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WO2006032952A1 - Oligonucleotides aleatoires therapeutiques generes a partir d'adn genomique - Google Patents

Oligonucleotides aleatoires therapeutiques generes a partir d'adn genomique Download PDF

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Publication number
WO2006032952A1
WO2006032952A1 PCT/IB2004/051842 IB2004051842W WO2006032952A1 WO 2006032952 A1 WO2006032952 A1 WO 2006032952A1 IB 2004051842 W IB2004051842 W IB 2004051842W WO 2006032952 A1 WO2006032952 A1 WO 2006032952A1
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dna
gdna
ddna
degradation
oligonucleotides
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Yun Oh
Zhifang Zhu
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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/08—Reducing the nucleic acid content
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • This invention relates generally to the field of oligonucleotide-based medical therapies. Specifically, it is a reduction to practice of concepts presented in PCT/ US03/20696 which demonstrates that synthetic random sequence oligonucleotides might be used to treat cancer and other proliferative disorders in humans.
  • This invention describes different methods to produce from a biologic source of DNA random sequence oligonucleotides that also can inhibit cellular proliferation. This invention further creates random sequence oligonucleotides much more economically and as random sizes that appear to be more effective at growth suppression and in addition to induce apoptosis in proliferating cells.
  • This present invention is both a reduction to practice of the first invention and an improvement of the original concept and involves the production of random-sequence AND random-size DNA fragments from a biologic source of DNA.
  • DNA fragments are herein defined as constituents or fragments of synthetic or biologic source DNA that are the result of chemical or mechanical degradation.
  • This degraded DNA (dDNA) prepared and aliquoted as described below has been demonstrated to inhibit growth of various cancer cell lines in vitro to varying degrees. In sensitive cell lines, the dDNA was more potent and had a greater maximal effect on growth than the random sequence 7 mer oligonucleotides that have been presented under patent PCT/ US03/20696. Disclosure of Invention
  • gDNA biologic source genomic DNA
  • dDNA degraded DNA
  • Various methods for this degradation process such as sonication, au- toclaving, boiling with or without acid or alkaline hydrolysis or DNAse digestion are discussed within this application.
  • Methods for monitoring the degradation process to quantitate and qualify the size distribution and yield of dDNA using gel elec ⁇ trophoresis and mass spectroscopy are also discussed.
  • the dDNA can be further processed to obtain rapidly different size fractions that might be used for different therapeutic effects.
  • This present invention is both a reduction to practice of the first invention and an improvement of the original concept and involves the production of random-sequence AND random-size DNA fragments from a biologic souxce of DNA.
  • DNA fragments are herein defined as constituents or fragments of synthetic or biologic source DNA that are the result of chemical or mechanical degradation.
  • the rationales for using a biologic source are four-fold. First, this would allow rapid production of a large amount of random sequence oligonucleotide at a substantial cost savings over what could be synthesized de novo. Second, the representative mixture of DlSfA sequences found in vivo, including those from non-coding repeat sequences, may have biologic effects that are unattainable from synthetic oligos.
  • oligos and DNA fragments produced from a biologic source could generate a wide size range from 2 to thousands of nu ⁇ cleotides. Although smaller oligonucleotides are expected to enter cells more ef ⁇ ficiently, they may not be able to bind efficiently to many single-stranded DNA binding proteins, including RPA, and may lack many additional biologic effects.
  • biologic source oligonucleotides and DNA fragments may contain trace amounts of unspecified DNA-binding proteins, such as histones, chapexone proteins, and RNAase-resistant RNA (e.g., double-stranded RNA or RNA complexed to other molecules) that may also exert favorable biologic effects.
  • DNA fragments (dDNA) degraded from biologic source DNA shares the potential therapeutic effects of synthetic random sequence oligonucletoide combinations described in patent PCT/US03/20696, but in addition dDNA appears to exert additional favorable biologic effects.
  • the dDNA was able to reduce the incorporation of BrdU into cancer cells (A549), suggesting that its biologic effect at least in part is due to inhibition of DNA replication (Fig 3).
  • Differential mRNA expression in A549 cells treated with dDNA versus the synthetic 7 mer and 25 mer oligonucleotide combination were compared by Affymetrix microarray analysis.
  • Transcripts from several genes involved in malignant invasion and cell cycle progression were reduced in common for both the synthetic oligonucleotide-treated and dDNA-treated cells.
  • the dDNA treatment inhibited transcription of more genes than the synthetic oligonucleotides, even at less than half the DNA dose by weight.
  • Additional transcripts suppressed included CDC42 (GTPase involved in en ⁇ dothelial morphogenesis during angiogenesis) and XRCC5 (implicated in DNA repair).
  • dDNA incubation with trypsin was as effective as incubation with DNAse in reducing the inhibitory effect of dDNA on proliferating cell lines, suggesting that an unspecified trace protein in dDNA may responsible for the added biologic effect it has over synthetic oligonucleotides (Fig 5).
  • FIG. 1 Combination Treatment with 25mer palindromic ODN and random sequence ODN in vivo, (a) Improved Survival in B16 Murine Melanoma Model. Mice were inoculated subcutaneously with Bl 6 Fl cells and given subcutaneous injections of oligos or vehicle controls daily for 4 weeks into the cancer inoculation site and later intratumorally. The random sequence ODN used were 25mers. Animals were sacrificed when tumors reached a maximal dimension of 2 cm. In reference to this endpoint, median survival for oligo-treated mice was doubled relative to control mice, (b) Complete response in p388 Murine Leukemia Ascites Model. In this model, the random sequence ODN were changed from 25mers to 7mers.
  • mice were inoculated in- traperitoneally with p388 cells, and all treatments were intravenously administered daily for 4 weeks.
  • a 100% cure rate was seen in mice treated with the combination of random 7mer ODN and 25mer palindromic ODN at 300 mg/kg, each.
  • 60%, 90%, and 0% cure rates were seen in mice treated with random 7mer ODN alone at 600 mg/kg, positive control treatment cyclophosphamide, and PBS, respectively.
  • HL60 cells treated at 10 -6 M for 96 hours demonstrated a reduction in cell cycle S phase, and increase in Gl, as well as a nearly 4-fold increase in subGl DNA suggestive of apoptosis.
  • Percentage of diploid cells in Gl, G2, and S phase were calculated using ModFit cell cycle analysis software. SubGl is calculated as a percentage of total events.
