E23. The method of any one of embodiments 16 to 22, wherein the polynucleotide is an antisense oligonucleotide (i.e., oligomer) of about 10 to about 50 nucleotides in length. E24. The method of embodiment 23, wherein the antisense oligonucleotide modulates an expression of a target protein. E31. The method of any one of embodiments 23 to 30, wherein the antisense oligonucleotide is complementary to an mRNA or a pre-mRNA of the target gene. The term "complement" as used herein indicates a sequence that is complementary to a reference sequence. 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. The term "design" or "oligomer design" or "ASO Sequence" as used herein refers to a pattern of nucleotides (e.g., DNA) and nucleotide analogs (e.g., LNA) in a given sequence. Exemplary polynucleotides can comprise a nucleotide sequence having two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides, ten nucleotides, oligonucleotides, 50 nucleotides, 51 nucleotides, or more. The term "nucleic acids," "nucleotides," "nucleotide sequence," or "nucleic acid sequence" is intended to encompass plural nucleic acids (e.g., two or more, three or more, etc.).



Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. PK's LIVE My cams - Check out who's online Right NOW! An "effective amount" of a therapeutic molecule as disclosed herein is an amount sufficient to carry out a specifically stated purpose. The term "oligomer" in the context of the present invention, refers to a molecule formed by covalent linkage of two or more nucleotides (i.e., an oligonucleotide). E74. The method of any one of Embodiments 63 to 73, wherein the molecule is an antisense oligonucleotide. E37. The method of any one of embodiments 34 to 36, wherein the tolerability category comprises at least one, at least two, at least three, at least four, or at least five tolerability categories. E36. A method of testing or determining in vivo tolerability of a molecule comprising administering the molecule to a mammal and grading the mammal's tolerability in a tolerability category.



The disclosure also provides an in vivo tolerability assay that can be used separately or combined with the calcium oscillation assay and/or the sequence score method. In other aspects, the disclosure provides a combined method of using a calcium oscillation assay and a sequence score method. In other embodiments, the calcium oscillations measured in the present methods are the combination of the oscillation frequency and magnitude. In one embodiment, My Cams the calcium oscillations measured in the present methods are the cumulative increase in calcium oscillations within a culture of neuronal cells, whereby the time to reach the maximum fluorescence signal constitutes the magnitude of the calcium response. E73. The method of any one of Embodiments 63 to 72, further comprising measuring tublin intensity in a culture of neuronal cells. E87. In the method of any one of Embodiments 81 to 85, the improvement further comprising measuring an in vivo tolerability of the molecule.



E33. The method of any one of embodiments 1 to 32, further comprising measuring an in vivo tolerability of the molecule. E44. The method of any one of embodiments 41 to 43, wherein the behavioral test is a short term memory test, a spatial learning and memory test, a gait analysis test, or any combination thereof. The term "oscillation frequency" refers to the time between oscillations. In some embodiments, the calcium oscillations are gamma-aminobutyric acid (GABA)-dependent calcium oscillations. E13. The method of one of embodiments 1 to 12, wherein the calcium oscillations are determined by measuring fluorescence of a calcium dye. E66. The method of any one of Embodiments 63 to 65, wherein the calcium oscillations are AMPA receptor my cams dependent. In some embodiments, calcium oscillations are measured in the present method through the use of fluorescent probes which detect the fluctuations of intracellular calcium levels. In another embodiment, the calcium oscillations measured in the present methods are the frequency of calcium oscillations.