U.S. patent application number 14/943096 was filed with the patent office on 2016-04-21 for method for inhibiting hiv replication in mammal and human cells.
The applicant listed for this patent is Centro De Ingenieria Genetica Y Biotechnologia. Invention is credited to Dionne Casillas Casanova, Lila Rosa Castellanos Serra, Carlos Antonio Duarte Cano, Marta Dubed Echevarria, Viviana Falcon Cama, Celia Berta Fernandez Ortega, Leonor Margarita Navea Leyva, Taimi Emelia Paneque Guerrero, Anna Caridys Ramirez Suarez, Osvaldo Reyes Acosta, Raimundo Ubieta Gomez.
Application Number | 20160106810 14/943096 |
Document ID | / |
Family ID | 44168763 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160106810 |
Kind Code |
A1 |
Fernandez Ortega; Celia Berta ;
et al. |
April 21, 2016 |
Method for Inhibiting HIV Replication in Mammal and Human Cells
Abstract
The present invention describes a method to inhibit replication
of the human immunodeficiency virus (HIV) by negatively modulating
or altering the cytoskeleton, more precisely the proteins forming
the intermediate cytoskeletal filaments, wherein the said proteins
are vimentin and/or keratin-10. The replication of the virus is
inhibited in human cells by intervening in the structure of these
proteins. The present invention is also related to the use of
agents, which comprise peptides and/or interfering RNA and/or
lipidic compounds, said agents producing a negative modulation or
alteration of the cytoskeleton to prevent or to treat the HIV
infection. The invention provides means and methods for altering
the cytoskeleton/filament structure of cells, as a result of which
the infection of human cells by HIV is disturbed and can even be
completely inhibited. The cytoskeleton is altered by reducing the
amount of vimentin and/or keratin (e.g. by transcriptional control
using interfering RNA) or by using peptides that disrupt the
cytoskeleton.
Inventors: |
Fernandez Ortega; Celia Berta;
(La Habana, CU) ; Ramirez Suarez; Anna Caridys;
(La Habana, CU) ; Casillas Casanova; Dionne; (La
Habana, CU) ; Paneque Guerrero; Taimi Emelia; (La
Habana, CU) ; Ubieta Gomez; Raimundo; (La Habana,
CU) ; Dubed Echevarria; Marta; (La Habana, CU)
; Navea Leyva; Leonor Margarita; (La Habana, CU) ;
Castellanos Serra; Lila Rosa; (La Habana, CU) ;
Duarte Cano; Carlos Antonio; (La Habana, CU) ; Falcon
Cama; Viviana; (La Habana, CU) ; Reyes Acosta;
Osvaldo; (La Habana, CU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Centro De Ingenieria Genetica Y Biotechnologia |
La Habana |
|
CU |
|
|
Family ID: |
44168763 |
Appl. No.: |
14/943096 |
Filed: |
November 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13637845 |
Jan 22, 2013 |
9205128 |
|
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PCT/CU2011/000001 |
Apr 1, 2011 |
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14943096 |
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Current U.S.
Class: |
514/3.8 |
Current CPC
Class: |
A61K 31/5575 20130101;
C12N 15/113 20130101; A61K 45/06 20130101; A61K 38/1748 20130101;
C12N 2310/531 20130101; C12N 2310/14 20130101; C12N 15/1131
20130101; C07K 14/47 20130101; A61K 31/19 20130101; C07K 14/4741
20130101; A61K 31/713 20130101; A61K 38/10 20130101; A61K 38/1709
20130101; A61P 43/00 20180101; A61P 31/18 20180101; C12N 5/0636
20130101; A61K 31/7105 20130101; C12N 15/1132 20130101; C12N
2320/31 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 38/10 20060101 A61K038/10 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 1, 2010 |
CU |
CU 2010/0056 |
Claims
1-31. (canceled)
32. A method of preventing HIV infection in a mammal in need
thereof, comprising administering to said mammal a therapeutically
effective dose of a pharmaceutical composition, said composition
comprising a peptide selected from the group consisting of SEQ ID
NO: 1 to SEQ ID NO: 10 and homologues thereof, said homologues
having at least 85% sequence identity with any one of SEQ ID NO: 1
to SEQ ID NO: 10 and having the ability to disrupt, negatively
modulate or modify the cytoskeletal IFs in cells of said mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/637,845, filed on Jan. 22, 2013, which is a U.S. National
Phase application of International Application No.
PCT/CU2011/000001 filed Apr. 1, 2011, which claims priority based
on Cuban Patent Application No. 2010-0056 filed Apr. 1, 2010, all
of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention is related to the field of
biomedicine, and more precisely to therapies against infections,
and particularly against the infection of the human
immunodeficiency virus (HIV). The present invention describes a
method to inhibit HIV replication by altering the cytoskeleton,
specifically those proteins forming the cytoskeletal intermediate
filaments (IFs). The present invention also relates to the use of
agents which negatively modulate or modify the cytoskeleton for the
purpose of manufacturing drugs to prevent and to treat HIV
infection.
BACKGROUND OF THE INVENTION
[0003] The emergence of the HIV/acquired immunodeficiency syndrome
pandemic is among the most significant health problems arising
worldwide in the last thirty years. It has led to the development
of antiretroviral treatments able to stop the progression of
infection and reducing mortality (De Cock K, Crowley S P, Lo Y R,
Granich R M, Williams B G. Boletin de la Organizacion Mundial de la
Salud 2009; 87: 488-488). UNAIDS estimated in its 2008 report that
there were 33 million people infected with HIV, 2.7 million of them
were new cases detected in that year. It is estimated that 67% of
global infections occur in sub-Saharan Africa (Report on the global
AIDS epidemic 2008. Geneva, UNAIDS). According to a bulletin issued
by the World Health Organization in 2009, there are two major
tendencies worldwide: the total affection of the sub-Saharan
African population and the concentration of infection in specific
risk groups throughout the rest of the world, respectively. (De
Cock K, Crowley S P, Lo Y R, Granich R M, Williams B G 2009.
Boletin de la Organizacion Mundial de la Salud 87: 488-489).
[0004] The annual incidence of HIV infection peaked in the middle
of the 1990's (Bongaarts J, Buettner T, Heilig G, Pelletier F.
Popul Dev Rev 2008; 34: 199-224). However, the total number of
HIV-infected people continues to increase in Africa, due to a
persistently high incidence of the virus and the population growth
rate (De Cock K, Crowley S P, Lo Y R, Granich R M, Williams B G
2009. Boletin de la Organizacion Mundial de la Salud 87:
488-489).
[0005] The appearance of HIV variants that are resistant against
the currently available anti-HIV drugs and the poor adherence to
treatment by patients remain as the main causes for therapeutic
failure. Viral resistance was observed since the start of
antiretroviral monotherapy, leading to the appearance of the
combined anti-HIV therapy with two or more anti-HIV agents, each of
them with a different mechanism of action. Morbidity and mortality
rates significantly decreased among treated patients with the
introduction of the highly active antiretroviral therapy (HAART).
This therapy combines nucleosidic and non-nucleosidic reverse
transcriptase inhibitors and protease (PR) inhibitors.
Nevertheless, the multi-drug therapy does not eliminate HIV
completely, with long-term treatment generally resulting in
resistance to several drugs. Half of the patients receiving
combined anti-HIV therapy do not completely respond to treatment,
mainly due to viral resistance to one or more of the drugs applied.
Additionally, viral resistance has been detected in recently
infected patients, significantly limiting the therapeutic options
for those patients.
[0006] The success of combined anti-HIV therapy gave an outlook on
a possible eradication of the virus. However, existence of viral
reservoirs has been described in latently infected cells and also
in tissues where the virus persists regardless of therapy. It has
been estimated that more than 70 years of continuous treatment are
required to eradicate viral reservoirs, a fact considered
improbable since therapy implies secondary effects and occasionally
fatal metabolic complications such as lactic acidosis, diabetes
mellitus, lipodystrophy, pancreatitis and others (Iglesias E 2009.
Biotecnologia Aplicada 26: 189-194).
[0007] The adherence to HIV antiretroviral therapy is one of the
most debatable issues regarding HAART, and specifically PR
(protease) inhibitors, due to the fast appearance of viral
resistance if the drugs are irregularly taken or the treatment is
interrupted. There are several factors for non-adherence to
treatment, including drug intolerance, complex administration
regimes, therapeutic failure, drug interactions, social-economical
problems, and others.
[0008] Combination therapy delays progression to AIDS, but does not
cure the infected patients (Marsden M D, Zack J A 2009. J
Antimicrob Chemoth 63: 7-10). Even when therapy has transformed
this infection into a chronic disease rather than a fatal disease
and also increased the life expectancy among patients to levels
similar to those of the general population, it still represents
unsolved serious problems which require the search for new
strategies to decrease the used of antiretrovirals. That is the
goal of the search for new therapeutic variants.
[0009] All the disadvantages of the available anti-HIV therapies
support the need for new anti-HIV drugs differing mostly on their
mechanisms and/or targets of action. An object of the invention is
to provide a method to inhibit the replication and/or infection of
the HIV. It is a further object of the invention to provide a
method to inhibit the replication and/or infection of the HIV that
has a different mechanism than inhibiting HIV polymerase or HIV
protease. Another object of the invention is to provide a method to
inhibit the replication and/or infection of the HIV by targeting
the host cell and not the virus. Specifically, an object of the
invention is to target the cytoskeleton, and more specifically the
intermediate filaments (IFs) from the host cell. Specifically,
another object of the invention is to target the host proteins
vimentin and keratin-10. By targeting the host cell it is believed
that it will be must more difficult for HIV to produce escape
mutants. Vimentin and keratin-10 are important for the structure of
the IFs and the present invention showed that they are a suitable
target to inhibit HIV. Another object of the present invention is
to provide agents and pharmaceutical compositions that disrupt the
IFs of a cell or to decrease the amount of vimentin and/or
keratin-10 host proteins.
[0010] At least one of the above mentioned objects is attained by
the present invention.
SUMMARY OF THE INVENTION
[0011] The present invention is related to altering the
cytoskeleton of a mammalian cell as a method to inhibit HIV
replication y/o infection. The cytoskeleton is a tridimensional
scaffold which contributes to the cellular integrity and plays
several roles for the cell. It is formed by three main structures:
microtubules, microfilaments and the intermediate filaments (IFs).
The IFs comprise a set of proteins specific for each cell type, the
vimentin and keratin-10 proteins among them.
[0012] Vimentin is a 58 kDa-molecular weight (MW) protein forming
the IFs and commonly expressed on blood vessels endothelial cells,
in certain epithelial cells and in mesenchymal cells (Alberts B,
Johnson A, Lewis J, Raff M, Roberts K, Walter P 2002. Molecular
Biology of the Cell, 4th ed., Garland Publishing, New York). It is
known that vimentin is a substrate of the HIV PR, and it is
proposed that the action of PR affects vimentin thereby could
affect the cytoskeletal structure (Blanco R, Carrasco L, Ventoso I
2003. J Biol Chem 278: 1086-1093). It has been also demonstrated
that treatment with vimentin N-terminal peptides obtained by
proteolytic processing are able to rearrange the cell nucleus
architecture. This nuclear architecture rearrangement is also
observed in HIV-infected cells (Shoeman R L, Huttermann C, Hartig
R, Traub P 2001. Mol Biol Cell 12:143-154). Previous evidences
suggest that HIV depends on vimentin excision for its lifecycle.
