WO1998017284A2

WO1998017284A2 - Inhibition of cap-independent protein synthesis and hiv-tar translation by heparin or heparin mimetics, and methods for gene therapy

Info

Publication number: WO1998017284A2
Application number: PCT/CA1997/000794
Authority: WO
Inventors: Daniel Favre
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-10-22
Filing date: 1997-10-21
Publication date: 1998-04-30
Anticipated expiration: 1999-04-22
Also published as: WO1998017284A3; AU4769197A

Abstract

The present invention relates to a method of inhibiting cap-independent protein synthesis of eukaryotic cells, which comprises adding heparin or heparin mimetics thereof to said eukaryotic cells. There is also disclosed a method of inhibiting translation of HIV TAR-containing messenger ribonucleic acids (mRNA) of HIV infected eukaryotic cells, which comprises adding heparin or heparin mimetics thereof to said eukaryotic cells. There is also disclosed a composition for gene therapy using glycosaminoglycan N-acetylglycosaminyl N-deacetylase/N-sulfotransferase cDNA.

Description

INHIBITION OF CAP-INDEPENDENT PROTEIN SYNTHESIS BY HEPARIN OR HEPARIN MIMETICS THEREOF

BACKGROUND OF THE INVENTION

( ) Field of the Invention The invention relates to the use of heparin and heparin mimetic compounds thereof to inhibit cap-independent protein synthesis (or translation) in addition to cap-dependent protein synthesis and to inhibit translation of HIV TAR-containing messenger ribonucleic acids (mRNA) mediated.

(b) Description of Prior Art

Heparin is among the best studied glycosamino- glycans. This compound is known for its involvement in a variety of physiological processes. It is involved in the control of homeostasis, smooth muscle proliferation, growth factors activity, extracellular matrix integrity, among others (Margalit, H. et al . (1993) Journal of Biological Chemistry 268: 19228-19231). Heparin is a negatively charged polymer of a regular disaccharide repeat sequence with a high degree of sul- fatation. Thus, many proteins are expected to bind heparin via electrostatic interactions, but electrostatic forces by themselves are probably not sufficient (Margalit, H. et al. (1993) Journal of Biological Chem- istry 268: 19228-19231).

Recently, the substitution of heparin for double-stranded (ds) RNA in the autophosphorylation of the interferon-inducible, RNA-dependent eIF-2α protein kinase (PKR) has been analyzed in detail (George, C.X. et al. (1996) Virology 221: 180-188). Phosphorylation of the alpha subunit of eIF-2 leads to the inhibition of the cap-dependent translation ( In : Translational control (1996). Edited by John W.B. Hershey et al., Cold Spring Harbor Laboratory Press, Cold Spring Har- bor, N.Y. USA. pp. 31-69 and 139-172). Internal initiation, or cap-independent translation, is known to occur with picornaviruses (such as poliovirus or encephalomyocarditis virus) mRNAs ( In : Translational control (1996) Edited by John W.B. Her- shey et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA. pp. 549-573), other RNAs such as BiP mRNA or Antennapedia mRNA ( In : Translational control (1996) Edited by John W.B. Hershey et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA. pp. 95-96), and might be involved during human immunodeficiency virus (HIV) infection (Svitkin et al. (1994) Journal of Virology 68, 7001-7007). Internal translation requires an internal ribosome entry site (IRES) on the mRNA. Recently, the inhibi- tion of IRES-mediated translation of poliovirus has been shown to be inhibited by a 60-nucleotide long yeast RNA (Das., S. et al. (1996) Journal of Virology 70: 1624-1632). To my knowledge, this is the only scientific report that has been published to date showing the specific and direct inhibition of cap-independent translation by a molecule, here an RNA.

Moreover, translation of mRNAs of human immunodeficiency virus type 1 (HIV-1) has been shown to be mediated by cis-acting sequences responsive to the tat gene product, the trans-acting responsive (TAR) region, which is located immediately adjacent to the site of transcription initiation (Rosen, CA. et al. (1985) Cell , 41:813-823). The TAR sequence is therefore located at the 5' end of all viral mRNAs. It has been proposed that the TAR sequence and flanking 3' region played a role in the regulation of translation of HIV-1 mRNAs by inhibiting this translation (Parkin, N.T. et al. (1988) EMBO Journal , 7:2831-2837). However, in the latter study, the authors, by translating HIV-1 TAR- containing mRNAs in a rabbit reticulocyte lysate or in a cytoplasmic extract of eukaryotic HeLa cells that have grown in suspension cultures, or by microinjecting HIV-1 TAR-containing mRNAs in Xenopus oocytes, predict that the block to translation of viral mRNAs by their 5' untranslated region (UTR) must somehow be overcome to allow for efficient viral structural protein synthesis and viral replication during viral infection (Parkin, N.T. et al . (1988) EMBO Journal , 7:2831-2837). It would be highly desirable to be provided with means to inhibit cap-independent protein synthesis or translation.

