WO2006043809A1 - Protection of biologically active molecules using amphiphiles - Google Patents

Protection of biologically active molecules using amphiphiles Download PDF

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Publication number
WO2006043809A1
WO2006043809A1 PCT/NL2005/000752 NL2005000752W WO2006043809A1 WO 2006043809 A1 WO2006043809 A1 WO 2006043809A1 NL 2005000752 W NL2005000752 W NL 2005000752W WO 2006043809 A1 WO2006043809 A1 WO 2006043809A1
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Prior art keywords
biologically active
active compound
saint
molecule
protection
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PCT/NL2005/000752
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French (fr)
Inventor
Marcel Herman Josef Ruiters
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Synvolux Ip B.V.
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Publication date
Priority claimed from NL1027311A external-priority patent/NL1027311C2/en
Priority claimed from NL1027417A external-priority patent/NL1027417C2/en
Application filed by Synvolux Ip B.V. filed Critical Synvolux Ip B.V.
Priority to DK05795379T priority Critical patent/DK1805307T3/en
Priority to PL05795379T priority patent/PL1805307T3/en
Priority to EP05795379A priority patent/EP1805307B1/en
Priority to DE602005012420T priority patent/DE602005012420D1/en
Publication of WO2006043809A1 publication Critical patent/WO2006043809A1/en
Priority to NO20071601A priority patent/NO20071601L/en
Priority to US11/737,588 priority patent/US20070224589A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • the present invention relates to a process to protect a biologically active compound against the effects of for instance enzymes, chemicals, oxygen, radicals or sunlight. Further the invention relates to a vehicle to protect a biological active compound against the effects of for instance enzymes, chemicals, oxygen, radicals or sunlight. The invention also relates to the application of a SAINT-molecule to protect a biological active compound against the effects of for instance enzymes, chemicals, oxygen, radicals or sunlight.
  • biologically active compounds such as siRNA, RNA, DNA, proteins and peptides are sensitive for the effects (e.g. degradation) of for instance enzymes, chemicals, oxygen, radicals or sunlight.
  • biologically active compounds such as siRNA, RNA, DNA, oligonucleotides or derivates thereof
  • proteins and peptides can only be transported as dry matter, or as a stabilized, glycerol or DMSO containing solution.
  • the solutions to be transported or dry matters are kept on dry ice, or are cooled in another way. Consequently, the transportation of these compounds is very expensive.
  • Another disadvantage of transporting biologically active compounds as dry matter is the fact that during the dissolution of the biologically active compound a conformational change may occur. As a result, not all biologically active matter will regain its original function.
  • glycerol (or DMSO) has shown to have a negative effect on the activity of the biologically active compound when the biologically active compound is diluted before application.
  • the solution found in the state of the art to circumvent these disadvantages is to replace the glycerol (or DMSO) by threhalose.
  • Threhalose is a sugar. Although in theory no conformational change occurs when dissolving the biologically active compound in threhalose, there are some other disadvantages when using threhalose. A particular disadvantage is that threhalose has an adverse effect on the desired enzymatic activity of the proteins.
  • threhalose is not used to package DNA, RNA, siRNA en oligonucleotides or derivatives thereof. After complexation with threhalose (and other sugars) DNA,RNA etc, are laboriously to be dissolved, as they can also be characterized as sugars.
  • the present invention aims at solving the disadvantages mentioned above.
  • the invention aims to provide for a process to protect a biologically active compound against the effects of for instance enzymes, chemicals, oxygen, radicals or sunlight.
  • the invention also aims to provide for a vehicle to protect a biologically active compound against the effects of enzymes, chemicals, oxygen, radicals or sunlight whereby the disadvantages mentioned above can be circumvented.
  • transport is understood to mean both the packaging of the biologically active compound in a transportation container, and the subsequent conveyance of this container by means of transportation means, such as carriers, etc., as well as the transport of individual biologically active molecules, which are each separately enwrapped by the protecting compounds.