Keratins comprise a set of IFs proteins (of about 30 members)
within a range of molecular weight from 10 to 68 kDa. They has been
classified and numbered according to their MW and their
electrophoretic behavior in acidic (pKi=4-6; type I) and
neutral-basic (pKi=6-8; type II). Keratin-10 is a type-I keratin of
approximately 60 kDa, found in the IFs of completely differentiated
epidermal cells mainly (Zhou X M 1988. J Biol Chem 263: 15584-9).
The present invention is directed to a method to inhibit the
replication and/or infection of the HIV in a mammalian cell
comprising disrupting the (structure of) cytoskeletal IFs in said
mammalian cell. Furthermore the present invention is also directed
to an agent that disrupts cytoskeletal IFs to prevent or to treat
HIV infection.
[0013] In a first aspect, the present invention provides a method
to inhibit the replication of the HIV in a mammalian cell, said
method comprising disrupting or negatively modulating the
(structure of) cytoskeletal IFs in a mammalian cell. In particular
said mammalian cell is the target cell for infection by an HIV
virus.
[0014] In a preferred embodiment of said method, the IFs comprise
vimentin and/or keratin-10 proteins.
[0015] In another preferred embodiment of said method, the method
comprises decreasing the amount of vimentin and/or keratin-10 in
said IF to disrupt or negatively modulate the (structure of)
cytoskeletal intermediate filaments and/or decreasing the amount of
free vimentin and/or keratin-10 available to make new IF.
[0016] In yet another preferred embodiment of said method, the
method comprises decreasing the expression of the genes encoding
the vimentin and/or keratin-10 proteins to disrupt or negatively
modulate the (structure of) cytoskeletal intermediate
filaments.
[0017] In yet another preferred embodiment of said method, the
disruption of said IF is achieved by administering to said
mammalian cell a therapeutically effective dose of an agent
selected from a group consisting of polypeptides, peptides, nucleic
acids and chemical compounds. In one preferred embodiment, said
agent is a peptide selected from the group of peptides identified
as SEQ ID NOs: 1-10, and homologues thereof. In another preferred
embodiment, said agent is an interfering RNA or an antisense
oligonucleotide targeting vimentin and/or keratin-10 genes or their
transcripts. In yet another preferred embodiment, said agent is a
chemical compound or a lipidic derivative.
[0018] In another aspect, the present invention provides an agent
that disrupts or negatively modulates cytoskeletal IFs to prevent
or to treat HIV infection.
[0019] In a preferred embodiment of this aspect, said IFs comprise
vimentin and/or keratin-10.
[0020] In another preferred embodiment of an agent according to the
invention, said agent induces/achieves a decrease in the amount of
vimentin and/or keratin-10 in said IFs.
[0021] In another preferred embodiment of such an agent, the agent
decreases the expression of the genes encoding vimentin and/or
keratin-10.
[0022] In another preferred embodiment of an agent according to the
invention, said agent is selected from a group consisting of
polypeptides, peptides, nucleic acids and chemical compounds.
[0023] In another preferred embodiment of an agent according to the
invention, said agent comprises a peptide selected from the group
of peptides identified as SEQ ID No. 1-SEQ ID No. 10, and
homologues thereof.
[0024] In another preferred embodiment of an agent according to the
invention, said agent is an interfering RNA or an antisense
oligonucleotide, targeting vimentin and/or keratin-10 genes, or
their transcripts.
[0025] In another preferred embodiment of an agent according to the
invention, said agent is an interfering RNA selected from a group
consisting of a siRNA, shRNA and miRNA. Preferably, said
interfering RNA comprises a sequence of 15 to 50 nucleotides
complementary to a region of a messenger RNA of the vimentin and/or
keratin-10 proteins, preferably 18 to 25 nucleotides.
[0026] In another preferred embodiment of an agent according to the
invention, said agent is a chemical compound and said compound is a
lipidic compound or a lipidic derivative. Preferably said lipidic
compound is prostaglandin cyclopentane 15
deoxy-.DELTA.-.sup.12,14-PGJ2 (15d-PGJ2).
[0027] In another aspect, the present invention provides a
pharmaceutical composition for treating or preventing HIV
infection, said composition comprising an agent according to the
present invention as described above that disrupts or negatively
modulates cytoskeletal IFs according to the present invention as
described above, and a pharmaceutically acceptable carrier or
excipient.
[0028] In a preferred embodiment of a composition according to the
invention, said agent is selected from the group consisting of
polypeptides, peptides, nucleic acids and chemical compounds that
disrupt IFs that comprise vimentin and/or keratin-10.
[0029] In a preferred embodiment of a composition according to the
invention, said agent is a peptide selected from the group
consisting of the peptides identified as SEQ ID No. 1-SEQ ID No.
10, and homologues thereof.
[0030] Preferably, said agent is an interfering RNA or an antisense
oligonucleotide, targeting vimentin and/or keratin-10 genes or
their transcripts.
[0031] In a highly preferred embodiment of an agent according to
the invention, said agent is for use in the treatment or prevention
of HIV infection, or for use in the manufacture of a medicament for
the treatment or prevention of HIV infection. Treatment or
prevention of HIV infection includes reference to inhibition or
blockage of viral replication.
[0032] In a preferred embodiment of said pharmaceutical composition
the interfering RNA is selected from a group consisting of siRNA,
shRNA or miRNA.
[0033] In a preferred embodiment of said pharmaceutical composition
the chemical compound is a lipidic compound or a lipidic
derivative. Preferably, said lipidic compound is prostaglandin
cyclopentane 15 deoxy-.DELTA.-.sup.12,14-PGJ2.
[0034] In another aspect, the present invention provides a
pharmaceutical combination comprising an agent that disrupts
cytoskeletal IFs in accordance with the present invention as
described herein above, and at least one anti-HIV drug. Examples of
anti-HIV drugs suitable for use in aspects of the present invention
include an HIV protease inhibitor, most preferably the protease
inhibitor that is selected from the group consisting of: atazanavir
(Reyataz.TM.), amprenavir (Agenerase.TM.), darunavir
(Prezista.TM.), nelfinavir (Viracept.TM.), saquinavir (Invirase.TM.
or Fortovase.TM.), indinavir (Crixivan.TM.), fosamprenavir
(Lexiva.TM. or Telzir.TM.), lopinavir (Aluvia.TM.), ritonavir
(Norvir.TM.), tipranavir (Aptivus.TM.), functional derivatives of
these drugs, and combinations thereof, such as: lopinavir+ritonavir
(Kaletra.TM.). Other antiretroviral drugs that can be used in
aspects of the present invention are non-nucleoside reverse
transcriptase inhibitors (nNRTI) such as: efavirenz (Stocrin.TM.)
and nevirapine (Viramune.TM.), etravirine (Intelence.TM.),
rilpivirine (TMC-278), loviride (R89439), delavirdine
(Rescriptor.TM.), functional derivatives of these drugs and
combinations thereof. Other antiretroviral drugs that can be used
in aspects of the present invention are nucleoside reverse
transcriptase inhibitors (NRTIs) or nucleoside analogue reverse
transcriptase inhibitors (NARTIs) such as: lamivudine (3TC or
Epivir.TM.), abacavir (Ziagen.TM.), zidovudine (AZT or Retrovir
AZT.TM.), stavudine (d4T or Zerit.TM.), zalcitabine (ddC or
Hivid.TM.), didanosine (ddl or Videx.TM.) emtricitabine (FTC or
Emtriva.TM.), tenofovir (Viread.TM.), apricitabine (AVX754),
stampidine, elvucitabine (L-Fd4C), racivir, amdoxovir, functional
derivatives of these drugs, and combinations thereof, such as:
emtricitabine+tenofovir (Truvada.TM.), zidovudine+lamivudine
(Combivir.TM.), and abacavir+lamivudine+zidovudine
(Trizivir.TM.).
[0035] In addition to the above-mentioned antiretroviral drugs, the
pharmaceutical combination of the present invention may comprise
combinations of various classes of antiretroviral drugs listed
above, such as the combinations: efavirenz+zidovudine+lamivudine,
efavirenz+tenofovir+emtricitabine, lopinavir boosted with
ritonavir+zidovudine+lamivudine, and lopinavir boosted with
ritonavir+tenofovir+emtricitabine.
[0036] In a preferred embodiment of a pharmaceutical combination
according to the invention the agents and drugs are administered
simultaneously, separately or sequentially, as part of a dosage
regime.
[0037] In another aspect, the present invention provides a method
of treating or preventing HIV infection in subject in need thereof,
comprising administering to said subject a therapeutically
effective dose of a pharmaceutical composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1. Relative intensity of the human vimentin and
keratin-10 proteins as identified by comparative proteomics. Panel
A shows the decrease of vimentin protein in the cultures treated
with an anti-HIV activity-bearing extract. Panel B shows the
decrease of the keratin-10 protein in cultures treated with the
anti-HIV activity-bearing extract. Error bars stand for standard
deviations.
[0039] FIG. 2. Detection of vimentin and keratin-10 proteins in
cultures stably silenced for each of both proteins. Vimentin (A)
and keratin-10 (B) were assessed by western blot in the MT4 cell
line subjected to silencing for each protein, MT4.sub.vim(s) and
MT4.sub.K-10(s) respectively. MT4.sub.vim(s) and MT4.sub.K-10(s)
cultures showed a decreased expression of the respective protein
compared to the MT4 cell culture. The .beta.-actin protein was used
as control to normalize the western blot analysis. Each variant was
analyzed in duplicate lanes. In this figure, K-10 stands for
keratin-10.
[0040] FIG. 3. Inhibition of HIV-1 replication in MT4.sub.vim(s)
and MT4.sub.K-10(s) cell cultures as evaluated by assessing the p24
antigen. MT4, MT4.sub.vim(s) and MT4.sub.K-10(s) cell cultures were
challenged with the HIV-1 strain Bru, at a multiplicity of
infection (m.o.i.) of 0.01. Viral replication was inhibited in more
than 90% in MT4.sub.vim(s) and MT4.sub.K-10(s) cell cultures. Error
bars stand for standard deviations.
[0041] FIG. 4. Challenge assay with the pLGW lentiviral vector in
MT4, MT4.sub.vim(s) and MT4.sub.K-10(s) cell cultures. Cultured
cells were transduced with a lentiviral vector which resembles the
first stages of the HIV-1 viral replication cycle after entry, also
carrying the GFP reporter gene. A) MT4.sub.vim(s) and
MT4.sub.K-10(s) cell cultures showing a decreased percent of
fluorescent cells once transduced with the lentivirus, compared to
the unsilenced MT4 cells. Error bars stand for standard deviation.
