Patent Application
20230323421
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The present invention relates to oligonucleotides which induce expression of an XBP1 splice variant. Such oligonucleotides can enhance the level and/or quality of protein expression in cells and have utility in mammalian protein expression systems, such as heterologous protein expression systems. The oligonucleotides also have therapeutic utilities including the treatment or prevention of proteopathological diseases.
XBP1, X-box binding protein 1, is a transcription factor which mediates adaptation to ER stress by inducing genes that are involved in protein folding and quality control.
The XBP1 transcript exists in different splice forms, including a splice variant whose expression is regulated by IRE1α (inositol requiring-enzyme 1 alpha). In mammalian cells, IRE1α excises a 26 nucleotide fragment from the XBP1 mRNA under endoplasmic reticulum (ER) stress to generate a splice variant that encodes the functionally active XBP1s protein.
The excision of the 26 nucleotide fragment results in a +2 out of frame event, resulting in the expression of the active XBP1 transcription factor (XBP-1S). The 26 nucleotide fragment is present in exon 4 of XBP1 mature mRNA.
Cain et al., (Biotechnol Prog 2013; 29(3):697-706) reports on Chinese hamster ovary (CHO) cells engineered to express both X-box binding protein (XBP-1S) and endoplasmic reticulum oxidoreductase (ERO1-Lα) (CHOS-XE. CHOS-XE cells) which provide increased antibody yields (5.3-6.2 fold) in comparison to CHOS cells.
Tong et al., (Neurochem. 2012 November; 123(3): 406-416), reports on the over-expression of mutant TDP-43 in transgenic rats, which resulted in prominent aggregation of ubiquitin and loss of fragmentation of Golgi complexes, prior to neuronal loss. Notably the aggregation of ubiquitin and loss of fragmentation of Golgi complexes was further preceded by depletion of XBP1 and inactivation of the unfolded protein response (UPR) This indicates that there is a need for restoring or up-regulating the XBP1 mediated UPR in diseases associated with aberrant protein folding (proteopathological diseases), such as neurodegenerative diseases, including TDP-43 pathologies, e.g. frontotemporal lobar degeneration (FTLD) and ALS.
In WO 2003/89622 novel genes, compositions, and methods for modulating the unfolded protein response are disclosed.
In WO 2019/004939 antisense oligonucleotides for modulating the function of a t cell are disclosed.
In WO 2008/016356 the genemap of the human genes associated with psoriasis is disclosed.
The inventors have surprisingly determined that an active XBP1 splice variant has applications in methods of protein production as well as in therapeutic methods, primarily relating to the treatment of proteopathological diseases.
The inventors have surprisingly determined that an active XBP1 spice variant can be produced using an antisense oligonucleotide which is complementary, such as fully complementary, to a portion of the XBP1 pre-mRNA transcript. This XPB1 splice variant may be an XBP1Δ4 splice variant (XBP1 splice variant with deleted exon 4). XBP1 exon 4 comprises the 26 nucleotide fragment which is excised by IRE1α in vivo, and as with the in vivo IRE1α 26 nucleotide excision event, the skipping of exon 4 introduces a +2 out of frame event.
The current invention is based, at least in part, on the finding that the generation or expression of the XBP1Δ4 variant in recombinant mammalian cells results in an enhanced expression of heterologously expressed proteins, such as monoclonal antibodies, particularly heterologously expressed proteins which are otherwise difficult to express. With the expression of the XBP1Δ4 variant, protein expression with enhanced quality in mammalian cells can be obtained.
The current invention is based, at least in part, on the finding that compounds, such as antisense oligonucleotides, which induce the generation or expression of XBP1Δ4 in mammalian cells, are useful in enhancing the recombinant expression of heterologously expressed proteins in mammalian cells. In particular, compounds, such as antisense oligonucleotides, which induce the expression of XBP1Δ4 in mammalian cells, are useful in enhancing the recombinant expression of correctly folded heterologously expressed proteins in mammalian cells.
The current invention is based, at least in part, on the finding that antisense oligonucleotides which induce the expression of XBP1Δ4 in mammalian cells are useful for the treatment of proteopathological diseases.
According to one aspect, the invention provides an antisense oligonucleotide for use in the generation or expression of a XBP1 splice variant in a cell which expresses XBP1, wherein the antisense oligonucleotide is 8-40 nucleotides in length and comprises a contiguous nucleotide sequence of 8-40 nucleotides in length which is complementary to a mammalian XBP1 pre-mRNA transcript.
The XBP1 splice variant may be a XBP1Δ4 variant.
The contiguous nucleotide sequence may be complementary to at least 10 contiguous nucleotides of the hamster XBP1 pre-mRNA transcript (SEQ ID NO 1), such as at least 10 contiguous nucleotides from nucleotides 2960-3113 of SEQ ID NO 1 or at least 10 contiguous nucleotides from nucleotides 2986-3018 of SEQ ID NO 1.
The contiguous nucleotide sequence may be complementary to a sequence selected from the group consisting of SEQ ID NO 299, SEQ ID NO 301, SEQ ID NO 302, SEQ ID NO 304, SEQ ID NO 305, SEQ ID NO 306, SEQ ID NO 307, SEQ ID NO 308, SEQ ID NO 309, SEQ ID NO 310, SEQ ID NO 314, SEQ ID NO 316, SEQ ID NO 317, SEQ ID NO 318, SEQ ID NO 319, SEQ ID NO 323, SEQ ID NO 325, SEQ ID NO 327, SEQ ID NO 328, SEQ ID NO 330, SEQ ID NO 331, SEQ ID NO 332, SEQ ID NO 333, SEQ ID NO 334, SEQ ID NO 336, SEQ ID NO 337, SEQ ID NO 385, SEQ ID NO 386, SEQ ID NO 387, SEQ ID NO 388, SEQ ID NO 390, SEQ ID NO 391, SEQ ID NO 392, SEQ ID NO 393, SEQ ID NO 394, SEQ ID NO 395, SEQ ID NO 396 397, SEQ ID NO 398, SEQ ID NO 399, SEQ ID NO 401, SEQ ID NO 402, SEQ ID NO 419, SEQ ID NO 431, SEQ ID NO, SEQ ID NO 432, SEQ ID NO 433, SEQ ID NO 434, SEQ ID NO 438, SEQ ID NO 439, SEQ ID NO 440, SEQ ID NO 441, SEQ ID NO 442, SEQ ID NO 449, SEQ ID NO 484, SEQ ID NO 485, SEQ ID NO 486, SEQ ID NO 487, SEQ ID NO 488, SEQ ID NO 489, SEQ ID NO 490, SEQ ID NO 491, SEQ ID NO 492, SEQ ID NO 493, SEQ ID NO 494, SEQ ID NO 495, SEQ ID NO 496, SEQ ID NO 497, SEQ ID NO 498, SEQ ID NO 499, SEQ ID NO 500, SEQ ID NO 501, SEQ ID NO 502, SEQ ID NO 503, SEQ ID NO 505, SEQ ID NO 506, SEQ ID NO 507, SEQ ID NO 508, SEQ ID NO 509, SEQ ID NO 510, SEQ ID NO 511, SEQ ID NO 512, SEQ ID NO 513, SEQ ID NO 515, SEQ ID NO 517, SEQ ID NO 520, SEQ ID NO 572, SEQ ID NO 573, SEQ ID NO 576, SEQ ID NO 577, SEQ ID NO 588 and SEQ ID NO 589.
The contiguous nucleotide sequence may be selected from the group consisting of SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 128, SEQ ID NO 140, SEQ ID NO 141, SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151, SEQ ID NO 158, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 198, SEQ ID NO 199, SEQ ID NO 200, SEQ ID NO 201, SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 204, SEQ ID NO 205, SEQ ID NO 206, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211, SEQ ID NO 212, SEQ ID NO 214, SEQ ID NO 215, SEQ ID NO 216, SEQ ID NO 217, SEQ ID NO 218, SEQ ID NO 219, SEQ ID NO 220. SEQ ID NO 221, SEQ ID NO 222, SEQ ID NO 224, SEQ ID NO 226, SEQ ID NO 229, SEQ ID NO 281, SEQ ID NO 282, SEQ ID NO 285, SEQ ID NO 286, SEQ ID NO 297 and SEQ ID NO 298.
The contiguous nucleotide sequence may be complementary to at least 10 contiguous nucleotides of the mouse XBP1 pre-mRNA transcript (SEQ ID NO 590).
The contiguous nucleotide sequence may be complementary to a sequence selected from the group consisting of SEQ ID NO 699, SEQ ID NO 700, SEQ ID NO 703, SEQ ID NO 710, SEQ ID NO 713, SEQ ID NO 724, SEQ ID NO 729, SEQ ID NO 739, SEQ ID NO 743, SEQ ID NO 744, SEQ ID NO 745, SEQ ID NO 749, SEQ ID NO 750, SEQ ID NO 751, SEQ ID NO 752, SEQ ID NO 753, SEQ ID NO 754, SEQ ID NO 755, SEQ ID NO 756, SEQ ID NO 757, SEQ ID NO 758, SEQ ID NO 759, SEQ ID NO 760, SEQ ID NO 761, SEQ ID NO 762, SEQ ID NO 763, SEQ ID NO 773, SEQ ID NO 776, SEQ ID NO 778, SEQ ID NO 781, SEQ ID NO 783, SEQ ID NO 784, SEQ ID NO 785, SEQ ID NO 787, SEQ ID NO 789, SEQ ID NO 790, SEQ ID NO 791, SEQ ID NO 792, SEQ ID NO 793, SEQ ID NO 794, SEQ ID NO 795, SEQ ID NO 796, SEQ ID NO 797, SEQ ID NO 798, SEQ ID NO 799 and SEQ ID NO 800.
The contiguous nucleotide sequence may be selected from the group consisting of SEQ ID NO 597, SEQ ID NO 598, SEQ ID NO 601, SEQ ID NO 608, SEQ ID NO 611, SEQ ID NO 622, SEQ ID NO 627, SEQ ID NO 637, SEQ ID NO 641, SEQ ID NO 642, SEQ ID NO 643, SEQ ID NO 647, SEQ ID NO 648, SEQ ID NO 649, SEQ ID NO 650, SEQ ID NO 651, SEQ ID NO 652, SEQ ID NO 653, SEQ ID NO 654, SEQ ID NO 655, SEQ ID NO 656, SEQ ID NO 657, SEQ ID NO 658, SEQ ID NO 659, SEQ ID NO 660, SEQ ID NO 661, SEQ ID NO 671, SEQ ID NO 674, SEQ ID NO 676, SEQ ID NO 679, SEQ ID NO 681, SEQ ID NO 682, SEQ ID NO 683, SEQ ID NO 685, SEQ ID NO 687, SEQ ID NO 688, SEQ ID NO 689, SEQ ID NO 690, SEQ ID NO 691, SEQ ID NO 692, SEQ ID NO 693, SEQ ID NO 694, SEQ ID NO 695, SEQ ID NO 696, SEQ ID NO 697 and SEQ ID NO 697.
The contiguous nucleotide sequence may be complementary to at least 10 contiguous nucleotides of the human XBP1 pre-mRNA transcript (SEQ ID NO 801).
The contiguous nucleotide sequence may be complementary to a sequence selected from the group consisting of SEQ ID NO 947, SEQ ID NO 948, SEQ ID NO 949, SEQ ID NO 950, SEQ ID NO 951 and SEQ ID NO 988.
The contiguous nucleotide sequence may be selected from the group consisting of SEQ ID NO 854, SEQ ID NO 855, SEQ ID NO 856, SEQ ID NO 857, SEQ ID NO 858 and SEQ ID NO 895.
The antisense oligonucleotide or contiguous nucleotide sequence thereof may be fully complementary to a mammalian XBP1 pre-mRNA transcript.
The contiguous nucleotide sequence may be the same length as the antisense oligonucleotide.
The antisense oligonucleotide may be isolated, purified or manufactured.
The antisense oligonucleotide or contiguous nucleotide sequence thereof may comprise one or more modified nucleotides or one or more modified nucleosides.
The antisense oligonucleotide or contiguous nucleotide sequence thereof may be or comprises an antisense oligonucleotide mixmer or totalmer.
The invention includes conjugates and pharmaceutically acceptable salts of the antisense oligonucleotides of the invention as well as compositions and pharmaceutical compositions comprising the antisense oligonucleotides of the invention.
In another aspect, the invention provides an isolated XBP1Δ4 protein.
The isolated XBP1Δ4 protein of the invention may comprise the sequence of SEQ ID NO: 7, SEQ ID NO: 596 or SEQ ID NO 807.
In another aspect, the invention provides an isolated mRNA encoding the XBP1Δ4 protein of the invention.
The isolated mRNA of the invention may comprise the sequence of SEQ ID NO: 7, SEQ ID NO: 595 or SEQ ID NO: 806.
In another aspect, the invention provides a method for producing a polypeptide comprising the steps of:
Within the invention, the method may comprise the steps of:
Within the method of the invention, the antisense oligonucleotide may be added to a final concentration of 25 μM or more.
Within the method of the invention the cells resulting in the first cell population may be cultivated at a starting cell density of 0.5*10E6 to 4*10E6 cells/mL.
Within the method of the invention, the second cell population may have a cell density of 0.5*10E6 to 10*10E6 cells/mL.
Within the method of the invention, the mammalian cell may be a CHO cell.
Within the method of the invention, the polypeptide may be an antibody.
One aspect of the invention is a method for the recombinant production of a multimeric polypeptide comprising the steps of:
One further aspect of the invention is a method for the recombinant production of a multimeric polypeptide comprising the steps of:
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the method comprises the steps of:
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the method comprises the steps of:
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the nucleic acid according to the invention is an antisense oligonucleotide.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the nucleic acid according to the invention is complementary to at least 10 contiguous nucleotides of the hamster XBP1 pre-mRNA transcript (SEQ ID NO 1), such as at least 10 contiguous nucleotides from nucleotides 2960-3113 of SEQ ID NO 1 or at least 10 contiguous nucleotides from nucleotides 2986-3018 of SEQ ID NO 1.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the nucleic acid according to the invention is complementary to at least 10 contiguous nucleotides of the human XBP1 pre-mRNA transcript (SEQ ID NO 801).
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the nucleic acid according to the invention is complementary to a sequence selected from the group consisting of SEQ ID NO 23 or SEQ ID NO 24.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the nucleic acid according to the invention is complementary to a sequence selected from the group consisting of SEQ ID NO 947, SEQ ID NO 948, SEQ ID NO 949, SEQ ID NO 950, SEQ ID NO 951 and SEQ ID NO 988.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the nucleic acid according to the invention is selected from the group consisting of SEQ ID NO 854, SEQ ID NO 855, SEQ ID NO 856, SEQ ID NO 857, SEQ ID NO 858 and SEQ ID NO 895.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the XBP1 variant comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 596 or SEQ ID NO 807.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the XBP1 variant is encoded by the sequence of SEQ ID NO: 7, SEQ ID NO: 595 or SEQ ID NO: 806.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the nucleic acid according to the invention is be added to a final concentration of 25 μM or more.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the cells resulting in the first cell population are cultivated with a starting cell density of 0.5*10E6 to 4*10E6 cells/mL.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the second cell population has a starting cell density of 0.5*10E6 to 10*10E6 cells/mL.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the mammalian cell is a CHO cell.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the mammalian cell is a HEK cell.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the mammalian cell is a SP2/0 cell.
In certain embodiments of all aspects and embodiments of the method for the recombinant production of a multimeric polypeptide, the multimeric polypeptide is an antibody. In certain embodiments, the antibody is a bispecific antibody. In certain embodiments, the bispecific antibody is a full-length antibody with domain exchange or an antibody-multimer-fusion. In certain embodiments, the bispecific antibody is a trivalent, bispecific antibody. In certain embodiments, the bispecific, trivalent antibody is a full-length antibody with domain exchange and additional heavy chain C-terminal binding site or a full-length antibody with an additional heavy chain C-terminal binding site with domain exchange or a T-cell bispecific antibody. In certain embodiments, the antibody is bi- or trivalent.
One aspect of the invention is the use of the nucleic acid according to the invention to enhance the yield or the quality of multimeric polypeptides produced by recombinant protein expression systems, for example in the manufacture of antibodies, such as monoclonal antibodies.
In certain embodiments of all aspects and embodiments of the use of the nucleic acid according to the invention, the nucleic acid according to the invention is an antisense oligonucleotide.
In certain embodiments of all aspects and embodiments of the use of the nucleic acid according to the invention, the nucleic acid according to the invention is complementary to at least 10 contiguous nucleotides of the hamster XBP1 pre-mRNA transcript (SEQ ID NO 1), such as at least 10 contiguous nucleotides from nucleotides 2960-3113 of SEQ ID NO 1 or at least 10 contiguous nucleotides from nucleotides 2986-3018 of SEQ ID NO 1.
In certain embodiments of all aspects and embodiments of the use of the nucleic acid according to the invention, the nucleic acid according to the invention is complementary to at least 10 contiguous nucleotides of the human XBP1 pre-mRNA transcript (SEQ ID NO 801).
In certain embodiments of all aspects and embodiments of the use of the nucleic acid according to the invention, the nucleic acid according to the invention is complementary to a sequence selected from the group consisting of SEQ ID NO 947, SEQ ID NO 948, SEQ ID NO 949, SEQ ID NO 950, SEQ ID NO 951 and SEQ ID NO 988.
In certain embodiments of all aspects and embodiments of the use of the nucleic acid according to the invention, the nucleic acid according to the invention is selected from the group consisting of SEQ ID NO 854, SEQ ID NO 855, SEQ ID NO 856, SEQ ID NO 857, SEQ ID NO 858 and SEQ ID NO 895.
One further aspect of the invention is the use of an)(BPI variant obtained from an XBP1 mRNA wherein exon 4 is skipped and +2 out of frame event is introduced to enhance the yield or the quality of multimeric polypeptides produced by recombinant protein expression systems, for example in the manufacture of antibodies, such as monoclonal antibodies.
One further aspect of the invention is the use of an XBP1 variant comprising the sequence of SEQ ID NO: 7, SEQ ID NO: 596 or SEQ ID NO 807 to enhance the yield or the quality of multimeric polypeptides produced by recombinant protein expression systems, for example in the manufacture of antibodies, such as monoclonal antibodies.
In certain embodiments of all aspects and embodiments of the before outlined uses, the nucleic acid according to the invention is used at a final concentration of 25 μM or more.
In another aspect, the invention provides a therapeutic application for the antisense oligonucleotides, compositions, pharmaceutical compositions, proteins and/or isolated mRNAs of the invention.
In one aspect, the invention provides an antisense oligonucleotide, composition, pharmaceutical composition, protein and/or isolated mRNA of the invention for use in medicine or therapy.
In another aspect, the invention provides the use of an antisense oligonucleotide, composition, pharmaceutical composition, protein and/or isolated mRNA of the invention in the manufacture of a medicament for the treatment of proteopathological disease.
In another aspect, the invention provides a method of treating a proteopathological disease, the method comprising administering an antisense oligonucleotide, composition, pharmaceutical composition, protein and/or isolated mRNA of the invention.
Throughout the therapeutic applications of the invention, the proteopathological disease may be a TDP-43 pathology, such as motor neuron disease or frontotemporal lobar degeneration.
Useful methods and techniques for carrying out the current invention are described in e.g. Ausubel, F. M. (ed.), Current Protocols in Molecular Biology, Volumes I to Ill (1997); Glover, N. D., and Hames, B. D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R. I. (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, J. D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E. L., From Genes to Clones; N.Y., VCH Publishers (1987); Cells, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R. I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987).
The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B. D., and Higgins, S. G., Nucleic acid hybridization—a practical approach (1985) IRL Press, Oxford, England).
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.
The term “about” denotes a range of +1-20% of the thereafter following numerical value. In one embodiment, the term about denotes a range of +1-10% of the thereafter following numerical value. In one embodiment the term “about” denotes a range of +/−5% of the thereafter following numerical value.
The term “comprising” also encompasses the term “consisting of”.
Herein, in the context of compounds of the present invention the term “compound” means any molecule capable of modulating the expression or activity of XBP1, particularly any molecule capable of modulating the splicing of the XBP1 pre-mRNA to increase the level of expression of XBP1 an XBP1 splice variant, such as an mRNA which lacks XBP1 exon 4. Particular compounds of the invention are nucleic acid molecules, such as antisense oligonucleotides, and conjugates comprising such a nucleic acid molecule.
The term “recombinant mammalian cell” as used herein denotes a mammalian cell comprising an exogenous nucleotide sequence capable of expressing a polypeptide. Such a polypeptide can be a polypeptide endogenous or heterologous (exogeneous) to said mammalian cell. Such recombinant mammalian cells are cells into which one or more exogenous nucleic acid(s) have been introduced, including the progeny of such cells. Thus, the term “a mammalian cell comprising a nucleic acid encoding a heterologous polypeptide” denotes cells comprising an exogenous nucleotide sequence integrated in the genome of the mammalian cell and capable of expressing the heterologous polypeptide. In one embodiment the mammalian cell comprising an exogenous nucleotide sequence is a cell comprising an exogenous nucleotide sequence integrated at a single site within a locus of the genome of the host cell, wherein the exogenous nucleotide sequence comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, and all the recombination recognition sequences are different.
Such “recombinant mammalian cells” can be used for the production of said homologous or heterologous polypeptide of interest at any scale.
A mammalian cell comprising an exogenous nucleotide sequence is a “transformed cell”. This term includes the primary transformed cell as well as progeny derived therefrom without regard to the number of passages. Progeny may, e.g., not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are encompassed.