  • FIG. 14 MTT Cell Viability Assay of dDNA-sensitive cell line N417 after 48 hour exposure to dDNA. Cell Viability is measured as an arbitrary optical density at 562 nm. On the far left is viability of control cells not treated with dDNA. On the far right is a greater than 80% reduction in cell viability of cells exposed to dDNA. Pre- treatment of dDNA with DNAse I modestly inhibited the reduction in viability resulting from dDNA exposure. Pretreatment of dDNA with trypsin, however, almost completely abrogated the reduction in viability from dDNA exposure.
  • DNAse I followed by trypsin pretreatment of dDNA did not significantly enhance the effect of trypsin alone, suggesting that an unknown protein component of dDNA may be more important in inhibiting the growth of N417 than the oligonucleotide constituents themselves.
  • gDNA genomic DNA
  • 2 kB e.g., Sigma Chemicals Company, catalog # D- 1626
  • This lyophilized gDNA was resuspended in double-distilled, carbon-filtered, UV-irradiated (polymerase chain reaction-grade, or PCR- grade) water to a concentration of 10 mg/ml.
  • This gDNA was then used directly or further purified by twice extracting with phenol/chloroform and precipitating in absolute ethanol with standard Sodium Perchlorate (NaClO4) or Ammonium Chloride (NH4C1) solution, followed by washing the DNA pellet in 75% Ethanol. The resulting DNA pellet was dried and resuspended again in PCR-grade water at 10 mg/ml. Both of these solutions of gDNA were subjected to several different degradation methods described below, but the simplest, most reproducible method for small scale production of dDNA was repetitive autoclaving.
  • gDNA genomic DNA
  • Other methods for mechanical degradation of genomic DNA might be more cost effective and safer for large-scale commercial manufacture of dDNA than the methods utilized by us to date. Some of these methods are already employed to manufacture commercial preparations of 'sheared' DNA, which is a common reagent used to reduce nonspecific interactions in nucleic acid hybridization experiments. Such alternative methods could include, but are not limited to, the following.
  • Carbon dioxide may facilitate the degradation of DNA by lowering the pH of the DNA solution and enhancing hydrolysis of the DNA deoxyriobose backbone.
  • Inert gases such as nitrogen, may be favorable if later gDNA might be modified to a chemically labile intermediate form prior to degradation.
  • gases containing specific con ⁇ centrations of oxygen may be favorable for differently modified chemical variants of gDNA depending on the optimal redox conditions for it.
  • glass or metallic beads of varying size can be mixed in with an aqueous solution of genomic DNA and agitated within a closed container, in a process that can be referred to as bead-shearing. After a brief centrifugation of the mixture to sediment any particulate matter from the beads or precipitated components of the DNA solution, supernatant collected from the mixture would contain sheared DNA.
  • gDNA solutions can be injected into or extruded through a small orifice to shear it into smaller fragments.
  • gDNA solutions could be subjected to ultrasonic energy (without neb ⁇ ulization). Tubes or vials of gDNA solutions can be immersed in sonicating water baths. The ultrasonic waves could be partially transmitted through the container into the gDNA solution. High-intensity focused ultrasound (HIFU) techniques also could be used to transduce ultrasound energy directly into a solution of gDNA. The gDNA solution could be continuously mixed or stirred during the ultrasonication process to ensure homogeneity of the DNA shearing.
  • HIFU high-intensity focused ultrasound
  • DNA degradation method including the ones discussed above could be repeated sequentially on the same gDNA specimen to produce the smaller size therapeutic DNA fragments described in this patent.
  • the degree of DNA degradation and the size of DNA fragments generated during any of these methods could be monitored crudely using DNA-binding fluorescent dyes. This method is effective because smaller fragments of DNA below a threshold size are intercalated by these dyes less efficiently.
  • the dDNA binding fluorescence could then be correlated to 260 nm absorbance of the dDNA sample by spectroscopy (representing total nucleoside content) and used as a semi-quantitative index of gDNA degradation.
  • Any DNA-intercalating fluorescent dye might be used for quantification, including, but not limited to, propidium iodide, acridine orange, 7-amino Actinomycin D (7- AAD), and ethidium bromide.
  • Flourescence of dDNA solutions containing dye could be measured directly, subtracting background flourescence from dye solution alone. Densitometry measurements of ethidium bromide-stained dDNA at specified size bands on agarose gel electrophoresis would give even more detailed information on the degree of dDNA degradation and the distribution of resulting sizes.
  • the source of gDNA may also influence the biologic activity of the processed dDNA. If random sequence DNA fragments of greater than 16 bases have therapeutic activity, they may be acting by sequence-specific hybridization to or other interactions with genes in cells under treatment. Such target sequences might be protein-coding regions of genes or repetitive sequences in non-translated regulatory areas of the genome. This would suggest that the ideal source of gDNA from which to derive therapeutic dDNA would be the target tumor or organ itself, or at least cells of human origin. Likewise, therapeutic activity that might be the result of trace proteins (e.g., histones) or other molecules associated with the gDNA also might be greater if the gDNA is derived from the same cancer, patient, or species under treatment.
  • trace proteins e.g., histones
  • dDNA might be later derived from gDNA of cultured immortalized human cells (such as epithelial cells transformed with oncogenes or cancer cell lines) or resected tumors from individual patients who could be then treated with dDNA derived from their own tumors.
  • a potential advantage of using salmon testes DNA is that it contains both haploid as well as diploid gDNA, since larger oligonucleotides derived from haploid DNA may tend to stay more readily denatured.
  • any given solution of dDNA or partially degraded synthetic random sequence oligonucleotides could also be passed through size exclusion columns or membranes to isolate DNA fragments within a specific size range.
  • size exclusion columns or membranes By isolating and testing different size ranges of dDNA, we would hope to identify optimal size fractions for selected biologic activities. For example, the optimal size range of dDNA to inhibit DNA pro ⁇ liferation may be smaller than that to bind RPA and inhibit DNA repair or tran ⁇ scription. Size exclusion columns or membranes could be applied sequentially to DNA samples to isolate different size ranges of DNA that may have different therapeutic potentials.
  • Biologic source DNA either before or after degradation might be chemically modified to improve biologic half-life, pharmacokinetics, and other pharmacologic properties.
  • modifications could include, but are not limited to, oxidation or reduction, addition of ester, ether, or amide groups to random or specific free hydroxyl groups on the DNA backbone or on nucleotide bases.
  • dDNA preparation may be given by different routes including, but not limited to, intravenous, intramuscular, sub ⁇ cutaneous, transmucosal, inhalational, oral, topical, gargle, or enema administration.