B) Flow cytometry histograms of each culture.
[0042] FIG. 5. IFs structural analysis in MT4 cell cultures. MT4,
MT4.sub.vim(s) and MT4.sub.K-10(s) cell cultures were analyzed by
transmission electron microscopy. Silenced cultures (B, C) showed
shortened IFs, instead of the long filaments observed in unsilenced
MT4 cell cultures used as control (A). Panel D shows fragmented IFs
by the action of the peptide identified as SEQ ID No. 1 on MT4
cells.
[0043] FIG. 6. Inhibition of HIV-1 replication in MT4 cells by
peptides. A) Cells were incubated with the peptide for 24 h and
further challenged with the HXB1 HIV-1 strain at m.o.i. of 0.05.
Viral replication was inhibited at high percents, which also
increased together with peptide concentration. Error bars stand for
standard deviation. B) Cells were incubated with peptides for 24 h
and further incubated with the HIV Bru strain at m.o.i. of 0.01.
The inhibitory concentration 50 (IC50) was at the nanomolar level
for all the peptides.
[0044] FIG. 7. Inhibition of HIV-1 replication by the different
peptides in peripheral blood mononuclear cells (PBMCs). These cells
were pre-stimulated, treated with different concentrations of the
peptides and further infected with the HIV-1 Bru strain. The
peptides inhibited HIV replication in a dose-dependent manner.
[0045] FIG. 8. Inhibition of HIV-2 replication by the peptide
identified as SEQ ID No. 1,2,3,4,5,6,7,8,9 y 10. PBMCs were
pre-stimulated, treated with the different concentration of the
peptides and further infected with the HIV-2 CBL-20 strain. The
peptides inhibited HIV-2 replication in a dose-dependent
manner.
[0046] FIG. 9. Decreased vimentin in the presence of the peptides
identified as SEQ ID No. 1, 4, 5, 7, 8 and 9. The MT4 cell line was
incubated with said peptides at 50 .mu.M each for 24 h. Vimentin
was detected by the western blot technique. Vimentin bands showed a
decreased intensity in the cultures treated with the peptides.
[0047] FIG. 10. Assessment of internalization of the peptides
identified as SEQ ID No. 1 (A) and SEQ ID No. 3 (B) in the MT4 cell
line. The graph represents the percent of fluorescent cells
corresponding to peptide penetration at 5, 10, 20 and 40 .mu.M
concentrations and at different time points in the MT4 cell line.
Cc: Untreated cells. Error bars stand for standard deviations.
[0048] FIG. 11. Inhibition of HIV-1 replication by a lipidic
derivative. MT4 cells were incubated with different concentrations
of the prostaglandin cyclopentane 15 deoxy-.DELTA.-.sup.12,14-PGJ2
(15d-PGJ2), and further challenged with the HIV-1 (Bru strain) at
m.o.i. of 0.01. The 15d-PGJ2 prostaglandin inhibited the HIV-1
replication. Error bars represent standard deviations.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention solves the problem mentioned above,
and describes a method to inhibit the HIV replication by disrupting
the cytoskeleton, more precisely the proteins forming cytoskeletal
IF.
[0050] For the purpose of the invention, said IF can be composed by
acidic keratin, basic keratin, vimentin, desmin, glial fibrillary
acidic factor, peripherin, neurofilament (NF) protein, internexin,
filensin, phakinin, and lamin.
[0051] In an embodiment of the invention, said IF can be composed
by vimentin and keratin proteins. More particularly, said IF can be
formed by the vimentin and keratin-10 proteins. The altered
cytoskeleton inhibits the HIV viral replication in human cells. By
disrupting the cytoskeletal IFs is meant that the structure of the
IFs is modified or altered in such a way that down regulate the
proteins forming cytoskeletal IF and/or structural breakage of
cytoskeletal IF in shorter subunits and/or change the structural
form of the cytoskeletal network and/or that the IF are
disassembled. Cytoskeleton as described herein refers to cellular
"scaffolding" or "skeleton" contained within the cytoplasm and is
made out of protein. The cytoskeleton is present in all cells; it
plays important roles in both intracellular transport and cellular
division. It is formed by three main structures: microtubules,
microfilaments and the intermediate filaments (IFs). The
cytoskeleton provides the cell with structure and shape.
Cytoskeletal elements interact extensively and intimately with
cellular membranes.
[0052] The IFs as defined herein are a family of related proteins
that share common structural and sequence features. Intermediate
filaments have an average diameter of 10 nanometers, which is
between that of actin (microfilaments) and microtubules. Most types
of intermediate filaments are cytoplasmatic, but one type, the
lamins, are nuclear. There are about 70 different genes coding for
various intermediate filament proteins, specific for each cell
type, the vimentin and keratin-10 proteins among them.
[0053] The term "vimentin", as used herein, refers to the member of
the intermediate filament family of proteins identified by NCBI
Reference Sequence: NP_003371.2, having the sequence as given in
SEQ ID NO.11. Vimentin proteins form filamentous polymers in a
series of assembly steps starting from antiparallels,
half-staggered double dimmers (or tetramers) to form unit-length
filaments (ULF) that are assembled longitudinally to form the
complete filament.
[0054] The term "keratin", as used herein, refers to the family of
fibrous structural proteins or intermediate filaments. Keratin
proteins form filamentous polymers in a series of assembly steps
beginning with dimerization; dimers assemble into tetramers and
octamers and eventually into ULF capable of annealing end-to-end
into long filaments. Each type I keratin is coexpressed with a
specific type II keratin partner, and each keratin pair that is
formed as coassembly of a specific preferred and predetermined
pairs is characteristic and indicative of differentiation and
specialization of a particular type of epithelial cell.
[0055] The term "keratin-10", as used herein, refers to Keratin,
type I cytoskeletal 10, the member of the intermediate filament
family of proteins identified by Swiss-Prot accession number:
Q6EIZ0.1, having the sequence as given in SEQ ID NO: 12.
[0056] The term "gene", as used herein refers to a DNA sequence
including but not limited to a DNA sequence that can be transcribed
into mRNA which can be translated into polypeptide chains,
transcribed into rRNA or tRNA or serve as recognition sites for
enzymes and other proteins involved in DNA replication,
transcription and regulation. The term refers to any DNA sequence
comprising several operably linked DNA fragments such as a promoter
region, a 5' untranslated region (the 5' UTR), a coding region
(which may or may not code for a protein), and an untranslated 3'
region (3' UTR) comprising a polyadenylation site. Typically, the
5'UTR, the coding region and the 3'UTR are transcribed into an RNA
of which, in the case of a protein encoding gene, the coding region
is translated into a protein. The gene usually comprises introns
and exons.
[0057] The term "vimentin gene", as used herein, refers to the gene
encoding the protein vimentin or a homologue thereof.
[0058] The term "keratin-10 gene", as used herein, refers to the
gene encoding the protein keratin-10 or a homologue thereof.
[0059] The term "disrupting" as used herein with reference to
disruption of the intermediate filaments as indicated herein refers
to interference with function or structural organization. In
particular, disruption may involve structural breakage, inhibition
of polymerization, inhibition of formation and biosynthesis,
including inhibition of formation of primary, secondary and
tertiary protein structures, etc.
[0060] The term "negatively modulating" as used herein with
reference to negatively modulating the intermediate filaments as
indicated herein, refers to changing or altering function or
structural organization in a manner that results in loss or
decreasing of biological function of said filaments.
[0061] The term "cytoskeletal intermediate filaments (IFs)" as used
herein, refers to Intermediate filaments as a type of cytoskeletal
elements, and their size is intermediate compared with actin and
microtubules. Together these three enhance the structural
integrity, cell shape, and cell and organelle motility.
Cytoskeletal intermediate filaments are regularly divided into five
types: Types I and II: Acidic Keratin and Basic Keratin. Keratins
also have subtypes that are unique to different epithelial cells;
Type III: Vimentin in fibroblasts, endothelial cells and
leukocytes; desmin in muscle; glial fibrillary acidic factor in
astrocytes and other types of glia, and peripherin in peripheral
nerve fibers; Type IV Neurofilament (NF) proteins H (heavy), M
(medium) and L (low), internexin filensin and phakinin; and Type V:
Lamins.
[0062] The term "structure of cytoskeletal intermediate filaments
(IFs)" as used herein, refers to the helical organization of
tetramers of the filaments. Each intermediate filament monomer
consists of an alpha helical rod domain which connects the amino
(head) and carboxyl (tail) terminals. The rods coil around another
filament to form a dimer. The N and C terminals of each filament
are aligned. Some Intermediate filaments form homodimers; other
form heterodimers. The dimers then form staggered tetramers that
line up head-tail. This tetramer is considered the basic subunit of
the intermediate filament. The final intermediate filament is a
helical array of these tetramers.
[0063] In the context of this specification, the terms "treatment"
and "treating" refer to any and all uses which remedy a condition
or disease or symptoms thereof, prevent the establishment of a
condition or disease or symptoms thereof, or otherwise prevent or
hinder or reverse the progression of a condition or disease or
other undesirable symptoms in any way whatsoever.
[0064] The term "therapeutically effective dose", as used herein
refers to a non-toxic amount of the therapeutic agent sufficient to
provide the desired therapeutic effect, e.g. to treat, ameliorate,
or prevent a desired disease or condition, or to exhibit a
detectable therapeutic or preventative effect. The effect can be
detected by, for example, chemical markers or antigen levels.
Therapeutic effects also include reduction in physical symptoms.
The precise effective amount for a subject will depend upon the
subject's size and health, the nature and extent of the condition,
and the therapeutics or combination of therapeutics selected for
administration. Thus, it is not useful to specify an exact
effective amount in advance. However, the effective amount for a
given situation can be determined by routine experimentation and is
within the judgment of the clinician.
[0065] For purposes of the present invention, an effective dose
will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10
mg/kg of the polynucleotide or polypeptide compositions in the
individual to which it is administered.
[0066] The term "pharmaceutically acceptable carrier or excipient",
as used herein refers to a carrier for administration of a
therapeutic agent, such as a polypeptide, polynucleotide, and other
therapeutic agents. The term refers to any pharmaceutical carrier
that does not itself induce the production of antibodies harmful to
the individual receiving the composition, and which may be
administered without undue toxicity. Suitable carriers may be
large, slowly metabolized macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, and inactive virus particles.
Such carriers are well known to those of ordinary skill in the art.
Pharmaceutically acceptable carriers in therapeutic compositions
may contain liquids such as water, saline, glycerol and ethanol.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present in
such vehicles. A thorough discussion of pharmaceutically acceptable
excipients is available in Remington's Pharmaceutical Sciences
(Mack Pub. Co., N.J. 1991). Typically, the therapeutic compositions
are prepared as injectables, either as liquid solutions or
suspensions, or in solid forms, either suitable for solution in, or
suspension in, liquid vehicles or for direct intake. Liposomes are
included within the definition of a pharmaceutically acceptable
carrier, as are arosols.
