Abstract
The present invention is in the field of a compound for use as a medicament for treatment of tRNA deficiencies in living cells, a dosage comprising said compound, and an in vivo and in vitro method for treatment of tRNA deficiencies, as well as for prevention, mitigation of symptoms, and regeneration of cells.
Claims
1. A compound for overexpression of cognate tRNA for use in a medicament for treating a heterozygous mutated cell, wherein the compound comprises a transfer RNA, and a vector, wherein the vector is coupled to the wherein the tRNA is selected from tRNA.sup.Ala, tRNA.sup.Gly, tRNA.sup.Tyr, tRNA.sup.His, tRNA.sup.Met, tRNA.sup.Trp, and combinations thereof.
2. The compound according to claim 1, wherein the heterozygous mutated cell is a neuron.
3. The compound according to claim 2, wherein the peripheral neuropathy is selected from an inherited neuromuscular disorder, a central nervous system disorder, a brain disorder, a motoric nerve disorder, a sensory nerve disorder, or a combination thereof.
4. The compound according to claim 1, wherein the vector is an adeno-associated viral (AAV) vector, preferably an AAV9 (serotype 9) vector.
5. The compound according to claim 1, wherein the promotor is an RNA polymerase III promotor.
6. The compound according to claim 1, wherein the compound is for overexpressing tRNA.
7. Compound according to claim 1, wherein the compound is in a form selected from a viral vector, a synthetic tRNA, and combinations thereof.
8. The compound according to claim 1, wherein the medicament is for an application selected from intrathecal application, cerebral application, the Peripheral Nervous System, for systemic application, and combinations thereof.
9. The compound according to claim 1, wherein the compound is selected froni partially embedded and fully embedded.
10. A dosage comprising a compound according to claim 1, wherein in a viral gene transfer the compound comprises >10.sup.12 vg/kg body mass.
11. The dosage according to claim 10, wherein the dosage is selected from a single dosage, and a multiple dosage.
12. An method selected from an iln vivo method and an in vitro method of treating a heterozygous mutated cell, of preventing a heterozygous mutated cell, of preventing symptoms thereof, of mitigating symptoms thereof, of regeneration of impaired cells, of gene therapy, of RNA therapy, and a combination thereof, comprising providing a dosage according to claim 10, and applying the dosage, wherein applying is selected from intrathecal application, from cerebral application, from application to the Peripheral Nervous System, or and from systemic application.
13. The method according to claim 12, wherein the method is repeated.
14. A method of introducing a sequence selected from a cognate tRNA and tRNA encoding sequence into a heterozygous mutated cell, comprising providing the heterozygous mutated cell, providing the tRNA or tRNA encoding sequence in a suitable form, wherein the tRNA or tRNA encoding sequence is selected from tRNA.sup.Ala, tRNA.sup.Gly, tRNA.sup.Tyr, tRNA.sup.His, tRNA.sup.Met, tRNA.sup.Trp, and combinations thereof, and introducing the tRNA or tRNA encoding sequence into the heterozygous mutated cell.
15. The method according to claim 14, wherein the tRNA or tRNA encoding sequence is obtained from a mammal.
16. The method according to claim 14, wherein the tRNA or tRNA encoding sequence is selected from a natural or-sequence and a synthetic sequence.
17. The method according to claim 14, wherein the tRNA comprises an anticodon.
18. The compound according to claim 1, wherein the compound comprises a promotor, wherein the vector is coupled to the tRNA promotor, and wherein the promotor is coupled to the tRNA.
19. The compound according to claim 2, wherein the heterozygous mutated cell is selected from a motor neuron and a sensory neuron.
20. The compound according to claim 3, wherein the peripheral neuropathy is Charcot-Marie-Tooth peripheral neuropathy.
Description
SUMMARY OF FIGURES
[0033] FIGS. 1, 2A-G, 3A_H, 4A-I, and 5A-I show details of the present invention.