An “isolated” composition is one that has been separated from a component of its natural environment. In some embodiments, a composition is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatographic (e.g., size exclusion chromatography or ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of e.g. antibody purity, see, e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.
An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but wherein the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
An “isolated” polypeptide or antibody refers to a polypeptide molecule or antibody molecule that has been separated from a component of its natural environment.
The term “integration site” denotes a nucleic acid sequence within a cell's genome into which an exogenous nucleotide sequence is inserted. In certain embodiments, an integration site is between two adjacent nucleotides in the cell's genome. In certain embodiments, an integration site includes a stretch of nucleotide sequences. In certain embodiments, the integration site is located within a specific locus of the genome of a mammalian cell. In certain embodiments, the integration site is within an endogenous gene of a mammalian cell. The terms “vector” or “plasmid”, which can be used interchangeably, as used herein, refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
As used herein, the term “selection marker” denotes a gene that allows cells carrying the gene to be specifically selected for or against, in the presence of a corresponding selection agent. For example, but not by way of limitation, a selection marker can allow the host cell transformed with the selection marker gene to be positively selected for in the presence of the respective selection agent (selective cultivation conditions); a non-transformed host cell would not be capable of growing or surviving under the selective cultivation conditions. Selection markers can be positive, negative or bi-functional. Positive selection markers can allow selection for cells carrying the marker, whereas negative selection markers can allow cells carrying the marker to be selectively eliminated. A selection marker can confer resistance to a drug or compensate for a metabolic or catabolic defect in the host cell. In prokaryotic cells, amongst others, genes conferring resistance against ampicillin, tetracycline, kanamycin or chloramphenicol can be used. Resistance genes useful as selection markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid. Further marker genes are described in WO 92/08796 and WO 94/28143.
Beyond facilitating a selection in the presence of a corresponding selection agent, a selection marker can alternatively be a molecule normally not present in the cell, e.g., green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. Cells expressing such a molecule can be distinguished from cells not harbouring this gene, e.g., by the detection or absence, respectively, of the fluorescence emitted by the encoded polypeptide.
As used herein, the term “operably linked” refers to a juxtaposition of two or more components, wherein the components are in a relationship permitting them to function in their intended manner. For example, a promoter and/or an enhancer is operably linked to a coding sequence if the promoter and/or enhancer acts to modulate the transcription of the coding sequence. In certain embodiments, DNA sequences that are “operably linked” are contiguous and adjacent on a single chromosome. In certain embodiments, e.g., when it is necessary to join two protein encoding regions, such as a secretory leader and a polypeptide, the sequences are contiguous, adjacent, and in the same reading frame. In certain embodiments, an operably linked promoter is located upstream of the coding sequence and can be adjacent to it. In certain embodiments, e.g., with respect to enhancer sequences modulating the expression of a coding sequence, the two components can be operably linked although not adjacent. An enhancer is operably linked to a coding sequence if the enhancer increases transcription of the coding sequence. Operably linked enhancers can be located upstream, within, or downstream of coding sequences and can be located at a considerable distance from the promoter of the coding sequence. Operable linkage can be accomplished by recombinant methods known in the art, e.g., using PCR methodology and/or by ligation at convenient restriction sites. If convenient restriction sites do not exist, then synthetic oligonucleotide adaptors or linkers can be used in accord with conventional practice. An internal ribosomal entry site (IRES) is operably linked to an open reading frame (ORF) if it allows initiation of translation of the ORF at an internal location in a 5′-end-independent manner.
As used herein, the term “exogenous” indicates that a nucleotide sequence does not originate from a specific cell and is introduced into said cell by DNA delivery methods, e.g., by transfection, electroporation, or transformation methods. Thus, an exogenous nucleotide sequence is an artificial sequence wherein the artificiality can originate, e.g., from the combination of subsequences of different origin (e.g. a combination of a recombinase recognition sequence with an SV40 promoter and a coding sequence of green fluorescent protein is an artificial nucleic acid) or from the deletion of parts of a sequence (e.g. a sequence coding only the extracellular domain of a membrane-bound receptor or a cDNA) or the mutation of nucleobases. The term “endogenous” refers to a nucleotide sequence originating from a cell. An “exogenous” nucleotide sequence can partly have an “endogenous” counterpart that is identical in base compositions, but where the “exogenous” sequence is introduced into the cell, e.g., via recombinant DNA technology.
As used herein, the term “heterologous” indicates that a polypeptide does not originate from a specific cell and the respective encoding nucleic acid has been introduced into said cell by DNA delivery methods, e.g., by transfection, electroporation, or transformation methods. Thus, a heterologous polypeptide is a polypeptide that is artificial to the cell expressing it, whereby this is independent of whether the polypeptide is a naturally occurring polypeptide originating from a different cell/organism or is a man-made polypeptide.
The term “oligonucleotide” as used herein is defined as it is generally understood by the skilled person, as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides can also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. In some embodiments, the oligonucleotides of the invention are man-made, and are chemically synthesized, and are typically purified or isolated. The oligonucleotides of the invention can comprise one or more modified nucleosides, also referred to as nucleoside analogues, such as 2′ sugar modified nucleosides. The oligonucleotides of the invention can comprise one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.
The term “antisense oligonucleotide” or “ASO,” as used herein, is defined as an oligonucleotide capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. Antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs. In some embodiments, the antisense oligonucleotides of the present invention can be single stranded. It is understood that single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity is less than approximately 50% across the full length of the oligonucleotide.
In some embodiments, the single stranded antisense oligonucleotides of the invention do not contain RNA nucleosides. As described elsewhere in the present disclosure, in some embodiments, antisense oligonucleotides of the disclosure comprise one or more modified nucleosides or nucleotides, such as 2′ sugar modified nucleosides. In certain embodiments, the non-modified nucleosides of an antisense oligonucleotide disclosed herein are DNA nucleosides.
In certain contexts, the antisense oligonucleotides of the invention may be referred to as oligonucleotides.
The term “contiguous nucleotide sequence” refers to the region of an antisense oligonucleotide which is complementary to the target nucleic acid. The term is used interchangeably herein with the term “contiguous nucleobase sequence” and the term oligonucleotide “sequence motif.” As used herein, the term “sequence motif” represents the sequence of nucleobases, independent of the nucleoside sugar chemistry and/or design. In some embodiments, the nucleobases A, T, C and G can be modified, for example, capital C can be 5-methyl cytosine beta-D-oxy LNA nucleoside, and in RNA sequences, T can be U. In some embodiments, ail the nucleosides of an antisense oligonucleotide constitute the contiguous nucleotide sequence. The contiguous nucleotide sequence is the sequence of nucleotides in the antisense oligonucleotide which is complementary to, and in some instances fully complementary to, the target nucleic acid or target sequence.
As described herein, in some embodiments, an antisense oligonucleotide comprises the contiguous nucleotide sequence, and can optionally comprise further nucleotide(s), for example a nucleotide linker region which can be used to attach a functional group (e.g. a conjugate group) to the contiguous nucleotide sequence. In some embodiments, the nucleotide linker region can be complementary to the target nucleic acid. In some embodiments, the nucleotide linker region is not complementary to the target nucleic acid. It is understood that the contiguous nucleotide sequence of an antisense oligonucleotide cannot be longer than the antisense oligonucleotide as such, and that the antisense oligonucleotide cannot be shorter than the contiguous nucleotide sequence.
The term “nucleic acids” or “nucleotides” is intended to encompass plural nucleic acids. In some embodiments, the term “nucleic acids” or “nucleotides” refers to a target sequence, e.g., pre-mRNAs, mRNAs, or DNAs in vivo or in vitro.
When the term refers to the nucleic acids or nucleotides in a target sequence, the nucleic acids or nucleotides can be naturally occurring sequences within a cell. In some embodiments, “nucleic acids” or “nucleotides” refer to a sequence in the antisense oligonucleotide of the invention. When the term refers to a sequence in the antisense oligonucleotide, the nucleic acids or nucleotides are not naturally occurring, i.e., chemically synthesized, enzymatically produced, recombinantly produced, or any combination thereof. In some embodiments, the nucleic acids or nucleotides in the antisense oligonucleotide are produced synthetically or recombinantly, but are not a naturally occurring sequence or a fragment thereof. In some embodiments, the nucleic acids or nucleotides in the antisense oligonucleotide are not naturally occurring because they contain at least one nucleotide analog that is not naturally occurring in nature.
The term “nucleic acid” or “nucleotide” refers to a single nucleic acid segment, e.g., a DNA, an RNA, or an analog thereof, in isolated form or present in a polynucleotide. “Nucleic acid” or “nucleotide” includes naturally occurring nucleic acids or non-naturally occurring nucleic acids. In some embodiments, the terms “nucleotide”, “unit” and “monomer” are used interchangeably. It will be recognized that when referring to a sequence of nucleotides or monomers, what is referred to is the sequence of bases, such as A, T, G, C or U, and analogs thereof.
When the term refers to the nucleic acid or nucleic acids encoding a polypeptide or protein, the nucleic acids or nucleotides can be naturally occurring sequences within a cell or an artificial sequence. In some embodiments, the nucleic acid(s) are produced synthetically or recombinantly.
The term “nucleotide,” as used herein, refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate internucleotide linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as “nucleotide analogs” herein. Herein, a single nucleotide (unit) can also be referred to as a monomer or nucleic acid unit. In certain embodiments, the term “nucleotide analogs” refers to nucleotides having modified sugar moieties. Non-limiting examples of the nucleotides having modified sugar moieties (e.g., LNA) are disclosed elsewhere herein. In some embodiments, the term “nucleotide analogs” refers to nucleotides having modified nucleobase moieties. The nucleotides having modified nucleobase moieties include, but are not limited to, 5-methyl-cytosine, isocytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, pseudoisocytosine, 5-bromouracil, 5-propynyl-uracil, thiazolo-uracil, 2-thio-uracil, 2-thiothymine, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine. As one of ordinary skill in the art would recognize, the 5′ terminal nucleotide of an oligonucleotide (e.g., an antisense oligonucleotide disclosed herein) does not comprise a 5′ internucleotide linkage group, although it can comprise a 5′ terminal group.
The term “nucleoside,” as used herein, is used to refer to a glycoside comprising a sugar moiety and a base moiety, and can therefore be used when referring to the nucleotide units, which are covalently linked by the internucleotide linkages between the nucleotides of the antisense oligonucleotide. In the field of biotechnology, the term “nucleotide” is often used to refer to a nucleic acid monomer or unit. In the context of an antisense oligonucleotide, the term “nucleotide” can refer to the base alone, i.e., a nucleobase sequence comprising cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), in which the presence of the sugar backbone and internucleotide linkages are implicit. Likewise, particularly in the case of oligonucleotides where one or more of the internucleotide linkage groups are modified, the term “nucleotide” can refer to a “nucleoside.” For example, the term “nucleotide” can be used, even when specifying the presence or nature of the linkages between the nucleosides.
The term “nucleotide length” or the “length” of an antisense oligonucleotide, or contiguous nucleotide sequence thereof, as used herein means the total number of the nucleotides (monomers) in a given sequence. Nucleotides and nucleosides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present disclosure include both naturally occurring and non-naturally occurring nucleotides and nucleosides (nucleo(s/t)ide analogs). In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides can also interchangeably be referred to as “units” or “monomers”.
The term “modified nucleoside” or “nucleoside modification”, or “nucleoside analog” as used herein, refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. In some embodiments, one or more of the modified nucleosides of the antisense oligonucleotide of the invention comprise a modified sugar moiety. The term modified nucleoside can also be used herein interchangeably with the term “nucleoside analogue,” modified “units,” or modified “monomers.” Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. In some embodiments, nucleosides with modifications in the base region of the DNA or RNA nucleoside are still termed DNA or RNA if they allow Watson Crick base pairing. Non-limiting examples of modified nucleosides which can be used in the antisense oligonucleotides of the invention include LNA, 2′-O-MOE and morpholino nucleoside analogues. Examples of other modified nucleosides are provided elsewhere in the present disclosure.
A “high affinity modified nucleoside,” as used herein, is a modified nucleotide which, when incorporated into the oligonucleotide, enhances the affinity of the oligonucleotide for its complementary target, for example, as measured by the melting temperature (Tm). A high affinity modified nucleoside of the present disclosure can result in an increase in melting temperature between +0.5 to +12° C., in some instances between +1.5 to +10° C. and in others between +3 to +8° C. per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include, for example, many 2′ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213).
The term “modified internucleoside linkage” is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages that covalently couple two nucleosides together. In some embodiments, the oligonucleotides of the invention can therefore comprise one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkage.
In some embodiments, at least about 50% of the internucleoside linkages of the antisense oligonucleotide (e.g., disclosed herein), or contiguous nucleotide sequence thereof, are phosphorothioate, such as at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90% or more of the internucleoside linkages of the antisense oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments, all of the internucleoside linkages of the antisense oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.
In some embodiments, ail the internucleoside linkages of the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate, or all the internucleoside linkages of the antisense oligonucleotide are phosphorothioate linkages.
The term “nucleobase” includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases which can differ from naturally occurring nucleobases, but which are functional during nucleic acid hybridization. In this context, “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are, for example, described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.
In some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2′thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.
The nucleobase moieties can be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter can optionally include modified nucleobases of equivalent function. For example, in certain embodiments, the nucleobase moieties of the antisense oligonucleotides disclosed herein are selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNA gapmers, 5-methyl cytosine LNA nucleosides can be used.
The term “modified oligonucleotide,” as used herein, describes an oligonucleotide (e.g., an antisense oligonucleotide) comprising one or more modified nucleosides (e.g., sugar modified nucleosides) and/or modified internucleoside linkages. The term “chimeric oligonucleotide” is a term that has been used in the literature to describe oligonucleotides comprising modified nucleosides (e.g., sugar modified nucleosides) and DNA nucleosides. In some embodiments, the ASO of the disclosure is a chimeric oligonucleotide.
As used herein, the term “alkyl”, alone or in combination, signifies a straight-chain or branched-chain alkyl group with 1 to 8 carbon atoms (C1-8), particularly a straight or branched-chain alkyl group with 1 to 6 carbon atoms (C1-6) and more particularly a straight or branched-chain alkyl group with 1 to 4 carbon atoms (C1-4). Examples of straight-chain and branched-chain C1-C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyls, the isomeric hexyls, the isomeric heptyls and the isomeric octyls, particularly methyl, ethyl, propyl, butyl and pentyl. Particular examples of alkyl are methyl. Further examples of alkyl are mono, di or trifluoro methyl, ethyl or propyl, such as cyclopropyl (cPr), or mono, di or tri fluoro cyclopropyl.
The term “alkoxy”, alone or in combination, signifies a group of the formula alkyl-O— in which the term “alkyl” has the previously given significance, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.butoxy and tert.butoxy. Particular “alkoxy” are methoxy.
As used herein, the term “bicyclic sugar” refers to a modified sugar moiety comprising a 4 to 7 membered ring comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In some embodiments, the bridge connects the C2′ and C4′ of the ribose sugar ring of a nucleoside (i.e., 2′-4′ bridge), as observed in SNA nucleosides.
As used herein, the term “exons” or “exonic regions” or “exonic sequences”, which can be used interchangeably herein, refer to nucleic acid molecules containing a sequence of nucleotides that is transcribed into RNA and is represented in a mature form of RNA, such as mRNA (messenger RNA), after splicing and other RNA processing. An mRNA contains one or more exons operatively linked. In some embodiments, exons can encode polypeptides or a portion of a polypeptide. In some embodiments, exons can contain non-translated sequences, for example, translational regulatory sequences.
The term “introns” or “intronic regions” or “intronic sequences”, which can be used interchangeably, refer to nucleic acid molecules containing a sequence of nucleotides that is transcribed into RNA and is then typically removed from the RNA by splicing to create a mature form of an RNA, for example, an mRNA. In some embodiments, nucleotide sequences of introns are not incorporated into mature RNAs, nor are intron sequences or portions thereof translated and incorporated into a polypeptide. Splice signal sequences, such as splice donors and acceptors, are used by the splicing machinery of a cell to remove introns from RNA. In some embodiments, an intron in one splice variant can be an exon (i.e., present in the spliced transcript) in another variant. Hence, spliced mRNA encoding an intron fusion protein can include an exon(s) and introns.
As used herein, the term “splicing” refers to a process of RNA maturation in which introns in the pre-mRNA are removed and exons are operatively linked to create a messenger RNA (mRNA).
As used herein, the term “alternative splicing” refers to the process of producing multiple mRNAs from a gene. In some embodiments, alternate splicing can include operatively linking less than all the exons of a gene, and/or operatively linking one or more alternate exons that are not present in all transcripts derived from a gene.
The term “splice modulation,” as used herein, refers to a process that can be used to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown. In the context of the present invention, a splice modulation can be used to modulate alternative splicing of XBP1 pre-mRNA to generate a splice variant. For example, a splice modulation can be used to modulate alternative splicing of XBP1 pre-mRNA to generate XBP1Δ4 mRNA and thereby enhance expression of XBP1Δ4 protein. Splice modulation can be assayed by RNA sequencing (RNA-Seg), which allows for a quantitative assessment of the different splice products of a pre-mRNA. In some embodiments of the invention, the antisense oligonucleotides modulate the splicing of the XBP1 pre-mRNA so as to reduce the level of mature XBP1 mRNA which comprises an exon 4 (mRNA), and to increase the expression of the level of mature XBP1 mRNA which lacks exon 4 (XBP1Δ4 mRNA).
As used herein, a “coding region” or “coding sequence”, which can be used interchangeably, is a portion of polynucleotide which consists of codons translatable into amino acids, Although a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, untranslated regions (“UTRs”), and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5′ terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3′ terminus, encoding the carboxyl terminus of the resulting polypeptide.
The term “non-coding region” as used herein means a nucleotide sequence that is not a coding region. Examples of non-coding regions include, but are not limited to, promoters, ribosome binding sites, transcriptional terminators, introns, untranslated regions (“UTRs”), non-coding exons and the like. Some of the exons can be wholly or part of the 5′ untranslated region (5′ UTR) or the 3° untranslated region (3′ UTR) of each transcript. The untranslated regions are important for efficient translation of the transcript and for controlling the rate of translation and half-life of the transcript.
The term “region” when used in the context of a nucleotide sequence refers to a section of that sequence. For example, the phrase “region within a nucleotide sequence” or “region within the complement of a nucleotide sequence” refers to a sequence shorter than the nucleotide sequence, but longer than at least 10 nucleotides located within the particular nucleotide sequence or the complement of the nucleotides sequence, respectively. The term “sub-sequence” or “subsequence” can also refer to a region of a nucleotide sequence.
The term “downstream,” when referring to a nucleotide sequence, means that a nucleic acid or a nucleotide sequence is located 3′ to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
The term “upstream” refers to a nucleotide sequence that is located 5′ to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that precede the starting point of transcription. For example, the promoter sequence of a gene is located upstream of the start site of transcription.
As used herein, the term “regulatory region” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, UTRs, and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
The term “target sequence,” as used herein, refers to a sequence of nucleotides present in the target nucleic acid which comprises the nucleobase sequence which is complementary to the antisense oligonucleotides of the invention, i.e. in the context of the present invention, a mammalian XBP1 pre-mRNA sequence is a target nucleic acid, and the target sequence is a region of the target nucleic acid which can be effectively targeted to modulate the splicing of exon 4, and includes, for example XBP1 exon 4, and the regions adjacent 5′ and/or 3′ to exon 4, of a XBP1 pre-mRNA transcript.
For example, for the present invention the target nucleic acid may be the hamster XBP1 pre-mRNA (SEQ ID NO 1, and particularly nucleotides 2960-3113 of SEQ ID NO 1), the mouse XBP1 pre-mRNA (SEQ ID NO 590) or the human XBP1 pre-mRNA (SEQ ID NO 801).
In some embodiments, the target sequence consists of a region on the target nucleic acid with a nucleobase sequence that is complementary to the contiguous nucleotide sequence of the antisense oligonucleotide of the invention. This region of the target nucleic acid can interchangeably be referred to as the target nucleotide sequence, target sequence, or target region. In some embodiments, the target sequence is longer than the complementary sequence of a single antisense oligonucleotide, and can, for example, represent a preferred region of the target nucleic acid, which can be targeted by several oligonucleotides of the invention.
As used herein, the term “target cell” refers to a cell which expresses the target nucleic acid. In some embodiments, the target cell comprises a mammalian cell, such as a rodent cell, such as a mouse cell or a rat cell, or a hamster cell, such as a CHO cell, or a primate cell such as a monkey cell or a human cell. In some embodiments, the target cell is a transgenic mammalian cell which is expressing a XBP1 target nucleic acid. In some embodiments, the cell is a transgenic animal cell which is expressing a XBP1Δ4 mRNA, for example via heterologous expression.
Due to its general use in heterologous protein expression a preferred cell for use in protein expression methods is a hamster cell, such as a Chinese hamster ovary cell (CHO cell), especially preferred is a CHO-K1 cell growing in suspension.
Due to the therapeutic applications of the antisense oligonucleotides of the invention in neurodegenerative disorders, the target cell may be a neuronal cell.
Typically, the target cell of the present invention expresses the XBP1 pre-mRNA, which is processed in the cell to the mature XBP1 mRNA, resulting in the expression of the both XBP1-E4 protein (also referred to as XBPu) and the XBP1Δ4 transcript variant. As described herein, in some embodiments, the compounds of the invention modulate the splicing of the XBP1 pre-mRNA to increase the proportion of XBP1 mRNA which lacks XBP1 exon 4. Suitably, thereby the expression of XBP1Δ4 transcript variant can be increased, as compared to XBP1-E4 transcript variant.