[0067] The term "homologue" as used herein, and when referring to a
peptide refers to a the peptide comprising an amino acid sequence
sharing at least a 70% sequence identity as established by sequence
alignment with e.g. Blast etc, preferably at least 75%, more
preferably at least 85%, 90% or even 95%, most preferably at least
97%, sequence identity with SEQ ID No. 1 to SEQ ID No. 10
sequences, and with the ability to disrupt, negatively modulate or
modify the cytoskeleton, specifically the proteins forming the
cytoskeletal IFs and more precisely the vimentin and keratin-10
proteins. Suitable homologues are peptides with conservative amino
acid substitutions. Suitable less than 10% of the amino acids are
substituted, more suitable less than 5%, less than 3% and most
preferably less than 1% of the amino acids are substituted.
Suitable less than 10 amino acid residues are substituted, more
suitably less than 5, and most suitably less than 2 amino acids are
substituted. A conservative substitution is one in which an amino
acid is replaced by another very similar amino acid, which
substitution has little or no effect on the activity of the
protein. A "conservative substitution" is the replacement of an
amino acid with another amino acid that has the same net electronic
charge and approximately the same size and shape. Amino acids with
aliphatic or substituted aliphatic amino acid sidechains have
approximately the same size when the total number carbon and
heteroatoms in their side chains differs by no more than about
four. They have approximately the same shape when the number of
branches in their side chains differs by no more than one. Amino
acids with phenyl or substituted phenyl groups in their side chains
are considered to have about the same size and shape. Listed below
are five groups of amino acids. Replacing an amino acid in a
polypeptide with another amino acid from the same group results in
a conservative substitution: Group I: glycine, alanine, valine,
leucine, isoleucine, serine, threonine, cysteine, and non-naturally
occurring amino acids with C1 C4 aliphatic or C1 C4 hydroxyl
substituted aliphatic sidechains (straight chained or
monobranched). Group II: glutamic acid, aspartic acid and
non-naturally occurring amino acids with carboxylic acid
substituted C1 C4 aliphatic side chains (unbranched or one branch
point). Group III: lysine, ornithine, arginine and non-naturally
occurring amino acids with amine or guanidine substituted C1 C4
aliphatic side chains (unbranched or one branch point). Group IV:
glutamine, asparagine and non-naturally occurring amino acids with
amide substituted C1 C4 aliphatic side chains (unbranched or one
branch point). Group V: phenylalanine, phenylglycine, tyrosine and
tryptophan.
[0068] The term "% sequence identity" is defined herein as the
percentage of nucleotides in a nucleic acid sequence that is
identical with the nucleotides in a nucleic acid sequence of
interest, after aligning the sequences and optionally introducing
gaps, if necessary, to achieve the maximum percent sequence
identity. Methods and computer programs for alignments are well
known in the art. As used herein, the terms "nucleic acid sequence"
and "nucleotides" also encompass non-natural molecules based on
and/or derived from nucleic acid sequences, such as for instance
artificially modified nucleic acid sequences, peptide nucleic
acids, as well as nucleic acid sequences comprising at least one
modified nucleotide and/or non-natural nucleotide such as for
instance inosine.
[0069] The term "RNA interference", refers to the process where an
interfering RNA (iRNA) causes intracellular degradation of specific
mRNA and can be used to interfere with the translation of a desired
mRNA target.
[0070] The term "interfering RNA" refers to a double or simple
stranded RNA (iRNA) agent, by which is meant a small nucleic acid
molecule used for RNA interference. Short iRNA agents that are
about 15-30 nucleotides in length are referred to as
"small-interfering RNA" or "siRNA." Longer iRNA agents are
generally referred to as "double-stranded RNA" or "dsRNA", other
forms of iRNA agents are microRNA (miRNA) and short hairpin RNA
(shRNA) molecules. The iRNA agents can be unmodified or
chemically-modified nucleic acid molecules. The iRNA agents can be
chemically synthesized or expressed from a vector or enzymatically
synthesized. The use of a chemically-modified iRNA agent can
improve one or more properties of an iRNA agent through increased
resistance to degradation, increased specificity to target
moieties, improved cellular uptake, and the like. A DNA molecule
that transcribes dsRNA or siRNA (for instance, as a hairpin duplex)
also provides RNA interference. DNA molecules for transcribing
dsRNA are disclosed in U.S. Pat. No. 6,573,099, and in U.S. Patent
Publication Nos. 20020160393 and 20030027783. DNA molecules for
transcribing siRNA are reviewed in Tuschl and Borkhardt, Molecular
Interventions, 2:158 (2002).
[0071] The term "antisense RNA" as used herein, refers to any RNA
that binds to mRNA with enough affinity to decrease the amount of
protein translated from the mRNA. The amount of protein translated
from the mRNA is preferably decreased by more than 20%; more
preferably decreased by more than 50%, 70%, and 80%; and most
preferably decreased by more than 90%. Antisense RNA materials and
methods are well known in the art.
[0072] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide. "Antisense inhibition" refers to the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein.
[0073] By the term "inhibiting the expression" is meant silencing
or downregulation of a gene or nucleic acid which refers to a
detectable decrease of transcription and/or translation of a target
nucleic acid sequence, i.e., the sequence targeted by the iRNA, or
a decrease in the amount or activity of the target sequence or
protein in comparison to the normal level that is detected in the
absence of the interfering RNA or other nucleic acid sequence. A
detectable decrease can be as small as about 5% or 10%, or as great
as about 80%, 90% or 100%. More typically, a detectable decrease is
about 20%, 30%, 40%, 50%, 60%, or 70%.
[0074] The term "lipidic compound" as used herein, refers to fatty
acid analogues derived from e.g. monounsaturated fatty acids,
polyunsaturated fatty acids and lipids comprising 1-6 triple
bonds.
[0075] "HIV" is the retrovirus Human Immunodeficiency Virus, a
virus that causes immunodeficiency by attacking CD4+ cells in the
body. The term "HIV", as used herein, includes any HIV, including
all groups and subtypes (clades) of HIV-1 and HIV-2, for example
HIV-1 M and HIV-1 O groups; the invention embraces each of the
known clades; HIV-1 is preferred.
[0076] The term "replication" as used herein, refers to the process
in which a complementary strand of a nucleic acid molecule is
synthesized by a polymerase enzyme. In the particular context of
the present invention, the term replication as used herein in
reference to a virus, refers to the completion of a complete or
entire viral life cycle, wherein infectious viral particles or
virions attach to the surface of the host cell (usually binding to
a specific cell surface molecule that accounts for the specificity
of the infection). Once inside the cell, the virions are uncoated
and viral genes begin to express proteins needed for replication of
the genome and synthesis of new proteins to make new capsids and
cores leading to the assembly of progeny infectious virus particles
which, themselves, are capable of infecting and replicating in new
host cells. Thus, a viral life cycle is only complete if within a
single cell, infection by one or more virus particles or virions
proceeds all the way to the production of fully infectious progeny
virus particles. In the particular case of retroviruses, a complete
viral life cycle involves infectious viral particles containing the
viral RNA entering a cell, the RNA being reverse transcribed into
DNA, the DNA being integrated into the host chromosome as a
provirus, and the infected cell producing virion proteins and
assembling them with full length viral genomic RNA into new,
equally infectious particles.
[0077] The term "host cell" as used herein, refers to a cell used
for the expression of a viral genome, or propagation of a vector or
virus.
[0078] The term "CD4+ cells" as used herein, refers to a major
classification of T lymphocytes, referring to those that carry the
CD4 antigen.
[0079] In particular, the present invention refers to methods
negatively modulating, modifying or disrupting the cytoskeletal
IFs. Preferred IFs contain vimentin and/or keratin-10 proteins. In
a preferred embodiment, the method comprises decreasing the amount
of vimentin and/or keratin-10 in the IFs. The decrease of vimentin
and or keratin-10 may be affected in several ways. A preferred
embodiment is decreasing or inhibiting coding genes expression for
vimentin and/or keratin-10. More preferably, the expression levels
of vimentin and/or keratin-10 in the IFs is decreased or the
structure of the cytoskeletal vimentin and/or keratin-10--is
altered. The structure of cytoskeleton IFs may be altered by
modifying the structure of the IF proteins, e.g. by cleavage or
misfolding, preferably the structure of vimentin and/or
keratin-10.
[0080] The evidences referred in the technical literature suggest
that HIV requires vimentin excision during its lifecycle.
Surprisingly, in the present invention, viral replication is
inhibited through what seems to be a natural mechanism present
during viral infection. It is not obvious to try to disrupt the
cytoskeleton and/or take away vimentin as a means to inhibit HIV
infection, in fact one would expect that the infection would be
much faster, as the disruption of the cytoskeleton also happens
during normal infection.
[0081] The applicants identified vimentin and keratin-10
cytoskeletal proteins by a comparative proteomic analysis of MT4
cells treated with a fraction of a human leukocyte extract showing
anti-HIV activity. It was found that leukocyte extract having
anti-HIV activity showed a decrease and/or destabilization of
vimentin and/or keratin-10 and/or the IFs. Previously, Thomas et
al. demonstrated that an anti-vimentin antibody was able to block
the binding of the HIV-1 gp120 glycoprotein to the cell surface
vimentin, preventing the cell entry of the virus (Thomas E K,
Connelly R J, Pennathur S, Dubrousky L, Haffar O K, Bukrinsky M I
1996. Viral Immunol 9: 73-87). Surprisingly, very low inhibition
levels of viral replication were detected under the experimental
conditions tested for the present invention, by using an antibody
against vimentin (aimed to decrease the HIV infection). Thomas et
al. used an antibody against vimentin thus blocking vimentin that
is accessible to the antibody. This is very different from the
action proposed in the present invention. In the present invention,
vimentin is altered and/or decreased so to disrupt the IFs. By
blocking the vimentin as done in Thomas et al. the IFs are not
altered. The experimental data also confirms the difference mode of
action in methods of the present invention. Very high percent of
inhibition of viral replication, up to 100%, were obtained when
vimentin levels are decreased and/or vimentin structure
destabilized in the target cell, as observed in the Example 2,
FIGS. 3 and 4, while in Thomas et al. a maximum of 47% of
inhibition was observed.
[0082] Moreover, there are no reports on the binding of keratin-10
to the viral gp120 protein and, precisely, HIV replication was also
inhibited by inhibiting and/or destabilizing keratin-10, as shown
in Example 2, FIGS. 3 and 4.
[0083] To further gain insight of the mechanism of HIV replication
inhibition, an experimental system was used which cellular entry is
not mediated by the gp120 viral protein. That system comprises an
HIV-1 based non-replicating lentiviral vector that is devoid of
gp120 and also expressing the green fluorescent protein (GFP). As
shown in Example 2, there was an efficient "infection" of MT4 cells
by this lentivirus, and the cultures showed high percent of GFP
expression indicative of an efficient penetration of the cell and
integration into the cellular genome of this lentiviral vector. In
this HIV-1-based lentiviral "infection" system, a decrease of
vimentin generates a dramatic decrease in the lentiviral
"infection", as shown in Example 2. This demonstrates that the
method used in the present invention to inhibit HIV-1 infection is
not related to the binding of gp120 to vimentin as proposed by
Thomas et al. Hence, this invention is related to a method not
previously reported for inhibiting HIV infection.