DETAILED DESCRIPTION OF FIGURES
[0034] FIG. 1: Molecular mechanism underlying CMT-aaRS. (A) In the wild type situation, the tRNA synthetase (aaRS) binds the cognate tRNA and amino acid, activates the amino acid, and aminoacylates the tRNA. The aminoacylated (‘charged’) tRNA is transferred to the eukaryotic elongation factor 1A (eEF1A), which delivers the tRNA to the ribosome for use during translation elongation. (B) In CMT-aaRS, both wild type and CMT-mutant aaRSs are present, derived from the wild type and CMT-mutant AARS alleles, respectively. The CMT-mutant aaRS binds the cognate tRNA and possibly also the amino acid, may or may not activate the amino acid and aminoacylate the tRNA, but fails to release the tRNA or releases at a very slow pace. As a consequence, the cellular pool of the cognate tRNA is reduced under a critical threshold, and insufficient cognate tRNA is available for aminoacylation by the wild type aaRS. This results in insufficient supply of the aminoacylated tRNA to the ribosome, and stalling of the ribosome on cognate codons.
[0035] FIG. 2A-G: tRNA.sup.Gly-GCC overexpression rescues inhibition of protein synthesis and peripheral neuropathy phenotypes in Drosophila CMT2D models. (A) Schematic overview 5 of the genomic region contained in the BAC used to generate tRNA.sup.Gly-GCC transgenic Drosophila. (B,C) Relative translation rate (% of driver-only control) as determined by FUNCAT in motor neurons (OK371-GAL4) of larvae expressing G240R (B), E71G (C), or G526R (C) GlyRS mutants (2×: two transgene copies), in the presence or absence of the 10× tRNA.sup.Gly-GCCtransgene. n=10-17 animals per genotype; ***p<0.0001 by Brown-Forsythe and Welch ANOVA. (D) Percentage of larvae with innervated muscle 24. GlyRS transgenes were selectively expressed in motor neurons (OK371-GAL4), in the presence or absence of 10× tRNA .sup.Gly-GCC Control larvae are driver-only. n=19-26 animals per genotype; *p<0.05; ***p<0.005 by Chi-square test. (E) Climbing speed (mm/s) in an automated negative geotaxis assay of 7-day-old male flies that selectively express GlyRS transgenes in motor neurons (OK371-GAL4), in the presence or absence of 10× tRNA.sup.Gly-GCC. Control flies are driver-only. n=13 groups of 10 flies per genotype; **p<0.01; * * *p<0.0001 by two-way ANOVA. (F) Dendritic coverage (% of driver-only control) of class IV multidendritic sensory neurons in the larval body wall upon selective expression of GlyRS transgenes in these sensory neurons (ppk-GAL4), in the presence or absence of 10× tRNA.sup.Gly-GCC. n=13 animals per genotype; ***p<0.005 by two-way ANOVA. (G) Life span of flies ubiquitously expressing GlyRS transgenes from the adult stage onwards (tub-GAL80.sup.ts; tub-GAL4), in the presence or absence of 10× tRNA.sup.Gly-GCC Control flies are driver-only. n=79-126 flies per genotype; p<0.0001 for each GlyRS mutant versus GlyRS mutant+10× tRNA.sup.Gly-GCC by Log-rank (Mantel-Cox) test.
[0036] FIG. 3A-H: tRNA.sup.Gly-TCC overexpression rescues inhibition of protein synthesis and peripheral neuropathy phenotypes in Drosophila CMT2D models. (A,B) Relative translation rate (% of driver-only control) as determined by FUNCAT in motor neurons (OK371-GAL4) of larvae expressing E71G (A) or G240R (B) GlyRS mutants, in the presence or absence of the 12× tRNA.sup.Gly-TCC transgene. n=10-17 (A) and 4-20 (B) animals per genotype; ***p<0.005 by Brown-Forsythe and Welch ANOVA. (C) Percentage of larvae with innervated muscle 24. G240R or G526R GlyRS was selectively expressed in motor neurons (OK371-GAL4), in the presence or absence of 12× tRNA.sup.Gly-TCC Control flies are driver-only. n=12-27 animals per genotype; *p<0.05; **p<0.01; ***p<0.0001 by Chi-square test. (D) Synapse length on distal muscle 1/9 of larvae selectively expressing GlyRS _G240R in motor neurons (OK371-GAL4), in the presence or absence of 12× tRNA.sup.Gly-TCC. Control flies are driver-only. n=11-14 animals per genotype; *p<0.05; ***p<0.0001 by one-way ANOVA. (E,F) Climbing speed (mm/s) of 7-day-old male flies. E71G (E), G240R (F), or G526R (F) GlyRS was selectively expressed in motor neurons (OK371-GAL4), in the presence or absence of 12× tRNA.sup.Gly-TCC. Control flies are driver-only. n=6-19 groups of 10 flies per genotype; ***p<0.005 by Brown-Forsythe and Welch ANOVA. (G) Dendritic coverage (% of driver-only control) of class IV multidendritic sensory neurons, in which GlyRS transgenes were selectively expressed (ppk-GAL4), in the presence or absence of 12× tRNA.sup.Gly.sup.−TCC. n=8-15 animals per genotype; ***p<0.0001 by one-way ANOVA. (H) Life span of flies ubiquitously expressing GlyRS transgenes from the adult stage onwards (GAL80.sup.ts; tub-GAL4), in the presence or absence of 12× tRNA.sup.Gly-TCC. Control flies are driver-only. n=85-193 flies per genotype; p<0.0001 for each GlyRS mutant versus GlyRS mutant+12× tRNA.sup.Gly-TCC by Log-rank (Mantel-Cox) test.