The term “complementarity” or “nucleobase complementarity”, which can be used interchangeably herein, describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U).
It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarily encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).
The term “% complementary” as used herein, refers to the proportion of nucleotides (in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which across the contiguous nucleotide sequence, are complementary to a reference sequence (e.g. a target sequence or sequence motif). The percentage of complementarity is thus calculated by counting the number of aligned nucleobases that are complementary (from Watson Crick base pairs) between the two sequences (when aligned with the target sequence 5′-3′ and the oligonucleotide sequence from 3′-5′), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch. Insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5′-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).
Within the present invention the term “complementary” requires the antisense oligonucleotide to be at least about 80% complementary, or at least about 90% complementary, to a XBP1 pre-mRNA transcript. In some embodiments the antisense oligonucleotide may be at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% complementary to a hamster (SEQ ID NO 1), mouse (SEQ ID NO 590) or human (SEQ ID NO 801) XBP1 pre-mRNA transcript. Put another way, for some embodiments, an antisense oligonucleotide of the invention may include one, two, three or more mis-matches, wherein a mis-match is a nucleotide within the antisense oligonucleotide of the invention which does not base pair with its target.
The term “fully complementary” refers to 100% complementarity.
The term “complement,” as used herein, indicates a sequence that is complementary to a reference sequence. It is well known that complementarity is the base principle (Watson-Crick base pairing) of DNA replication and transcription as it is a property shared between two DNA or RNA sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, much like looking in the mirror and seeing the reverse of things. Therefore, for example, the complement of a sequence of 5′-ATGC-3′ can be written as 3′-TACG-5′ or 5′-GCAT-3′. The terms “reverse complement”, “reverse complementary”, and “reverse complementarity” as used herein are interchangeable with the terms “complement”, “complementary”, and “complementarity.”
The term “identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g. a sequence motif).
The percentage of identity is thus calculated by counting the number of aligned nucleobases that are identical (a Match) between two sequences (in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. Therefore, Percentage of Identity=(Matches×100)/Length of aligned region (e.g. the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).
As used herein, the terms “homologous” and “homology” are interchangeable with the terms “identity” and “identical.”
The term “naturally occurring variant thereof” refers to variants of the XBP1 polypeptide sequence or XBP1 nucleic acid sequence (e.g., transcript) which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, rat, Chinese hamster, monkey, and human. Typically, when referring to “naturally occurring variants” of a polynucleotide the term also can encompass any allelic variant of the XBP1-encoding genomic DNA by chromosomal translocation or duplication, and the RNA, such as mRNA derived therefrom. “Naturally occurring variants” can also include variants derived from alternative splicing of the XBP1 mRNA. When referenced to a specific polypeptide sequence, e.g., XBP1 the term also includes naturally occurring forms of the protein, which can therefore be processed, e.g., by co- or post-translational modifications, such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc. In some embodiments, the naturally occurring variants have at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homology to a mammalian XBP1 target nucleic acid, such as that set forth in SEQ ID NO: 1 (hamster), SEQ ID NO 590 (mouse) or SEQ ID NO 801 (human). In some embodiments, the naturally occurring variants have at least 99% homology to the hamster XBP1 target nucleic acid of SEQ ID NO: 1. In some embodiments, the naturally occurring variants have at least 99% homology to the mouse XBP1 target nucleic acid of SEQ ID NO: 590. In some embodiments, the naturally occurring variants have at least 99% homology to the human XBP1 target nucleic acid of SEQ ID NO: 801.
The terms “corresponding to” and “corresponds to,” which can be used interchangeably herein, when referencing two separate nucleic acid or nucleotide sequences can be used to clarify regions of the sequences that correspond or are similar to each other based on homology and/or functionality, although the nucleotides of the specific sequences can be numbered differently. For example, different isoforms of a gene transcript can have similar or conserved portions of nucleotide sequences whose numbering can differ in the respective isoforms based on alternative splicing and/or other modifications. In addition, it is recognized that different numbering systems can be employed when characterizing a nucleic acid or nucleotide sequence (e.g., a gene transcript and whether to begin numbering the sequence from the translation start codon or to include the 5′UTR). Further, it is recognized that the nucleic acid or nucleotide sequence of different variants of a gene or gene transcript can vary. As used herein, however, the regions of the variants that share nucleic acid or nucleotide sequence homology and/or functionality are deemed to “correspond” to one another. For example, a nucleotide sequence of a XBP1 transcript corresponding to nucleotides X to Y of SEQ ID NO: 1 (“reference sequence”) refers to an XBP1 transcript sequence (e.g., XBP1 pre-mRNA or mRNA) that has an identical sequence or a similar sequence to nucleotides X to Y of SEQ ID NO: 1, wherein X is the start site and Y is the end site. A person of ordinary skill in the art can identify the corresponding X and Y residues in the XBP1 transcript sequence by aligning the XBP1 transcript sequence with SEQ ID NO: 1.
The terms “hybridizing” or “hybridizes” as used herein are to be understood as two nucleic acid strands (e.g. an antisense oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (Tm) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions, Tm is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° is a more accurate representation of binding affinity and is related to the dissociation constant (Kd) of the reaction by ΔG°=−RTIn(Kd), where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG° of the reaction between an oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and target nucleic acid. ΔG° is the energy associated with a reaction where aqueous concentrations are 1M, the pH is 7, and the temperature is 37° C. The hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions ΔG° is less than zero. ΔG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for ΔG° measurements. ΔG° can also be estimated numerically by using the nearest neighbour model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et at, 2004, Biochemistry 43:5388-5405.
In some embodiments, antisense oligonucleotides of the present invention hybridize to a target nucleic acid with estimated ΔG° values below −10 kcal for oligonucleotides that are 10-30 nucleotides in length.
In some embodiments the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotides may hybridize to a target nucleic acid with estimated ΔG° values below the range of −10 kcal, such as below −15 kcal, such as below −20 kcal and such as below −25 kcal for oligonucleotides that are 8-30 nucleotides in length. In some embodiments the oligonucleotides hybridize to a target nucleic acid with an estimated ΔG° value of −10 to −60 kcal, such as −12 to −40, such as from −15 to −30 kcal, or −16 to −27 kcal such as −18 to −25 kcal.
The term “transcript” as used herein can refer to a primary transcript that is synthesized by transcription of DNA and becomes a messenger RNA (mRNA) after processing, i.e., a precursor messenger RNA (pre-mRNA), and the processed mRNA itself. The term “transcript” can be interchangeably used with “pre-mRNA” and “mRNA.” After DNA strands are transcribed to primary transcripts, the newly synthesized primary transcripts are modified in several ways to be converted to their mature, functional forms to produce different proteins and RNAs such as mRNA, tRNA, rRNA, lncRNA, miRNA and others. Thus, the term “transcript” can include exons, introns, 5′-DTRs, and 3′-DTRs.
The term “expression” as used herein refers to a process by which a polynucleotide produces a gene product, for example, a RNA or a polypeptide. It includes, without limitation, transcription of the polynucleotide into messenger RNA (mRNA) and the translation of an mRNA into a polypeptide. Expression produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
The term “Compound Number” or “Comp No.” as used herein refers to a unique number given to a nucleotide sequence having the detailed chemical structure of the components, e.g., nucleosides (e.g., DNA), nucleoside analogs (e.g., LNA, e.g., beta-D-oxy-LNA), nucleobase (e.g., A, T, G, C, U, or MC), and backbone structure (e.g., phosphorothioate or phosphorodiester).
A reference to a SEQ ID number includes a particular nucleic acid sequence but does not include any design or full chemical structure. Furthermore, the antisense oligonucleotide sequences disclosed in the examples herein show a representative design but are not limited to the specific design shown unless otherwise indicated.
By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on. In some embodiments, the subject is a human.
In some embodiments, the subject is a human who is suffering from a proteopathological diseases, or is at risk of developing a proteopathological disease.
The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. Such compositions can be sterile.
Proteopathological diseases (also known as proteopathies, proteinopathies, protein conformational disorders, or protein misfolding diseases) include such diseases as prion diseases e.g. Creutzfeldt-Jakob disease; tauopathies, such as Alzheimer's disease; synucleinopathies such as Parkinson's disease; amyloidosis, multiple system atrophy; and TDP-43 pathologies, such as amyotrophic lateral sclerosis (ALS) frontotemporal lobar degeneration (FTLD); CAG repeat indications, such as spinocerebellar ataxies, such as spinocerebellar ataxia type 1, Spinocerebellar ataxia type 2 (SCA2), and Spinocerebellar ataxia type 3 (SCA3, Machado-Joseph disease).
An “effective amount” of a composition disclosed herein (e.g., a composition comprising a compound, such as an antisense oligonucleotide, or conjugate or salt thereof) refers to an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder, such as a proteopathological disease. Thus, those in need of treatment include those already with the disorder, those prone to have the disorder and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully “treated” for a disease or condition disclosed elsewhere herein according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.
General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).
As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and is referred to as “numbering according to Kabat” herein. Specifically, the Kabat numbering system (see pages 647-660) of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domain CL of kappa and lambda isotype, and the Kabat EU index numbering system (see pages 661-723) of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the constant heavy chain domains (CH1, hinge, CH2 and CH3, which is herein further clarified by referring to “numbering according to Kabat EU index” in this case).
The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to full length antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-antibody fragment-fusions as well as combinations thereof.
The term “native antibody” denotes naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a heavy chain variable region (VH) followed by three heavy chain constant domains (CH1, CH2, and CH3), whereby between the first and the second heavy chain constant domain a hinge region is located. Similarly, from N- to C-terminus, each light chain has a light chain variable region (VL) followed by a light chain constant domain (CL). The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.
The term “full length antibody” denotes an antibody having a structure substantially similar to that of a native antibody. A full length antibody comprises two full length antibody light chains each comprising in N- to C-terminal direction a light chain variable region and a light chain constant domain, as well as two full length antibody heavy chains each comprising in N- to C-terminal direction a heavy chain variable region, a first heavy chain constant domain, a hinge region, a second heavy chain constant domain and a third heavy chain constant domain. In contrast to a native antibody, a full length antibody may comprise further immunoglobulin domains, such as e.g. one or more additional scFvs, or heavy or light chain Fab fragments, or scFabs conjugated to one or more of the termini of the different chains of the full length antibody, but only a single fragment to each terminus. These conjugates are also encompassed by the term full-length antibody.
The term “antibody binding site” denotes a pair of a heavy chain variable domains and a light chain variable domain. To ensure proper binding to the antigen these variable domains are cognate variable domains, i.e. belong together. An antibody binding site comprises at least three HVRs (e.g. in case of a VHH) or three-six HVRs (e.g. in case of a naturally occurring, i.e. conventional, antibody with a VH/VL pair). Generally, the amino acid residues of an antibody that are responsible for antigen binding form the binding site. These residues are normally contained in a pair of an antibody heavy chain variable domain and a corresponding antibody light chain variable domain. The antigen-binding site of an antibody comprises amino acid residues from the “hypervariable regions” or “HVRs”. “Framework” or “FR” regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the regions FR1, HVR1, FR2, HVR2, FR3, HVR3 and FR4. Especially, the HVR3 region of the heavy chain variable domain is the region, which contributes most to antigen binding and defines the binding specificity of an antibody. A “functional binding site” is capable of specifically binding to its target. The term “specifically binding to” denotes the binding of a binding site to its target in an in vitro assay, in one embodiment in a binding assay. Such binding assay can be any assay as long the binding event can be detected. For example, an assay in which the antibody is bound to a surface and binding of the antigen(s) to the antibody is measured by Surface Plasmon Resonance (SPR). Alternatively, a bridging ELISA can be used.
The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antibody variable domain comprising the amino acid residue stretches which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”), and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs; three in the heavy chain variable domain VH (H1, H2, H3), and three in the light chain variable domain VL (L1, L2, L3).
HVRs Include
Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.
The “class” of an antibody refers to the type of constant domains or constant region, preferably the Fc-region, possessed by its heavy chains. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
The term “heavy chain constant region” denotes the region of an immunoglobulin heavy chain that contains the constant domains, i.e. the CH1 domain, the hinge region, the CH2 domain and the CH3 domain. In one embodiment, a human IgG constant region extends from Ala118 to the carboxyl-terminus of the heavy chain (numbering according to Kabat EU index). However, the C-terminal lysine (Lys447) of the constant region may or may not be present (numbering according to Kabat EU index). The term “constant region” denotes a dimer comprising two heavy chain constant regions, which can be covalently linked to each other via the hinge region cysteine residues forming inter-chain disulfide bonds.
The term “heavy chain Fc-region” denotes the C-terminal region of an immunoglobulin heavy chain that contains at least a part of the hinge region (middle and lower hinge region), the CH2 domain and the CH3 domain. In one embodiment, a human IgG heavy chain Fc-region extends from Asp221, or from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain (numbering according to Kabat EU index). Thus, an Fc-region is smaller than a constant region but in the C-terminal part identical thereto. However, the C-terminal lysine (Lys447) of the heavy chain Fc-region may or may not be present (numbering according to Kabat EU index). The term “Fc-region” denotes a dimer comprising two heavy chain Fc-regions, which can be covalently linked to each other via the hinge region cysteine residues forming inter-chain disulfide bonds.
The constant region, more precisely the Fc-region, of an antibody (and the constant region likewise) is directly involved in complement activation, C1q binding, C3 activation and Fc receptor binding. While the influence of an antibody on the complement system is dependent on certain conditions, binding to C1q is caused by defined binding sites in the Fc-region. Such binding sites are known in the state of the art and described e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16 (1979) 907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434. Such binding sites are e.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat). Antibodies of subclass IgG1, IgG2 and IgG3 usually show complement activation, C1q binding and C3 activation, whereas IgG4 do not activate the complement system, do not bind C1q and do not activate C3. An “Fc-region of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.
The term “valent” as used within the current application denotes the presence of a specified number of binding sites in an antibody. As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding site, four binding sites, and six binding sites, respectively, in an antibody.
A “monospecific antibody” denotes an antibody that has a single binding specificity, i.e. specifically binds to one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab′)2) or combinations thereof (e.g. full length antibody plus additional scFv or Fab fragments), A monospecific antibody does not need to be monovalent, i.e. a monospecific antibody may comprise more than one binding site specifically binding to the one antigen. A native antibody, for example, is monospecific but bivalent.
A “multispecific antibody” denotes an antibody that has binding specificities for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab′)2 bispecific antibodies) or combinations thereof (e.g. full length antibody plus additional scFv or Fab fragments). A multispecific antibody is at least bivalent, i.e. comprises two antigen binding sites. In addition, a multispecific antibody is at least bispecific. Thus, a bivalent, bispecific antibody is the simplest form of a multispecific antibody. Engineered antibodies with two, three or more (e.g. four) functional antigen binding sites have also been reported (see, e.g., US 2002/0004587).
In certain embodiments, the antibody is a multispecific antibody, e.g. at least a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens or epitopes. In certain embodiments, one of the binding specificities is for a first antigen and the other is for a different second antigen. In certain embodiments, multispecific antibodies may bind to two different epitopes of the same antigen. Multispecific antibodies may also be used to localize cytotoxic agents to cells, which express the antigen.
Multispecific antibodies can be prepared as full-length antibodies or antibody-antibody fragment-fusions.
Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, and Traunecker, A., et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S. A., et al., J. Immunol. 148 (1992) 1547-1553); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98/50431); using specific technology for making bispecific antibody fragments (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and preparing trispecific antibodies as described, e.g., in Tuft, A., et al., J. Immunol. 147 (1991) 60-69).
Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-Ig are also included herein (see, e.g., WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792, and WO 2013/026831. The bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting Fab” or “DAF” (see, e.g., US 2008/0069820 and WO 2015/095539).
Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH/VL domains (see, e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL domains (see, e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al., Proc. Natl. Acad. Sci. USA 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-1020). In one aspect, the multispecific antibody comprises a Cross-Fab fragment. The term “Cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A Cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CH1), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e.g., WO 2016/172485.
The antibody or fragment can also be a multispecific antibody as described in WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, or WO 2010/145793.
The antibody or fragment thereof may also be a multispecific antibody as disclosed in WO 2012/163520.
Various further molecular formats for multispecific antibodies are known in the art and are included herein (see e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).
Bispecific antibodies are generally antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two epitopes on different antigens.
Complex (multispecific) antibodies are
The “knobs into holes” dimerization modules and their use in antibody engineering are described in Carter P.; Ridgway J. B. B.; Presta Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73(1).
The CH3 domains in the heavy chains of an antibody can be altered by the “knob-into-holes” technology, which is described in detail with several examples in e.g. WO 96/027011, Ridgway, J. B., et al, Protein Eng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of these two CH3 domains and thereby of the polypeptide comprising them. Each of the two CH3 domains (of the two heavy chains) can be the “knob”, while the other is the “hole”. The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.
The mutation T366W in the CH3 domain (of an antibody heavy chain) is denoted as “knob-mutation” or “mutation knob” and the mutations T366S, L368A, Y407V in the CH3 domain (of an antibody heavy chain) are denoted as “hole-mutations” or “mutations hole” (numbering according to Kabat EU index). An additional inter-chain disulfide bridge between the CH3 domains can also be used (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681) e.g. by introducing a S354C mutation into the CH3 domain of the heavy chain with the “knob-mutation” (denotes as “knob-cys-mutations” or “mutations knob-cys”) and by introducing a Y349C mutation into the CH3 domain of the heavy chain with the “hole-mutations” (denotes as “hole-cys-mutations” or “mutations hole-cys”) (numbering according to Kabat EU index).
The term “domain crossover” as used herein denotes that in a pair of an antibody heavy chain VH-CH1 fragment and its corresponding cognate antibody light chain, i.e. in an antibody Fab (fragment antigen binding), the domain sequence deviates from the sequence in a native antibody in that at least one heavy chain domain is substituted by its corresponding light chain domain and vice versa. There are three general types of domain crossovers, (i) the crossover of the CH1 and the CL domains, which leads by the domain crossover in the light chain to a VL-CH1 domain sequence and by the domain crossover in the heavy chain fragment to a VH-CL domain sequence (or a full length antibody heavy chain with a VH-CL-hinge-CH2-CH3 domain sequence), (ii) the domain crossover of the VH and the VL domains, which leads by the domain crossover in the light chain to a VH-CL domain sequence and by the domain crossover in the heavy chain fragment to a VL-CH1 domain sequence, and (iii) the domain crossover of the complete light chain (VL-CL) and the complete VH-CH1 heavy chain fragment (“Fab crossover”), which leads to by domain crossover to a light chain with a VH-CH1 domain sequence and by domain crossover to a heavy chain fragment with a VL-CL domain sequence (all aforementioned domain sequences are indicated in N-terminal to C-terminal direction).
As used herein the term “replaced by each other” with respect to corresponding heavy and light chain domains refers to the aforementioned domain crossovers. As such, when CH1 and CL domains are “replaced by each other” it is referred to the domain crossover mentioned under item (i) and the resulting heavy and light chain domain sequence. Accordingly, when VH and VL are “replaced by each other” it is referred to the domain crossover mentioned under item (ii); and when the CH1 and CL domains are “replaced by each other” and the VH and VL domains are “replaced by each other” it is referred to the domain crossover mentioned under item (iii). Bispecific antibodies including domain crossovers are reported, e.g. in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254 and Schaefer, W., et al, Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192. Such antibodies are generally termed CrossMab.
Multispecific antibodies also comprise in one embodiment at least one Fab fragment including either a domain crossover of the CH1 and the CL domains as mentioned under item (i) above, or a domain crossover of the VH and the VL domains as mentioned under item (ii) above, or a domain crossover of the VH-CH1 and the VL-VL domains as mentioned under item (iii) above. In case of multispecific antibodies with domain crossover, the Fabs specifically binding to the same antigen(s) are constructed to be of the same domain sequence. Hence, in case more than one Fab with a domain crossover is contained in the multispecific antibody, said Fab(s) specifically bind to the same antigen.
A “humanized” antibody refers to an antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., the CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.
The term “recombinant antibody”, as used herein, denotes all antibodies (chimeric, humanized and human) that are prepared, expressed, created or isolated by recombinant means, such as recombinant cells. This includes antibodies isolated from recombinant cells such as NS0, HEK, BHK, amniocyte or CHO cells.
As used herein, the term ‘antibody fragment’ refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds, i.e. it is a functional fragment. Examples of antibody fragments include but are not limited to Fv; Fab; Fab′; Fab′-SH; F(ab′)2; bispecific Fab; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or scFab).
Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. For these methods, one or more isolated nucleic acid(s) encoding an antibody are provided.
In one aspect, a method of making an antibody is provided, wherein the method comprises culturing a host cell comprising nucleic acid(s) encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium), wherein at least one cultivation step is in the presence of a compound according to the invention.
For recombinant production of an antibody, nucleic acids encoding the antibody, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) or produced by recombinant methods or obtained by chemical synthesis.
Generally, for the recombinant large-scale production of a polypeptide of interest, such as e.g. a therapeutic antibody, a cell stably expressing and secreting said polypeptide is required.
This cell is a “recombinant mammalian cell” or “recombinant production cell” and the process used for generating such a cell is termed “cell line development”. In the first step of the cell line development process, a suitable host cell, such as e.g. a CHO cell, is transfected with a nucleic acid sequence suitable for expression of said polypeptide of interest. In a second step, a cell stably expressing the polypeptide of interest is selected based on the co-expression of a selection marker, which had been co-transfected with the nucleic acid encoding the polypeptide of interest.