It would be highly desirable to be provided with means to inhibit translation of HIV TAR-containing messenger ribonucleic acids (mRNA).

SUMMARY OF THE INVENTION

Heparin inhibits cap-independent translation, in addition to bind to and activate PKR and therefore inhibit cap-dependent translation. This inhibition of cap-independent translation does not necessarily imply the involvement of PKR.

One aim of the present invention is to provide means to inhibit cap-independent protein synthesis or translation. One aim of the present invention is to provide means to inhibit translation of HIV TAR-containing messenger ribonucleic acids (mRNA) .

Another aim of the present invention is to provide means to inhibit cap-independent protein synthesis or translation in addition to cap-dependent protein synthesis .

In accordance with the present invention there is provided the use of heparin and heparin mimetic compounds thereof to inhibit cap-independent protein syn- thesis or translation in addition to cap-dependent protein synthesis. In accordance with the present invention there is provided the use of heparin and heparin mimetic compounds thereof to inhibit translation of HIV TAR-containing messenger ribonucleic acids (mRNA) . In accordance with the present invention there is provided a method of inhibiting cap-independent protein synthesis of eukaryotic cells, which comprises adding heparin or heparin mimetics thereof to the eukaryotic cells. In accordance with the present invention there is provided a method of inhibiting translation of HIV TAR-containing messenger ribonucleic acids (mRNA) of HIV infected eukaryotic cells, which comprises adding heparin or heparin mimetics thereof to the infected eukaryotic cells. Such a heparin mimetic include, without limitation, sulfated polysaccharides .

In accordance with the present invention there is provided a pharmaceutical composition for the therapeutical inhibition of cap-independent protein synthe- sis in a patient suffering from a viral infection, which comprises a therapeutic amount of heparin and/or heparin mimetics thereof in association with a pharmaceutical carrier. The carrier may be a liposome or a biovector . In accordance with the present invention there is provided a pharmaceutical composition for the therapeutical inhibition of translation of HIV TAR-containing messenger ribonucleic acids (mRNA) in a patient HIV-infected, which comprises a therapeutic amount of heparin and/or heparin mimetics thereof in association with a pharmaceutical carrier. The carrier may be a liposome or a biovector.

In accordance with the present invention there is provided a composition for gene therapy of patients infected with a virus, which comprises administering to — b —

the patient an expression vector consisting of a cDNA sequence coding for enzyme glycosaminoglycan N-acetyl- glucosaminyl N-deacetylase/N-sulfotransferase with a constitutive or inducible promoter operatively linked upstream of the cDNA sequence, the expression vector is adapted to express the enzyme thereby reducing viral protein synthesis in the patient.

The expression vector may be a recombinant virus selected from the group consisting of adenovirus and retrovirus for targeting of the infected cells.

The delivery vector may be a liposome for targeting of the infected cells.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates a fluorography of radiola- belled polypeptides synthesized in vi tro in a rabbit reticulocyte lysate in accordance with the method of the present invention;

Fig. 2 illustrates a fluorography of radiola- belled polypeptides synthesized in vi tro in a Krebs ascites fluid in accordance with the method of the present invention;

Fig. 3 illustrates a fluorography of radiola- belled polypeptides synthesized in vi tro in a cytoplas- mic extract obtained from eukaryotic cells in accordance with the method of the present invention;

Fig. 4 illustrates a fluorography of radiola- belled polypeptides synthesized in vi tro in a cytoplas- mic extract obtained from eukaryotic cells that have grown as monolayers; and

Fig. 5 illustrates a scheme of potential targeting of virally infected cells using heparin or heparin mimetics thereof in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, the use of heparin inhibits:

(i) cap-independent translation and, (ii) translation of mRNAs containing the TAR structure from HIV-1 at their 5' end, in addition to bind to and activate the double-stranded (ds) RNA-dependent protein kinase PKR. This inhibition of cap-independent translation and HIV TAR-mediated translation does not necessarily imply the involvement of PKR.

In accordance with the present invention there is provided a method of inhibiting cap-independent protein synthesis of eukaryotic cells, which comprises adding heparin or heparin mimetics thereof to the eukaryotic cells.

In accordance with the present invention there is provided a method of inhibiting translation of mRNAs containing the TAR structure from HIV-1 at their 5 ' end, which comprises adding heparin or heparin mimetics thereof to the eukaryotic cells.