[0037] FIG. 4A-I: tRNA.sup.Gly-GCC overexpression rescues peripheral neuropathy in Gars.sup.C201R/+ mice. (A) Schematic overview of the mouse genomic fragment used for generation of tRNA.sup.Gly-GCC transgenic mice. (B) Hanging time in the inverted grid test of a cohort of male WT, tRNA.sup.Gly-high, Gars.sup.C201R/+, and Gars.sup.C201R/+; tRNA.sup.Gly-high littermate mice at 4, 8, and 12 weeks of age. n=8-9 mice per genotype; ***p<0.0005 by one-sample t-test (theoretical mean of WT) and two-tailed unpaired t-test with Bonferroni's multiple comparisons test per time point. (C) 4-paw grip strength of the same cohort of mice as measured by dynamometer. n=8-9 mice per genotype; ***p<0.001 by two-way ANOVA with Tukey's multiple comparisons test per time point. (D,E) l Analysis of the same cohort of mice at 12 weeks of age by electromyography (EMG). (D) Latency time between stimulation of the sciatic nerve at the sciatic notch level and detection of a compound muscle action potential (CMAP) in the gastrocnemius muscle. n=8-9 mice per genotype; ***p<0.0001 by two-way ANOVA with Tukey's multiple comparisons test. (E) CMAP amplitude in the gastrocnemius muscle. n=8-9 mice per genotype; ***p<0.0005 by Brown-Forsythe and Welch ANOVA. (F,G) Weight of the tibialis anterior (F) and gastrocnemius (G) muscles of the same cohort of mice at 12 weeks of age. n=8-9 mice per genotype; ***p<0.0001 by two-way ANOVA with Tukey's multiple comparisons test. (H,I) Representative images (H) and quantification (I) of NMJ innervation status in the plantaris muscle. In (H), the presynaptic nerve ending was visualized by immunostaining for neurofilament (NF) and SV2, while postsynaptic acetylcholine receptors were visualized by TRITC-conjugated bungarotoxin (BTX). n=5 mice per genotype; ***p<5×10.sup.−6 by Fisher's Exact test. Scale bar: 25 μm.
[0038] FIG. 5A-I: Mechanism underlying rescue of CMT2D phenotypes by tRNA.sup.Gly overexpression. (A) Size-exclusion chromatography of purified recombinant human GlyRS proteins reveals different partitioning between dimer and monomer forms of WT, E71G, C157R (equivalent to mouse C201R), G240R and G526R variants. Dimer:monomer (D:M) ratio of each GlyRS variant is indicated. (B) K.sub.on and K.sub.off values of Drosophila tRNA.sup.Gly-GCC binding and release to the indicated human GlyRS variants. K.sub.on and K.sub.off values are shown for dimer and monomer fractions. (C) Hanging time in the inverted grid test of male Gtpbp2.sup.+/? or −/−; Gars.sup.+/+ (control), Gtpbp2.sup.+/?; Gars.sup.C201R/+, and Gtpbp2.sup.−/−; Gars.sup.C201R/+ littermate mice at 4, 5, 6, 7 and 8 weeks of age. n=15-28 mice per genotype group; ***p<0.0005 by one-sample t-test (theoretical mean of Gtpbp2.sup.+/? or −/−, Gars.sup.+/+, and two-tailed unpaired t-test with Bonferroni's multiple comparisons test per time point. (D) Nerve conduction velocity of the sciatic nerve of Gtpbp2.sup.+/? or −/−; Gars.sup.+/+, Gtpbp2.sup.+/?; Gars.sup.C201R/+, and Gtpbp2.sup.−/−; Gars.sup.C201R/+ littermate mice at 8 weeks of age. n=13-20 mice per genotype group; ***p<0.0001 by Brown-Forsythe and Welch ANOVA. (E) Axon number in the motor branch of the femoral nerve Gtpbp2.sup.+/? or −/−; Gars.sup.+/+, Gtpbp2.sup.+/?; Gars.sup.C201R/+, and Gtpbp2.sup.−/−; Gars.sup.C201R/+ littermate mice at 8 weeks of age. n=8-13 per genotype group; ***p<0.0001 by one-way ANOVA. (F-I) Representative images (F) and quantification of fluorescent in situ hybridization for the ATF4 target genes Gdf15 (G), Adm2 (H), and B4galnt2 (I). Scale bar: 50 μm. n=5-6 mice per genotype; ***p<0.05 by two-tailed Welch's t-test with Bonferroni's multiple comparisons correction.