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Abstract

L'invention concerne des procédés de production d'oligonucléotides en vrac de séquence aléatoire, dont les dimensions s'étendent sur une plage étendue, à partir D'ADN génomique biologique. Cette invention se rapporte également à un procédé pour surveiller et réguler le degré de dégradation d'un échantillon d'ADN. La présente invention concerne par ailleurs un procédé d'utilisation d'ADN génomique dégradé pour supprimer la prolifération de cellules humaines de culture, et induire en outre l'apoptose de cellules sensibles. Ainsi, l'ADN génomique dégradé constitue potentiellement un traitement anti-prolifération efficace.
PCT/IB2004/051842 2004-09-23 2004-09-23 Oligonucleotides aleatoires therapeutiques generes a partir d'adn genomique Ceased WO2006032952A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9127306B2 (en) 2008-02-15 2015-09-08 Life Technologies Corporation Methods and apparatuses for nucleic acid shearing by sonication

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6329146B1 (en) * 1998-03-02 2001-12-11 Isis Pharmaceuticals, Inc. Mass spectrometric methods for biomolecular screening
US20020025522A1 (en) * 2000-01-03 2002-02-28 Yakubov Leonid A. Compositions comprising genome segments and methods of using the same
US20020115089A1 (en) * 1997-03-28 2002-08-22 Orasure Technologies, Inc. Simultaneous collection of DNA and non-nucleic analytes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020115089A1 (en) * 1997-03-28 2002-08-22 Orasure Technologies, Inc. Simultaneous collection of DNA and non-nucleic analytes
US6329146B1 (en) * 1998-03-02 2001-12-11 Isis Pharmaceuticals, Inc. Mass spectrometric methods for biomolecular screening
US20020025522A1 (en) * 2000-01-03 2002-02-28 Yakubov Leonid A. Compositions comprising genome segments and methods of using the same

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9127306B2 (en) 2008-02-15 2015-09-08 Life Technologies Corporation Methods and apparatuses for nucleic acid shearing by sonication
US10246736B2 (en) 2008-02-15 2019-04-02 Life Technologies Corporation Methods and apparatuses for nucleic acid shearing by sonication
US10329598B2 (en) 2008-02-15 2019-06-25 Life Technologies Corporation Methods and apparatuses for nucleic acid shearing by sonication

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