Compositions and methods for treating oculopharyngeal muscular dystrophy (OPMD)
A technology of transcripts and viruses, applied in botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve problems such as death, dysphagia, and progressive degeneration of the pharyngeal muscle system that cannot be corrected
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Embodiment 1
[0490] Example 1 - Design of shmiR targeting PABPN1
[0491] Sequences representing potential targets for siRNA construct design were identified from the PABPN1 mRNA sequence using publicly available siRNA design algorithms (including Ambion, Promega, Invitrogen, Origene, and MWG): the selected sequences were expressed in human, nonhuman primate Conserved in human, bovine and mouse species. Sequences encoding candidate siRNAs were incorporated into the pre-miR30a scaffold to generate sequences encoding short hairpin microRNAs (shmiRs) including 5' flanking regions (SEQ ID NO: 41), siRNA sense strand sequence (effector complement), stem / loop junction sequence (SEQ ID NO: 40), siRNA antisense strand (effector sequence) and 3' flanking region (SEQ ID NO: 42). The predicted secondary structures of representative shmiRs are shown in figure 1 C. The target regions of the PABPN1 mRNA transcripts of the designed shmiRs are shown in Table 1, and the corresponding shmiR effector seq...
Embodiment 2
[0492] Example 2 - Generation of a single "silencing and replacement construct" for simultaneous gene silencing of endogenous PABPN1 and replacement with codon-optimized PABPN1.
[0493] A single-stranded adeno-associated virus type 2 (ssAAV2) plasmid expressing shmiR17 and shmiR13 (eg, as described in Tables 3 and 4) and the optPABPN1 sequence was created.
[0494] The silencing and replacement constructs (hereinafter referred to as "SR-constructs") were obtained by subcloning the DNA sequences encoding shmiR17 and shmiR13 (as in Table 4) into optPABPN1 in the pAAV2 vector backbone (pAAV-shmiR viral plasmid) produced in the 3' untranslated region of the transcript. Expression of optPABPN1 and both shmiRs in a single transcript is driven by the muscle-specific promoter Spc512. figure 1 (A), figure 1 (B) and figure 2 A schematic of the SR construct is provided in .
[0495] A recombinant pseudotyped AAV vector stock is then generated. Briefly, HEK293T cells were culture...
Embodiment 3
[0496] Example 3 - In vivo studies using a single vector "silence and replace" approach.
[0497] To test the in vivo efficacy of the SR-constructs described in Example 2 in an OPMD-associated disease model, the SR-constructs were administered alone in a range of doses by intramuscular injection into the tibiae of 10-12 week-old A17 mice in the anterior muscle (TA). The doses administered were 7.5x10 per muscle 11 , 2.5x10 11 , 5x10 10 , 1x10 10 , 2x10 9 and 4x10 8 A vector genome (vg). Age-matched A17 mice were injected with saline as untreated group. Mice were sacrificed 14 or 20 weeks after treatment.
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