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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/33—Chemical structure of the base
  • C12N2310/337—Chemical structure of the base in alpha-anomeric form

Definitions

• IgM, IgG, IgA, and IgE are synthesized.
• An antibody monomer contains two
• Antibodies have enormously diverse binding specificity characterized by
• Each heavy chain or light chain has its own unique variable region segment, as well as constant region segments which
• a mature, immunocompetent, resting B cell has a rearranged genes encoding for the heavy and light chain variable regions and, therefore, a defined antigen specificity.
• a B cell can further mature and differentiate into a cell expressing one or more immunoglobulins of the five classes, all of which
• IgM exists as a pentamer and is multivalent, and fixes
• IgG generally is most effective in mediating antibody- dependent cellular cytotoxicity and crosses the placenta to the fetus; IgA is most important in mucosal surface; IgE is dominant in sensitizing mast cells and basophils in pharmacological mediator release; IgD is on the B cell surface and
• IgG isotype are most conspicuously involved as autoantibodies in autoimmune diseases, such as in rheumatoid arthritis, system lupus erythematosus, Graves' disease, Hashimoto's
• IgE is mainly responsible for immediate type
• IgG is responsible for many autoimmune diseases, and IgE is mainly responsible for immediate-hypersensitivities, it is desirable that the antibody suppression be isotype-specific. By doing so, the adverse effects to the immune system will be minimized.
• lymphokines involved in isotype-specific switching e.g. experiments have been
• the present invention employs anti-sense oligonucleotides for targeting the mRNA precursors of the specific isotypes and even of particular subclasses of isotypes.
• Anti-sense technology is based on the principle that a properly designed
• oligonucleotide or analogue capable of binding to a key segment of an RNA or
• a DNA can enter a cell, binding to the crucial element of the mRNA, or its precursor RNA, or DNA, and effectively inhibit the function of the RNA or DNA
• DNA In mammals, DNA generally exist as double helices of two complementary strands, complexing with nuclear proteins such as histones, forming extremely complex but orderly structural units that compact together to form a chromosome.
• the DNA strand is a genetically inherited sequence of millions of four deoxyribonucleotides distinguished by their base side chains, adenine ("A"), thymine (“T”), cytosine ("C”), and guanine ("G”).
• A adenine
• T thymine
• C cytosine
• G guanine
• the DNA strand is functionally a linear stretch of thousand of genes, some of which encode structural polypeptides and some of which carry regulatory information or
• Messenger RNA is a transcribed product of a segment, generally a gene, in DNA.
• Messenger RNA is a sequence of ribonucleotides with A, C, G, and
• RNA is generally single stranded and hence is
• a naturally occurring mRNA and its complementary DNA strand is a "sense” strand.
• RNA polymerase RNA polymerase to assemble an RNA molecule according to the DNA sequence in that gene.
• the newly synthesized RNA precursor is processed by another set of proteins and enzymes to form a mature mRNA species, which is then transported from the
• nucleus to the cytosol.
• a set of regulatory proteins and enzymes bind to the control segment near the 5' end of the mRNA and read the information along the mRNA sequence (from the 5' end to the 3' end) to assemble amino acids to form a unique polypeptide chain.
• oligonucleotides for inhibiting the production of crucial viral proteins or products or oncogenic proteins are used.
• the first group target the control region of mRNA or its precursor RNA and interfere the proper expression of the messenger species.
• RNA transcripts can interfere with proper maturation of the precursor RNA transcripts
• the other group of anti-sense compounds target the control region of a
• the oligonucleotide binds to one strand of the DNA as the two strands of DNA open up, and forms an apparent "triplex" structure.
• oligonucleotides made from the native nucleotides are highly charged and penetrate the cellular plasma membrane very poorly. They are also very sensitive to nuclease digestion in the blood.
• the modified oligonucleotides include phosphorothioate oligomers, methylphosphonate oligomers, ⁇ -anomeric
• oligomers and others. These oligonucleotides hybridize with the complementary RNA or DNA segments equally well. However, since they are less hydrophilic and more lipophilic, they can get into cells more effectively. They are also much more resistant to nuclease cleavage than the native oligonucleotides.
• coding regions can provide targets for anti-sense compounds.
• One widely used process would be to synthesize several oligonucleotides of 10 to 20 nucleotides in length covering portions of the regions mentioned above, and to test the effects on inhibiting the expression of the encoded protein.
• a DNA gene consists of not only
• RNA which is initially
• RNA transcribed corresponds to the entire sequence, including the regulatory regions at the 5' end, the exons, and the introns.
• This RNA precursor is processed in a
• mRNA which contains a contiguous coding sequence of a polypeptide, along with regulatory elements flanking the 5' and 3' ends.
• the mechanism of mRNA splicing is complex. It may involve Ul- and U2- containing small RNA-protein complexes (Ul, U2 snRNPs).
• Ul, U2 snRNPs small RNA-protein complexes
• the Ul RNA has a sequence at its 5' end that is precisely complementary to the nine-nucleotide consensus for the 5' splice site of an exon. Binding of Ul-snRNP to the 5' splice site results in a cut at the 5' splice junction. The cut occurs through ATP
• the 5' junctions extends three residues upstream and six residues downstream
• the C at the 5' end of SEQ ID NO: 1 can also be an A, and the 6th A from the 5' end
• the 3' splice junction includes a pyrimidine-
• the C can be a T.