  • the present invention provides for a process as mentioned in the preamble, which process is characterised by the measures according to claim 1.
  • the biologically active compound has become completely protected from effects of enzymes, chemicals, oxygen, radicals or sunlight. Furthermore, completely no or no functional inhibitory conformational changes of the biological compound occur.
  • the invention provides for a vehicle as mentioned in the pre-amble, characterized by the measures as described in claim 4.
  • the vehicle of the present invention provides a very reliable and simple solution for transporting a biologically active compound in such a way that the biologically active compound will not become damaged, for instance by the effect of enzymes, chemicals, oxygen, radicals or sunlight, or a conformational change occurs.
  • a SAINT-molecule protects a biologically active compound against the effect of for instance enzymes, chemicals, oxygen, radicals or sunlight, which is characterized in that the biologically active compound is contacted with the biologically active compound, causing the biologically active compounds to interact with the SAINT-molecule.
  • the biologically active compound which can be chosen from, for instance, siRNA, RNA, DNA, oligonucleotides or derivatives thereof, proteins and peptides, is actually enwrapped by one ore more SAINT-molecules.
  • the saint molecules may all be of the same kind, but it is also possible that a mixture of different SAINT-molecules is applied, which can be connected to the biological active compound. This interaction is a hydrogen-bond.
  • the vehicle of the present invention consisting of the biologically active compound and the SAINT-molecule or SAINT-molecules enwrapping it, can be kept without occurring a separation of the biologically active compound and the SAINT-molecules.
  • the biologically active compound when the biologically active compound has to fulfil its function, it is not suffering any functional hindrance from the SAINT-molecules present. Therefore the biologically active compounds can freely operate and can easily be applied in basically any biological assay.
  • a remarkably great advantage of the present invention is that no separation of the SAINT-molecule (s) from the biological active compound is needed, because the saint-molecule (s) have no inhibitory effect in the buffer. The molecule will be diluted to such a great extent, that no inhibitory effect will be shown. Furthermore it is biologically degradable, without forming any toxic compounds.
  • siRNA when directed to a specific gene, is able to silence the gene-expression by inhibition of the mRNA translation.
  • SiRNA needs to be stored as dried powder and, after dissolution, aliquots are stored at -2O 0 C. Regardless of aliquots being prepared, the reproducibility of (the results obtained over time using) such aliquots is weak.
  • the delivery of siRNA to cells has been described in patents EP-0755924 and US 5,853,694.
  • siRNA 25 micrograms of siRNA were complexed with 0,5 ml of SAINT-MIX. Per transfection 20 ⁇ l of this (preservation) -complex is used. Transfection was performed as described in detail in EP-0755924.
  • the efficacy of the siRNA-transfection is validated by measuring the enzyme activity of the silenced gene according to a standard enzyme assay. Lifetime of the enzyme is approximately 5 days. Therefore, after one single transfection with 1 Dg siRNA, about 50% enzyme activity can be measured after 48 hours and almost no enzyme activity can be measured after 10 days, as shown in the figure 1 below.
  • siRNA complexed with SAINT-RED is active over a 9 month- period while the aliguoted siRNA has lost almost all its activity within 2 days.
  • results show that the siRNA/SAINT prevention complex is even more active, up to 5 months, when compared with the freshly prepared siRNA/SAINT complex. This indicates the strong protective character of SAINT-molecules towards biologically active compounds.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Preparation (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The invention relates to a process for the protection of a biologically active compound against the effect of for instance enzymes, chemicals, oxygen, radicals or sunlight. The process is characterised in comprising the step of bringing the biologically active compound in contact with at least one SAINT-molecule . Thereby a good protection from the biologically active compound is achieved.