In accordance with the present invention, the method of inhibiting translation of mRNAs containing the TAR structure is intended to be used also for HIV-2, since HIV-2 has also a TAR structure.

In accordance with the present invention there is provided a pharmaceutical composition for the therapeutical inhibition of cap-independent protein synthesis in a patient suffering from a viral infection, which comprises a therapeutic amount of heparin and/or heparin mimetics thereof in association with a pharmaceutical carrier. The carrier may be a liposome or a biovector.

In accordance with the present invention there is provided a pharmaceutical composition for the thera- peutical inhibition of TAR (HIV-1) synthesis in a HIV-1 infected patient, which comprises a therapeutic amount of heparin and/or heparin mimetics thereof in association with a pharmaceutical carrier. The carrier may be a liposome or a biovector. Materiel and methods A) Inhibition of cap-independent translation

In vi tro protein syntheses were performed by employing a rabbit reticulocyte lysate from commercial source (Promega), and a Krebs ascites fluid, by following the manufacturer's instructions or currently employed procedures, respectively. Furthermore, a cyto- plasmic extract obtained from eukaryotic cells has been generated by following a method described by Skup, D. et al. ((1977) Nucleic Acids Research 4: 3581-3587). To label the newly-synthesized polypeptides during 1-hour- incubation reactions, ^^s-methionine (Amersham; >1200 Ci/mmol) was employed. Heparin, which was resuspended in phosphate-buffered saline (PBS) and stored at room temperature, was obtained from a commercial source (Gibco BRL; catalog number 15077-019; 100000 units at 164 units per mg). Messenger RNAs were either a) transcribed in vi tro for the generation of a capped CAT (chloramphenycol)-EMC (IRES of encephalomyocarditis virus) -LUC (luciferase) mRNA as previously described (Pause, A. et al. (1994) Nature 371: 762-767), or b) from viral source, i.e.: from poliovirus (Mahoney strain) and from encephalomyocarditis virus. Analysis of the labeled polypeptides was performed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Gels were treated for fluorography with En3Hance (Dupont). B) Inhibition of translation of mRNAs containing the TAR structure from HIV-1 at their 5 ' end

The cytoplasmic extract employed for translation was obtained from monkey Cos-1 cells that have grown as monolayers.

To label the newly-synthesized polypeptides during 1-hour-incubation reactions, -^^S-methionine (Amersham; >1200 Ci/mmol) was employed. Heparin, which was resuspended in phosphate-buffered saline (PBS) and stored at room temperature, was obtained from a commercial source (Gibco BRL; catalog number 15077-019).

HIV-1 TAR-containing mRNA is capped TAR(+111)CAT RNA described by Parkin, N.T. et al . ((1988) EMBO Journal , 7:2831-2837). The 5' region of the latter mRNA corresponds to nucleotides +1 to +111 of HIV mRNAs and is transcribed from plasmid pSP64/TAR( +111 )CAT.

Analysis of the labeled polypeptides was performed by sodium dodecyl sulfate ( SDS)-polyacrylamide gel electrophoresis (PAGE). Gels were treated for fluorography with En3Hance (Dupont). Results

A) Translation in rabbit reticulocyte lysate and in Krebs ascites fluid. As shown in the fluorography of Fig. 1 (rabbit reticulocyte lysate translation) and Fig. 2 (translation in Krebs ascites fluid), inhibition of both cap-dependent translation (as seen with incorporation of 35g-πιethionine into the CAT polypeptide) and cap-independent translation (as seen with incorporation of 5g-_methionine into the LUC polypeptide) is reduced, an event dependent of the heparin concentration present in the reaction. Furthermore, translation of both the poliovirus and encephalomyocarditis virus polypeptide precursors are drastically reduced when 5 μg per μl of heparin is present in the translation reaction, when compared to the reactions that contain the viral RNAs alone. Both extracts have been previously treated with micrococcal nuclease to hydrolyze the endogenous mRNAs, and the micrococcal nuclease has been inhibited with EGTA or pTp ( 2 ' deoxythymidine, 3 ' -5 ' -diphosphate) prior to the use of the extracts. 0.4 μg of CAT-EMC-LUC RNA and 0.2 μg of either EMC or poliovirus RNA were employed in 40 μl translation reactions.