[0084] Furthermore we demonstrated with the aid of transmission
electron microscopy (TEM) that IFs are destabilized in MT4 cells
silenced for vimentin (MT4.sub.vim(s)), as well as in cells
silenced for keratin-10 (MT4.sub.K-10(s)), resulting in a inhibited
"infection" of the HIV-1-based lentiviral vector (Example 3).
MT4.sub.vim(s) and MT4.sub.K-10(s) cells were obtained by means of
introducing RNA hairpins specific for each of the protein coding
genes.
[0085] The disruption of the IFs may be achieved by an agent
selected from a group consisting of polypeptides, peptides, nucleic
acids and chemical compounds. In a preferred embodiment the agent
is a peptide, more preferably the peptide is a peptide selected
from the group consisting of peptides identified as SEQ ID No. 1 to
SEQ ID No. 10, and homologues thereof.
[0086] In another preferred embodiment the agent is an interfering
RNA or an antisense oligonucleotide targeting vimentin and/or
keratin-10 genes.
[0087] In another preferred embodiment the agent is a chemical
compound or a lipidic derivative. A suitable lipidic compound is
said lipidic compound is prostaglandin cyclopentane 15
deoxy-.DELTA.-.sup.12,14-PGJ2.
[0088] The present invention describes methods to treat and/or
prevent the infection of human cells by HIV. Those methods involve
the disruption of IFs and in particular the disruption of vimentin
and/or keratin-10 in the cell, in order to prevent or treat the HIV
infection in the cell.
[0089] The negative regulation occurs within an HIV host cell of a
given subject, for the means of preventing or inhibiting the
effective infection of the host cells of the subject. Thus, the
present invention similarly comprises methods to treat and/or to
prevent the infection of a subject with HIV.
[0090] The inhibition of infection with HIV by using the method
described in the present invention is applied both at cellular
level and for the whole organism. The term inhibition implies
complete or partial inhibition of the infection.
[0091] The present invention describes the manipulation of IFs and
in particular vimentin and/or keratin-10 cytoskeletal proteins to
inhibit HIV replication. This strategy provides the advantage of
minimal or inexistent viral resistance over the antiretroviral
drugs currently available, since these proteins are endogenous
cellular proteins rather than viral. The mechanisms of action of
the drugs of the present invention operate through pathways
different to those already described and showing a high inhibition
capacity. Therefore, its combination with drugs currently available
against the HIV infection could enhance the effectiveness of
anti-HIV treatments. Moreover, the use of the therapeutic agents of
the present invention could be combined with the novel therapeutic
strategies already proposed in the state of the art, as the
transplantation of stem cells bearing modified endogenous genes.
The therapeutic modality of the present invention provides a new
option for those patients showing multiple drug resistance, who
represent a high percent among the patients treated with the
currently available therapy.
[0092] In spite of a possible damage of the IFs structure leading
to toxicity or even cellular death, a surprising major achievement
of this invention comprises inhibiting the HIV infection without
affecting cellular viability, all these adding even more novelty
for the treatment of patients infected with HIV.
[0093] The present invention also comprises the use of agents that
negatively modulate, modify, or disrupt the cytoskeleton, more
precisely the proteins forming the cytoskeletal IFs and
specifically vimentin and/or keratin-10, to produce a
pharmaceutical to prevent or to treat the HIV infection. Such
agents can be fused and/or conjugated to other molecules. Such
agents include peptide-like compounds, interfering RNA and lipidic
compounds producing the negative modulation or modifying the
cytoskeleton, IFs, and particularly those agents negatively
modulating vimentin and/or keratin-10.
[0094] Vimentin and/or keratin-10 can be negatively modulated by
putting the cell into contact with the agent that negatively
modulates vimentin and/or keratin-10. The agent can be formulated
to increase its capacity for cell penetration as required. The
negative modulation can be achieved by administering an agent to a
subject, such agent negatively modulating vimentin and/or
keratin-10 in the cells of the subject.
[0095] The agent is administered in such a way that it is contacted
with the cells of the subject, which are already infected with HIV
or which could be potentially infected. Such cells are referred
herein as HIV host cells. The administration of the agent comprises
the agent getting into contact with the host cell. Administration
routes include the parenteral route and those by which the agent is
delivered through the mucosae of the subject. In a specific
embodiment of aspects of the invention, the host cell is a CD4+
cell.
[0096] In an embodiment of the invention, the negative modulation
or modification can be achieved by directly affecting vimentin
and/or keratin-10, either by reducing the expression of the gene or
protein synthesis, by modifying the structure of the filaments
formed by these proteins, by destabilizing filament structure or
reducing its activity/function.
[0097] Within the context of the present invention, negative
modulation (or modification) comprises the inhibition of the level
of vimentin and/or keratin-10 proteins in the cell, or
modification, destabilization, disassembly or even destruction of
the structure of the IFs containing these proteins within the
cell.
[0098] Any agent known as inhibiting or negatively modulating IFs
and in particular vimentin and/or keratin-10 can be used to inhibit
the HIV infection, according to the method explained in the present
invention.
[0099] Similarly, the HIV infection can also be inhibited by using
peptide- or polypeptide-like agents that negatively modulate or
destabilize IFs and in particular vimentin and/or keratin-10. Such
agents comprise endogenous proteins or proteins that are not
normally present within the host cell. They could be, for example,
mutated proteins, genetically engineered proteins, peptides,
synthetic peptides, recombinant proteins, chimeric proteins,
antibody fragments, humanized proteins, humanized antibodies,
chimeric antibodies, modified proteins and fragments of all of
them.
[0100] In the present invention, the use of peptides capable of
disrupting the structure of IFs, in particular those which contain
vimentin and/or keratin-10, was found to strongly inhibited the
infection of MT4 cells with HIV, corroborating the results obtained
in MT4.sub.vim(s) and MT4.sub.K-10(s) cells. This supports the use
of those peptides to prevent or to treat the HIV infection, as part
of the present invention.
[0101] In a preferred embodiment of aspects of the invention, the
peptides are those identified in the sequence listing as SEQ ID No.
1 to SEQ ID No. 10. The invention also comprises the use of
homologues of those peptides. The peptides can be fused to another
molecule, for example, can be fused to a penetrating peptide.
[0102] An agent useful to prevent or to treat the HIV, according to
the present invention, is that agent able to inhibit the expression
of vimentin and/or keratin-10 genes, or its protein synthesis, or
the structure of IFs which contain vimentin and/or keratin-10. A
preferred agent, according to the present invention, comprises an
agent that silences the vimentin and/or keratin-10 genes or
transcripts thereof by using an iRNA, such as a small interfering
RNA (siRNA), a short hairpin RNA (shRNA) or a micro RNA
(miRNA).
[0103] RNA interference refers to a type of selective
posttranscriptional gene silencing process which destroys the
specific messenger RNA (mRNA) by means of a molecule which binds
and inhibits the mRNA processing. For example, it can inhibit
translation of the mRNA or degrade it. Within the context of the
present invention, iRNA refers to any type of interfering RNA,
including, but not restrained to, a siRNA, shRNA, endogenous miRNA
and an artificial miRNA.
[0104] The term siRNA used herein refers to a nucleic acid forming
a double strand of RNA which is able to reduce or inhibit the
expression of the vimentin and/or keratin-10 genes. The siRNA
sequence can correspond to the entire sequence of the vimentin
and/or keratin-10 genes. The typical siRNA is at least 15 to 50
nucleotides long, preferentially being 19 to 30 nucleotides long. A
siRNA can be chemically synthesized, produced by in vitro
transcription or be produced within a cell which is specifically
used to produce it.
[0105] The term shRNA is used herein as a type of siRNA. These
shRNA are made up of a short antisense strand of, for example, 19
to 25 nucleotides and followed by a loop of 5 to 9 nucleotides and
the analogous sense strand. Alternatively, the sense strand can
precede the nucleotide loop and the following antisense strand.
shRNAs function as siRNA and/or siRNA species, but they differ in
that shRNAs show particular hairpin-like structures for increased
stability. These shRNAs as other agents described herein, can be
delivered as plasmids, retroviruses and lentiviruses and be
expressed from promoters such as the U6 polimerase III promoter or
others.
[0106] Delivery methods for the interfering RNA type agents to the
target cell can include, for example, the injection of a
composition containing the agent, or the said composition getting
into direct contact with the cell, for example, a hematopoietic
cell getting into contact with a composition containing the
interfering RNA. In another case, the interfering RNA type agent
can be directly injected by any route for direct inoculation into
the bloodstream, such as a venous or arterial route, for example,
by hydrodynamic injection or catheterization. In some cases, the
interfering RNA agent can be delivered to specific organs or
systemically. The colloidal dispersion systems can be used as
delivery vehicles to increase the in vivo stability of the
agents.
[0107] The agents may inhibit the expression of the vimentin and/or
keratin-10 genes through mechanisms similar to those used by, for
example, an oligonucleotide or a nucleic acid analogue. They
include, for example, a peptide-nucleic acid (PNA), a pseudo
complementary PNA (pc-PNA), locked nucleic acids (LNA) and their
derivatives. The nucleic acid sequences code for proteins which act
as transcriptional repressors, antisense molecules, ribozymes,
small inhibitory nucleic acid sequences such as interfering RNA,
shRNA, siRNA, miRNA and antisense oligonucleotides.
[0108] The agents can resemble the shape of any entity normally
present or not, at the levels being administered to the cell or the
organism. Agents such as chemicals, small molecules, aptamers, can
be identified or generated to negatively modulate IFs and in
particular vimentin and/or keratin-10. Within the context of the
present invention, aptamers are single-stranded nucleic acids
showing well defined tridimensional structures to facilitate its
binding to target molecules in a manner conceptually similar to
that of antibodies. Aptamers combine the optimal properties of both
small molecules and antibodies, including their high specificity
and affinity, chemical stability, low immunogenicity and the
capacity to attack protein-protein interactions. The agent can
function directly as administered, but it could be also modified or
used intracellularly to generate the negative modulation of
vimentin and/or keratin-10. For example, the introduction of a
nucleic acid sequence into the cell and its transcription results
in the production of the nucleic acid and/or the protein which
inhibits IFs protein and in particular vimentin and/or keratin-10
within the cell.
[0109] The agent may comprise a vector. Vectors can be episomal,
for example, plasmids, vectors derived from viruses such as
cytomegaloviruses, adenoviruses, etc., or can be integrated into
the genome of the target cell, for example, vectors derived from
retroviruses such as the Moloney Murine Leukemia Virus, HIV-1, the
avian leukosis virus, and others. Vectors based on HIV or the
Feline Leukemia Virus can be used to transfect non-dividing cells.
Vectors combining different retroviruses can be used.
