Methods of selecting therapeutic molecules

1. A method of identifying a molecule having tolerable in vivo acute neurotoxicity before administering the molecule to a laboratory animal, comprising:

(a) selecting a molecule comprising a polynucleotide having a sequence score of greater than or equal to 0.2, comprising calculating the sequence score of the polynucleotide by formula (I):

(number of C nucleotides or analogs thereof in the polynucleotide−number of G nucleotides or analogs thereof in the polynucleotide)/total nucleotide length (number) of the polynucleotide  (I),

wherein the polynucleotide is single-stranded or one strand of a double-stranded molecule,

(b) adding the molecule to a culture of neuronal cells,

(c) measuring calcium oscillations in vitro in the neuronal cells which are in contact with the molecule,

(d) identifying the molecule as having tolerable in vivo acute neurotoxicity when the neuronal cells in contact with the molecule exhibit calcium oscillations at a level between about 30% less than and the same as that of vehicle control cells, or at a level higher than that of vehicle control cells,

(e) administering the molecule identified as having tolerable in vivo acute neurotoxicity to a laboratory animal, and

(f) measuring an in vivo tolerability of the molecule in the laboratory animal.

2. The method of claim 1 , wherein the calcium oscillations in the neuronal cells in contact with the molecule are about 70% or higher compared to the calcium oscillations in the vehicle control cells.

3. The method of claim 1 , wherein the calcium oscillations are AMPA receptor-dependent calcium oscillations.

4. The method of claim 1 , wherein the calcium oscillations are measured in the presence of Mg 2+ ions.

5. The method of claim 1 , wherein the in vivo tolerability is a tolerability category selected from the group consisting of: 1) hyperactivity; 2) decreased activity and arousal; 3) motor dysfunction and/or ataxia; 4) abnormal posture and breathing; 5) tremor and/or convulsions, and two or more combinations thereof.

6. The method of claim 5 , wherein the molecule exhibits a sum of in vivo tolerability scores between 0 and 8.

7. The method of claim 1 , wherein the method further comprises measuring a behavioral test score of the laboratory animal.

8. The method of claim 7 , wherein the behavioral test is a short term memory test, a spatial learning and memory test, a gait analysis test, or any combination thereof.

9. The method of claim 1 , further comprising measuring tubulin intensity of the molecule in a culture of neuronal cells.

10. The method of claim 9 , wherein the molecule reduces less than about 25% of the tubulin intensity in the culture of neuronal cells.

11. The method of claim 1 , wherein the polynucleotide comprises DNA.

12. The method of claim 1 , wherein the polynucleotide comprises RNA.

13. The method of claim 1 , wherein the polynucleotide is single-stranded.

14. The method of claim 1 , wherein the polynucleotide comprises an antisense oligonucleotide.

15. The method of claim 14 , wherein the antisense oligonucleotide comprises at least one nucleotide analog.

16. The method of claim 15 , wherein the at least one nucleotide analog is a Locked Nucleic Acid (LNA), 2′-O-alkyl-RNA, 2′-amino-DNA, 2′-fluoroDNA, arabino nucleic acid (ANA), 2′-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt), 2′-O-methyl nucleic acid (2′-OMe), 2′-O-methoxyethyl nucleic acid (2′-MOE), or any combination thereof.

17. The method of claim 14 , wherein the antisense oligonucleotide comprises an internucleoside linkage that is a phosphodiester linkage, a phosphotriester linkage, a methylphosphonate linkage, a phosphoramidate linkage, a phosphorothioate linkage, or any combination thereof.

18. The method of claim 14 , wherein the antisense oligonucleotide is a gapmer, blockmer, mixmer, or a wingmer.

19. The method of claim 14 , wherein the antisense oligonucleotide is a headmer, tailmer, or totalmer.