Splice-Switching RNA: Modulating Interactions in the Spliceosome, In Vivo Therpeutics
Luke McLaughlin, Biotech Digital Marketer, Business Developer and Life Science Content Creator
In the realm of genetic medicine, splice-switching oligonucleotides (SSOs) represent a burgeoning frontier that promises to redefine the approach to treating a myriad of genetic disorders. This innovative class of therapeutics is designed to modulate RNA splicing, the critical process by which precursor messenger RNA (pre-mRNA) is edited to produce mature mRNA templates for protein synthesis. By redirecting splicing events, SSOs hold the potential to correct gene expression errors at their source, offering a beacon of hope for conditions previously deemed intractable.
The interaction of SSOs with the spliceosome, and their ability to influence splicing outcomes, involves several key steps:
Targeting Specific Pre-mRNA Sequences: SSOs are designed to hybridize with complementary sequences within the pre-mRNA. These sequences can be located in exons or introns and are often near or within splicing regulatory elements like splice sites, branch points, polypyrimidine tracts, or exonic or intronic splicing enhancers or silencers.
Blocking Spliceosome Components or Regulatory Proteins: By binding to their target sequences, SSOs can physically block the components of the spliceosome or various regulatory proteins from accessing their usual binding sites on the pre-mRNA. This blockage can prevent the recognition of a splice site or alter the assembly of the spliceosomal complex, leading to the skipping of an exon or the inclusion of an otherwise skipped exon.
Modifying Splicing Patterns: The primary mechanism by which SSOs alter splicing is through steric hindrance. By binding to specific sequences, SSOs can sterically hinder the spliceosome and associated factors from accessing critical regions of the pre-mRNA necessary for normal splicing. This hindrance can result in the exclusion of targeted exons (exon skipping) or the inclusion of exons that would otherwise be excluded (exon inclusion), depending on the design of the SSO and its target site.
Correcting Genetic Mutations: In cases where genetic mutations cause diseases by disrupting normal splicing, SSOs can be used to redirect the splicing machinery to produce more functional proteins. For example, in certain types of muscular dystrophy, SSOs have been used to promote the skipping of exons that contain mutations, resulting in the production of a shorter, but still functional, dystrophin protein.
Enhancing or Repressing Splicing: Beyond correcting mutations, SSOs can also be designed to enhance or repress the splicing of specific exons to upregulate or downregulate the production of certain protein isoforms. This approach can be used in research and therapeutic contexts to understand the function of specific exons or to modulate the activity of proteins for therapeutic benefit.
The spliceosome
The spliceosome is a complex molecular machine found within the nucleus of eukaryotic cells. It plays a critical role in the process of pre-mRNA splicing, wherein introns (non-coding regions) are removed from a pre-messenger RNA (pre-mRNA) transcript, and exons (coding regions) are joined together to form a mature messenger RNA (mRNA) molecule. The spliceosome consists of several core components:
Small Nuclear Ribonucleoproteins (snRNPs): These are the primary building blocks of the spliceosome. Each snRNP consists of small nuclear RNA (snRNA) and protein components. The major snRNPs involved in splicing are U1, U2, U4, U5, and U6.
Small Nuclear RNAs (snRNAs): These RNA molecules are part of snRNPs and play crucial roles in the splicing process. They participate in recognizing splice sites, catalyzing the splicing reactions, and providing the structural framework for the spliceosome. The major snRNAs are U1, U2, U4, U5, and U6, corresponding to their snRNP counterparts.
Splicing Factors: These proteins assist in the assembly, function, and regulation of the spliceosome. They include the serine/arginine-rich (SR) proteins and various other proteins that interact with the snRNPs or the pre-mRNA substrate.
Pre-mRNA Substrate: The pre-mRNA molecule that is to be spliced. It contains exons (which will be joined together) and introns (which will be removed).
Dynamic Protein Complexes: Several dynamic protein complexes assist in the assembly, activation, and disassembly of the spliceosome. These include the Prp19 complex (also known as the Nineteen Complex or NTC), which is involved in stabilizing the active site and ensuring the fidelity of splicing.
Regulatory Proteins and RNAs: These include proteins and non-coding RNAs that modulate the spliceosome's activity, ensuring that splicing occurs with precision and in response to cellular conditions or signals.
The spliceosome undergoes significant conformational changes throughout the splicing process, transitioning through various assembly stages (E, A, B, C complexes, etc.), which correspond to different steps in the splicing reaction. Each stage is characterized by specific interactions among the snRNPs, splicing factors, and the pre-mRNA substrate, facilitating the accurate removal of introns and joining of exons.