A nucleic acid encoding a polypeptide, i.e. the coding sequence, is denoted as a structural gene. Such a structural gene is pure coding information. Thus, additional regulatory elements are required for expression thereof. Therefore, normally a structural gene is integrated in a so-called expression cassette. The minimal regulatory elements needed for an expression cassette to be functional in a mammalian cell are a promoter functional in said mammalian cell, which is located upstream, i.e. 5′, to the structural gene, and a polyadenylation signal sequence functional in said mammalian cell, which is located downstream, i.e. 3′, to the structural gene. The promoter, the structural gene and the polyadenylation signal sequence are arranged in an operably linked form.
In case the polypeptide of interest is a heteromultimeric polypeptide that is composed of different (monomeric) polypeptides, such as e.g. an antibody or a complex antibody format, not only a single expression cassette is required but a multitude of expression cassettes differing in the contained structural gene, i.e. at least one expression cassette for each of the different (monomeric) polypeptides of the heteromultimeric polypeptide. For example, a full-length antibody is a heteromultimeric polypeptide comprising two copies of a light chain as well as two copies of a heavy chain. Thus, a full-length antibody is composed of two different polypeptides. Therefore, two expression cassettes are required for the expression of a full-length antibody, one for the light chain and one for the heavy chain. If, for example, the full-length antibody is a bispecific antibody, i.e. the antibody comprises two different binding sites specifically binding to two different antigens, the two light chains as well as the two heavy chains are also different from each other. Thus, such a bispecific, full-length antibody is composed of four different polypeptides and therefore, four expression cassettes are required.
The expression cassette(s) for the polypeptide of interest is(are) generally integrated into one or more so called “expression vector(s)”. An “expression vector” is a nucleic acid providing all required elements for the amplification of said vector in bacterial cells as well as the expression of the comprised structural gene(s) in a mammalian cell. Typically, an expression vector comprises a prokaryotic plasmid propagation unit, e.g. for E. coli, comprising an origin of replication, and a prokaryotic selection marker, as well as a eukaryotic selection marker, and the expression cassettes required for the expression of the structural gene(s) of interest. An “expression vector” is a transport vehicle for the introduction of expression cassettes into a mammalian cell.
The more complex the polypeptide to be expressed is the higher also the number of required different expression cassettes is. Inherently with the number of expression cassettes also the size of the nucleic acid to be integrated into the genome of the host cell increases. Concomitantly also the size of the expression vector increases. However, there is a practical upper limit to the size of a vector in the range of about 15 kbps above which handling and processing efficiency profoundly drops. This issue can be addressed by using two or more expression vectors. Thereby the expression cassettes can be split between different expression vectors each comprising only some of the expression cassettes resulting in a size reduction.
Cell line development (CLD) for the generation of recombinant cell expressing a heterologous polypeptide, such as e.g. a multispecific antibody, employs either random integration (RI) or targeted integration (TI) of the nucleic acid(s) comprising the respective expression cassettes required for the expression and production of the heterologous polypeptide of interest.
Using RI, in general, several vectors or fragments thereof integrate into the cell's genome at the same or different loci.
Using TI, in general, a single copy of the transgene comprising the different expression cassettes is integrated at a predetermined “hot-spot” in the host cell's genome.
Unlike RI CLD, targeted integration (TI) CLD introduces the transgene comprising the different expression cassettes at a predetermined “hot-spot” in a cell's genome. Also the introduction is with a defined ratio of the expression cassettes. Thereby, without being bound by this theory, all the different polypeptides of the heteromultimeric polypeptide are expressed at the same (or at least a comparable and only slightly differing) rate and at an appropriate ratio.
Also, given the defined copy number and the defined integration site, recombinant cells obtained by TI should have better stability compared to cells obtained by RI. Moreover, since the selection marker is only used for selecting cells with proper TI and not for selecting cells with a high level of transgene expression, a less mutagenic marker may be applied to minimize the chance of sequence variants (SVs), which is in part due to the mutagenicity of the selective agents like methotrexate (MTX) or methionine sulfoximine (MSX).
Suitable host cells for the expression of an (glycosylated) antibody are generally derived from multicellular organisms such as e.g. vertebrates.
Any mammalian cell line that is adapted to grow in suspension can be used in the method according to the current invention. In addition, independent from the integration method, i.e. for RI as well as TI, any mammalian host cell can be used.
Examples of useful mammalian host cell lines are human amniocyte cells (e.g. CAP-T cells as described in Woelfel, J. et al., BMC Proc. 5 (2011) P133); monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (HEK293 or HEK293T cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells, Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0.
For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, NJ (2004), pp, 255-268.
In one embodiment, the mammalian host cell is, e.g., a Chinese Hamster Ovary (CHO) cell (e.g. CHO K1, CHO DG44, etc.), a Human Embryonic Kidney (HEK) cell, a lymphoid cell (e.g., Y0, NS0, Sp2/0 cell), or a human amniocyte cells (e.g. CAP-T, etc.). In one preferred embodiment, the mammalian (host) cell is a CHO cell.
Targeted integration allows exogenous nucleotide sequences to be integrated into a pre-determined site of a mammalian cell's genome. In certain embodiments, the targeted integration is mediated by a recombinase that recognizes one or more recombination recognition sequences (RRSs), which are present in the genome and in the exogenous nucleotide sequence to be integrated. In certain embodiments, the targeted integration is mediated by homologous recombination.
A “recombination recognition sequence” (RRS) is a nucleotide sequence recognized by a recombinase and is necessary and sufficient for recombinase-mediated recombination events. A RRS can be used to define the position where a recombination event will occur in a nucleotide sequence.
In certain embodiments, a RRS can be recognized by a Cre recombinase. In certain embodiments, a RRS can be recognized by a FLP recombinase. In certain embodiments, a RRS can be recognized by a Bxb1 integrase. In certain embodiments, a RRS can be recognized by a φC31 integrase.
In certain embodiments when the RRS is a LoxP site, the cell requires the Cre recombinase to perform the recombination. In certain embodiments when the RRS is a FRT site, the cell requires the FLP recombinase to perform the recombination. In certain embodiments when the RRS is a Bxb1 attP or a Bxb1 attB site, the cell requires the Bxb1 integrase to perform the recombination. In certain embodiments when the RRS is a φC31 attP or a φC31 attB site, the cell requires the φC31 integrase to perform the recombination. The recombinases can be introduced into a cell using an expression vector comprising coding sequences of the enzymes or as protein or a mRNA.
With respect to TI, any known or future mammalian host cell suitable for TI comprising a landing site as described herein integrated at a single site within a locus of the genome can be used in the current invention. Such a cell is denoted as mammalian TI host cell. In certain embodiments, the mammalian TI host cell is a hamster cell, a human cell, a rat cell, or a mouse cell comprising a landing site as described herein. In one preferred embodiment, the mammalian T1 host cell is a CHO cell. In certain embodiments, the mammalian TI host cell is a Chinese hamster ovary (CHO) cell, a CHO K1 cell, a CHO K1SV cell, a CHO DG44 cell, a CHO DUKXB-11 cell, a CHO K1S cell, or a CHO K1M cell comprising a landing site as described herein integrated at a single site within a locus of the genome.
In certain embodiments, a mammalian TI host cell comprises an integrated landing site, wherein the landing site comprises one or more recombination recognition sequence (RRS). The RRS can be recognized by a recombinase, for example, a Cre recombinase, an FLP recombinase, a Bxb1 integrase, or a φC31 integrase. The RRS can be selected independently of each other from the group consisting of a LoxP sequence, a LoxP L3 sequence, a LoxP 2L sequence, a LoxFas sequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence, a FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and a φC31 attB sequence. If multiple RRSs have to be present, the selection of each of the sequences is dependent on the other insofar as non-identical RRSs are chosen.
In certain embodiments, the landing site comprises one or more recombination recognition sequence (RRS), wherein the RRS can be recognized by a recombinase. In certain embodiments, the integrated landing site comprises at least two RRSs. In certain embodiments, an integrated landing site comprises three RRSs, wherein the third RRS is located between the first and the second RRS. In certain preferred embodiments, all three RRSs are different. In certain embodiments, the landing site comprises a first, a second and a third RRS, and at least one selection marker located between the first and the second RRS, and the third RRS is different from the first and/or the second RRS. In certain embodiments, the landing site further comprises a second selection marker, and the first and the second selection markers are different. In certain embodiments, the landing site further comprises a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked to the third selection marker. The third selection marker can be different from the first or the second selection marker.
Although the invention is exemplified with a CHO cell hereafter, this is presented solely to exemplify the invention but shall not be construed in any way as limitation. The true scope of the invention is set forth in the claims.
An exemplary mammalian TI host cell that is suitable for use in a method according to the current invention is a CHO cell harboring a landing site integrated at a single site within a locus of its genome wherein the landing site comprises three heterospecific loxP sites for Cre recombinase mediated DNA recombination.
In this example, the heterospecific loxP sites are L3, LoxFas and 2L (see e.g. Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), whereby L3 and 2L flank the landing site at the 5′-end and 3′-end, respectively, and LoxFas is located between the L3 and 2L sites. The landing site further contains a bicistronic unit linking the expression of a selection marker via an IRES to the expression of the fluorescent GFP protein allowing to stabilize the landing site by positive selection as well as to select for the absence of the site after transfection and Cre-recombination (negative selection), Green fluorescence protein (GFP) serves for monitoring the RMCE reaction.
Such a configuration of the landing site as outlined in the previous paragraph allows for the simultaneous integration of two vectors, e.g. of a so called front vector harboring an L3 and a LoxFas site and a back vector harboring a LoxFas and an 2L site. The functional elements of a selection marker gene different from that present in the landing site can be distributed between both vectors: promoter and start codon can be located on the front vector whereas coding region and poly A signal are located on the back vector. Only correct recombinase-mediated integration of said nucleic acids from both vectors induces resistance against the respective selection agent.
Generally, a mammalian TI host cell is a mammalian cell comprising a landing site integrated at a single site within a locus of the genome of the mammalian cell, wherein the landing site comprises a first and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence located between the first and the second recombination recognition sequence, and all the recombination recognition sequences are different.
The selection marker(s) can be selected from the group consisting of an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol 0), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid. The selection marker(s) can also be a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced GFP (eGFP), a synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald6, CyPet, mCFPm, Cerulean, and T-Sapphire.
An exogenous nucleotide sequence is a nucleotide sequence that does not originate from a specific cell but can be introduced into said cell by DNA delivery methods, such as, e.g., by transfection, electroporation, or transformation methods. In certain embodiments, a mammalian TI host cell comprises at least one landing site integrated at one or more integration sites in the mammalian cell's genome. In certain embodiments, the landing site is integrated at one or more integration sites within a specific a locus of the genome of the mammalian cell.
In certain embodiments, the integrated landing site comprises at least one selection marker. In certain embodiments, the integrated landing site comprises a first, a second and a third RRS, and at least one selection marker. In certain embodiments, a selection marker is located between the first and the second RRS. In certain embodiments, two RRSs flank at least one selection marker, i.e., a first RRS is located 5′ (upstream) and a second RRS is located 3′ (downstream) of the selection marker. In certain embodiments, a first RRS is adjacent to the 5′-end of the selection marker and a second RRS is adjacent to the 3′-end of the selection marker. In certain embodiments, the landing site comprises a first, second, and third RRS, and at least one selection marker located between the first and the third RRS.
In certain embodiments, a selection marker is located between a first and a second RRS and the two flanking RRSs are different. In certain preferred embodiments, the first flanking RRS is a LoxP L3 sequence and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, a LoxP L3 sequence is located 5′ of the selection marker and a LoxP 2L sequence is located 3′ of the selection marker. In certain embodiments, the first flanking RRS is a wild-type FRT sequence and the second flanking RRS is a mutant FRT sequence. In certain embodiments, the first flanking RRS is a Bxb1 attP sequence and the second flanking RRS is a Bxb1 attB sequence. In certain embodiments, the first flanking RRS is a φC31 attP sequence and the second flanking RRS is a φC31 attB sequence. In certain embodiments, the two RRSs are positioned in the same orientation. In certain embodiments, the two RRSs are both in the forward or reverse orientation. In certain embodiments, the two RRSs are positioned in opposite orientation.
In certain embodiments, the integrated landing site comprises a first and a second selection marker, which are flanked by two RRSs, wherein the first selection marker is different from the second selection marker. In certain embodiments, the two selection markers are both independently of each other selected from the group consisting of a glutamine synthetase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In certain embodiments, the integrated landing site comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection maker is selected from the group consisting of an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid, and the second selection maker is selected from the group consisting of a GFP, an eGFP, a synthetic GFP, a YFP, an eYFP, a CFP, an mPlum, an mCherry, a tdTomato, an mStrawberry, a J-red, a DsRed-monomer, an mOrange, an mKO, an mCitrine, a Venus, a YPet, an Emerald, a CyPet, an mCFPm, a Cerulean, and a T-Sapphire fluorescent protein. In certain embodiments, the first selection marker is a glutamine synthetase selection marker and the second selection marker is a GFP fluorescent protein. In certain embodiments, the two RRSs flanking both selection markers are different.
In certain embodiments, the selection marker is operably linked to a promoter sequence. In certain embodiments, the selection marker is operably linked to an SV40 promoter. In certain embodiments, the selection marker is operably linked to a human Cytomegalovirus (CMV) promoter.
One method for the generation of a recombinant mammalian cell according to the current invention is targeted integration (TI).
In targeted integration, site-specific recombination is employed for the introduction of an exogenous nucleic acid into a specific locus in the genome of a mammalian TI host cell. This is an enzymatic process wherein a sequence at the site of integration in the genome is exchanged for the exogenous nucleic acid. One system used to effect such nucleic acid exchanges is the Cre-lox system. The enzyme catalyzing the exchange is the Cre recombinase. The sequence to be exchanged is defined by the position of two lox(P)-sites in the genome as well as in the exogenous nucleic acid. These lox(P)-sites are recognized by the Cre recombinase. Nothing more is required, i.e. no ATP etc. Originally, the Cre-lox system has been found in bacteriophage P1.
The Cre-lox system operates in different cell types, like mammals, plants, bacteria and yeast.
In one embodiment, the exogenous nucleic acid encoding the heterologous polypeptide has been integrated into the mammalian TI host cell by single or double recombinase mediated cassette exchange (RMCE). Thereby a recombinant mammalian cell, such as a recombinant CHO cell, is obtained, in which a defined and specific expression cassette sequence has been integrated into the genome at a single locus, which in turn results in the efficient expression and production of the heterologous polypeptide.
The Cre-LoxP site-specific recombination system has been widely used in many biological experimental systems. Cre recombinase is a 38-kDa site-specific DNA recombinase that recognizes 34 bp LoxP sequences. Cre recombinase is derived from bacteriophage P1 and belongs to the tyrosine family site-specific recombinase. Cre recombinase can mediate both intra and intermolecular recombination between LoxP sequences. The LoxP sequence is composed of an 8 bp non-palindromic core region flanked by two 13 bp inverted repeats. Cre recombinase binds to the 13 bp repeat thereby mediating recombination within the 8 bp core region. Cre-LoxP-mediated recombination occurs at a high efficiency and does not require any other host factors. If two LoxP sequences are placed in the same orientation on the same nucleotide sequence, Cre recombinase-mediated recombination will excise DNA sequences located between the two LoxP sequences as a covalently closed circle. If two LoxP sequences are placed in an inverted position on the same nucleotide sequence, Cre recombinase-mediated recombination will invert the orientation of the DNA sequences located between the two sequences. If two LoxP sequences are on two different DNA molecules and if one DNA molecule is circular, Cre recombinase-mediated recombination will result in integration of the circular DNA sequence.
The term “matching RRSs” indicates that a recombination occurs between two RRSs. In certain embodiments, the two matching RRSs are the same. In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matching RRSs are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matching RRS is a Bxb1 attP sequence and the second matching RRS is a Bxb1 attB sequence. In certain embodiments, the first matching RRS is a φC31 attB sequence and the second matching RRS is a φC31 attB sequence.
A “two-plasmid RMCE” strategy or “double RMCE” is employed in the method according to the current invention when using a two-vector combination. For example, but not by way of limitation, an integrated landing site could comprise three RRSs, e.g., an arrangement where the third RRS (“RRS3”) is present between the first RRS (“RRS1”) and the second RRS (“RRS2”), while a first vector comprises two RRSs matching the first and the third RRS on the integrated exogenous nucleotide sequence, and a second vector comprises two RRSs matching the third and the second RRS on the integrated exogenous nucleotide sequence. The two-plasmid RMCE strategy involves using three RRS sites to carry out two independent RMCEs simultaneously. Therefore, a landing site in the mammalian TI host cell using the two-plasmid RMCE strategy includes a third RRS site (RRS3) that has no cross activity with either the first RRS site (RRS1) or the second RRS site (RRS2). The two plasmids to be targeted require the same flanking RRS sites for efficient targeting, one plasmid (front) flanked by RRS1 and RRS3 and the other (back) by RRS3 and RRS2. In addition, two selection markers are needed in the two-plasmid RMCE. One selection marker expression cassette was split into two parts. The front plasmid would contain the promoter followed by a start codon and the RRS3 sequence. The back plasmid would have the RRS3 sequence fused to the N-terminus of the selection marker coding region, minus the start-codon (ATG). Additional nucleotides may need to be inserted between the RRS3 site and the selection marker sequence to ensure in frame translation for the fusion protein, i.e. operable linkage. Only when both plasmids are correctly inserted, the full expression cassette of the selection marker will be assembled and, thus, rendering cells resistance to the respective selection agent.
Two-plasmid RMCE involves double recombination cross-over events, catalyzed by a recombinase, between the two heterospecific RRSs within the target genomic locus and the donor DNA molecule, Two-plasmid RMCE is designed to introduce a copy of the DNA sequences from the front- and back-vector in combination into the pre-determined locus of a mammalian TI host cell's genome. RMCE can be implemented such that prokaryotic vector sequences are not introduced into the mammalian TI host cell's genome, thus, reducing and/or preventing unwanted triggering of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.
In certain embodiments, targeted integration is achieved by two RMCEs, wherein two different DNA sequences, each comprising at least one expression cassette encoding a part of a heteromultimeric polypeptide and/or at least one selection marker or part thereof flanked by two heterospecific RRSs, are both integrated into a pre-determined site of the genome of a RRSs matching mammalian TI host cell. In certain embodiments, targeted integration is achieved by multiple RMCEs, wherein DNA sequences from multiple vectors, each comprising at least one expression cassette encoding a part of a heteromultimeric polypeptide and/or at least one selection marker or part thereof flanked by two heterospecific RRSs, are all integrated into a predetermined site of the genome of a mammalian TI host cell. In certain embodiments the selection marker can be partially encoded on the first the vector and partially encoded on the second vector such that only the correct integration of both by double RMCE allows for the expression of the selection marker.
In certain embodiments, targeted integration via recombinase-mediated recombination leads to selection marker and/or the different expression cassettes for the multimeric polypeptide integrated into one or more pre-determined integration sites of a host cell genome free of sequences from a prokaryotic vector.
It has to be pointed out that, as in one embodiment, knockout can be performed either before introduction of the exogenous nucleic acid encoding the heterologous polypeptide or thereafter.
XBP1 exon 4 comprises a 26 nucleotide fragment which is excised by IRE1α in vivo to introduce a +2 out of frame event and produce XBP1s. The present inventors have determined that skipping of exon 4 also introduces a +2 out of frame event and produces a functional protein. Skipping of exon 4 can be accomplished using antisense oligonucleotides of the invention. By skipping exon 4 in accordance with the invention, a much larger nucleotide fragment, of 146 bp, is removed from the pre-mRNA as compared to the 26 nucleotide fragment excised by IRE1α. Thus, XBP1Δ4 according to the invention is not equal to in vivo spliced XBP1.
The present inventors have also identified that the generation or expression of the XBP1Δ4 variant in mammalian cells results in an enhanced recombinant expression of heterologously expressed proteins, such as monoclonal antibodies, particularly of heterologously expressed proteins which are otherwise difficult to express. This indicates that the generation or expression of the XBP1Δ4 variant results in an enhanced quality of protein expression in mammalian cells.
The present invention discloses and utilizes specific antisense oligonucleotides, which are complementary, such as fully complementary, to a portion of the XBP1 pre-mRNA transcript. The antisense oligonucleotides of the invention are capable of reducing the inclusion (enhancing the excision) of XBP1 exon 4 in XBP1 transcripts. The antisense oligonucleotides of the invention thereby result in the expression of, or enhanced expression of, an XBP1Δ4 variant.
The inventors have identified that the generation or expression of the XBP1Δ4 variant in mammalian cells results in enhanced protein expression. The antisense oligonucleotides of the invention may therefore be used to enhance the yield or the quality of proteins produced from heterologous protein expression systems, for example in the manufacture of antibodies, such as monoclonal antibodies.
The antisense oligonucleotides of the invention also have therapeutic utilities in the treatment and prevention of proteopathological disease.
In one aspect, the present invention relates to an antisense oligonucleotide for use in the expression of a XBP1 splice variant in a cell which expresses XBP1, wherein the antisense oligonucleotide is 8-40 nucleotides in length and comprises a contiguous nucleotide sequence of 8-40 nucleotides in length which is complementary to a mammalian XBP1 pre-mRNA transcript.
In certain embodiments of the present invention, the XBP1 splice variant has a +2 out of frame event.
In certain embodiments, the XBP1 splice variant is XBP1Δ4.