B) Inhibition of cap-dependent translation in a cell extract obtained from eukaryotic cells that have grown in suspension cultures

A cytoplasmic extract from BHK (baby hamster kidney) cells has been generated and in vitro translation reactions have been performed by following the method described by Skup and Millward (Skup, D. et al . (1977) Nucleic Acids Research 4: 3581-3587). The cytoplasmic extract has not been treated with micrococcal nuclease. Again, a drastic inhibition of both cap- dependent and cap-independent translation is observed, as mentioned above in section A) : as seen on this autoradiogram (Fig. 3), 5 μg per μl of heparin totally abolishes translation of the CAT (translated in a cap- dependent fashion) and the LUC polypeptides (translated in a cap-independent fashion) in the translation reac- tion. This reveals that the initiation of their translation is inhibited. Translation of the latter polypeptides is recovered accordingly when the concentration of heparin is reduced in the reactions. 0.4 μg of CAT- EMC-LUC RNA was employed in 40 μl translation reac- tions.

C) Inhibition of HIV-1 TAR-mediated translation in a cytoplasmic extract obtained from cells that have grown as monolayers A cytoplasmic extract from monkey Cos-1 cells has been generated. The cytoplasmic extract has not been treated with micrococcal nuclease. Interestingly, the capped TAR(+111)CAT RNA is very efficiently translated in this system (Fig. 4; lane 2 ) , when compared to the control reaction that was performed without exogenously added RNA (lane 1). This observed efficient synthesis of capped TAR(+111)CAT RNA is thus in contrast to the results obtained by others (Parkin, N.T. et al . (1988) EMBO Journal , 7:2831-2837). However, the translation of the capped TAR(+111) CAT RNA is drastically inhibited by heparin (lane 3: 1.25 μg per μl of translation reaction; lane 4: 0.125 μg per μl; lane 5: 0.0125 μg per μl; lane 6: 0.00125 μg per μl; in the latter case, translation of capped TAR(+111)CAT RNA is inhibited by more than 90% when compared to the translation performed in absence of heparin as shown in lane 2). Translation of capped TAR(+111)CAT RNA recovers when heparin is present at a concentration of 0.000125 μg per μl (lane 7).

0.4 μg of capped TAR(+111)CAT RNA was employed in 40 μl translation reactions (lanes 2 to 7). Discussion

To date, virus replication inhibition by heparin or heparin mimetic compounds thereof have been shown to be restricted to the virus adsorption stage of the virus to the target cells (Taylor, D.L. et al. (1995) Antiviral Research 28: 159-173; Thormar, H. et al. (1995) Antiviral Research 27: 49-57; Banfield, B.W. et al. (1995) Virology 208: 531-539; Ida, H. et al. (1994) Antiviral Research 23: 143-159; Barzu, T. et al. (1993) Journal of medicinal Chemistry 36: 3546-3555; Hanssens, F.P. et al. (1993) Journal of Virology 67: 4492-4496). One report mentions the in vi tro inhibition of translation of brome mosaic virus RNA in a wheat germ extract in presence of heparin, however an effect mediated by the plant homologue of PKR (pPKR) (Langland, J.O. et al. (1996) Plant Physiology and Biochemistry 34: 521- 526). To my knowledge, no report has been thus far published showing inhibition of cap-independent translation and HIV TAR-mediated translation by heparin or heparin mimetics either in vi tro or in vivo . Thus, my discovery has a great potential when one considers the fact that heparin or heparin mimetics can be targeted by different means to cells that have been infected by viruses, the latter showing a cap- independent translation initiation and HIV TAR-mediated of their RNA. This targeting of heparin might be performed and mediated by biovectors or liposomes, for example, the latter vectors containing heparin or heparin mimetics. This targeting can be specific when one considers the strategy depicted in Fig. 5. Perspectives

1) Potentially, any kind of cap-independent translation resulting on the attachment on RNA of ribosomes by internal ribosome entry could be reduced or inhibited with adequate concentrations of heparin or heparin mimetics thereof.

2) The effect of small heparin oligosaccharides of define lengths (as described in George, C.X. et al. (1996) Virology 221: 180-188) should be analyzed for their ability to reduce or inhibit the extent of cap- independent translation.

3 ) The effect of heparin or heparin mimetics on HIV-2 TAR-mediated translation of RNA should be analyzed.

4) Some experiments should be performed in vivo as well as in si tu: heparin should be incorporated into liposomes or biovectors which are then targeted to cells infected with various viruses by following the general procedure depicted in Fig. 5. 5) Targeting of any kind cell, not necessarily virus-infected, in which the cap-independent translation of any protein has to be inhibited.

The present invention will be more readily un- derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I

Therapeutic treatment of virus-infected cells As shown in Fig. 5A, target cells are infected by viruses through ligand-receptor interactions. As depicted, receptors are on the surface of the virus- infected cells and are recognized by the ligand of the virus . To generate newly synthesized virus particles, the ligand(s) of the virus has(ve) to be exposed at the cell surface of the infected cells (Fig. 5B).

