be determined, and appropriate amount
of
viral particles may be introduced into the patients or hosts. It is understood
that the viral
vector may already contain a therapeutic gene or nucleic acid in addition to a-
MSH and/or
its derivatives.
Once the recombinant virus is introduced into cells, the cells may express a-
MSH
and/or its derivatives, which in turn, inhibits inflammation. The anti-
inflammatory effect
of a-MSH expression in cells through inhibition of NF-~cB activation have been
reported
in Ichiyama, et. al., Autoc~iyze a Melanocyte-Stimulating Hof°fnofZe
Inhibits NP xB
Activatio~z iya Humafz Glioma, J. Neurosci. Res. 58:684-689 (1999), and is
hereby
incorporated by reference as if fully set forth herein.
It is understood that modifying the examples above does not depart from the
spirit
of the invention. It is further understood that each example can be applied on
its own or in
combination with each other.