The invention provides an antisense oligonucleotide, wherein the antisense oligonucleotide is 8-40 nucleotides in length and comprises a contiguous nucleotide sequence of at least 12 nucleotides in length which is complementary, such as fully complementary, to a mammalian XBP1 pre-mRNA transcript.
The invention provides an antisense oligonucleotide, wherein the antisense oligonucleotide is 8-40 nucleotides in length and comprises a contiguous nucleotide sequence of 12-16 nucleotides in length which is complementary, such as fully complementary, to a mammalian XBP1 pre-mRNA transcript.
The invention provides an antisense oligonucleotide, wherein the antisense oligonucleotide is 12-16 nucleotides in length and comprises a contiguous nucleotide sequence of 12-16 nucleotides in length which is complementary, such as fully complementary, to a mammalian XBP1 pre-mRNA transcript.
The invention provides an antisense oligonucleotide, wherein the antisense oligonucleotide is 8-40 nucleotides in length and comprises a contiguous nucleotide sequence of 12-18 nucleotides in length which is complementary, such as fully complementary, to a mammalian XBP1 pre-mRNA transcript.
The antisense oligonucleotide may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
In some embodiments, the antisense oligonucleotide is 8-40, 12-40, 12-20, 10-20, 14-18, 12-18 or 16-18 nucleotides in length.
The contiguous nucleotide sequence may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length. In some embodiments, the contiguous nucleotide sequence is at least 12 nucleotides in length, such as 12-16 or 12-18 nucleotides in length.
In some embodiments, the contiguous nucleotide sequence is the same length as the antisense oligonucleotide.
In some embodiments, the antisense oligonucleotide consists of the contiguous nucleotide sequence.
In some embodiments, the antisense oligonucleotide is the contiguous nucleotide sequence.
In some embodiments, the antisense oligonucleotide comprises a contiguous sequence of 8 to 40 nucleotides in length, which is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more complementary with a region of the target nucleic acid or a target sequence. Put another way, in some embodiments, an antisense oligonucleotide of the invention may include one, two, three or more mis-matches, wherein a mis-match is a nucleotide within the antisense oligonucleotide of the invention which does not base pair with its target.
It is advantageous if the oligonucleotide, or contiguous nucleotide sequence thereof, is fully complementary (100% complementary) to a region of the target sequence.
In some embodiments, the antisense oligonucleotide is isolated, purified, or manufactured.
In some embodiments, the antisense oligonucleotide comprises one or more modified nucleotides or one or more modified nucleosides.
In some embodiments, the antisense oligonucleotide is a morpholino modified antisense oligonucleotide.
In some embodiments, the antisense oligonucleotide comprises one or more modified nucleosides, such as one or more modified nucleotides independently selected from the group consisting of 2′-O-alkyl-RNA; 2′-O-methyl RNA (2′-OMe); Z-alkoxy-RNA; 2′-O-methoxyethyl-RNA (2′-MOE); 2′-amino-DNA; 2′-fluoro-RNA; 2′-fluoro-DNA; arabino nucleic acid (ANA); 2′-fluoro-ANA; bicyclic nucleoside analog (LNA); or any combination thereof.
In some embodiments, one or more of the modified nucleosides is a sugar modified nucleoside.
In some embodiments, one or more of the modified nucleosides comprises a bicyclic sugar.
In some embodiments, one or more of the modified nucleosides is an affinity enhancing 2′ sugar modified nucleoside.
In some embodiments, one or more of the modified nucleosides is an LNA nucleoside.
In some embodiments, the antisense oligonucleotide, or contiguous nucleotide sequence thereof, comprises one or more 5′-methyl-cytosine nucleobases.
In some embodiments, one or more of the internucleoside linkages within the contiguous nucleotide sequence of the antisense oligonucleotide is modified.
In some embodiments, the one or more modified internucleoside linkages comprises a phosphorothioate linkage.
In some embodiments, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the internucleoside linkages of the antisense oligonucleotide or contiguous nucleotide sequence thereof are modified.
In some embodiments, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the internucleoside linkages of the antisense oligonucleotide or contiguous nucleotide sequence thereof are phosphorothioate internucleoside linkages.
In some embodiments, the antisense oligonucleotides of the invention are in solid powdered form, such as in the form of a lyophilized powder.
Additional disclosures regarding the above antisense oligonucleotides are provided throughout the present disclosure.
As Described Herein, the Antisense Oligonucleotides of the Invention Target the XBP1 mRNA sequence in order to cause expression of an XBP1 splice variant, such as a XBP1Δ4 variant.
As used herein, the term “XBP1Δ4” refers to a XBP1 transcript which lacks exon 4 (a XBP1Δ4 variant), or a XBP1 protein which lacks the amino acids encoded by XBP1 exon 4. A key feature of the XBP1Δ4 variant is that the deletion of exon 4 and the introduction of a +2 frame shift in the XBP1 coding sequence has occurred, which results in the expression of a XBP1Δ4 variant with a C-terminal region which is homologous to the C-terminal region of the XBP1s variant of XBP1 (induced by IRE1).
In certain embodiments, a XBP1Δ4 protein lacks all or essentially all of the peptide sequence encoded by XBP1 exon 4.
The term “target”, as used herein, is used to refer to the transcript of the gene that the antisense oligonucleotides of the present invention specifically hybridizes/binds to (i.e., “XBP1”).
XBP1 is also known as X-box binding protein 1, TREB-5, TREB5, XBP-1, and XBP2.
The target for oligonucleotides of the present invention is an XBP1 pre-mRNA transcript. The XBP1 pre-mRNA transcript is preferably a mammalian XBP1 pre-mRNA transcript
In some embodiments, the mammalian XBP1 pre-mRNA transcript is a hamster XBP1 pre-mRNA transcript.
The hamster XBP1 pre-mRNA sequence is recited in SEQ ID NO 1.
In certain embodiments, the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides of the hamster XBP1 pre-mRNA transcript (SEQ ID NO 1).
In certain embodiments, the contiguous nucleotide sequence may be complementary to at least 10 contiguous nucleotides from nucleotides 2960-3113 of SEQ ID NO 1.
In other embodiments, the contiguous nucleotide sequence may be complementary to at least 10 contiguous nucleotides from nucleotides 2986-3018 of SEQ ID NO 1.
In some embodiments the contiguous nucleotide sequence is complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 or at least 17 contiguous nucleotides of the hamster XBP1 pre-mRNA transcript (SEQ ID NO 1).
In other embodiments the contiguous nucleotide sequence may be complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO 299, SEQ ID NO 301, SEQ ID NO 302, SEQ ID NO 304, SEQ ID NO 305, SEQ ID NO 306, SEQ ID NO 307, SEQ ID NO 308, SEQ ID NO 309, SEQ ID NO 310, SEQ ID NO 314, SEQ ID NO 316, SEQ ID NO 317, SEQ ID NO 318. SEQ ID NO 319, SEQ ID NO 323, SEQ ID NO 325, SEQ ID NO 327, SEQ ID NO 328, SEQ ID NO 330, SEQ ID NO 331, SEQ ID NO 332, SEQ ID NO 333, SEQ ID NO 334, SEQ ID NO 336, SEQ ID NO 337, SEQ ID NO 385, SEQ ID NO 386, SEQ ID NO 387, SEQ ID NO 388, SEQ ID NO 390, SEQ ID NO 391, SEQ ID NO 392, SEQ ID NO 393, SEQ ID NO 394, SEQ ID NO 395, SEQ ID NO 396 397, SEQ ID NO 398, SEQ ID NO 399, SEQ ID NO 401, SEQ ID NO 402, SEQ ID NO 419, SEQ ID NO 431, SEQ ID NO, SEQ ID NO 432, SEQ ID NO 433, SEQ ID NO 434, SEQ ID NO 438, SEQ ID NO 439, SEQ ID NO 440, SEQ ID NO 441, SEQ ID NO 442, SEQ ID NO 449, SEQ ID NO 484, SEQ ID NO 485, SEQ ID NO 486, SEQ ID NO 487, SEQ ID NO 488, SEQ ID NO 489, SEQ ID NO 490, SEQ ID NO 491, SEQ ID NO 492, SEQ ID NO 493, SEQ ID NO 494, SEQ ID NO 495, SEQ ID NO 496, SEQ ID NO 497, SEQ ID NO 498, SEQ ID NO 499, SEQ ID NO 500, SEQ ID NO 501, SEQ ID NO 502, SEQ ID NO 503, SEQ ID NO 505, SEQ ID NO 506, SEQ ID NO 507, SEQ ID NO 508, SEQ ID NO 509, SEQ ID NO 510, SEQ ID NO 511, SEQ ID NO 512, SEQ ID NO 513, SEQ ID NO 515, SEQ ID NO 517, SEQ ID NO 520, SEQ ID NO 572, SEQ ID NO 573, SEQ ID NO 576, SEQ ID NO 577, SEQ ID NO 588 and SEQ ID NO 589.
In other embodiments the contiguous nucleotide sequence may be complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO 305, SEQ ID NO 307, SEQ ID NO 314, SEQ ID NO 315, SEQ ID NO 316, SEQ ID NO 317, SEQ ID NO 319, SEQ ID NO 331, SEQ ID NO 332, SEQ ID NO 392, SEQ ID NO 394, SEQ ID NO 395, SEQ ID NO 440, SEQ ID NO 492, SEQ ID NO 497, SEQ ID NO 498, SEQ ID NO 499, SEQ ID NO 500, SEQ ID NO 501, SEQ ID NO 502, SEQ ID NO 513 and SEQ ID NO 576.
In other embodiments the contiguous nucleotide sequence may be complementary to SEQ ID NO 314 or SEQ ID NO 315.
In some embodiments the mammalian XBP1 pre-mRNA transcript is a mouse XBP1 pre-mRNA transcript.
The mouse XBP1 pre-mRNA is recited in SEQ ID NO 590.
In certain embodiments the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides of the mouse XBP1 pre-mRNA transcript (SEQ ID NO 590).
In certain embodiments the contiguous nucleotide sequence may be complementary to at least 10 contiguous nucleotides from nucleotides 3560-3783 of SEQ ID NO 590.
In some embodiments the contiguous nucleotide sequence is complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 or at least 17 contiguous nucleotides of the mouse XBP1 pre-mRNA transcript (SEQ ID NO 590).
In other embodiments the contiguous nucleotide sequence may be complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO 699, SEQ ID NO 700, SEQ ID NO 703, SEQ ID NO 710, SEQ ID NO 713, SEQ ID NO 724, SEQ ID NO 729, SEQ ID NO 739, SEQ ID NO 743, SEQ ID NO 744, SEQ ID NO 745, SEQ ID NO 749, SEQ ID NO 750, SEQ ID NO 751, SEQ ID NO 752, SEQ ID NO 753, SEQ ID NO 754, SEQ ID NO 755, SEQ ID NO 756, SEQ ID NO 757, SEQ ID NO 758, SEQ ID NO 759, SEQ ID NO 760, SEQ ID NO 761, SEQ ID NO 762, SEQ ID NO 763, SEQ ID NO 773, SEQ ID NO 776, SEQ ID NO 778, SEQ ID NO 781, SEQ ID NO 783, SEQ ID NO 784, SEQ ID NO 785, SEQ ID NO 787, SEQ ID NO 789, SEQ ID NO 790, SEQ ID NO 791, SEQ ID NO 792, SEQ ID NO 793, SEQ ID NO 794, SEQ ID NO 795, SEQ ID NO 796, SEQ ID NO 797, SEQ ID NO 798, SEQ ID NO 799 and SEQ ID NO 800.
In other embodiments the contiguous nucleotide sequence may be complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO 710, SEQ ID NO 754, SEQ ID NO 756, SEQ ID NO 757, SEQ ID NO 758, SEQ ID NO 759, SEQ ID NO 760, SEQ ID NO 791, SEQ ID NO 792, SEQ ID NO 794, SEQ ID NO 795 and SEQ ID NO 797.
In some embodiments, the mammalian XBP1 pre-mRNA transcript is a human XBP1 pre-mRNA transcript.
The human XBP1 pre-mRNA is recited in SEQ ID NO 801.
In certain embodiments the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides of the human XBP1 pre-mRNA transcript (SEQ ID NO 801).
In certain embodiments, the contiguous nucleotide sequence may be complementary to at least 10 contiguous nucleotides from nucleotides 4338-4563 of SEQ ID NO 801
In some embodiments the contiguous nucleotide sequence is complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 contiguous nucleotides of the human XBP1 pre-mRNA transcript (SEQ ID NO 801).
In other embodiments, the contiguous nucleotide sequence may be complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO 947, SEQ ID NO 948, SEQ ID NO 949, SEQ ID NO 950, SEQ ID NO 951 and SEQ ID NO 988.
In other embodiments, the contiguous nucleotide sequence may be complementary to SEQ ID NO 951.
The contiguous nucleotide sequence may be complementary to a portion of the hamster XBP1 pre-mRNA transcript (SEQ ID NO 1).
In certain embodiments, the contiguous nucleotide sequence may be selected from the group consisting of SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 128, SEQ ID NO 140, SEQ ID NO 141, SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151, SEQ ID NO 158, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 198, SEQ ID NO 199, SEQ ID NO 200, SEQ ID NO 201, SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 204, SEQ ID NO 205, SEQ ID NO 206, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211, SEQ ID NO 212, SEQ ID NO 214, SEQ ID NO 215, SEQ ID NO 216, SEQ ID NO 217, SEQ ID NO 218, SEQ ID NO 219, SEQ ID NO 220, SEQ ID NO 221, SEQ ID NO 222, SEQ ID NO 224, SEQ ID NO 226, SEQ ID NO 229, SEQ ID NO 281, SEQ ID NO 282, SEQ ID NO 285, SEQ ID NO 286, SEQ ID NO 297 and SEQ ID NO 298.
In certain embodiments, the contiguous nucleotide sequence may be selected from the group consisting of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 101, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 149, SEQ ID NO 201, SEQ ID NO 206, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211, SEQ ID NO 222 and SEQ ID NO 285.
In certain embodiments, the contiguous nucleotide sequence may be SEQ ID NO 23 or SEQ ID NO 24.
The contiguous nucleotide sequence may be complementary to a portion of the mouse XBP1 pre-mRNA transcript (SEQ ID NO 590).
In certain embodiments, the contiguous nucleotide sequence may be selected from the group consisting of SEQ ID NO 597, SEQ ID NO 598, SEQ ID NO 601, SEQ ID NO 608, SEQ ID NO 611, SEQ ID NO 622, SEQ ID NO 627, SEQ ID NO 637, SEQ ID NO 641, SEQ ID NO 642, SEQ ID NO 643, SEQ ID NO 647, SEQ ID NO 648, SEQ ID NO 649, SEQ ID NO 650, SEQ ID NO 651, SEQ ID NO 652, SEQ ID NO 653, SEQ ID NO 654, SEQ ID NO 655, SEQ ID NO 656, SEQ ID NO 657, SEQ ID NO 658, SEQ ID NO 659, SEQ ID NO 660, SEQ ID NO 661, SEQ ID NO 671, SEQ ID NO 674, SEQ ID NO 676, SEQ ID NO 679, SEQ ID NO 681, SEQ ID NO 682, SEQ ID NO 683, SEQ ID NO 685, SEQ ID NO 687, SEQ ID NO 688, SEQ ID NO 689, SEQ ID NO 690, SEQ ID NO 691, SEQ ID NO 692, SEQ ID NO 693, SEQ ID NO 694, SEQ ID NO 695, SEQ ID NO 696, SEQ ID NO 697 and SEQ ID NO 698.
In certain embodiments, the contiguous nucleotide sequence may be selected from the group consisting of SEQ ID NO 608, SEQ ID NO 652, SEQ ID NO 654, SEQ ID NO 655, SEQ ID NO 656, SEQ ID NO 657, SEQ ID NO 658, SEQ ID NO 689, SEQ ID NO 690, SEQ ID NO 692, SEQ ID NO 693 and SEQ ID NO 695.
The contiguous nucleotide sequence may be complementary to a portion of the human XBP1 pre-mRNA transcript (SEQ ID NO 801).
In certain embodiments, the contiguous nucleotide sequence may be selected from the group consisting of SEQ ID NO 854, SEQ ID NO 855, SEQ ID NO 856, SEQ ID NO 857, SEQ ID NO 858 and SEQ ID NO 895.
In certain embodiments, the contiguous nucleotide sequence may be SEQ ID NO 858.
In some embodiments, the contiguous nucleotide sequence is the same length as the antisense oligonucleotide.
In some embodiments, the antisense oligonucleotide consists of the contiguous nucleotide sequence.
In some embodiments, the antisense oligonucleotide is the contiguous nucleotide sequence.
The invention also contemplates fragments of the contiguous nucleotide sequence, including fragments of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 or at least 17 contiguous nucleotides thereof.
In some embodiments, the antisense oligonucleotides of the present invention modulate the splicing of a mammalian XBP1 pre-mRNA transcript, such as that described herein. In some embodiments, modulating the splicing of a mammalian XBP1 pre-mRNA transcript can regulate the expression and/or activity of certain XBP1 variants.
Without wishing to be bound by theory, splice modulating oligonucleotides typically operate via an occupation-based mechanism rather than via a degradation mechanism (such as RNaseH or RISC mediated inhibition).
In some embodiments, the antisense oligonucleotides of the invention are capable of reducing or inhibiting the expression (e.g., number) of a XBP1 mRNA transcript comprising exon 4 in a cell. Herein a XBP1 mRNA transcript comprising exon 4 is referred to as XBP1-E4.
The term “reducing” or “inhibiting” the expression of a transcript as used herein is to be understood as an overall term for an antisense oligonucleotide's ability to inhibit or reduce the amount or the activity of XBP1-E4 protein in a target cell (e.g., by reducing or inhibiting the expression of XBP1-E4 mRNA and thereby reducing the expression of a XBP1-E4 protein).
Inhibition of activity can be determined by measuring the level (e.g., number) of XBP1-E4 mRNA, or by measuring the level (e.g., number) or activity of XBP1-E4 protein in a cell. Inhibition of expression can therefore be determined in vitro or in viva. It will be understood that splice modulation can result in an inhibition of expression (e.g., number) of XBP1-E4 transcript (e.g., mRNA), or the protein encoded thereof, in the cell. In certain embodiments, the expression (e.g., number) of XBP1-E4 transcript (e.g., mRNA) is reduced by at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% or more compared to a corresponding cell that is not exposed to the antisense oligonucleotide.
As used herein, the term “corresponding cell that is not exposed to the antisense oligonucleotide” can refer to the same cell prior to the treatment with an antisense oligonucleotide of the invention, or to the same cell type (but not the same cell).
Accordingly, in some embodiments treating a cell with an antisense oligonucleotide of the present invention reduces (e.g., by at least about 10% or by at least about 20%) the expression of XBP1-E4 transcript (e.g., mRNA) in the cell compared to the expression of XBP1-E4 transcript (e.g., mRNA) in the same cell prior to the antisense oligonucleotide treatment.
In other embodiments treating a cell with an antisense oligonucleotide of the present invention reduces (e.g., by at least about 10% or by at least about 20%) the expression of XBP1-E4 transcript (e.g., mRNA) in the cell compared to the expression of XBP1-E4 transcript (e.g., mRNA) in the same cell type which has not undergone antisense oligonucleotide treatment.
In some embodiments, the antisense oligonucleotides of the invention are capable of increasing or enhancing the expression (e.g., number) of a XBP1 mRNA transcript lacking exon 4 in a cell. Herein a XBP1 mRNA transcript lacking exon 4 is referred to as XBP1Δ4.
The term “increasing” the expression of a transcript as used herein is to be understood as an overall term for an antisense oligonucleotide's ability to increase or enhance the amount or the activity of XBP1Δ4 protein in a target cell (e.g., by increasing the expression of XBP1 mRNA and thereby increasing the expression of a XBP1Δ4 protein).
Increases in activity can be determined by measuring the level (e.g., number) of XBP1Δ4 mRNA, or by measuring the level (e.g., number) or activity of XBP1Δ4 protein in a cell. Increases in expression can therefore be determined in vitro or in vivo. It will be understood that splice modulation can result in an increase in expression (e.g., number) of XBP1Δ4 transcript (e.g., mRNA), or the protein encoded thereof, in the cell. In certain embodiments, the expression (e.g., number) of XBP1Δ4 transcript (e.g., mRNA) is increased or enhanced by at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% or more compared to a corresponding cell that is not exposed to the antisense oligonucleotide. It is preferred that the expression (e.g., number) of XBP1114 transcript (e.g., mRNA) is increased or enhanced by at least about 1% or at least about 5% compared to a corresponding cell that is not exposed to the antisense oligonucleotide.
As used herein, the term “corresponding cell that is not exposed to the antisense oligonucleotide” can refer to the same cell prior to the treatment with an antisense oligonucleotide of the invention, or to the same cell type (but not the same cell).
Accordingly, in some embodiments treating a cell with an antisense oligonucleotide of the present invention increases or enhances (e.g., by at least about 10% or by at least about 20%) the expression of XBP1Δ4 transcript (e.g., mRNA) in the cell compared to the expression of XBP1Δ4 transcript (e.g., mRNA) in the same cell prior to the antisense oligonucleotide treatment.
In other embodiments treating a cell with an antisense oligonucleotide of the present invention increases or enhances (e.g., by at least about 10% or by at least about 20%) the expression of XBP1Δ4 transcript (e.g., mRNA) in the cell compared to the expression of XBP1Δ4 transcript (e.g., mRNA) in the same cell type which has not undergone antisense oligonucleotide treatment.