The Mechanism of Action
Splicing is a fundamental cellular process whereby non-coding sequences (introns) are excised from pre-mRNA transcripts, and the remaining coding sequences (exons) are joined together. The intricacy of splicing allows for a single gene to encode multiple protein variants through alternative splicing. However, mutations that disrupt the normal splicing process can lead to disease.
SSOs are synthetic nucleic acid molecules designed to bind to specific sequences within a pre-mRNA transcript, thereby influencing the spliceosome's assembly and modifying the splicing pattern. They can promote the inclusion or exclusion of specific exons, correcting splicing defects at their root. This precision targeting opens up avenues for addressing genetic diseases caused by splicing abnormalities, including certain muscular dystrophies, spinal muscular atrophy (SMA), and some inherited blood disorders.
Components and general process of action of the spliceosome:
Pre-mRNA: The substrate that contains both introns and exons.
U1 snRNP: Recognizes and binds to the 5' splice site of the pre-mRNA.
U2 snRNP: Binds to the branch point, a sequence near the 3' end of the intron.
U4 snRNP: Initially binds to U6 snRNP, keeping it inactive until the right time.
U5 snRNP: Binds to exons at both ends of the intron, positioning them for splicing.
U6 snRNP: Interacts with the 5' splice site and catalyzes the splicing reaction after release from U4.
Branch Point Binding Protein (BBP) and U2AF: Help in recognizing the branch point and the polypyrimidine tract at the intron's 3' end, aiding in the assembly of U2 snRNP.
Numerous other proteins: Facilitate spliceosome assembly, splicing reaction, and spliceosome recycling.
The splicing process involves several steps:
Complex E (Early) Assembly: U1 snRNP binds to the 5' splice site, and BBP and U2AF help recruit U2 snRNP to the branch point.
A Complex Formation: U2 snRNP binding creates the A complex.
B Complex Formation: U4/U6.U5 tri-snRNP complex is recruited to form the B complex.
Catalytic Activation: U1 and U4 are released, allowing U6 and U2 to catalyze the splicing reaction through two transesterification steps, forming the C complex.
Ligation and Release: Exons are ligated together, and the intron lariat is released along with the disassembly of the spliceosome components for recycling.
Advancements in SSO Technology
Recent years have witnessed significant advancements in SSO technology, driven by improvements in oligonucleotide chemistry, delivery mechanisms, and our understanding of the splicing machinery. Chemical modifications such as 2'-O-methyl (2'-OMe), phosphorothioates (PS), and locked nucleic acids (LNAs) have enhanced the stability, affinity, and specificity of SSOs, reducing their susceptibility to degradation by nucleases and improving their pharmacokinetic properties.
Delivery, however, remains a formidable challenge, as SSOs must traverse the cell membrane and reach the nucleus to exert their effects. Recent strategies include the use of lipid nanoparticles (LNPs), cell-penetrating peptides (CPPs), and conjugation with specific ligands to facilitate targeted delivery and cellular uptake.
Clinical Applications and Successes
The therapeutic potential of SSOs is vast, with several drugs already approved by regulatory agencies. Nusinersen (Spinraza®), approved for the treatment of SMA, is a landmark SSO drug that targets the SMN2 gene, modulating its splicing to produce more functional SMN protein, essential for motor neuron survival.
Another example is eteplirsen (Exondys 51®), designed for Duchenne muscular dystrophy (DMD) patients with specific gene mutations. It induces exon skipping in the dystrophin gene, enabling the production of a truncated but partially functional dystrophin protein, which is crucial for muscle function.
Challenges and Future Directions
Despite their promise, SSOs face challenges, including off-target effects, immune responses, and the need for repeated administrations due to their transient nature. Advances in delivery technologies and the development of more stable and specific SSOs are crucial for overcoming these hurdles.
The future of SSO research also lies in expanding their applications beyond neuromuscular disorders. Potential targets include cancer, where alternative splicing plays a role in tumor progression, and neurodegenerative diseases, where splicing alterations contribute to pathology.
Moreover, the integration of bioinformatics and artificial intelligence (AI) for predicting splicing outcomes and designing more effective SSOs is an exciting area of growth. These tools can streamline the identification of therapeutic targets and optimize SSO design, accelerating the pace of drug development.
Conclusion
Splice-switching oligonucleotides stand at the confluence of genetics, molecular biology, and medicine, offering a versatile platform for correcting genetic disorders at the RNA level. As research continues to unravel the complexities of RNA splicing and the technologies for SSO delivery evolve, the potential for SSOs to provide targeted, effective treatments for a wide range of diseases is becoming increasingly tangible. This innovative class of therapeutics not only underscores the power of precision medicine but also illuminates the path toward conquering genetic diseases that have long eluded effective treatment.