Description

Protection of biologically active molecules using amphiphiles
The present invention relates to a process to protect a biologically active compound against the effects of for instance enzymes, chemicals, oxygen, radicals or sunlight. Further the invention relates to a vehicle to protect a biological active compound against the effects of for instance enzymes, chemicals, oxygen, radicals or sunlight. The invention also relates to the application of a SAINT-molecule to protect a biological active compound against the effects of for instance enzymes, chemicals, oxygen, radicals or sunlight.
In general, biologically active compounds, such as siRNA, RNA, DNA, proteins and peptides are sensitive for the effects (e.g. degradation) of for instance enzymes, chemicals, oxygen, radicals or sunlight. For example, in many cases biologically active compounds, such as siRNA, RNA, DNA, oligonucleotides or derivates thereof, proteins and peptides can only be transported as dry matter, or as a stabilized, glycerol or DMSO containing solution. The solutions to be transported or dry matters are kept on dry ice, or are cooled in another way. Consequently, the transportation of these compounds is very expensive.
Another disadvantage of transporting biologically active compounds as dry matter is the fact that during the dissolution of the biologically active compound a conformational change may occur. As a result, not all biologically active matter will regain its original function.
Also the transport of the biologically active compound in a (buffered) glycerol (or DMSO) containing solution has disadvantages. Glycerol (or DMSO) has shown to have a negative effect on the activity of the biologically active compound when the biologically active compound is diluted before application. The solution found in the state of the art to circumvent these disadvantages is to replace the glycerol (or DMSO) by threhalose. Threhalose is a sugar. Although in theory no conformational change occurs when dissolving the biologically active compound in threhalose, there are some other disadvantages when using threhalose. A particular disadvantage is that threhalose has an adverse effect on the desired enzymatic activity of the proteins. In the state of the art threhalose is not used to package DNA, RNA, siRNA en oligonucleotides or derivatives thereof. After complexation with threhalose (and other sugars) DNA,RNA etc, are laboriously to be dissolved, as they can also be characterized as sugars.
The present invention aims at solving the disadvantages mentioned above.
In particular, the invention aims to provide for a process to protect a biologically active compound against the effects of for instance enzymes, chemicals, oxygen, radicals or sunlight. The invention also aims to provide for a vehicle to protect a biologically active compound against the effects of enzymes, chemicals, oxygen, radicals or sunlight whereby the disadvantages mentioned above can be circumvented. The word "transport" is understood to mean both the packaging of the biologically active compound in a transportation container, and the subsequent conveyance of this container by means of transportation means, such as carriers, etc., as well as the transport of individual biologically active molecules, which are each separately enwrapped by the protecting compounds.
To meet at least one of the aforementioned aims, the present invention provides for a process as mentioned in the preamble, which process is characterised by the measures according to claim 1. Herewith the advantage is reached that the biologically active compound has become completely protected from effects of enzymes, chemicals, oxygen, radicals or sunlight. Furthermore, completely no or no functional inhibitory conformational changes of the biological compound occur.
Furthermore the invention provides for a vehicle as mentioned in the pre-amble, characterized by the measures as described in claim 4. The vehicle of the present invention provides a very reliable and simple solution for transporting a biologically active compound in such a way that the biologically active compound will not become damaged, for instance by the effect of enzymes, chemicals, oxygen, radicals or sunlight, or a conformational change occurs.
According to a further aspect of the invention an application is provided in which a SAINT-molecule protects a biologically active compound against the effect of for instance enzymes, chemicals, oxygen, radicals or sunlight, which is characterized in that the biologically active compound is contacted with the biologically active compound, causing the biologically active compounds to interact with the SAINT-molecule. ■ By means of the invention, the biologically active compound, which can be chosen from, for instance, siRNA, RNA, DNA, oligonucleotides or derivatives thereof, proteins and peptides, is actually enwrapped by one ore more SAINT-molecules. The saint molecules may all be of the same kind, but it is also possible that a mixture of different SAINT-molecules is applied, which can be connected to the biological active compound. This interaction is a hydrogen-bond.
It has been shown that the vehicle of the present invention, consisting of the biologically active compound and the SAINT-molecule or SAINT-molecules enwrapping it, can be kept without occurring a separation of the biologically active compound and the SAINT-molecules. Nevertheless, it has also been shown that, when the biologically active compound has to fulfil its function, it is not suffering any functional hindrance from the SAINT-molecules present. Therefore the biologically active compounds can freely operate and can easily be applied in basically any biological assay. A remarkably great advantage of the present invention is that no separation of the SAINT-molecule (s) from the biological active compound is needed, because the saint-molecule (s) have no inhibitory effect in the buffer. The molecule will be diluted to such a great extent, that no inhibitory effect will be shown. Furthermore it is biologically degradable, without forming any toxic compounds.
The invention has been described in essence in the above. Based on the description above and the attached claims, a person skilled in the art will easily be able to develop further embodiments, which all will fall within the scope of the present invention.
EXAMPLE
One of the most sensitive and at this moment frequently used biologically active compounds is siRNA. SiRNA, when directed to a specific gene, is able to silence the gene-expression by inhibition of the mRNA translation.
SiRNA needs to be stored as dried powder and, after dissolution, aliquots are stored at -2O0C. Regardless of aliquots being prepared, the reproducibility of (the results obtained over time using) such aliquots is weak. The delivery of siRNA to cells has been described in patents EP-0755924 and US 5,853,694.
To illustrated the present invention we here show data concerning the preservation of siRNA by the use of SAINT-molecules.
Method:
25 micrograms of siRNA were complexed with 0,5 ml of SAINT-MIX. Per transfection 20 μl of this (preservation) -complex is used. Transfection was performed as described in detail in EP-0755924.
The efficacy of the siRNA-transfection is validated by measuring the enzyme activity of the silenced gene according to a standard enzyme assay. Lifetime of the enzyme is approximately 5 days. Therefore, after one single transfection with 1 Dg siRNA, about 50% enzyme activity can be measured after 48 hours and almost no enzyme activity can be measured after 10 days, as shown in the figure 1 below.
Remaining enzyme activity after transfection of 1 ug siRNA with SAINT-RED
□Enzymeactivity
Figure imgf000006_0001
Oday 2days 10days
Figure 1.
Over a 9 month-period, we compared the siRNA stored at -20°C degrees versus the siRNA/SAINT-RED preservation complex. Enzyme activity was measured 48 hours after transfection. Results have been depicted in Figure 2.
Figure imgf000007_0001
Figure imgf000007_0002
0 1 day 2 days 5 months 9 months storage time
Figure 2 .
The figure clearly shows that siRNA complexed with SAINT-RED is active over a 9 month- period while the aliguoted siRNA has lost almost all its activity within 2 days. Moreover the results show that the siRNA/SAINT prevention complex is even more active, up to 5 months, when compared with the freshly prepared siRNA/SAINT complex. This indicates the strong protective character of SAINT-molecules towards biologically active compounds.