In some embodiments, the antisense oligonucleotides of the invention can change the ratio of alternative XBP1 splice variants expressed in a cell. For instance, increased or enhanced expression of XBP1Δ4 will result in an increase in the ratio of expression of XBP1Δ4/XBP1E4 transcripts.
Accordingly, in some embodiments, the antisense oligonucleotides disclosed herein can increase the ratio of expression of XBP1Δ4/XBP1E4 mRNA transcripts compared to a corresponding ratio of a cell that is not exposed to the antisense oligonucleotides of the present invention. In certain embodiments, the ratio of the expression of XBP1Δ4 mRNA transcript to the expression of XBP1-E4 mRNA transcript is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold or more compared to a corresponding ratio of a cell that is not exposed to the antisense oligonucleotides of the present invention
In some embodiments, the antisense oligonucleotides disclosed herein can increase the ratio of expression of XBP1Δ4/XBP1E4 protein compared to a corresponding ratio of a cell that is not exposed to the antisense oligonucleotides of the present invention. In certain embodiments, the ratio of the expression of XBP1Δ4 protein to the expression of XBP1-E4 protein is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold or more compared to a corresponding ratio of a cell that is not exposed to the antisense oligonucleotides of the present invention
In some embodiments, the antisense oligonucleotides of the invention are capable of both i) increasing the amount of XBP1Δ4 mRNA or XBP1Δ4 protein in the target cell and ii) decreasing the amount of XBP1-E4 mRNA and XBP1-E4 protein in a target cell.
The change in ratio of different transcript products (e.g., XBP1-E4 vs. XBP1Δ4) can be measured by comparing mRNA levels, or levels of the corresponding protein products. Anti-XBP1 antibodies which can be used for assaying the protein levels of XBP1-E4 and XBP1Δ4 including monoclonal or polyclonal antibodies raised against XBP1.
The antisense oligonucleotides of the invention can comprise a nucleotide sequence which comprises both nucleosides and nucleoside analogs, and can be in the form of a gapmer, blockmer, mixmer, headmer, tailmer, or totalmer.
In one embodiment, the antisense oligonucleotide comprises at least 1 modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 16, at least 16 or at least 17 modified nucleosides.
The term “gapmer” as used herein refers to an antisense oligonucleotide which comprises a region of RNase H recruiting oligonucleotides (gap) which is flanked 5 and 3′ by one or more affinity enhancing modified nucleosides (flanks). The terms “headmers” and “tailmers” are oligonucleotides capable of recruiting RNase H where one of the flanks is missing, i.e., only one of the ends of the oligonucleotide comprises affinity enhancing modified nucleosides. For headmers, the 3° flank is missing (i.e., the 5′ flank comprise affinity enhancing modified nucleosides) and for tailmers, the 5′ flank is missing (i.e., the 3′ flank comprises affinity enhancing modified nucleosides). The term “LNA gapmer” is a gapmer oligonucleotide wherein at least one of the affinity enhancing modified nucleosides is an LNA nucleoside. The term “mixed wing gapmer” refers to an LNA gapmer wherein the flank regions comprise at least one LNA nucleoside and at least one DNA nucleoside or non-LNA modified nucleoside, such as at least one 2′ substituted modified nucleoside, such as, for example, 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, 2′-Fluro-DNA, arabino nucleic acid (ANA), and 2′-Fluoro-ANA nucleoside(s).
Other “chimeric” antisense oligonucleotides, called “mixmers”, consist of an alternating composition of (i) DNA monomers or nucleoside analog monomers recognizable and cleavable by RNase, and (ii) non-RNase recruiting nucleoside analog monomers.
A “totalmer” is a single stranded ASO which only comprises non-naturally occurring nucleotides or nucleotide analogs.
A high affinity modified nucleoside is a modified nucleotide which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (Tm). A high affinity modified nucleoside of the present invention preferably results in an increase in melting temperature between +0.5 to +12° C., more preferably between +1.5 to +10° C. and most preferably between +3 to +8° C. per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include for example, many 2′ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213).
The antisense oligonucleotides of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.
Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2′-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2′, 3′, 4′ or 5′ positions.
A 2′ sugar modified nucleoside is a nucleoside which has a substituent other than H or —OH at the 2′ position (2′ substituted nucleoside) or comprises a 2′ linked biradicle capable of forming a bridge between the 2′ carbon and a second carbon in the ribose ring, such as LNA (2′-4′ biradicle bridged) nucleosides.
Indeed, much focus has been given to developing 2′ sugar substituted nucleosides, and numerous 2′ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, the 2′ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. Examples of 2′ substituted modified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, and 2′-F-ANA nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl, Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937, Below in Scheme 1 are illustrations of some 2′ substituted modified nucleosides.
In relation to the present invention 2′ substituted sugar modified nucleosides does not include 2′ bridged nucleosides like LNA.
A “LNA nucleoside” is a 2′-modified nucleoside which comprises a biradical linking the C2′ and C4′ of the ribose sugar ring of said nucleoside (also referred to as a “2′-4′ bridge”), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.
Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.
Further non limiting, exemplary LNA nucleosides are disclosed in Scheme 2.
Particular LNA nucleosides are beta-D-oxy-LNA, 6′-methyl-beta-D-oxy LNA such as (S)-6′-methyl-beta-D-oxy-LNA (ScET) and ENA.
A particularly advantageous LNA is beta-D-oxy-LNA.
In some embodiments, the antisense oligonucleotide of the invention comprises or consists of Morpholino nucleosides (i.e. is a Morpholino oligomer and as a phosphorodiamidate Morphol no oligomer (PMO)). Splice modulating morpholino oligonucleotides have been approved for clinical use—see for example eteplirsen, a 30 nt morpholino oligonucleotide targeting a frame shift mutation in DMD, used to treat Duchenne muscular dystrophy. Morpholino oligonucleotides have nucleobases attached to six membered morpholine rings rather ribose, such as methylenemorpholine rings linked through phosphorodiamidate groups, for example as illustrated by the following illustration of 4 consecutive morpholino nucleotides (Scheme 3):
In some embodiments, morpholino oligonucleotides of the invention may be, for example 20-40 morpholino nucleotides in length, such as morpholino 25-35 nucleotides in length.
The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. Typically an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10%, at least 20% or more than 20%, of the initial rate determined when using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Examples 91-95 of WO01/23613 (hereby incorporated by reference). For use in determining RHase H activity, recombinant RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland.
DNA oligonucleotides are known to effectively recruit RNaseH, as are gapmer oligonucleotides which comprise a region of DNA nucleosides (typically at least 5 or 6 contiguous DNA nucleosides), flanked 5′ and 3′ by regions comprising 2′ sugar modified nucleosides, typically high affinity 2′ sugar modified nucleosides, such as 2-O-MOE and/or LNA. For effective modulation of splicing, degradation of the pre-mRNA is not desirable, and as such it is preferable to avoid the RNaseH degradation of the target. Therefore, the antisense oligonucleotides of the invention are not RNaseH recruiting gapmer oligonucleotide.
RNaseH recruitment may be avoided by limiting the number of contiguous DNA nucleotides in the oligonucleotide—therefore mixmers and totalmer designs may be used. Advantageously the antisense oligonucleotides of the invention, or the contiguous nucleotide sequence thereof, do not comprise more than 3 contiguous DNA nucleosides. Further, advantageously the antisense oligonucleotides of the invention, or the contiguous nucleotide sequence thereof, do not comprise more than 4 contiguous DNA nucleosides. Further advantageously, the antisense oligonucleotides of the invention, or contiguous nucleotide sequence thereof, do not comprise more than 2 contiguous DNA nucleosides.
For splice modulation it is often advantageous to use antisense oligonucleotides which do not recruit RNAaseH. As RNaseH activity requires a contiguous sequence of DNA nucleotides, RNaseH activity of antisense oligonucleotides may be achieved by designing antisense oligonucleotides which do not comprise a region of more than 3 or more than 4 contiguous DNA nucleosides. This may be achieved by using antisense oligonucleotides or contiguous nucleoside regions thereof with a mixmer design, which comprise sugar modified nucleosides, such as 2′ sugar modified nucleosides, and short regions of DNA nucleosides, such as 1, 2 or 3 DNA nucleosides. Mixmers are exemplified herein by every second design, wherein the nucleosides alternate between 1 LNA and 1 DNA nucleoside, e.g. LDLDLDLDLDLDLDLL, with 5′ and 3′ terminal LNA nucleosides, and every third design, such as LDDLDDLDDLDDLDDL, where every third nucleoside is a LNA nucleoside.
A totalmer is an antisense oligonucleotide or a contiguous nucleotide sequence thereof which does not comprise DNA or RNA nucleosides, and may for example comprise only 2′-O-MOE nucleosides, such as a fully MOE phosphorothioate, e.g. MMMMMMMMMMMMMMMMMMMM, where M=2′-O-MOE, which are reported to be effective splice modulators for therapeutic use.
Alternatively, a mixmer may comprise a mixture of modified nucleosides, such as MLMLMLMLMLMLMLMLMLML, wherein L=LNA and M=2′-O-MOE nucleosides.
Advantageously, the internucleoside nucleosides in mixmers and totalmers may be phosphorothioate, or a majority of nucleoside linkages in mixmers may be phosphorothioate. Mixmers and totalmers may comprise other internucleoside linkages, such as phosphodiester or phosphorodithioate, by way of example.
The antisense oligonucleotide of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequence of the oligonucleotide which is complementary to the target nucleic acid, such as a mixmer or totalmer region, and further 5′ and/or 3′ nucleosides. The further 5′ and/or 3′ nucleosides may or may not be complementary, such as fully complementary, to the target nucleic acid. Such further 5′ and/or 3′ nucleosides may be referred to as region D′ and D″ herein.
The addition of region D′ or D″ may be used for the purpose of joining the contiguous nucleotide sequence, such as the mixmer or totalmer, to a conjugate moiety or another functional group. When used for joining the contiguous nucleotide sequence with a conjugate moiety it can serve as a biocleavable linker. Alternatively, it may be used to provide exonuclease protection or for ease of synthesis or manufacture.
Region D′ or D″ may independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. The nucleotide adjacent to the F or F′ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these. The D′ or D″ region may serve as a nuclease susceptible biocleavable linker (see definition of linkers). In some embodiments the additional 5′ and/or 3′ end nucleotides are linked with phosphodiester linkages, and are DNA or RNA. Nucleotide based biocleavable linkers suitable for use as region D′ or D″ are disclosed in WO2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide. The use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO2015/113922, where they are used to link multiple antisense constructs within a single oligonucleotide.
In one embodiment the antisense oligonucleotide of the invention comprises a region D′ and/or D″ in addition to the contiguous nucleotide sequence which constitutes a mixmer or a totalmer.
In some embodiments the internucleoside linkage positioned between region D′ or D″ and the mixmer or totalmer region is a phosphodiester linkage.
The invention encompasses an antisense oligonucleotide covalently attached to at least one conjugate moiety. In some embodiments this may be referred to as a conjugate of the invention.
The term “conjugate” as used herein refers to an antisense oligonucleotide which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region). The conjugate moiety may be covalently linked to the antisense oligonucleotide, optionally via a linker group, such as region D′ or D″.
Oligonucleotide conjugates and their synthesis has also been reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S. T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.
In some embodiments, the conjugate moiety may comprise a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer or any combination thereof.
In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates (e.g. GalNAc), cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.
In some embodiments, the antisense oligonucleotide conjugate of the invention is a prodrug. Here the conjugate moiety may be cleaved off the nucleic acid molecule once the prodrug is delivered to the site of action, e.g. the target cell.
A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the antisense oligonucleotide directly or through a linking moiety (e.g. linker or tether), Linkers serve to covalently connect a third region, e.g. a conjugate moiety (Region C), to a first region, e.g. an oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A).
In some embodiments of the invention the conjugate or antisense oligonucleotide conjugate of the invention may optionally comprise a linker region (second region or region B and/or region Y) which is positioned between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).
Region B refers to biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. Conditions under which physiologically labile linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases. In one embodiment the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments the nuclease susceptible linker comprises between 1 and 5 nucleosides, such as DNA nucleoside(s) comprising at least two consecutive phosphodiester linkages. Phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195.
Region Y refers to linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an oligonucleotide (region A or first region). The region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. The antisense oligonucleotide conjugates of the present invention can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments the linker (region Y) is an amino alkyl, such as a C2-C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In some embodiments the linker (region Y) is a C6 amino alkyl group.
The invention provides for an antisense oligonucleotide according to the invention wherein the antisense oligonucleotide is in the form of a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the antisense oligonucleotides of the present invention.
In some embodiments, the pharmaceutically acceptable salt may be a sodium salt, a potassium salt or an ammonium salt.
The invention provides for a pharmaceutically acceptable sodium salt of the antisense oligonucleotide according to the invention, or the conjugate according to the invention.
The invention provides for a pharmaceutically acceptable potassium salt of the antisense oligonucleotide according to the invention, or the conjugate according to the invention.
The invention provides for a pharmaceutically acceptable ammonium salt of the antisense oligonucleotide according to the invention, or the conjugate according to the invention.
The invention provides for a pharmaceutical composition comprising the antisense oligonucleotide of the invention, or the conjugate of the invention, or the salt of the invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments, the nucleic acid molecule is used in the pharmaceutically acceptable diluent at a concentration of 50 to 300 μM solution.
Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990). WO 2007/031091 provides further suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in WO2007/031091.
The invention provides for a pharmaceutical composition comprising the antisense oligonucleotide of the invention, or the conjugate of the invention, and a pharmaceutically acceptable salt. For example, the salt may comprise a metal cation, such as a sodium salt, a potassium salt or an ammonium salt.
The invention provides for a pharmaceutical composition according to the invention, wherein the pharmaceutical composition comprises the antisense oligonucleotide of the invention or the conjugate of the invention, or the pharmaceutically acceptable salt of the invention; and an aqueous diluent or solvent.
In some embodiments, the antisense oligonucleotide of the invention, the conjugate of the invention, or pharmaceutically acceptable salt thereof is in a solid form, such as a powder, such as a lyophilized powder.
The antisense oligonucleotide of the invention, conjugate of the invention or salt of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
In one aspect the present invention provides a composition comprising an antisense oligonucleotide according to the invention or the conjugate according to the invention, or the salt according to the invention: and a diluent, solvent, carrier, salt and/or adjuvant.
The composition may be a pharmaceutical composition.
In a further aspect, the invention provides methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phophoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313).
In a further embodiment, the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach a conjugate moiety to the oligonucleotide.
In a further embodiment, a method is provided for manufacturing the composition of the invention, comprising mixing the oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
In one aspect, the invention includes an isolated XBP1Δ4 protein.
The isolated XBP1Δ4 protein may be a mammalian protein. In some embodiments the XBP1Δ4 protein may be a hamster, mouse or human protein.
In certain embodiments, the isolated XBP1Δ4 protein is a hamster protein and is encoded by SEQ ID NO 7.
In certain embodiments, the isolated XBP1Δ4 protein is a mouse protein and is encoded by SEQ ID NO 596.
In certain embodiments, the isolated XBP1Δ4 protein is a human protein and is encoded by SEQ ID NO 807.
The invention also contemplates fragments of the isolated XBP1Δ4 protein.
XBP1Δ4 mRNA
In one aspect, the invention includes an isolated mRNA encoding the isolated XBP1Δ4 protein of the invention.
The isolated XBP1Δ4 mRNA may be a mammalian protein. In some embodiments, the XBP1Δ4 mRNA may be a hamster, mouse or human mRNA.
In certain embodiments, the isolated XBP1Δ4 mRNA is a hamster mRNA and is encoded by SEQ ID NO 6.
In certain embodiments, the isolated XBP1Δ4 mRNA is a mouse mRNA and is encoded by SEQ ID NO 595.
In certain embodiments, the isolated XBP1Δ4 mRNA is a human mRNA and is encoded by SEQ ID NO 806.
The invention also contemplates fragments of the isolated XBP1Δ4 mRNA.
The present inventors have identified that compounds, which induce the expression of XBP1Δ4 in mammalian cells, are useful in enhancing the recombinant expression of heterologously expressed proteins in mammalian cells, especially of multimeric polypeptides, such as antibodies.
As explained above, XBP1s is a functionally active protein which functions to enhance correct protein folding. The inventors have surprisingly determined that an XBP1 splice variant, such as XBP1Δ4, can enhance the production of correctly folded proteins in recombinant polypeptide production methods.
In one aspect the invention provides a method for (recombinantly) producing a polypeptide comprising the steps of:
In one preferred embodiment, the cultivating comprises a pre- and a main-cultivating step, wherein at least the pre-cultivating step is performed in the presence of an oligonucleotide of the invention.
In certain embodiments, the method comprises the steps of:
In certain embodiments, the antisense oligonucleotide is added to a final concentration of at least about 5 μM, at least about 10 μM, at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 35 μM, at least about 40 μM, at least about 45 μM, at least about 50 μM or more. In one preferred embodiment, the antisense oligonucleotide is added to a final concentration of about 25 μM.
In certain embodiments, the propagating of the mammalian cell is performed at a starting cell density of at least about of 0.5*10E6 cells/mL, at least about of 1*10E6 cells/mL, at least about of 2*10E6 cells/mL, at least about of 3*10E6 cells/mL, at least about of 4*10E6 cells/mL, at least about of 5*10E6 cells/mL or more. In certain embodiments, the cultivation is performed at a starting cell density of 1*10E6 to 2*10E6 cells/mL.
In certain embodiments, the cultivation of the second cell population is performed at a starting cell density of at least about of 0.5*10E6 cells/mL, at least about of 1*10E6 cells/mL, at least about of 2*10E6 cells/mL, at least about of 3*10E6 cells/mL, at least about of 4*10E6 cells/mL, at least about of 5*10E6 cells/mL, at least about 10*10E6 cells/mL or more. In certain embodiments, the cultivation is performed at a starting cell density of 1*10E6 to 2*10E6 cells/mL.
In certain embodiments, the cell is a mammalian cell.
In certain embodiments, the cell is a hamster cell.
In certain embodiments, the cell is a CHO cell, such as a CHO-K1 cell. Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of therapeutic proteins, such as monoclonal antibodies.
In some embodiments, the cell may be a human cell
In some embodiments, the cell may be a neuronal cell or a brain cell.
In some embodiments, the cell may be in vitro. The in vitro cell may for example be a iPSC cell.
In certain embodiments, the polypeptide is a Fab, preferably a bispecific Fab, an Fc-region comprising fusion polypeptide, a human therapeutic polypeptide, or a cytokine.
In certain embodiments, the polypeptide is an antibody. Here the antibody may take any form, as discussed in the definition of “antibody” provided herein.
In certain embodiments, the method of the invention provides for an increase in protein yield by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 1000%, at least about 200%, at least about 300%, at least about 400%, at least about 500% or more, relative to the protein yield obtained in the absence of an antisense oligonucleotide of the invention.
In certain embodiments, the increase in yield represents an increase in the absolute amount of polypeptide. In other embodiments, the increase in yield represents an increase in the amount of correctly folded polypeptide. Herein a polypeptide can be defined as correctly folded either by viewing the structure of the polypeptide or by determining the polypeptide's activity.
The term ‘treatment’ as used herein refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic.
In one aspect, the invention relates to an antisense oligonucleotide, composition or pharmaceutical composition of the invention for use in medicine or therapy.
In some embodiments the therapy relates to the treatment or prevention of proteopathological disease.
In another aspect, the invention relates to use of an antisense oligonucleotide, composition or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of proteopathological disease.
In another aspect, the invention relates to a method for treating a proteopathological disease in a patient, the method comprising administering to the patient an antisense oligonucleotide, composition or pharmaceutical composition of the invention.
In certain embodiments, the invention relates to the treatment or prevention of proteopathological diseases. Proteopathological diseases are also known as proteopathies, proteinopathies, protein conformational disorders, or protein mis-folding diseases.
In certain embodiments, the proteopathological disease may be selected from prion diseases, tauopathies, synucleinopathies, amyloidosis, multiple system atrophy, TDP-43 pathologies and CAG repeat indications.
In certain embodiments, the proteopathological disease may be selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, Parkinson's disease, Autism, Hippocampal sclerosis dementia, Down syndrome, Huntington's disease, polyglutamine diseases, such as spinocerebellar ataxia 3, myopathies and Chronic Traumatic Encephalopathy.
In certain embodiments the prior disease may be Creutzfeldt-Jakob disease.
In certain embodiments the tauopathy may be Alzheimer's disease.
In certain embodiments the synucleinopathy may be Parkinson's disease.
In certain embodiments the TDP-43 pathology may be amyotrophic lateral sclerosis (ALS) frontotemporal lobar degeneration (FTLD).
In certain embodiments the CAG repeat indication may be spinocerebellar ataxics, including spinocerebellar ataxia type 1, Spinocerebellar ataxia type 2 (SCA2), and Spinocerebellar ataxia type 3 (SCA3, Machado-Joseph disease),
The compounds, antisense oligonucleotides, compositions, pharmaceutical compositions, proteins or nucleic acids of the present invention may be administered topically or enterally or parenterally (such as, intravenous, subcutaneous, or intra-muscular).
In certain embodiments it is the antisense nucleic acid or pharmaceutical composition which is administered for therapy.
In a preferred embodiment, the antisense oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion.
In one embodiment, the antisense nucleic acid or pharmaceutical composition are administered intravenously.
In another embodiment, the antisense nucleic acid or pharmaceutical composition is administered subcutaneously.
In some embodiments, the antisense nucleic acid or pharmaceutical composition of the invention is administered at a dose of 0.1-15 mg/kg, such as from 0.2-10 mg/kg, such as from 0.25-5 mg/kg. The administration can be once a week, every second week, every third week or even once a month.