• This pyrimidine-rich region is followed by a short consensus sequence extending only three residues upstream and one residue downstream from the splice point, which is between the two Gs at the 3' end.
• branch point A residue occurs either 32 residues upstream (in the case of the
• IgGs, IgD, and IgE are 35 residues upstream (in the case of the IgAs) or 34 residues upstream (for IgM) from the 3' splice junction.
• IgGs, IgD, and IgE are considerably non-homologous among immunoglobulins of different isotypes. Since the intron sequences between the 5' splice junction and the branch point are not needed for splicing reactions, oligonucleotide sequences which would successfully interfere with
• splicing would be complementary to the region about 30 nucleotides upstream of the 3' splice junction.
• variable (V) gene family of 50 or more genes and the cluster of heavy chain constant region genes are in separate chromosomes.
• V gene is
• the fused VDJ segment may be switched to one of the ⁇ 's or a, or e, this switching being regulated by certain T cell factors and some other yet-to-be characterized factors.
• the VDJ for a B cell expressing a particular isotype and subclass, the VDJ
• VDJ segment is adjacent to the particular heavy chain constant region gene segment.
• the VDJ segment is thought to be adjacent to the constant region gene of ⁇ l.
• the leader sequence serves as secretion signal
• region domains including CHI, CH2, and CH3, and for ⁇ and e, CH4, and the membrane anchor peptide segments, all are encoded for by discrete exons separated by introns.
• the hinge region of the immunoglobulin is encoded by a separate exon.
• the control regions for transcription and translation are at the 5' end flanking the exon of the leader sequence.
• termination sequence is at the 3' end flanking the membrane exons.
• RNA transcript complementary to the entire length of the genomic structure from the 5' end control regions to the 3' end transcription termination signal sequence is made.
• This RNA precursor is then processed through a sequence of splicing steps, in which the introns are excised and the peptide-coding exons are spliced together to form mature mRNA.
• the 3' end of an exon is spliced to the 5' end of the next exon in sequence.
• the 3' end of the fused VDJ segment is spliced to the 5' end of the CHI exon.
• VDJ may be rearranged to the constant region genes of various heavy chain isotypes during B cell maturation.
• the splicing between the VDJ exon and an isotype specific CHI exon is inhibited by anti-sense oligonucleotides that bind to the splicing recognition sequence 5' to the CHI exon or that bind to the splicing junctional segment of CHI.
• anti-sense oligonucleotides that bind to the splicing recognition sequence 5' to the CHI exon or that bind to the splicing junctional segment of CHI.
• the invention includes oligonucleotides which are complementary to the
• immunoglobulin heavy chain in one or more of the CH regions of the
• the oligonucleotides are complementary to a portion of the sequence upstream of the 3' splice junction and towards the branch point A residue, or to a portion of the sequence upstream and downstream of the 3' splice junction.
• the oligonucleotides generally should be
• oligonucleotides of the invention would be complementary to some or all of the underlined portions of SEQ ID NOS: 3-33, and/or to some of these
• portions as well as some of the portions downstream of the 3' splice junction and in the exon region (shown in bold face type).
• the underlined portions represent the conserved portions of the ougonucleotides. In these sequences the exons are shown in bold face type, and the introns in normal type.
• the 3' splice point is the point of transition between these regions.
• the branch point A residue is also in bold face type.
• oligonucleotides will hybridize with the splicing recognition region
• oligonucleotides can be tested for activity in vitro on B cell lines or peripheral blood mononuclear cells (PBMCs), to determine their ability to inhibit
• oligonucleotide demonstrates significant activity, additional oligonucleotides of varying lengths covering the target segment can be synthesized and tested, with those of optimal length and sequence being selected as product candidates.
• the present disclosure focuses on the splice junctions of the various immunoglobulin domains as the target segments for designing anti-sense compounds.
• the 3'-end untranslated regions of each antibody isotype which are
• oligonucleotides of about 15 nucleotide long, covering the entire length of the 3'- end untranslated region. These oligonucleotides may overlap with adjacent
• oligonucleotides by up to 5 nucleotides on each end. Since oligonucleotide synthesis with synthesizers is a very established procedure, to make overlapping oligonucleotides covering a gene region of 1000 to 2000 nucleotide long is feasible. These oligonucleotides can then be tested in vitro against B cell lines or PBMCs, to determine their ability to inhibit the production of a particular class of antibodies. Those which inhibit production to the greatest extent would be
• immunoglobulin isotypes is suitable for treating autoimmune diseases and allergies.
• the immunoglobulins IgG and IgM have been associated with autoimmune diseases, including especially rheumatoid arthritis. Suppressing IgG and/or IgM production would be useful in treating
• rheumatoid arthritis and probably also in treating other autoimmune diseases including systemic lupus erythematosus, scleroderma, and myasthenia gravis.
• oligonucleotides of the invention are in production of monoclonal antibodies.