[0039] The figures are further detailed in the description.
EXAMPLES/EXPERIMENTS
[0040] Inventors generated Drosophila models for CMT-TyrRS and CMT-GlyRS, which recapitulate several hallmarks of the human disease. Loss of aminoacylation activity is not a common feature of CMT-mutant aaRSs and thus considered not to be required to cause CMT. Furthermore, a novel method which allows to cell-type-specifically monitor translation in Drosophila in vivo was developed. This ground-breaking approach revealed that each of six distinct GlyRS or TyrRS mutants substantially reduced global protein synthesis in motor and sensory neurons. Based on these unprecedented novel insights, it is considered that impaired translation constitutes a common pathogenic mechanism underlying CMT-aaRS. It is found that, strikingly, transgenic overexpression of tRNAGly in Drosophila CMT-GlyRS models fully rescued translation and partially but substantially rescued peripheral neuropathy phenotypes. Consistently, generation of tRNAGly overexpressing mice revealed that tRNAGly overexpression fully prevented peripheral neuropathy in a CMT-GlyRS mouse model. Finally, overexpression of Drosophila orthologs of the elongation factor eEF1A partially but significantly rescued peripheral neuropathy in Drosophila CMT-GlyRS models. Therefore, it is considered that CMT-mutant aaRSs bind the cognate tRNA, may or may not aminoacylate it, but fail to transfer the aminoacylated tRNA to eEF1A. Consequently, the supply of aminoacylated cognate tRNA to the ribosome may drop below a critical threshold, causing the ribosome to pause or stall on cognate codons, thus explaining the translation defect (FIG. 1).
[0041] Results
[0042] It is found that tRNA.sup.Gly overexpression rescues peripheral neuropathy in Gars.sup.C201/R+ mice. Also, tRNA.sup.Tyroverexpression rescued peripheral neuropathy in CMT-TyrRS Drosophila models. Further tRNA.sup.Tyr overexpressing mice may rescue peripheral neuropathy in a CMT-TyrRS mouse model. Also, translation may be inhibited in motor neurons of CMT-GlyRS and CMT-TyrRS mice. It is found that translation elongation is affected in CMT-GlyRS mice. The degree of phenotypic rescue is found to be dependent on the level of tRNAGly overexpression. tRNAGly overexpression induced full rescue of peripheral neuropathy in CMT-GlyRS mice versus partial rescue in Drosophila. Northern blotting revealed substantial tRNAGly overexpression in mice versus moderate overexpression in Drosophila. Drosophila lines with higher tRNAGly overexpression are generated to evaluate whether this results in more substantial/full rescue of peripheral neuropathy. It is found that only overexpression of cognate tRNA can rescue. It is confirmed that tRNAGly overexpression does not rescue CMT-TyrRS models and that tRNATyr overexpression does not rescue CMT-GlyRS models. Furthermore, tRNAGly with GCC anticodon rescued CMT-GlyRS models. Ribosome profiling/foot printing on spinal cord extracts from CMT-GlyRS and CMT-TyrRS mice is performed to detect ribosome stalling on Gly or Tyr codons, respectively.
[0043] For all above mentioned compounds similar results are achieved or found.
[0044] The invention although described in detailed explanatory context may be best understood in conjunction with the accompanying examples and figures as detailed above.
[0045] Some exemplary qualifications and quantifications are given below.