1. An antisense oligonucleotide for use in the expression of a XBP1 splice variant in a cell which expresses XBP1, wherein the antisense oligonucleotide is 8-40 nucleotides in length and comprises a contiguous nucleotide sequence of 8-40 nucleotides in length which is complementary to a mammalian XBP1 pre-mRNA transcript.
2. The antisense oligonucleotide according to embodiment 1, wherein the XBP1 splice variant is a XBP1Δ4 variant.
3. The antisense oligonucleotide according to embodiment 1 or embodiment 2, wherein the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides of the hamster XBP1 pre-mRNA transcript (SEQ ID NO 1).
4. The antisense oligonucleotide according to embodiment 3, wherein the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides from nucleotides 2960-3113 of SEQ ID NO 1.
5. The antisense oligonucleotide according to embodiment 4, wherein the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides from nucleotides 2986-3018 of SEQ ID NO 1.
6. The antisense oligonucleotide according to embodiment 3, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 299, SEQ ID NO 301, SEQ ID NO 302, SEQ ID NO 304, SEQ ID NO 305, SEQ ID NO 306, SEQ ID NO 307, SEQ ID NO 308, SEQ ID NO 309, SEQ ID NO 310, SEQ ID NO 314, SEQ ID NO 316, SEQ ID NO 317, SEQ ID NO 318, SEQ ID NO 319, SEQ ID NO 323, SEQ ID NO 325, SEQ ID NO 327, SEQ ID NO 328, SEQ ID NO 330, SEQ ID NO 331, SEQ ID NO 332, SEQ ID NO 333, SEQ ID NO 334, SEQ ID NO 336, SEQ ID NO 337, SEQ ID NO 385, SEQ ID NO 386, SEQ ID NO 387, SEQ ID NO 388, SEQ ID NO 390, SEQ ID NO 391, SEQ ID NO 392, SEQ ID NO 393, SEQ ID NO 394, SEQ ID NO 395, SEQ ID NO 396 397, SEQ ID NO 398, SEQ ID NO 399, SEQ ID NO 401, SEQ ID NO 402, SEQ ID NO 419, SEQ ID NO 431, SEQ ID NO, SEQ ID NO 432, SEQ ID NO 433, SEQ ID NO 434, SEQ ID NO 438, SEQ ID NO 439, SEQ ID NO 440, SEQ ID NO 441 SEQ ID NO 442, SEQ ID NO 449, SEQ ID NO 484, SEQ ID NO 485, SEQ ID NO 486, SEQ ID NO 487, SEQ ID NO 488, SEQ ID NO 489, SEQ ID NO 490, SEQ ID NO 491, SEQ ID NO 492, SEQ ID NO 493, SEQ ID NO 494, SEQ ID NO 495, SEQ ID NO 496, SEQ ID NO 497, SEQ ID NO 498, SEQ ID NO 499, SEQ ID NO 500, SEQ ID NO 501, SEQ ID NO 502, SEQ ID NO 503, SEQ ID NO 505, SEQ ID NO 506, SEQ ID NO 507, SEQ ID NO 508, SEQ ID NO 509. SEQ ID NO 510, SEQ ID NO 511, SEQ ID NO 512, SEQ ID NO 513, SEQ ID NO 515, SEQ ID NO 517, SEQ ID NO 520, SEQ ID NO 572, SEQ ID NO 573, SEQ ID NO 576, SEQ ID NO 577, SEQ ID NO 588 and SEQ ID NO 589.
7. The antisense oligonucleotide according to embodiment 6, wherein the contiguous nucleotide sequence is selected from the group consisting of SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 46. SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 110, SEQ ID NO 111. SEQ ID NO 128, SEQ ID NO 140, SEQ ID NO 141, SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151, SEQ ID NO 158, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 198, SEQ ID NO 199, SEQ ID NO 200, SEQ ID NO 201, SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 204, SEQ ID NO 205, SEQ ID NO 206, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211, SEQ ID NO 212, SEQ ID NO 214, SEQ ID NO 215, SEQ ID NO 216, SEQ ID NO 217, SEQ ID NO 218, SEQ ID NO 219, SEQ ID NO 220, SEQ ID NO 221, SEQ ID NO 222, SEQ ID NO 224, SEQ ID NO 226, SEQ ID NO 229, SEQ ID NO 281, SEQ ID NO 282, SEQ ID NO 285, SEQ ID NO 286, SEQ ID NO 297 and SEQ ID NO 298.
8. The antisense oligonucleotide according to embodiment 3, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 305, SEQ ID NO 307, SEQ ID NO 314, SEQ ID NO 315, SEQ ID NO 316, SEQ ID NO 317, SEQ ID NO 319, SEQ ID NO 331, SEQ ID NO 332, SEQ ID NO 392, SEQ ID NO 394, SEQ ID NO 395, SEQ ID NO 440, SEQ ID NO 492, SEQ ID NO 497, SEQ ID NO 498, SEQ ID NO 499, SEQ ID NO 500, SEQ ID NO 501, SEQ ID NO 502, SEQ ID NO 513 and SEQ ID NO 576.
9. The antisense oligonucleotide according to embodiment 8, wherein the contiguous nucleotide sequence is selected from the group consisting of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 101, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 149, SEQ ID NO 201, SEQ ID NO 206, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211, SEQ ID NO 222 and SEQ ID NO 285.
10. The antisense oligonucleotide according to embodiment 3, wherein the contiguous nucleotide sequence is complementary to SEQ ID NO 314 or SEQ ID NO 315.
11. The antisense oligonucleotide according to embodiment 10, wherein the contiguous nucleotide sequence is SEQ ID 23 or SEQ ID 24.
12. The antisense oligonucleotide according to embodiment 1 or embodiment 2, wherein the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides from the mouse XBP1 pre-mRNA transcript (SEQ ID NO 590).
13. The antisense oligonucleotide according to embodiment 12, wherein the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides from nucleotides 3560-3783 of SEQ ID NO 590.
14. The antisense oligonucleotide according to embodiment 12, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 699, SEQ ID NO 700, SEQ ID NO 703, SEQ ID NO 710, SEQ ID NO 713, SEQ ID NO 724, SEQ ID NO 729, SEQ ID NO 739, SEQ ID NO 743, SEQ ID NO 744, SEQ ID NO 745, SEQ ID NO 749, SEQ ID NO 750, SEQ ID NO 751, SEQ ID NO 752, SEQ ID NO 753, SEQ ID NO 754, SEQ ID NO 755, SEQ ID NO 756, SEQ ID NO 757, SEQ ID NO 758, SEQ ID NO 759, SEQ ID NO 760, SEQ ID NO 761, SEQ ID NO 762, SEQ ID NO 763, SEQ ID NO 773, SEQ ID NO 776, SEQ ID NO 778, SEQ ID NO 781, SEQ ID NO 783, SEQ ID NO 784, SEQ ID NO 785, SEQ ID NO 787, SEQ ID NO 789, SEQ ID NO 790, SEQ ID NO 791, SEQ ID NO 792, SEQ ID NO 793, SEQ ID NO 794, SEQ ID NO 795, SEQ ID NO 796, SEQ ID NO 797, SEQ ID NO 798, SEQ ID NO 799 and SEQ ID NO 800.
15. The antisense oligonucleotide according to embodiment 14, wherein the contiguous nucleotide sequence is selected from the group consisting of SEQ ID NO 597, SEQ ID NO 598, SEQ ID NO 601, SEQ ID NO 608, SEQ ID NO 611, SEQ ID NO 622, SEQ ID NO 627, SEQ ID NO 637, SEQ ID NO 641, SEQ ID NO 642, SEQ ID NO 643, SEQ ID NO 647, SEQ ID NO 648, SEQ ID NO 649, SEQ ID NO 650, SEQ ID NO 651, SEQ ID NO 652, SEQ ID NO 653, SEQ ID NO 654, SEQ ID NO 655, SEQ ID NO 656, SEQ ID NO 657, SEQ ID NO 658, SEQ ID NO 659, SEQ ID NO 660, SEQ ID NO 661, SEQ ID NO 671, SEQ ID NO 674, SEQ ID NO 676, SEQ ID NO 679, SEQ ID NO 681, SEQ ID NO 682, SEQ ID NO 683, SEQ ID NO 685, SEQ ID NO 687, SEQ ID NO 688, SEQ ID NO 689, SEQ ID NO 690, SEQ ID NO 691, SEQ ID NO 692, SEQ ID NO 693, SEQ ID NO 694, SEQ ID NO 695, SEQ ID NO 696, SEQ ID NO 697 and SEQ ID NO 698.
16. The antisense oligonucleotide according to embodiment 12, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 710, SEQ ID NO 754, SEQ ID NO 756, SEQ ID NO 757, SEQ ID NO 758, SEQ ID NO 759, SEQ ID NO 760, SEQ ID NO 791, SEQ ID NO 792, SEQ ID NO 794, SEQ ID NO 795 and SEQ ID NO 797.
17. The antisense oligonucleotide according to embodiment 16, wherein the contiguous nucleotide sequence is selected from the group consisting of SEQ ID NO 608, SEQ ID NO 652, SEQ ID NO 654, SEQ ID NO 655, SEQ ID NO 656, SEQ ID NO 657, SEQ ID NO 658, SEQ ID NO 689, SEQ ID NO 690, SEQ ID NO 692, SEQ ID NO 693 and SEQ ID NO 695.
18. The antisense oligonucleotide according to embodiment 1 or embodiment 2, wherein the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides of the human XBP1 pre-mRNA transcript (SEQ ID NO 801).
19. The antisense oligonucleotide according to embodiment 18, wherein the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides from nucleotides 4338-4563 of SEQ ID NO 801.
20. The antisense oligonucleotide according to embodiment 18, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 947, SEQ ID NO 948, SEQ ID NO 949, SEQ ID NO 950, SEQ ID NO 951 and SEQ ID NO 988.
21. The antisense oligonucleotide according to embodiment 21, wherein the contiguous nucleotide sequence is selected from the group consisting of SEQ ID NO 854, SEQ ID NO 855, SEQ ID NO 856, SEQ ID NO 857, SEQ ID NO 858 and SEQ ID NO 895.
22. The antisense oligonucleotide according to embodiment 18, wherein the contiguous nucleotide sequence is complementary to SEQ ID NO 951.
23. The antisense oligonucleotide according to embodiment 22, wherein the contiguous nucleotide sequence is SEQ ID NO 858.
24. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide or contiguous nucleotide sequence thereof is fully complementary to a mammalian XBP1 pre-mRNA transcript.
25. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the contiguous nucleotide sequence is at least 12 nucleotides in length.
26. The antisense oligonucleotide according to embodiment 25, wherein the contiguous nucleotide sequence is 12-16 or 12-18 nucleotides in length.
27. The antisense oligonucleotide according embodiment 25, wherein the contiguous nucleotide sequence is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
28. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the contiguous nucleotide sequence is the same length as the antisense oligonucleotide.
29. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide is isolated, purified or manufactured.
30. The antisense oligonucleotide according to any one of the preceding embodiment, wherein the antisense oligonucleotide or contiguous nucleotide sequence thereof comprises one or more modified nucleotides or one or more modified nucleosides.
31. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide or contiguous nucleotide sequence thereof comprises one or more modified nucleosides, such as one or more modified nucleotides independently selected from the group consisting of 2′-O-alkyl-RNA; 2′-O-methyl RNA (2′-OMe); 2′-alkoxy-RNA; 2′-O-methoxyethyl-RNA (2′-MOE); 2′-amino-DNA; 2′-fluro-RNA; 2′-fluoro-DNA; arabino nucleic acid (ANA); 2′-fluoro-ANA; bicyclic nucleoside analog (LNA); or any combination thereof.
32. The antisense oligonucleotide according to embodiment 30 or embodiment 31, wherein one or more of the modified nucleosides is a sugar modified nucleoside.
33. The antisense oligonucleotide according to any one of embodiments 30 to 32, wherein one or more of the modified nucleosides comprises a bicyclic sugar.
34. The antisense oligonucleotide according to any one of embodiments 30 to 32, wherein one or more of the modified nucleosides is an affinity enhancing 2′ sugar modified nucleoside.
35. The antisense oligonucleotide according to any one of embodiments 30 to 34, wherein one or more of the modified nucleosides is an LNA nucleoside, such as one or more beta-D-oxy LNA nucleosides.
36. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide or contiguous nucleotide sequence thereof comprises one or more 5′-methyl-cytosine nucleobases.
37. The antisense oligonucleotide according to any one of the preceding embodiments, wherein one or more of the internucleoside linkages within the contiguous nucleotide sequence of the antisense oligonucleotide are modified.
38. The antisense oligonucleotide according to embodiment 37, wherein at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the internucleoside linkages are modified.
39. The antisense oligonucleotide according to embodiment 37 or embodiment 38, wherein the one or more modified internucleoside linkages comprise a phosphorothioate linkage.
40. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide is a morpholino modified antisense oligonucleotide.
41. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide or contiguous nucleotide sequence thereof is or comprises an antisense oligonucleotide mixmer or totalmer.
42. An antisense oligonucleotide according to any one of the preceding embodiments covalently attached to at least one conjugate moiety.
43. The antisense oligonucleotide according to embodiment 42, wherein the conjugate moiety comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer or any combination thereof.
44. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide is in the form of a pharmaceutically acceptable salt.
45. The antisense oligonucleotide according to embodiment 44, wherein the salt is a sodium salt, a potassium salt or an ammonium salt.
46. A composition comprising the antisense oligonucleotide according to any one of the preceding embodiments.
47. A pharmaceutical composition comprising the antisense oligonucleotide according to any one of embodiments 1 to 45 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
48. The pharmaceutical composition according to embodiment 47, wherein the pharmaceutical composition comprises an aqueous diluent or solvent, such as phosphate buffered saline.
49. An isolated XBP1Δ4 protein.
50. The isolated XBP1Δ4 protein according to embodiment 49, wherein the protein comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 596 or SEQ ID NO 807.
51. An isolated mRNA encoding the XBP1Δ4 protein according to embodiment 49 or embodiment 50
52. The isolated mRNA according to embodiment 51, comprising the sequence, of SEQ ID NO: 6, SEQ ID NO: 595 or SEQ ID NO: 806.
53. A method for producing a polypeptide comprising the steps of:
54. The method according to embodiment 53, comprising the steps of;
55. The method according to embodiment 53 or embodiment 54, wherein the antisense oligonucleotide is added to a final concentration of 25 μM or more.
56. The method according to any one of embodiments 53 to 55, wherein the propagating and/or the cultivating is with a starting cell density of 1*10E6 to 2*10E6 cells/mL.
57. The method according to embodiment 56, wherein the starting cell density is about 2*10E6 cells/mL.
58. The method according to any one of embodiments 53 to 57, wherein the mammalian cell is a CHO cell.
59. The method according to any one of embodiments 53 to 58, wherein the polypeptide is an antibody.
60. An antisense oligonucleotide according to any one of embodiments 1 to 45, a composition according to embodiment 46 or a pharmaceutical composition according to embodiment 47 or embodiment 48 for use in medicine.
61. An antisense oligonucleotide according to any one of embodiments 1 to 45, a composition according to embodiment 46 or a pharmaceutical composition according to embodiment 47 or embodiment 48 for use in the treatment of patient with a proteopathological disease.
62. The antisense oligonucleotide for use according to embodiment 61, wherein the proteopathological disease has TDP-43 pathology.
63. The antisense oligonucleotide for use according to embodiment 61 or embodiment 62, wherein the proteopathological disease is motor neuron disease or frontotemporal lobar degeneration.
64. The use of an antisense oligonucleotide according to any one of embodiments 1 to 45, a composition according to embodiment 46 or a pharmaceutical composition according to embodiment 47 or embodiment 48 in the manufacture of a medicament for the treatment of proteopathological disease.
65. The use according to embodiment 64, wherein the disease has disease TDP-43 pathology.
66. The use according to embodiment 64 or embodiment 65, wherein the disease is motor neuron disease or frontotemporal lobar degeneration.
67. A method for treating a proteopathological disease in a patient, the method comprising administering to the patient an antisense oligonucleotide according to any one of embodiments 1 to 45, a composition according to embodiment 46 or a pharmaceutical composition according to embodiment 47 or embodiment 48.
68. The method according to embodiment 67, wherein the disease has TDP-43 pathology.
69. The method according to embodiment 67 or embodiment 68, wherein the disease is motor neuron disease or frontotemporal lobar degeneration.
Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, (1989). The molecular biological reagents were used according to the manufacturer's instructions.
Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).
DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).
The EMBOSS (European Molecular Biology Open Software Suite) software package and Invitrogen's Vector NTI version 11.5 or Geneious prime were used for sequence creation, mapping, analysis, annotation and illustration.
All commercial chemicals, antibodies and kits were used as provided according to the manufacturer's protocol if not stated otherwise.
The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al. Protein Science 4 (1995) 2411-1423.
The concentration of antibodies in cell culture supernatants was estimated by immunoprecipitation with protein A agarose-beads (Roche Diagnostics GmbH, Mannheim, Germany). Therefore, 60 μL protein A Agarose beads were washed three times in TBS-NP40 (50 mM Tris buffer, pH 7.5, supplemented with 150 mM NaCl and 1% Nonidet-P40). Subsequently, 1-15 mL cell culture supernatant was applied to the protein A Agarose beads pre-equilibrated in TBS-NP40. After incubation for at 1 hour at room temperature the beads were washed on an Ultrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twice with 0.5 mL 2× phosphate buffered saline (2×PBS, Roche Diagnostics GmbH, Mannheim, Germany) and briefly four times with 0.5 mL 100 mM Na-citrate buffer (pH 5.0). Bound antibody was eluted by addition of 35 μl NuPAGE® LDS sample buffer (Invitrogen). Half of the sample was combined with NuPAGE® sample reducing agent or left unreduced, respectively, and heated for 10 min at 70° C. Consequently, 5-30 μl were applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE gel (Invitrogen) (with MOPS buffer for non-reduced SOS-PAGE and MES buffer with NuPAGE® antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue.
The concentration of the antibodies in cell culture supernatants was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies that bind to protein A were applied to an Applied Biosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH 7.4 and eluted with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The eluted antibody was quantified by UV absorbance and integration of peak areas. A purified standard IgG1 antibody served as a standard.
Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by Sandwich-IgG-ELISA. Briefly, StreptaWell™ High Bind Streptavidin A-96 well microtiter plates (Roche Diagnostics GmbH, Mannheim, Germany) were coated with 100 μL/well biotinylated anti-human IgG capture molecule F(ab′)2<h-Fcγ> BI (Dianova) at 0.1 μg/mL for 1 hour at room temperature or alternatively overnight at 4° C. and subsequently washed three times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). Thereafter, 100 μL/well of a dilution series in PBS (Sigma) of the respective antibody containing cell culture supernatants was added to the wells and incubated for 1-2 hour on a shaker at room temperature. The wells were washed three times with 200 μL/well PBST and bound antibody was detected with 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detection antibody by incubation for 1-2 hours on a shaker at room temperature. Unbound detection antibody was removed by washing three times with 200 μL/well PBST. The bound detection antibody was detected by addition of 100 μL ABTS/well followed by incubation. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).
CHO host cells were cultivated at 37° C. in a humidified incubator with 85% humidity and 5% CO2, They were cultivated in a proprietary DMEM/F12-based medium containing 300 μg/ml Hygromycin B and 4 μg/ml of a second selection marker. The cells were split every 3 or 4 days at a concentration of 0.3×10E6 cells/ml in a total volume of 30 ml. For the cultivation 125 ml non-baffle Erlenmeyer shake flasks were used. Cells were shaken at 150 rpm with a shaking amplitude of 5 cm. The cell count was determined with Cedex HiRes Cell Counter (Roche). Cells were kept in culture until they reached an age of 60 days.
Transformation 10-Beta Competent E. coli Cells
For transformation, the 10-beta competent E. coli cells were thawed on ice. After that, 2 μl of plasmid DNA were pipetted directly into the cell suspension. The tube was flicked and put on ice for 30 minutes. Thereafter, the cells were placed into the 42° C.-warm thermal block and heat-shocked for exactly 30 seconds. Directly afterwards, the cells were chilled on ice for 2 minutes. 950 μl of NEB 10-beta outgrowth medium were added to the cell suspension. The cells were incubated under shaking at 37° C. for one hour. Then, 50-100 μl were pipetted onto a pre-warmed (37° C.) LB-Amp agar plate and spread with a disposable spatula. The plate was incubated overnight at 37° C. Only bacteria, which have successfully incorporated the plasmid, carrying the resistance gene against ampicillin, can grow on these plates. Single colonies were picked the next day and cultured in LB-Amp medium for subsequent plasmid preparation.
Cultivation of E. coli was done in LB-medium, short for Luria Bertani, which was spiked with 1 ml/L 100 mg/ml ampicillin resulting in an ampicillin concentration of 0.1 mg/ml. For the different plasmid preparation quantities, the following amounts were inoculated with a single bacterial colony.
E. coli cultivation volumes
For Mini-Prep, a 96-well 2 ml deep-well plate was filled with 1.5 ml LB-Amp medium per well. The colonies were picked and the toothpick was tuck in the medium. When all colonies were picked, the plate closed with a sticky air porous membrane. The plate was incubated in a 37° C. incubator at a shaking rate of 200 rpm for 23 hours.
For Mini-Preps a 15 ml-tube (with a ventilated lid) was filled with 3.6 ml LB-Amp medium and equally inoculated with a bacterial colony. The toothpick was not removed but left in the tube during incubation. Like the 96-well plate, the tubes were incubated at 37° C., 200 rpm for 23 hours.
