Splice Switching Oligonucleotides for NF-kB Knockdown
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Solution Overview
Problem
Current methods for studying B cell and plasma cell differentiation in humans are hindered by the challenges of gene knockdown in primary cells, particularly high mortality rates and poor penetrance with RNAi technology, and the need for tools that avoid DNA modifications and facilitate transient knockdown without mutagenic effects.
Innovation Solution
The use of splice switching oligonucleotides (SSO) for exon skipping-mediated knockdown of NF-κB components, such as c-REL and RELA, which allows for efficient and transient modification of gene expression without DNA mutagenesis, using morpholino SSO targeting specific splice sites to induce reading frameshifts and nonsense-mediated decay.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNAi technology is used for gene knockdown in primary B cells, then gene expression can be reduced, but high mortality rates and poor penetrance occur during transfection
Solution Approach 1:
The patent changes the chemical parameters of the oligonucleotide from standard RNAi (siRNA) to modified antisense oligonucleotides with specific chemistries (phosphorothioate backbone, 2'-O-methyl modifications). This parameter change allows the oligonucleotide to resist nuclease degradation and reduces off-target effects while improving cellular uptake and reducing mortality rates in primary B cells
Solution Approach 2:
The patent uses modified antisense oligonucleotides as an intermediary mechanism between the researcher and the target gene. These oligonucleotides bind to pre-mRNA splice sites or regulatory sequences, inducing exon skipping or modulating splicing patterns without requiring RNAi machinery, thereby avoiding the transfection-related toxicity associated with RNAi
2Reliability
If CRISPR-Cas9 technology is used for gene knockout, then efficient gene knockout can be achieved, but DNA modifications occur that could affect other genes
Solution Approach 1:
The patent segments the gene expression process by targeting specific pre-mRNA splice sites or regulatory sequences rather than the entire gene. By designing oligonucleotides that bind to specific exons or intronic regions, the invention achieves localized modulation of splicing patterns, allowing selective knockdown of specific isoforms or complete gene suppression without modifying the underlying DNA sequence
Solution Approach 2:
Instead of directly modifying DNA to achieve gene knockout (as with CRISPR-Cas9), the patent inverts the approach by targeting the RNA level through antisense oligonucleotides. This inversion allows transient and reversible gene suppression without permanent genomic alterations, avoiding off-target DNA modifications and potential genomic instability
3Reliability
If sufficient SSO amounts are delivered into intracellular compartments, then efficient gene knockdown can be achieved, but delivery remains challenging
Solution Approach 1:
The patent employs composite oligonucleotide structures combining different chemical modifications (phosphorothioate backbone for stability, 2'-O-methyl for affinity, amino-allyl groups for conjugation). These composite materials provide both the necessary intracellular delivery capabilities and the high-affinity binding required for efficient gene knockdown, overcoming the delivery challenge through integrated chemical design
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
This approach effectively reduces NF-κB component expression in B-lineage cells, altering signaling pathways and decreasing cell viability, providing a powerful tool for cancer treatment and avoiding off-target effects associated with RNAi and other antisense technologies.
Implementation Method 1
the oligonucleotide being complementary to a sequence within the pre-mRNA molecule
Implementation Method 2
the apparition of a premature stop codon (PTC) and ultimately the degradation of alternative mRNAs by NMD
Data Source
AI summary
The need to identify new therapeutic approaches in the treatment of cancers of the B lymphoid lineage is crucial. Here, the inventors provide evidence for efficient knockdown of c-REL and RELA expression after treatment with splice switching antisense oligonucleotides (SSO) inducing exon skipping and reading frameshift. For instance, treatments with morpholino SSO targeting c-REL exon 2 donor splice site or RELA exon 5 acceptor splice site elicited very efficient knockdown in diffuse large B cell lymphoma (DLBCL) cell lines and antibody-secreting cells derived from primary human B cells. Consistent with the clinical relevance of c-REL activation in DLBCL, treatment with c-REL SSO induced major alterations in NF-κB and TNF signalling pathways and strongly decreased cell viability. Altogether, SSO-mediated knockdown is a powerful approach to inhibit transiently the expression of a NF-κB component in B-lineage cells that should open new avenues for cancer treatments. Accordingly, the present invention relates to the use of splice switching oligonucleotides for exon skipping-mediated knockdown of a NF-κB component in B cells.