[0110] Several viral and virus-associated vectors have being
described in the state of the art. Such vectors can be used as
carriers for transferring a nucleic acid construct to the cell. The
constructs can be integrated in non-replicative viral genomes
similar to adenoviruses (adeno-associated viruses, AAV), herpes
simplex viruses, or others, including retroviral and lentiviral
vectors to infect or transduce the cells. An HIV-based vector can
be particularly useful in HIV host cells.
[0111] Another embodiment of the present invention comprises a
pharmaceutical composition comprising the agent according to the
present invention. The agents mentioned in the present invention
and contained within the said composition can be combined to each
other or be associated to other therapeutic agents such as, but not
limited to, the already known anti-HIV drugs (for example,
zidovudine (AZT).
[0112] In a preferred embodiment the negative modulation of IFs and
in particular vimentin and/or keratin-10 is applied in the present
invention to cells able to be infected by the HIV, for the purpose
of preventing or reducing the infection of HIV in that cell. In a
preferred embodiment, the human cell is a CD4+ cell. The
application of such negative modulation to a whole organism, a
human or a primate, may be an effective therapeutic treatment for
the organism against HIV infection.
[0113] Another embodiment of the present invention is a
pharmaceutical combination comprising an agent according to the
present invention and a anti-HIV drug. In the pharmaceutical
combination, the agents and drugs being part of it can be
administered simultaneously, separately or sequentially.
ADVANTAGES OF THE INVENTION
[0114] The present invention is advantageous over the currently
available antiretroviral drugs because it avoids viral resistance
or reduces its probabilities for occurrence to the minimum. This is
based on the cellular endogenous rather than viral origin of IFs
and in particular vimentin and keratin-10 proteins.
[0115] The drugs of the present invention act with a high
inhibition capacity through mechanisms different to those already
described in the prior art. Therefore, its combination with
currently available therapeutic drugs specific for the HIV
infection could enhance the effectiveness of anti-HIV
treatments.
[0116] On the other hand, the use of the therapeutic agents of the
present invention could be combined with novel therapeutic
alternatives proposed in the state of the art, such as
transplantation of stem cells bearing endogenous modified
genes.
[0117] The present invention offers a new therapy to patients
resistant to multiple drugs, which represent a high percent among
patients treated with the currently available therapy.
Delivery Methods
[0118] Once formulated, the pharmaceutical compositions of the
invention can be (1) administered directly to the subject; (2)
delivered ex vivo, to cells derived from the subject; or (3)
delivered in vitro for expression of recombinant proteins.
[0119] Direct delivery of the compositions will generally be
accomplished by injection, either subcutaneously,
intraperitoneally, intravenously or intramuscularly, or delivered
to the interstitial space of a tissue. The compositions can also be
administered into the nervous system. Other modes of administration
include topical, oral, suppositories, and transdermal applications,
needles, and particle guns or hyposprays. Dosage treatment may be a
single dose schedule or a multiple dose schedule.
[0120] Methods for the ex vivo delivery and re-implantation of
transformed cells into a subject are known in the art and described
in e.g., International Publication No. WO 93/14778. Examples of
cells useful in ex vivo applications include, for example, stem
cells, particularly hematopoietic, lymph cells, macrophages,
dendritic cells, or tumor cells.
[0121] Generally, delivery of nucleic acids for both ex vivo and in
vitro applications can be accomplished by, for example,
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, electroporation, encapsulation of
the polynucleotide(s) in liposomes, and direct microinjection of
the DNA, all well known in the art. Methods for introducing
polynucleotides (such as siRNAs) into a cell are known in the art.
Methods for introducing nucleic acid for instance comprise calcium
phosphate transfection, DEAE-Dextran, electroporation or
liposome-mediated transfection. Alternatively, direct injection of
the polynucleotide is employed. Preferably however, a nucleic acid
sequence is introduced into a cell by a vector, preferably a viral
vector. Said vector preferably comprises a retroviral, adenoviral,
adeno-associated viral (AAV), or lentiviral vector.
[0122] Various methods are used to administer the therapeutic
composition directly to a specific site in the body.
Receptor-mediated targeted delivery of therapeutic compositions
containing an antisense polynucleotide, subgenomic polynucleotides,
or antibodies to specific tissues is also used. Receptor-mediated
DNA delivery techniques are described in, for example, Findeis et
al., Trends in Biotechnol. (1993) 11:202-205; Wu et al., J. Biol.
Chem. (1994) 269:542-46.
[0123] Pharmaceutical compositions containing polynucleotides are
preferably administered in a range of about 100 ng to about 200 mg
of polynucleotides for local administration in a gene therapy
protocol. Concentration ranges of about 500 ng to about 50 mg,
about 1 .mu.g to about 2 mg, about 5 .mu.g to about 500 .mu.g, and
about 20 .mu.g to about 100 .mu.g of polynucleotides can also be
used during a gene therapy protocol. Factors such as mode of action
and efficacy of transformation and expression are considerations
which will affect the dosage required for ultimate efficacy of the
polynucleotides. Where greater expression is desired over a larger
area of tissue, larger amounts of polynucleotides or the same
amounts re-administered in a successive protocol of
administrations, or several administrations to different adjacent
or close tissue portions of, for example, a nerve ending or synaps,
may be required to affect a positive therapeutic outcome. In all
cases, routine experimentation in clinical trials will determine
specific ranges for optimal therapeutic effect. A more complete
description of gene therapy vectors, especially retroviral vectors,
is contained in WO 98/00542.
EXAMPLES
Example 1
Comparative Proteomics of MT4 Cells Treated with a Leukocyte
Extract Showing Anti-HIV Activity
[0124] The MT4 cell line was treated with a leukocyte extract
showing anti-HIV activity (Fernandez-Ortega C; Dubed M; Ruibal I;
Vilarrubia O L; Menendez J C; Navea L et al. 1996, Biotherapy 9:
33-40) and the resulting protein expression profile was compared to
a control of untreated cells. The cells were lysed and centrifuged
at 12 000 rpm for 20 min. The supernatant was collected and the
pellet was subjected to a second lysis procedure. After a second
centrifugation step under the same conditions, the second
supernatant was collected together with the first one, being
further delipidated with ethyl alcohol and alkylated with
polyacrylamide. Afterwards, the deoxyribonucleic acid (DNA) was
precipitated and a bidimensional electrophoresis of the sample was
carried out by using a 12.5 to 3% Tris-Tricine polyacrylamide gel
at 4.degree. C.
[0125] The images of analytical gels were analyzed by using the
Melanie 5 software. Spots to be identified were cut out from the
preparative gels and further digested with trypsin. The proteolytic
peptides were extracted for mass spectrometry analysis and mass
spectra were obtained by using a hybrid mass spectrometer with
QTOF-2 orthogonal geometry and equipped with a nanospray ionization
source.
[0126] ESI-MS/MS spectra were analyzed, and the respective searches
were carried out for protein identification in the non-redundant
protein sequence database of the National Center for Biotechnology
Information of the USA and in the European Molecular Biology
Laboratory database of Germany. A decrease in cytoskeletal proteins
was detected in the sample treated with the anti-HIV leukocyte
extract, particularly in those forming the IFs (vimentin and
keratin-10) (FIG. 1).
Example 2
Interfering RNA Against Vimentin and Keratin-10 Inhibits HIV
Infection
[0127] The MT4 cell line was transduced by using the
pLenti-shRNA.sub.vim or pLenti-shRNA.sub.K-10 lentiviral vectors,
which bear a sequence encoding a RNA hairpin which silences the
expression of the vimentin and keratin proteins, respectively.
These lentiviral vectors were assembled by packaging in the 293T
cell line transduced with four plasmids. The said plasmids were
pLP1, pLP2, pLP/VSVG and p-shRNA, this last specific for either
vimentin or keratin-10. The pLP1 vector codes for the gene products
of the gag/pol sequences of HIV-1. The pLP/VSVG codes for the
surface protein of the vesicular stomatitis virus and the p-shRNA
contains the genome of the lentiviral vector which bears the
sequences coding for the vimentin- or keratin-10-specific RNA
hairpins (Ui-Tei K, Naito Y, Takahashi F, Haraguchi T et al., 2004
Nucleic Acids Research 32: 936-948; Santa Cruz Biotechnology). All
the plasmids were amplified in the Escherichia coli XL-1 strain
under ampicillin selection. The four plasmid vectors were
transfection-quality purified by column chromatography and put
together into contact with the 293T packaging cell line in the
presence of polyethyleneimine. The cells were incubated for 48 h at
37.degree. C. under 5% CO.sub.2 atmosphere, and virions were
further purified by ultracentrifugation at 20 000.times.g. Once
purified the lentiviral vector, MT4 cells were transduced and the
recombinants were selected for blasticidin resistance. The
recombinant clones were isolated by the limiting dilution assay and
cultivated in RPMI medium supplemented at 10% with fetal bovine
serum (FBS) under 5% CO.sub.2 atmosphere and 95% relative humidity
at 37.degree. C. until harvest. Total proteins were extracted from
the cultures and silencing of vimentin was demonstrated by western
blot in MT4 (MT4.sub.vim(s)) cells, as well as for keratin-10 in
the MT4.sub.K-10(s) cells silenced for keratin-10. Transduced
cultures showed a decreased expression of vimentin or keratin-10
compared to the MT4 untransduced control, respectively (FIG.
2).
[0128] The anti-HIV activity was evaluated in two challenge
systems:
[0129] System A: Cells stably silenced for the vimentin
(MT4.sub.vim(s)) or keratin-10 (MT4.sub.K-10(s)) proteins were
cultured in RPMI medium supplemented at 10% FBS under a 5% CO.sub.2
atmosphere and relative humidity of 95% at 37.degree. C. The
challenge with total virus was carried out in MT4.sub.vim(s),
MT4.sub.K-10(s) and MT4 cell cultures. The Bru viral strain was
used at m.o.i. of 0.01, and replication was evaluated by
determining the p24 antigen concentration in culture supernatants
by the ELISA method. The MT4.sub.vim(s) and MT4.sub.K-10(s) cells
showed approximately a 90% inhibition of viral replication compared
to the MT4 cell cultures unsilenced for each of these proteins
(FIG. 3).
[0130] System B: The MT4.sub.vim(s), MT4.sub.K-10(s) and MT4 cell
cultures were challenged with a lentiviral vector bearing part of
the HIV-1 genome, lacking the genes involved in infectivity and
entry (pLGW). This vector was constructed by packaging the products
of four plasmids in the 293T cell line. The said plasmids were
pLP1, pLP2, pLP/VSVG and pLGFP, this last coding for the GFP. The
pLP1 plasmid codes for the gene products of the HIV-1gag/pol
sequences. The pLP2 plasmid bears the genetic sequence of the HIV-1
Rev protein and the pLP/VSVG codes for the surface protein of the
vesicular stomatitis virus. The pLGFP plasmid codes for the GFP,
also bearing the packaging sequence, the HIV Rev-responsive element
sequences (RRE) and also the 3'-deleted HIV-1 long terminal repeats
(LTRs), constituting the lentiviral vector genome. GFP expression
was followed as marker of completing the viral replication cycle
after entry and until integration. The results were followed by
fluorescence microscopy, and the number of fluorescent cells
decreased in the MT4.sub.vim(s) and MT4.sub.K-10(s) cultures,
compared to that on MT4 cell cultures (data not shown). The samples
were analyzed by flow cytometry and the number of fluorescent cells
because of GFP expression decreased nearly 70% as compared to the
unsilenced MT4 cell cultures (FIG. 4).