• the oligonucleotides can be used to selectively inhibit production of various immunoglobulin isotypes, either by B cells extracted from the immunized animal prior to fusion with an immortalized cell line, or by the hybridomas after the fusion. This allows production of immunoglobulins of a particular desired isotype, or the prevention of production of immunoglobulins of a particular isotype. As a result, no monoclonal antibodies of these isotypes
• the invention pertains to use of anti-sense compounds to achieve isotype-
• RNA are at the splice junctions and in the 3'-end untranslated regions.
• coding regions or other areas of the untranslated regions may also provide targets
• oligonucleotides at concentrations ranging from 0J to 200 ⁇ g/ml are tested for their effect on the synthesis of IgE by treating SKO-007 cells (ATCC, Rockville, Maryland). These IgE-expressing cells are incubated with the potential inhibitors for 1 to 3 days and the IgE secreted into the medium is assayed by a standard IgE ELISA, such as that described in Sun L. et al. /.
• oligonucleotides ranging from 10 to 12 nucleotides in length which overlap by only one or two nucleotides and covering the identified region, are then synthesized and tested for effect on IgE production. By doing so, the segment with the optimal sequence and length can be identified. Analogues of oligonucleotides with enhanced lipophilicity and resistance to RNase can also be
• anti-sense compounds to target the 3 '-end untranslated regions of the four subclasses of ⁇ chains.
• the region between the ATG stop codon and AATAA signal is 100 nucleotides long for ⁇ l (SEQ ID NO:35), and is 101 nucleotides long for ⁇ 2, ⁇ 3, and ⁇ 4, (SEQ ID NOS: 36, 37 and 38, respectively). See Huck S. et al. Nucl. Acids Res. 14:1779-1789 (1986) .
• the regions have only a few
• a set of 18 oligonucleotides can be synthesized for each subclass, with most of these oligonucleotides shared among the subclasses. These oligonucleotides can then be tested for abUity to
• anti-sense compounds of the invention also target the segments at the
• oligonucleotides are complementary to at least about a continuous 12 nucleotide segment of the underlined portions of the sequences of SEQ ID NOS:3-33.
• SEQ ID NOS:3-ll The sequences represented by SEQ ID NOS:3-ll are explained above.
• SEQ ID NOS: 12, 13, 14 and 15 represent, respectively, the sequences of the intron and
• SEQ ID NOS:16, 17, 18 and 19 represent, respectively, the sequences of the intron and exon near the 3' splice sites of the CH3 regions of ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4.
• SEQ ID NOS:20 and 21 represent, respectively, the sequences of the intron and exon near the 3' splice sites of the CH2 and CH3 regions of ⁇ .
• SEQ ID NOS:22 and 23 represent, respectively, the sequences of the intron and exon near the 3' splice sites of the hinge 1 and hinge 2 regions of ⁇ .
• SEQ ID NOS: 24 and 25 represent, respectively, the sequences of the intron and exon near the 3' splice sites of CH2 and CH3 of ⁇ l.
• SEQ ID NOS:26 and 27 represent, respectively, the sequences of the intron and exon near the 3' splice sites of CH2 and CH3 of ⁇ 2.
• NOS:28, 29 and 30 represent, respectively, the sequences of the intron and the
• the preferred length for the oligomers of the invention is 15
• nucleotides although they can be as short as about 12 nucleotides. As noted
• the oligomers or the invention hybridize with the segment of the nucleotide upstream of the 3' splice junction (underlined in the sequence listing portion), and/or with a portion of this segment as well as a continuous portion of the segment downstream of the 3' splice junction (shown in bold letters in the sequence listing).
• the ougonucleotides of the invention can therefore either be
• RNA (having a U instead of a T base nucleotide as in DNA) or DNA.
• oligonucleotides of the invention can include the entire 32 or 35 nucleotide portion upstream from the 3' splice point which they hybridize with,
• the shorter oligonucleotides can include just the portion upstream of the 3' splice point, or some of the upstream portion and some of the downstream (exon) portion.
• the disadvantage of segments much shorter than 12 nucleotides is that they may not hybridize in a stable fashion.
• the disadvantage of segments much longer than 15 nucleotides is the added cost of synthesizing longer oligonucleotides, and the fact that they may not readily permeate the cellular
• the oligonucleotides of the invention are useful in treating autoimmune diseases, allergies, and in causing humoral immun ⁇ suppression.
• oligonucleotide complementary to the portion of the hinge region sequence upstream of the hinge region 3' splice junction, or a portion upstream and a continuous downstream portion could also be used as an oligonucleotide of the invention.
• oligonucleotides complementary to the appropriate portion of SEQ ID NOS:3-33 as they would suppress production of all immunoglobulins.
• the oligonucleotides admmistered can be DNA or RNA, as long as they are complementary to the target segments, and hybridize with them.
• oligonucleotides of the invention can be used in vitro and in vivo in
• oligonucleotides a large excess of the oligonucleotides to the patient. They could be administered intravenously, or possibly even orally. The dosage could be readily calculated.
• oligonucleotide to attach to the appropriate gene segment in each B cell.
• oligonucleotides of the invention in vitro, i.e. , in production

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