To selectively target the virus-infected cells liposomes or biovectors containing heparin and/or hepa- rin mimetics and bearing the cell receptor at their surface will be generated. These liposomes or biovectors will selectively interact with the virus-infected cells through ligand-receptor interactions and will allow the transfer of the heparin and/or heparin mimet- ics into the virus-infected cells. This will inhibit the viral protein synthesis and render the virus to be avirulent (unable to synthesize the viral components, such as viral proteins).

The above-described therapeutic treatment of virus-infected cells will be effective for any virus wherein its protein synthesis is cap-independent. EXAMPLE II

Gene therapy using glycosaminoglycan N-acetylglucosami- nyl N-deacetylase/N-sulfotransferase cDNA

Heparin biosynthesis occurs only in connective tissue mast cells (Nader, H.B., and Dietrisch, in Heparin; Lane, D.A. and Lindahl, U, eds pp. 81-96, Arnold, London). The biosynthesis of heparin is initiated by glycosylation reactions that generate saccharide sequences composed of alternating D-glucuronic and N- acetylglucosamine (GlcNAc) units. N-deacetylation/N- sulfation of N-acetylglucosamine is a key event in the biosynthesis of heparin. This N-deacetylation/N sul- fation of GlcNAc is an obligatory step for the subsequent reactions for the biosynthesis of heparin. Recently, cDNAs coding for enzyme containing N- deacetylase/N-sulfotransferase activities have been cloned from a heparin-producing cell line MST from mouse (Orellana, A., Hirschberg, C.B., Wei, Z., Swiedler, S.J., and Ishihara M. , Journal of Biological Chemistry 21, pp. 2270-2276, 1994) and from a mouse mastocytoma cell line (Erikson, I., Sandback, D., Ek, B., Lindhal, U., and Kjellen, L. , Journal of Biological Chemistry 14, pp. 10438-10443, 1994).

In order to produce heparin in cell lines that do not normally produce heparin, it might be of great interest to express an enzyme that contains glycosaminoglycan N-acetylglucosaminyl N deacetylase/N-sulfo- transferase activities in these cells. For this, the cDNA coding for a glycosaminoglycan N-acetylglucosami- nyl N-deacetylase/N-sulfotransferase is incorporated into a plasmid or any suitable DNA sequence under the control of a viral or an eukaryotic promoter, thus allowing transcription of the corresponding RNA in these cells. This promoter can be an inducible promoter or a constitutive promoter. Targeting of the cells in order to incorporate the glycosaminoglycan N-acetylglu- cosaminyl N-deacetylase/N-sulfotransferase cDNA can be performed by gene therapy using retroviral vectors, adenoviral vectors, or liposome-mediated gene delivery. While the invention has been described in con- nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

I CLAIM;

1. A method of inhibiting cap-independent protein synthesis of eukaryotic cells, which comprises adding heparin or heparin mimetics thereof to said eukaryotic cells .

2. The method of claim 1, wherein said heparin mimetic is sulfated polysaccharides .

3. A method of inhibiting translation of HIV TAR- containing messenger ribonucleic acids (mRNA) of HIV infected eukaryotic cells, which comprises adding heparin or heparin mimetics thereof to said eukaryotic cells.

4. A pharmaceutical composition for the therapeutical inhibition of cap-independent protein synthesis in a patient suffering from a viral infection, which comprises a therapeutic amount of heparin and/or heparin mimetics thereof in association with a pharmaceutical carrier.

5. The composition of claim 4, wherein said carrier is a liposome or a biovector.

6. A pharmaceutical composition for the therapeutical inhibition of translation of HIV TAR-containing messenger ribonucleic acids (mRNA) in a HIV-infected patient, which comprises a therapeutic amount of heparin and/or heparin mimetics thereof in association with a pharmaceutical carrier.

7. The composition of claim 6, wherein said carrier is a liposome or a biovector.

8. A composition for gene therapy of patients infected with a virus, which comprises administering to said patient an expression vector consisting of a cDNA sequence coding for enzyme glycosaminoglycan N-acetyl- glucosaminyl N-deacetylase/N-sulfotransferase with a constitutive or inducible promoter operatively linked upstream of the cDNA sequence, said expression vector is adapted to express said enzyme thereby reducing viral protein synthesis in said patient.

9. The composition of claim 8, wherein the expression vector is a recombinant virus selected from the group consisting of adenovirus and retrovirus for targeting of the infected cells.

10. The composition of claim 8, wherein the delivery vector is a liposome for targeting of the infected cells.