Example 3
Changes in the Structure of Intermediate Filaments in MT4 Cells
[0131] Firstly, MT4.sub.vim(s), MT4.sub.K-10(s) and MT4 cells were
fixed in 3.2% glutaraldehyde for 1 h at 4.degree. C. and then fixed
in 2% osmium tetroxide for 1 h at 4.degree. C. They were
subsequently washed with 0.1 M phosphate buffered saline (PBS), pH
7.2, and dehydrated at increasing ethanol concentrations (30, 50,
70 and 100%) for 10 min each at 4.degree. C. Inclusion was carried
out and ultrathin 40-50 nm-width sections were taken in an
ultramicrotome (NOVA, LKB), which were placed on 400-orifices
nickel trays. Once the ultrathin cuts were taken and placed on the
trays, they were contrasted with saturated uranyl acetate and lead
citrate, and further examined under a JEOL JEM 2000 EX (JEOL)
microscope. Five microphotographs were analyzed at different
magnifications. MT4 cell intact IFs are shown in FIG. 5A, meanwhile
these structures appeared shortened in MT4.sub.vim(s) and
MT4.sub.K-10(s) cells (FIGS. 5B and C, respectively). Section D
shows that effect in IFs as caused by the action of a peptide
(peptide identified as SEQ ID No. 1) which disassembles the
structure of vimentin in MT4 cells. Inhibition of the viral
replication was observed under these conditions. Vimentin or
keratin-10 proteins were identified at IFs by immunomicroscopy.
Example 4
Synthetic Peptides which Inhibit HIV Replication in MT4 Cells
[0132] Peptides corresponding to amino acid sequences of the human
keratin-10, human keratin 1 and human vimentin were synthesized
(Goldman R D, Khuon S, Hao Chou Y, Opal P, Steinert P M 1996, J
Cell Biol 134: 971-983; Steinert P M, Yang J M, Bale S J, Compton J
G 1993, BBRC 197: 840-848). One of the peptides has a cell
penetrating peptide conjugated at its C terminus (Vallespi M G,
Fernandez J R, Torrens I, Garda I, Garay H, Mendoza O et al, 2009.
J Peptide Science 16: 40-47). The anti-HIV activity of said
peptides was evaluated by using a total virus challenge system, in
the presence of different viral strains: HXB1 (HIV-1 IIIB clone)
and Bru. The MT4 cell line was incubated with the peptide for 24 h
prior to viral challenge. The assays were done at m.o.i. values of
0.01 and 0.05, comprising nine replicas for each experimental
variant. The value of the p24 viral antigen was determined in cell
cultures by using an ELISA type assay, and results were expressed
as percent of viral inhibition or as percent of infection, both
versus peptide concentration. An important inhibition of viral
replication was observed in the presence of peptides, both when the
cultures were challenged at a high viral concentration (SEQ ID No.
1, FIG. 6A) and at a m.o.i. of 0.01 (FIG. 6B). The 1050 of peptides
was in the nanomolar range.
Example 5
Synthetic Peptides which Inhibit HIV Replication in Peripheral
Blood Mononuclear Cells
[0133] PBMCs were isolated from whole blood of healthy individuals
by cesium chloride Ficoll density gradients. Cells were
pre-stimulated for 2 days in RPMI medium supplemented at 20% FBS,
100 U/mL Interleukin 2 (IL-2) and 5 .mu.g/mL phytohemagglutinin
(PHA). Subsequently, they were kept in PHA-free medium and seeded
at 150 000 cells per well in 96-well plates. After 24 h, the
peptides were added at the different concentrations and the
cultures infected with the HIV-1 Bru strain at a m.o.i. of 0.01.
The cultures were kept for 7 days, with the medium being changed
and peptides added every 3 days. The cultures were harvested and
the supernatants collected to evaluate the presence of the p24
viral protein. The peptides inhibited the HIV-1 replication in a
dose-dependent manner (FIG. 7). Similar IC50 results in the
nanomolar range were obtained in cultures infected with the HIV-1
BaL1 strain.
Example 6
Inhibition of HIV-2 by Synthetic Peptides
[0134] PBMCs were isolated from whole blood of healthy individuals
by using Ficoll density gradients. The cells were pre-stimulated
for 2 days with RPMI medium supplemented at 20% FBS, 100 U/mL IL-2
and 5 .mu.g/mL PHA. Cells were subsequently maintained in PHA-free
medium and seeded at 150 000 cells per well in 96-well plates.
After 24 h, peptides were added at different concentrations and the
cultures were infected with the CBL-20 HIV-2 strain. The cultures
were kept for 7 days, being changed every 3 days the medium and the
peptides added. Cultures were harvested and supernatants collected
to evaluate the presence of the p24 viral protein. The peptides
inhibited the HIV-2 replication in a dose-dependent manner (FIG.
8).
Example 7
Decrease of Vimentin in the Presence of Peptides Identified as SEQ
ID No. 1, 4, 5, 7, 8 and 9
[0135] The MT4 cell line was incubated at 50 .mu.M of each peptide
for 24 h. The vimentin protein was detected by the western blot
technique. Cellular extracts were resuspended in 1% sodium dodecyl
sulphate (SDS) and applied in a 10% polyacrylamide gel, being
further transferred to a Hybond-P cellulose membrane. For
immunoidentification purposes, anti-vimentin and anti-.beta. actin
(as control) antibodies were used. An anti-mouse IgG
antibody-peroxidase conjugate was used as secondary antibody. The
activity of the peroxidase enzyme was visualized by using
diaminobenzidine in the presence of hydrogen peroxide and PBS. The
vimentin protein was decreased in MT4 cells treated with the
peptides (FIG. 9).
Example 8
Cellular Penetration of Peptides Identified as SEQ ID No. 1 and SEQ
ID No. 3
[0136] HeLa CD4+ cells were seeded in RPMI medium supplemented at
10% FBS and incubated until reaching 60% confluence of the
monolayer. MT4 cells were seeded at 50 000 cells per well in RPMI
medium at 10% FBS. Peptides identified as SEQ ID No. 1 and SEQ ID
No. 3 were resuspended in injection water and evaluated at 5, 10,
20 and 40 .mu.M concentrations. Peptides were incubated for 24 h at
37.degree. C. under a 5% CO.sub.2 atmosphere, and penetration was
evaluated after 15, 30 and 60 min. After each period of time, cells
were harvested and immediately analyzed by flow cytometry. Three
replicates were analyzed per experimental variant. The peptides
were able to penetrate by their own into the MT4 cells as shown in
FIG. 10.
Example 9
Lipidic Derivative which Binds to Vimentin and Inhibits HIV-1
Replication
[0137] It is known that the prostaglandin cyclopentane 15
deoxy-.DELTA.-.sup.2,14-PGJ2 (15d-PGJ2) binds to the vimentin
protein (Stamatakis K, Sanchez-Gomez F J, Perez-Sala D 2006, J Am
Soc Nephrol 17: 89-98). In the present invention, it was
demonstrated that 15d-PGJ2 inhibits the HIV replication in vitro.
The antiviral activity assay was carried out on MT4 cells
challenged with HIV-1 (Bru strain). The cells were incubated at
different concentrations of 15d-PGJ2, and further challenged with
HIV-1 at a m.o.i. of 0.01. After a 5-day incubation period, cell
cultures were harvested and the p24 protein was evaluated in the
supernatants (FIG. 11).
Example 10
Effect of Synthetic Peptides and 15d-PGJ2 on PBMCs of HIV-1
Infected Patients
[0138] PBMCs were isolated from whole blood of HIV-1 infected
individuals by Ficoll density gradients. Cells were pre-stimulated
and treated with the peptides, similarly as described in example 5,
or treated with 5 .mu.M of 15d-PGJ2. Replication was evaluated by
determining the p24 antigen concentration on culture supernatants
by the ELISA method. The p24 values significantly decreased in
PBMCs treated with the peptides or with the lipidic derivative,
compared to untreated cells (Table 1). This was indicative of the
inhibition of HIV-1 replication caused by treatment with these
compounds.
TABLE-US-00001 TABLE 1 Percent of HIV-1 inhibition in PBMCs of
infected individuals, which were treated ex vivo with the peptides
or 15d-PGJ2. Compound % of HIV-1 inhibition Peptide 1 (SEQ ID No.
1) 89.3 Peptide 2 (SEQ ID No. 2) 81.1 Peptide 3 (SEQ ID No. 3) 85.3
Peptide 4 (SEQ ID No. 4) 84.9 Peptide 5 (SEQ ID No. 5) 82.1 Peptide
6 (SEQ ID No. 6) 80.5 Peptide 7 (SEQ ID No. 7) 83.7 Peptide 8 (SEQ
ID No. 8) 86.7 Peptide 9 (SEQ ID No. 9) 81.3 Peptide 10 (SEQ ID No.
10) 83.4 15d-PGJ2 80.1
[0139] IFs structure was analyzed by transmission electron
microscopy, following the methodology described in Example 3. It
was shown that IFs were very short in those infected individuals
PBMCs which were treated with the peptides or with the lipidic
derivative, as compared to untreated cells.
Example 11
Interfering RNA Against Vimentin and Keratin-10 Inhibits HIV in
PBMCs of Infected Individuals
[0140] PBMCs were isolated from whole blood of HIV-1 infected
individuals by Ficoll density gradient. Cells were pre-stimulated
for 2 days in RPMI medium at 20% FBS, 100 U/mL IL-2 and 5 .mu.g/mL
PHA. Subsequently, they were kept in PHA-free medium and further
transduced with the lentiviral vectors pLenti-shRNA.sub.vim or
pLenti-shRNA.sub.K-10, which bear a sequence coding for a RNA
hairpin to silence the vimentin and the keratin-10 proteins,
respectively. Vectors were obtained as described in Example 2.
Replication was evaluated by determining the concentrations of the
p24 antigen on culture supernatants by the ELISA method. PBMCs
silenced for the vimentin or keratin-10 proteins showed a high
inhibition of viral replication as compared to the cultures
unsilenced for each of these proteins (Table 2).
TABLE-US-00002 TABLE 2 Percent of HIV-1 inhibition in PBMCs from
infected individuals, which were transduced with shRNA specific for
vimentin or keratin-10 PBMCs from infected individuals % of HIV-1
inhibition PBMCs transduced with pLenti-shRNA.sub.vim 89.5 PBMCs
transduced with pLenti-shRNA.sub.K-10 75.6
[0141] IFs structures were analyzed by transmission electron
microscopy, following the methodology described in Example 3. It
was shown that IFs structure was shorter in those PBMCs from
infected individuals which were transduced with the lentiviral
vectors, as compared to untransduced cells.
