Reagents for treatment of oculopharyngeal muscular dystrophy (OPMD) and use thereof
A muscular dystrophy and effector technology, applied in gene therapy, muscular system diseases, single-stranded DNA virus, etc., can solve problems such as death, progressive degeneration of pharyngeal muscle tissue that cannot be corrected, and dysphagia
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
example 1
[0672] Example 1 - Design of dsRNA and shRNA targeting PABPN1
[0673] Representative ddRNAi construct designs identified from PABPN1 mRNA sequences using disclosed siRNA design algorithms (including Ambion, Promega, Invitrogen, Origene, and MWG) Sequences of potential targets: select sequences that are conserved in human, bovine, and mouse. siRNAs were synthesized and tested in vitro, and three active siRNAs were selected for conversion into ddRNAi constructs. The mRNA transcripts corresponding to the regions selected as targets for the designed double-stranded RNA (dsRNA) and short hairpin RNA (shRNA) are shown in Table 1.
[0674] dsRNAs comprising effector sequences substantially complementary to the target regions described in Table 1 were designed and validated (Table 3). Screening for siRNA sequences that downregulate human PABPN1 was performed in HeLa cells in which endogenous human PABPN1 was constitutively expressed. Transfection of siRNA was performed using Oligo...
example 2
[0676] Example 2 - Generation of self-complementary AAV-based plasmid constructs and viruses expressing shRNA targeting PABPN1
[0677]Self-complementary adeno-associated virus type 2 (scAAV2) plasmids expressing one or three shRNAs targeting PABPN1 were generated by subcloning single or three shRNAs targeting PABPN1 into the scAAV2 backbone (as shown in Table 5). ).
[0678] Briefly, a single shRNA construct comprising DNA encoding shRNA5 (SEQ ID NO: 20) under the control of the H1 promoter was cloned into the pAAV2 vector backbone (pAAV-shRNA5). Two triple shRNA constructs were also produced, comprising encoding shRNA1 (SEQ ID NO: 16), shRNA3 (SEQ ID NO: 16) and shRNA3 (SEQ ID NO: 18) and the DNA of shRNA5 (SEQ ID NO: 20). These triple constructs are pAAV-shRNAx3-short (SEQ ID NO:22) and pAAV-shRNAx3-long (SEQ ID NO:23). Both variants included the described tricistronic shRNA construct, however, the construct in pAAV-shRNAx3-long also included a stuffer DNA sequence to ge...
example 3
[0683] Example 3 - Gene silencing of PABPN1 in vitro
[0684] This example demonstrates the ability of the PABPN1pAAV-shRNA plasmid generated in Example 2 to knock down PABPN1 expression in vitro.
[0685] cell
[0686] Human embryonic kidney cells (HEK293T, ATCC, Manassas, USA) were incubated with 20 mM HEPES, 10% fetal bovine serum (FBS) and 2 mM glutamine (PAA Laboratories, Yeovil, UK). Grow in Dulbecco's modified Eagle's medium (DMEM).
[0687] Primary mouse myoblasts (clone IM2) (transgenicly immortalized with the temperature-sensitive SV40 large T antigen (tsA58) and derived from Immorto-mouse H2kB-IM2 (parental cell line), H2kB-WTA (encoding human wild type PABPN1) and H2kB-D7e (encoding 7-alanine expanded PABPN1)) were kindly provided by Dr. Michael Antoniou, King's College London. Make IM2 cells contain 20mM HEPES, 2mM glutamine, and supplemented with 20% FBS, 0.5% chicken embryo extract, 100U / ml penicillin-streptomycin, 2mmol / L L-glutamine and 20U / ml interferenc...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More