Claims

1. A process for the protection of a biologically- active compound against the effect of for instance enzymes, chemicals, oxygen, radicals or sunlight, characterised in that it contains the step of bringing the biologically active compound in contact with a SAINT- molecule.
2. The process according to claim 1, characterised in that this step comprises the contacting of a biologically active compound with a mixture of SAINT- molecules.
3. The process according to claims 1 or 2, characterised in that the biologically active compound is bound to at least one SAINT-molecule by means of electrostatic interaction.
4. A vehicle for the protection of a biologically active compound against the effect of for instance, enzymes, chemicals, oxygen, radicals or sunlight, characterised in that the vehicle comprises of the biologically active compound an at least one SAINT- molecule bound to it.
5. The vehicle according to claim 4, characterised in that the at least one SAINT-molecule is bound to the biologically active compound by means of electrostatic interaction.
6. A process to preserve a biologically active compound, characterised in that it comprises the step of combining the biologically active compound with a SAINT- molecule or a mixture thereof.
7. The application of a SAINT-molecule for the protection of a biologically active compound against the effect of for instance enzymes, chemicals, oxygen, radical or sunlight, whereby the biologically active compound is brought in contact with a SAINT-molecule in such a way that the biologically active compound interacts with the SAINT-molecule through electrostatic interaction.
PCT/NL2005/000752 2004-10-21 2005-10-20 Protection of biologically active molecules using amphiphiles WO2006043809A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
DK05795379T DK1805307T3 (en) 2004-10-21 2005-10-20 Protection of biologically active molecules by amphiphiles
PL05795379T PL1805307T3 (en) 2004-10-21 2005-10-20 Protection of biologically active molecules using amphiphiles
EP05795379A EP1805307B1 (en) 2004-10-21 2005-10-20 Protection of biologically active molecules using amphiphiles
DE602005012420T DE602005012420D1 (en) 2004-10-21 2005-10-20 PROTECTION OF BIOLOGICALLY ACTIVE MOLECULES BY AMPHIPHILES
NO20071601A NO20071601L (en) 2004-10-21 2007-03-27 Protection of biologically active molecules using amphiphiles
US11/737,588 US20070224589A1 (en) 2004-10-21 2007-04-19 Protection of Biologically Active Molecules Using Amphiphiles

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
NL1027311A NL1027311C2 (en) 2004-10-21 2004-10-21 New vehicle, useful for the transport to the nucleus of a DNA-modifying enzyme/molecule which is combined with a SAINT-molecule or a combination of several entities
NL1027311 2004-10-21
NL1027417 2004-11-04
NL1027417A NL1027417C2 (en) 2004-10-21 2004-11-04 New vehicle, useful for the transport to the nucleus of a DNA-modifying enzyme/molecule which is combined with a SAINT-molecule or a combination of several entities
NL1027479 2004-11-10
NL1027479A NL1027479C2 (en) 2004-10-21 2004-11-10 Protection of biologically active molecules with the help of amphiphiles.

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PCT/NL2005/000754 WO2006043811A1 (en) 2004-10-21 2005-10-20 Vehicle for the transport of a chosen molecule to a cell

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EP (3) EP1805307B1 (en)
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AT (3) ATE420173T1 (en)
AU (1) AU2005296360B2 (en)
CA (2) CA2583860A1 (en)
DE (3) DE602005012303D1 (en)
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DE602005012303D1 (en) 2009-02-26
DK1805306T3 (en) 2009-04-20
ES2318548T3 (en) 2009-05-01
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NO20071599L (en) 2007-06-26
PL1805305T3 (en) 2009-06-30
CA2584633A1 (en) 2006-04-27
US20080085273A1 (en) 2008-04-10
ATE420954T1 (en) 2009-01-15
PT1805307E (en) 2009-04-13
AU2005296360A1 (en) 2006-04-27
EP1805305A1 (en) 2007-07-11
SI1805306T1 (en) 2009-06-30
EP1805305B1 (en) 2009-01-07
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PL1805307T3 (en) 2009-06-30
DE602005012420D1 (en) 2009-03-05
PT1805306E (en) 2009-04-13
SI1805307T1 (en) 2009-06-30
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JP2008517903A (en) 2008-05-29
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DE602005012304D1 (en) 2009-02-26
EP1805307A1 (en) 2007-07-11
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