Example 12
Treatment of HIV-1 Infected Patients with Formulations Containing
the Peptides Identified as SEQ ID No. 1 and 2
[0142] Eight HIV-1 seropositive patients having less than a year
from diagnosis and values of CD4+ T cells higher than 350
cells/mm.sup.3 were treated with a formulation containing the
peptide identified as SEQ ID No. 1 or a formulation containing the
peptide identified as SEQ ID No. 2. The peptides were administered
at 150 mg per day, and patients were followed up for 6 months
attending to viral load and CD4+ T cell counts. The viral load was
undetectable in two of the patients after treatment, and decreased
in more than 1.5 log in the other six patients. On the other hand,
seven patients registered an increase in CD4+ T cells higher than
50 cells/mm.sup.3, while the CD4+ T cell counts decreased in the
other patient. The IFs structure was analyzed by transmission
electron microscopy according to the methodology described in
Example 3, in two of the patients treated with the peptide
identified as SEQ ID No. 1 and three of the patients treated with
SEQ ID No. 2. IFs appeared shortened in all the cases.
INCORPORATION OF SEQUENCE LISTING
[0143] Incorporated herein by reference in its entirety is the
Sequence Listing for the above-identified Application. The Sequence
Listing is disclosed on a computer-readable ASCII text file titled
"SequenceListing_976-79PCTUSCON.txt", created on Nov. 17, 2015. The
sequence.txt file is 13 KB in size.
Sequence CWU 1
1
12118PRTHomo sapiens 1Arg Val Thr Gln Met Asn Leu Asn Asp Arg Leu
Ala Ser Leu Tyr Asp 1 5 10 15 Lys Val 218PRTHomo sapiens 2Asp Met
Glu Ile Ala Thr Tyr Arg Thr Leu Leu Glu Gly Glu Glu Ser 1 5 10 15
Arg Met 320PRTHomo sapiens 3Lys Val Glu Leu Gln Glu Leu Asn Asp Arg
Phe Ala Asn Tyr Ile Asp 1 5 10 15 Lys Val Arg Phe 20420PRTHomo
sapiens 4Glu Leu Asn Asp Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg
Phe Leu 1 5 10 15 Glu Gln Gln Asn 20520PRTHomo sapiens 5Phe Ala Asn
Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn Lys 1 5 10 15 Ile
Leu Leu Ala 20620PRTHomo sapiens 6Asp Lys Val Arg Phe Leu Glu Gln
Gln Asn Lys Ile Leu Leu Ala Glu 1 5 10 15 Leu Glu Gln Leu
20735PRTHomo sapiens 7Lys Val Glu Leu Gln Glu Leu Asn Asp Arg Phe
Ala Asn Tyr Ile Asp 1 5 10 15 Lys Val Arg Phe Leu Glu Gln Gln Asn
Lys Ile Leu Leu Ala Glu Leu 20 25 30 Glu Gln Leu 35835PRTHomo
sapiens 8Arg Val Thr Gln Met Asn Leu Asn Asp Arg Leu Ala Ser Leu
Tyr Asp 1 5 10 15 Lys Val Arg Ala Leu Glu Glu Ser Asn Tyr Glu Leu
Glu Gly Lys Ile 20 25 30 Lys Glu Trp 35938PRTArtificial
sequenceDescription of the Artificial Sequence Peptide derived from
human keratin-10 joined to a penetrating peptide 9Arg Val Thr Gln
Met Asn Leu Asn Asp Arg Leu Ala Ser Leu Tyr Asp 1 5 10 15 Lys Val
His Tyr Arg Ile Lys Pro Thr Phe Arg Arg Leu Lys Trp Lys 20 25 30
Tyr Lys Gly Lys Phe Trp 35 1035PRTHomo sapiens 10Ala Arg Leu Leu
Cys Asp Tyr His Glu Leu Met Asn Thr Lys Leu Ala 1 5 10 15 Leu Asp
Met Glu Ile Ala Thr Tyr Arg Thr Leu Leu Glu Gly Glu Glu 20 25 30
Ser Arg Met 3511466PRTHomo sapiens 11Met Ser Thr Arg Ser Val Ser
Ser Ser Ser Tyr Arg Arg Met Phe Gly 1 5 10 15 Gly Pro Gly Thr Ala
Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr 20 25 30 Thr Ser Thr
Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr 35 40 45 Ser
Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg 50 55
60 Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu
65 70 75 80Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr
Glu Phe 85 90 95 Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln
Glu Leu Asn Asp 100 105 110 Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg
Phe Leu Glu Gln Gln Asn 115 120 125 Lys Ile Leu Leu Ala Glu Leu Glu
Gln Leu Lys Gly Gln Gly Lys Ser 130 135 140 Arg Leu Gly Asp Leu Tyr
Glu Glu Glu Met Arg Glu Leu Arg Arg Gln 145 150 155 160Val Asp Gln
Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp 165 170 175 Asn
Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu 180 185
190 Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln
195 200 205 Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg
Lys Val 210 215 220 Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys
Leu His Glu Glu 225 230 235 240Glu Ile Gln Glu Leu Gln Ala Gln Ile
Gln Glu Gln His Val Gln Ile 245 250 255 Asp Val Asp Val Ser Lys Pro
Asp Leu Thr Ala Ala Leu Arg Asp Val 260 265 270 Arg Gln Gln Tyr Glu
Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu 275 280 285 Glu Trp Tyr
Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg 290 295 300 Asn
Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg 305 310
315 320Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly
Thr 325 330 335 Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu
Asn Phe Ala 340 345 350 Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly
Arg Leu Gln Asp Glu 355 360 365 Ile Gln Asn Met Lys Glu Glu Met Ala
Arg His Leu Arg Glu Tyr Gln 370 375 380 Asp Leu Leu Asn Val Lys Met
Ala Leu Asp Ile Glu Ile Ala Thr Tyr 385 390 395 400Arg Lys Leu Leu
Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro 405 410 415 Asn Phe
Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro 420 425 430
Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu 435
440 445 Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp
Asp 450 455 460 Leu Glu 465 12568PRTHomo sapiens 12Met Ser Val Arg
Tyr Ser Ser Ser Lys Gln Tyr Ser Ser Ser Arg Ser 1 5 10 15 Gly Gly
Gly Gly Gly Gly Gly Gly Gly Ser Ser Phe Arg Ile Ser Ser 20 25 30
Ser Lys Gly Ser Ile Gly Gly Gly Phe Ser Ser Gly Gly Phe Ser Gly 35
40 45 Gly Ser Phe Ser Arg Gly Ser Ser Gly Gly Gly Cys Phe Gly Gly
Ser 50 55 60 Ser Gly Gly Tyr Gly Gly Leu Gly Gly Gly Phe Gly Gly
Gly Asn Phe 65 70 75 80Gly Gly Gly Tyr Gly Ser Ser Ser Phe Gly Gly
Gly Tyr Gly Gly Val 85 90 95 Ser Phe Gly Gly Gly Ser Phe Gly Gly
Gly Ser Phe Gly Gly Gly Gly 100 105 110 Phe Ser Gly Gly Ser Phe Gly
Gly Tyr Gly Gly Gly Tyr Gly Gly Asp 115 120 125 Gly Gly Leu Leu Ser
Gly Asn Glu Lys Val Thr Met Gln Asn Leu Asn 130 135 140 Asp Arg Leu
Ala Ser Tyr Leu Asp Lys Val Arg Ala Leu Glu Glu Ser 145 150 155
160Asn Tyr Glu Leu Glu Gly Lys Ile Lys Glu Trp Tyr Glu Lys His Gly
165 170 175 Asn Ser Ser Gln Arg Ala Pro Arg Asp Tyr Ser Lys Tyr Tyr
Gln Thr 180 185 190 Ile Glu Asp Leu Lys Asn Gln Ile Leu Asn Leu Thr
Thr Asp Asn Ala 195 200 205 Asn Ile Leu Leu Gln Ile Asp Asn Ala Arg
Leu Ala Ala Asp Asp Phe 210 215 220 Arg Leu Lys Tyr Glu Asn Glu Val
Ala Leu Arg Gln Ser Val Glu Ala 225 230 235 240Asp Ile Asn Gly Leu
Arg Arg Val Leu Asp Glu Leu Thr Leu Thr Lys 245 250 255 Ala Asp Leu
Glu Met Gln Ile Glu Ser Leu Thr Glu Glu Leu Ala Tyr 260 265 270 Leu
Lys Lys Asn His Glu Glu Glu Met Arg Asp Leu Gln Asn Val Ser 275 280
285 Thr Gly Asp Val Asn Val Glu Met Asn Ala Ala Pro Gly Val Asp Leu
290 295 300 Thr Glu Leu Leu Asn Asn Met Arg Asn Gln Tyr Glu Gln Leu
Ala Glu 305 310 315 320Gln Asn Arg Lys Asp Ala Glu Ala Trp Phe Asn
Glu Lys Ser Lys Glu 325 330 335 Leu Thr Thr Glu Ile Asn Ser Asn Ile
Glu Gln Met Ser Ser His Lys 340 345 350 Ser Glu Ile Thr Glu Leu Arg
Arg Thr Val Gln Gly Leu Glu Ile Glu 355 360 365 Leu Gln Ser Gln Leu
Ala Leu Lys Gln Ser Leu Glu Gly Ser Leu Ala 370 375 380 Glu Thr Glu
Gly Arg Tyr Cys Val Gln Leu Ser Gln Ile Gln Ala Gln 385 390 395
400Ile Ser Ser Leu Glu Glu Gln Leu Gln Gln Ile Arg Ala Glu Thr Glu
405 410 415 Cys Gln Asn Ala Glu Tyr Gln Gln Leu Leu Asp Ile Lys Ile
Arg Leu 420 425 430 Glu Asn Glu Ile Gln Thr Tyr Arg Ser Leu Leu Glu
Gly Glu Gly Ser 435 440 445 Ser Gly Gly Gly Gly Tyr Gly Gly Gly Arg
Gly Gly Gly Ser Ser Gly 450 455 460 Gly Gly Tyr Gly Gly Ser Ser Gly
Gly Gly Tyr Gly Gly Ser Ser Gly 465 470 475 480Gly Gly Gly Tyr Gly
Gly Gly Ser Ser Gly Gly Gly Gly His Ile Gly 485 490 495 Gly His Ser
Gly Gly His Ser Gly Ser Ser Gly Gly Gly Tyr Gly Gly 500 505 510 Gly
Ser Ser Ser Gly Gly Gly Gly Tyr Gly Gly Gly Ser Ser Gly Gly 515 520
525 Gly Gly Ser His Gly Gly Ser Ser Gly Gly Gly Tyr Gly Gly Gly Ser
530 535 540 Ser Ser Ser Gly Gly His Lys Ser Ser Ser Ser Gly Ser Val
Gly Glu 545 550 555 560Ser Ser Ser Lys Gly Pro Arg Tyr565
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