For Maxi-Prep 200 ml of LB-Amp medium were filled into an autoclaved glass 1 L Erlenmeyer flask and inoculated with 1 ml of bacterial day-culture, which was roundabout 5 hours old. The Erlenmeyer flask was closed with a paper plug and incubated at 37° C., 200 rpm for 16 hours.
For Mini-Prep, 50 μl of bacterial suspension were transferred into a 1 mi deep-well plate. After that, the bacterial cells were centrifuged down in the plate at 3000 rpm, 4° C. for 5 min. The supernatant was removed and the plate with the bacteria pellets placed into an EpMotion. After approx. 90 minutes, the run was done and the eluted plasmid-DNA could be removed from the EpMotion for further use.
For Mini-Prep, the 15 ml tubes were taken out of the incubator and the 3.6 ml bacterial culture split into two 2 ml Eppendorf tubes. The tubes were centrifuged at 6,800×g in a table-top microcentrifuge for 3 minutes at room temperature. After that, Mini-Prep was performed with the Qiagen QIAprep Spin Miniprep Kit according to the manufacturer's instructions. The plasmid DNA concentration was measured with Nanodrop.
Maxi-Prep was performed using the Macherey-Nagel NucleoBond® Xtra Maxi EF Kit according to the manufacturer's instructions. The DNA concentration was measured with Nanodrop.
The volume of the DNA solution was mixed with the 2.5-fold volume ethanol 100%. The mixture was incubated at −20° C. for 10 min. Then the DNA was centrifuged for 30 min, at 14,000 rpm, 4° C. The supernatant was carefully removed and the pellet washed with 70% ethanol. Again, the tube was centrifuged for 5 min. at 14,000 rpm, 4° C. The supernatant was carefully removed by pipetting and the pellet dried. When the ethanol was evaporated, an appropriate amount of endotoxin-free water was added. The DNA was given time to re-dissolve in the water overnight at 4° C. A small aliquot was taken and the DNA concentration was measured with a Nanodrop device.
Antibodies were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 2.8 followed by immediate neutralization. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine buffer comprising 150 mM NaCl (pH 6.0). Monomeric antibody fractions were pooled, concentrated (if required) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at −20° C. or −80° C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.
The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturers instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer was used.
Purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA). Therefore, 5 μl of antibody solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according manufacturer's instructions and analyzed on Labchip GXII system using a HT Protein Express Chip. Data were analyzed using Labchip GX Software.
Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2PO4/K2HPO4 buffer (pH 7.5) on an Dionex Ultimate® system (Thermo Fischer Scientific), or to a Superdex 200 column (GE Healthcare) in 2×PBS on a Dionex HPLC-System. The eluted antibody was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.
This section describes the characterization of the bispecific antibodies with emphasis on their correct assembly. The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact antibody and in special cases of the deglycosylated/limited LysC digested antibody.
The antibodies were deglycosylated with N-Glycosidase F in a phosphate or Tris buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/ml. The limited LysC (Roche Diagnostics GmbH, Mannheim, Germany) digestions were performed with 100 μg deglycosylated antibody in a Tris buffer (pH 8) at room temperature for 120 hours, or at 37° C. for 40 min, respectively. Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).
CHOK1 cells were obtained from the ATCC cell bank, and were grown and maintained according to ATCC guidelines. 40 ASOs complementary to a region around exon 4 of the XBP1 mRNA NM_001244047.1 were tested for the ability to induce exon skipping of exon 4.
5000 cells CHOK1 cells were seeded in a 96 well plate, 6 hours later the ASOs were added directly to the cell medium at a final concentration of 5 μM and 25 μM. Cells were cultivated and harvested after 6 days and total RNA was isolated using the RNeasy 96 well kit from Qiagen according to manufacturer's instructions.
cDNA was generated using the iScript™ Advanced cDNA Synthesis Kit for RT-qPCR from Biorad. Relative mRNA expression was measured by droplet digital PCR using the QX200 ddPCR system from Biorad along with the automated droplet generator AutoDG from Biorad.
PCR was performed using the ddPCR supermix for probes (no UTP) from Biorad according to manufacturer's instructions.
The following primers and probes were used to measure the amount of mRNAs with exon skipping of exon 4 (XBP1 Δ 4 assay) and the amount of mRNA with normal joining of exon 4 and 5 (XBP1 WT) both purchased from IDT technologies. The XBP1 WT assay detected both the IRE-1 processed and unprocessed mRNA.
Data was analyzed using the QuantaSoft™ Analysis Pro software from Blared. The percentile of mRNAs containing skipping of exon 4, was calculated by (concΔ4/(concΔ14+concWT))*100. The normal percentile of mRNA with exon 4 skipping was calculated from the average of 14 control wells treated with PBS only. The average of PBS wells was 0.6%. The data are shown in Table 2.
CHOK1 cells were obtained from the ATCC cell bank, and were grown and maintained according to ATCC guidelines. 251 ASOs complementary to a region around exon 4 of the XBP1 mRNA NM_001244047.1 were tested for the ability to induce exon skipping of exon 4.
3000 CHOK1 cells were seeded in a 96 well plate, 24 hours later the ASOs were added directly to the cell medium at a final concentration of 5 μM and 25 μM. Cells were harvested after 6 days and total RNA was isolated using the RNeasy 96 well kit from Qiagen according to manufactures instructions.
cDNA was generated using the Script™ Advanced cDNA Synthesis Kit for RT-qPCR from Biorad. Relative mRNA expression was measured by droplet digital PCR using the QX200 ddPCR™ system from Biorad along with the automated droplet generator AutoDG from Biorad.
PCR was performed using the ddPCR supermix for probes (no UTP) from Biorad according to manufactories instructions.
The following primers and probes were used to measure the amount of mRNAs with exon skipping of exon4 (XBP1Δ4 assay) and the amount of mRNA with normal joining of exon 4 and 5 (XBP1 WT) both purchased from IDT technologies. The XBP1 WT assay detected both the IRE-1 processed and unprocessed mRNA.
Data was analyzed using the QuantaSoft™ Analysis Pro software from Biorad. The percentile of mRNAs containing skipping of exon 4, was calculated by (concΔ4/(concΔ4+concWT))*100. The normal percentile of mRNA with exon 4 skipping was calculated from the average of 170 control wells treated with PBS only. The average of PBS wells were 0.1%. The data is shown in Table 3.
Ltk-11 (ATCC® CRL-10422™) cells were obtained from the ATCC cell bank, and were grown and maintained according to ATCC guidelines. 102 ASOs complementary to a region around exon 4 of the XBP1 mRNA NM_013842.3 (SeqID 2) were tested for the ability to induce exon skipping of exon 4.
2000 cells LTK cells were seeded in a 96 well plate, 24 hours later the ASOs were added directly to the cell medium at a final concentration of 5 uM and 25 uM. Cells were harvested after 3 days and total RNA was isolated using the RNeasy 96 well kit from Qiagen according to manufactures instructions.
cDNA was generated using the iScrip™ Advanced cDNA Synthesis Kit for RT-qPCR from Biorad. Relative mRNA expression was measured by droplet digital PCR using the QX200 ddPCR system from Biorad along with the automated droplet generator AutoDG from Biorad. PCR was performed using the ddPCR supermix for probes (no UTP) from biorad according to manufactories instructions.
The following primers and probes were used to measure the amount of mRNAs with exon skipping of exon4 (XBP1 delta 4 assay) and the amount of mRNA with normal joining of exon 4 and 5 (XBP1 WT) both purchased from IDT technologies. The XBP1 WT assay detected both the IRE-1 processed and unprocessed mRNA.
Data was analyzed using the QuantaSoft™ Analysis Pro software from Biorad. The percentile of mRNAs containing skipping of exon 4, was calculated by (conc delta 4/(conc delta 4+concWT))*100. The normal percentile of mRNA with exon 4 skipping was calculated from the average of 61 control wells treated with PBS only. The average of PBS wells were 0.37% with a standard deviation of 0.17. The data is shown in Table 4.
A459 cells were obtained from the ATCC cell bank, and were grown and maintained according to ATCC guidelines. 100 ASOs complementary to a region around exon 4 of the XBP1 mRNA NM_005080.4 (SeqID 2) were tested for the ability to induce exon skipping of exon 4.
4000 A549 cells were seeded in a 96 well plate, 24 hours later the ASOs were added directly to the cell medium at a final concentration of 25 M. Cells were harvested after 3 days and total RNA was isolated using the RNeasy 96 well kit from Qiagen according to manufactures instructions.
cDNA was generated using the iScript™ Advanced cDNA Synthesis Kit for RT-qPCR from Biorad. Relative mRNA expression was measured by droplet digital PCR using the QX200 ddPCR system from Biorad along with the automated droplet generator AutoDG from Biorad. PCR was performed using the ddPCR supermix for probes (no UTP) from biorad according to manufactories instructions.
The following primers and probes were used to measure the amount of mRNAs with exon skipping of exon4 (XBP14 assay) and the amount of mRNA with normal joining of exon 4 and 5 (XBP1 WT) both purchased from IDT technologies. The XBP1 WT assay detected both the IRE-1 processed and unprocessed mRNA.
Data was analyzed using the QuantaSoft™ Analysis Pro software from Biorad. The percentile of mRNAs containing skipping of exon 4, was calculated by (concΔ4/(concΔ4+concWT))*100. The normal percentile of mRNA with exon 4 skipping was calculated from the average of 40 control wells treated with PBS only. The average of PBS wells was 0.03% with a standard deviation of 0.05. The data is shown in Table 5.
In general, to construct the plasmids for RMCE, the respective expression cassettes for the antibody light chain and heavy chain were cloned into a first vector backbone flanked by L3 and LoxFas sequences, and a second vector flanked by LoxFas and 2L sequences and also further including a selectable marker. A Cre recombinase plasmid (see, e.g., Wong, E. T., et al., Nucl. Acids Res. 33 (2005) e147; O'Gorman, S., et al., Proc. Natl. Acad. Sci. USA 94 (1997) 14602-14607) was used for all RMCE processes.
The cDNAs encoding the respective polypeptides were generated by gene synthesis (Geneart, Life Technologies Inc.). The synthesized cDNAs and backbone-vectors were digested with HindIII-HF and EcoRI-HF (NEB) at 37° C. for 1 h and separated by agarose gel electrophoresis. The bands comprising the DNA-fragment of the insert and backbone, respectively, were cut out from the agarose gel and extracted by QIAquick Gel Extraction Kit (Qiagen). The purified insert and backbone fragment was ligated via the Rapid Ligation Kit (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's protocol with an Insert/Backbone ratio of 3:1. The ligation approach was then transformed into competent E. coli DH5a via heat shock and incubated for 1 h at 37° C., Thereafter the cells were plated out on agar plates with ampicillin for selection. Plates were incubated at 37° C. overnight.
On the following day clones were picked and incubated overnight at 37° C. under shaking for the Mini or Maxi-Preparation, which was performed with the EpMotion® 5075 (Eppendorf) or with the QIAprep Spin Mini-Prep Kit (Qiagen)/NucleoBond Xtra Maxi EF Kit (Macherey & Nagel), respectively. All constructs were sequenced to ensure correctness of the sequences.
In the second cloning step, the generated vectors were digested with KpnI-HF/SalI-HF and SalI-HF/MfeI-HF with the same conditions as outlined above. The respective RMCE (TI) backbone vector was digested with KpnI-HF and MfeI-HF. Separation and extraction was performed as described above. Ligation of the purified insert and backbone was performed using T4 DNA Ligase (NEB) following the manufacturer's protocol with an Insert/Insert/Backbone ratio of 1:1:1 overnight at 4° C. Thereafter ligase was inactivated at 65° C. for 10 min. The following steps were performed as described above.
CHO TI host cells comprising a GFP expression cassette in the TI landing site were propagated in disposable 125 ml vented shake flasks under standard humidified conditions (95% rH, 37° C., and 5% CO2) at a constant agitation rate of 150 rpm in a DMEM/F12-based medium. Every 3-4 days the cells were seeded with a concentration of 3×10E5 cells/ml in chemically defined medium containing selection marker 1 and selection marker 2 in effective concentrations. Density and viability of the cultures were measured with a Cedex HiRes cell counter (F, Hoffmann-La Roche Ltd. Basel, Switzerland).
For stable transfection, equimolar amounts of first and second vector generated according to Example 5 were mixed. 1 μg Cre encoding nucleic acid was added per 5 μg of the mixture, i.e. 5 μg Cre expression plasmid or Cre mRNA was added to 25 μg of the vector mixture.
Two days prior to transfection TI host cells were seeded in fresh medium with a density of about 4×10E5 cells/ml, Transfection was performed with the Nucleofector device using the Nucleofector Kit V (Lonza, Switzerland), according to the manufacturer's protocol. 3×10E7 cells were transfected with a total of 30 μg nucleic acid mixture, i.e. with 30 μg plasmid (5 μg Cre plasmid and 25 μg vector mixture). After transfection, the cells were seeded in 30 ml medium without selection agents.
On day 5 after seeding the cells were centrifuged and transferred at a cell density of 6×10E5 cells/ml to 80 mL chemically defined medium containing selection agent 1 and selection agent 2 at effective concentrations for selection of recombinant cells. The cells were incubated at 37° C., 150 rpm, 5% CO2, and 85% humidity from this day on without splitting. Cell density and viability of the culture was monitored regularly. When the viability of the culture started to increase again, the concentrations of selection agents 1 and 2 were reduced to about half the amount used before. In more detail, to promote the recovering of the cells, the selection pressure was reduced if the viability is >40% and the viable cell density (VCD) is >0.5×10E6 cells/mL. Therefore, 4×10E5 cells/ml were centrifuged and re-suspended in 40 ml selection media II (chemically-defined medium, ½ selection marker 1 & 2). The cells were incubated with the same conditions as before and also not splitted.
Ten days after starting selection, the success of RMCE was checked by flow cytometry measuring the expression of intracellular GFP and extracellular heterologous polypeptide sticking to the cell surface. An APC antibody (allophycocyanin-labeled F(ab′)2 Fragment goat anti-human IgG) against human antibody light and heavy chain was used for FACS staining. Flow cytometry was performed with a BD FACS Canto II flow cytometer (BD, Heidelberg, Germany). Ten thousand events per sample were measured. Living cells were gated in a plot of forward scatter (FSC) against side scatter (SSC). The live cell gate was defined with non-transfected TI host cells and applied to all samples by employing the FlowJo™ 7.6.5 EN software (TreeStar, Olten, Switzerland). Fluorescence of GFP was quantified in the FITC channel (excitation at 488 nm, detection at 530 nm). Antibody was measured in the APC channel (excitation at 645 nm, detection at 660 nm). Parental CHO cells, i.e. those cells used for the generation of the TI host cell, were used as a negative control with regard to GFP and antibody expression. Fourteen days after the selection had been started, the viability exceeded 90% and selection was considered as complete.
FACS analysis was performed to check the transfection and RMCE efficiency. 4×10E5 cells of the transfected approaches were centrifuged (1200 rpm, 4 min.) and washed twice with 1 mL PBS. After the washing steps with PBS the pellet was re-suspended in 400 μL PBS and transferred into FACS tubes (Falcon 8 Round-Bottom Tubes with cell strainer cap; Corning). The measurement was performed with a FACS Canto II and the data were analyzed by the software FlowJo™.
All fed-batch cultures were performed in shake flasks or Ambr®15 vessels (Sartorius Stedim) with the same proprietary serum-free, chemically defined medium and under the same cultivation and feeding conditions.
The recombinant mammalian cells used in this example were obtained according to the procedure described in Example 6 and expressed a heterologous antibody (Protein 1: antibody-multimer-fusion).
The cell culture process consisted of a seed train cultivation, followed with inoculation train (N-2 and N-1 cultures; pre-fermentation) and main fermentation (N). The seed- and inoculation train for the Ambr®15 was performed in shake flasks with cell splits every 3 or 4 days.
The antisense oligonucleotides of SEQ ID NO 23 and SEQ ID NO 24 were chosen as the LNAs due to the high level of exon 4 skipping observed with these antisense oligonucleotides in initial studies (see Example 1).
The (main) cultivations (N) in Ambr®15 were performed with a starting cell density of about 2*10E6 cells/mi in a total volume of 13 mi. The cultivation temperature was controlled, the N2 gassing rate was set constant, oxygen supply was regulated via a PID controller to maintain a constant DO, the agitation rate was set to 1200-1400 rpm (down stirring), the pH was set to pH 7.0. The pH-control was performed by adding a 1 M sodium carbonate solution or sparging CO2 into the bioreactor. The pH spots of the bioreactors were recalibrated every other day with the integrated analysis module of the Ambr®15. Defoamer was added one day before inoculation and daily during the cultivation. The cells were cultivated in a 14 days fed batch process with glucose control and two different feeds, which were added as bolus at predefined time points. The cell count and viability measurements were performed at-line with a Cedex HiRes (Roche Diagnostics GmbH, Mannheim, Germany). A Cedex Bio HT Analyzer (Roche Diagnostics GmbH) was used to measure product and metabolite concentrations.
The LNA addition at the beginning of the N-1 pre-cultivation (N-1), inoculation day (d0) or three days after the inoculation (d3) were performed by the liquid handling system of the Ambr®15 by adding a defined volume of a high concentrated LNA stock solution.
The supernatant was harvested 14 days after start of fed-batch by centrifugation (10 min, 1000 rpm and 10 min, 4000 rpm) and cleared by filtration (0.22 μm). Day 14 titers were determined using protein A affinity chromatography with UV detection. Product quality was determined by Caliper's LabChip® (Caliper Life Sciences).
It appears as though any efficiency in exon4 skipping of the LNA is sufficient to generate the effect of increased recombinant titer.
All fed-batch cultures were performed in shake flasks or Ambr®15 vessels (Sartorius Stedim) with the same proprietary serum free, chemically defined medium.
The cell culture process consisted of a seed train cultivation, followed with inoculation train (N-2 and N-1 cultures; pre-fermentation) and main fermentation (N). The seed- and inoculation train for the Ambr®15 was performed in shake flasks with cell splits every 3 or 4 days.
The recombinant mammalian cells used in this example were obtained according to the procedure described in Example 6 and stably expressed a heterologous antibody as well as XBP1 splice variant XBP1Δ4 with an amino acid sequences as depicted in SEQ ID NO; 7.
The (main) cultivations (N) in Ambr®15 were performed with a starting cell density of about 2*10E6 cells/ml in a total volume of 13 ml. The cultivation temperature was controlled, the N2 gassing rate was set constant, oxygen supply was regulated via a PID controller to maintain a constant DO, the agitation rate was set to 1200-1400 rpm (down stirring), the pH was set to pH 7.0. The pH-control was performed by adding a 1 M sodium carbonate solution or sparging CO2 into the bioreactor. The pH spots of the bioreactors were recalibrated every other day with the integrated analysis module of the Ambr®15. Defoamer was added one day before inoculation and daily during the cultivation. The cells were cultivated in a 14 days fed batch process with glucose control and two different feeds, which were added as bolus at predefined time points. The cell count and viability measurements were performed at-line with a Cedex HiRes (Roche Diagnostics GmbH, Mannheim, Germany). A Cedex Bio HT Analyzer (Roche Diagnostics GmbH) was used to measure product and metabolite concentrations.
The supernatant was harvested 14 days after start of fed-batch by centrifugation (10 min, 1000 rpm and 10 min, 4000 rpm) and cleared by filtration (0.22 μm). Day 14 titers were determined using protein A affinity chromatography with UV detection. Product quality was determined by Caliper's LabChip® (Caliper Life Sciences).
The same conditions for the fed-batch cultivation as described in Example 8 above were also used herein. The only difference of the current Example 10 to Example 8 is with respect to the expressed protein and the addition time of the LNA.
Likewise, the recombinant CHO cells used in this Example were obtained with the method according to Example 6.
Protein 1: antibody-multimer-fusion
100%
127%
Protein 2: bispecific, trivalent antibody comprising a full-length antibody binding to human A-beta protein and additional heavy chain C-terminal Fab fragment with domain exchange binding to human transferrin receptor (see WO 2017/055540)
Protein 3: tetravalent, bispecific antibody with domain exchange
100%
indicates data missing or illegible when filed
VRPVAGSAESAAGAGPVVTSPEHLPMDSDTVDSSDSESDILLGILDKLDPVMFFKCPSPESANLEELPEVYPGPSSLP
4
ATGTGCACCTCTGCAGCAGGTGCAGGCCCAGTTGTCACCTCCCCAGAACATCTTCCCATGGACTCTGACACTGT
TGCCTCTTCAGATTCTGAGGTAGAGCTTATTCTGTAGCCTAAGTGGCGTGTGACACGCTTAGCCAGGCAAACG
AGTGAGGCCAGTGGCCGGGTCTGCTGAGTCCGCAGCACTCAGACTACGTGCACCTCTGCAGCAGGTGCAGGC
CCAGTTGTCACCCCTCCAGAACATCTCCCCATGGATTCTGGCGGTATTGACTCTTCAGATTCAGAGGTAGGGAT
| Number | Date | Country | Kind |
|---|---|---|---|
| 20216690.6 | Dec 2020 | EP | regional |
The present application is a continuation of International Application No. PCT/EP2021/086382, filed on Dec. 17, 2021, which claims the benefit of and the priority to European Patent Application No. 20216690.6, filed on Dec. 22, 2020, the entire contents of which are herein incorporated by reference in their entireties for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2021/086382 | Dec 2021 | US |
| Child | 18340016 | US |
