Antisense Oligonucleotides Targeting Natural Antisense Transcripts
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Solution Overview
Problem
Current methods for modulating the expression and function of GDNF polynucleotides are limited in their ability to specifically target and regulate the natural antisense transcripts, leading to inefficient up-regulation or down-regulation of GDNF gene expression.
Innovation Solution
The use of antisense oligonucleotides, specifically designed to have at least 50% sequence identity to the reverse complement of specific GDNF polynucleotide regions, is employed to inhibit natural antisense transcripts, thereby modulating GDNF expression in patient cells or tissues. These oligonucleotides can be administered subcutaneously, intramuscularly, or intravenously and may include modified nucleotides such as phosphorothioate, locked nucleic acids, or be encapsulated in liposomes for enhanced delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional antisense methods are used to target GDNF polynucleotides, then some modulation of GDNF expression is achieved, but the ability to specifically target and regulate natural antisense transcripts is limited, resulting in inefficient up-regulation or down-regulation
Solution Approach 1:
The patent applies inversion by designing antisense oligonucleotides that target the natural antisense transcript rather than directly targeting the GDNF sense transcript. By inhibiting the antisense transcript, the invention indirectly up-regulates GDNF expression, achieving the opposite effect of traditional antisense approaches. This is evidenced by the design of oligonucleotides complementary to antisense sequences (SEQ ID NOS: 2-4) to restore sense transcript expression.
Solution Approach 2:
The natural antisense transcript serves as an intermediary target in this invention. Instead of directly modulating GDNF expression, the invention uses antisense oligonucleotides to bind to and inhibit the antisense transcript, which then indirectly regulates GDNF expression levels. This intermediary approach allows for more specific and efficient control of GDNF modulation.
2Reliability
If antisense oligonucleotides are designed with high sequence identity to reverse complement of GDNF regions, then specific inhibition of natural antisense transcripts is achieved, but the complexity of oligonucleotide design and selection increases
Solution Approach 1:
The invention segments the GDNF polynucleotide into specific regions (nucleotides 1-237, 1-1246, 1-684, 1-400, 1-619, 1-813 of various SEQ ID NOS) and designs separate antisense oligonucleotides for each region. This segmentation allows for systematic evaluation of different target regions and facilitates the selection of optimal oligonucleotide sequences with at least 50% sequence identity to reverse complements of these specific regions.
Solution Approach 2:
The invention systematically varies parameters of the antisense oligonucleotides including length (5-30 nucleotides), sequence identity (at least 50% to reverse complement), and chemical modifications (phosphorothioate, LNA, methylphosphonate). This parameter optimization enables identification of oligonucleotides with optimal binding specificity while managing design complexity through structured variation of key parameters.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The approach effectively up-regulates or down-regulates GDNF expression by specifically targeting natural antisense transcripts, as demonstrated by real-time PCR results showing significant changes in GDNF mRNA levels in various cell types, indicating precise modulation of GDNF function.
Implementation Method 1
DNA-RNA and RNA-RNA hybridization are important to many aspects of nucleic acid function including DNA replication, transcription, and translation. Hybridization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. Antisense nucleotides, for example, disrupt gene expression by hybridizing to target RNA
Implementation Method 2
Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types
Data Source
Figure 1A~1B
Figure 1C
Figure 1D
AI summary
The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Glial cell derived neurotrophic factor (GDNF), in particular, by targeting natural antisense polynucleotides of Glial cell derived neurotrophic factor (GDNF). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of GDNF.