Effects Of Genetic Mutations Become Knowable With ‘Super Minigene’ System

By Deborah Borfitz 

April 11, 2024 | Molecular biologists at Iowa State University report the potential for the effects of genetic mutations to be known using a “super minigene” system developed in the lab of Ravindra Singh, Ph.D., professor of biomedical sciences. The approach essentially enables scientists to reconstruct pathogenic mutations in a test tube and then see the effect of those mutations in different cells—meaning, no genetically engineered mouse models requiring sophisticated equipment and highly skilled technical staff. 

In a proof-of-concept study that published recently in Nucleic Acids Research (DOI: 10.1093/nar/gkad1259), Singh’s group created a truncated version of the entire survival motor neuron 2 (SMN2) gene, associated with spinal muscular atrophy (SMA), a major genetic disease linked to infant mortality. They validated the utility of the super minigene in monitoring SMN protein levels upon splicing correction and showed how it could be employed to capture cell type-specific effects of a pathogenic SMN1 mutation. 

SMN is a multifunctional protein involved in many essential cellular processes and humans carry two nearly identical copies of it, known as SMN1 and SMN2, Singh explains. The SMN1 copy is the defective one in SMA and SMN2 on its own can only produce a small amount of fully functional SMN protein. “SMN2 is a normal copy except for the splicing problem.” 

Disease severity is influenced by the SMN2 copy number, with individuals having very low SMN levels suffering from severe SMA. Individuals with relatively low SMN levels suffer from mild SMA although sometimes remain symptom-free until their 50s or 60s, he says. However, mild SMA patients may have male infertility issues. On the flipside, highly expressed SMN has been associated with worse cancer outcomes, making it a gene of general interest. 

Drug Target Identified

Singh’s specialty is splicing, which removes non-coding sequences (introns) as messenger RNA forms. SMA is a rare, splicing-associated disease, believed to affect only one in about 10,000 child births, and has consequently attracted limited industry dollars. 

Before moving to Iowa State in 2007, Singh was on the faculty at the University of Massachusetts Medical School (Worcester) where he and his team found the target of an antisense drug—Spinraza (nusinersen), now a property of Biogen—attacking the source of SMA.  In 2016, Spinraza became the first such drug approved by the Food and Drug Administration for the treatment of SMA. The drug was subsequently approved in other countries, including Canada, Europe, and Japan.  

The drug is a small, synthetic oligonucleotide similar in size to the nucleic acid molecules used as primers for PCR assays, Singh says. It works by correcting the splicing of the SMN2 gene that is generally present in all SMA patients and binds to a specific intronic sequence in the gene to increase production of functional SMN protein. 

During their early work on the drug target, Singh and his team began deleting negative regulatory elements using a minigene-based assay—an extensively used approach for testing the effect of mutations in the splicing of a target sequence. Minigenes are short gene fragments that include the coding sections of an RNA transcript (exons) and the control regions necessary for the gene to express itself in the same way as a wild type gene fragment. 

Other marketed and investigational antisense drugs for neurological disorders have since come on the scene aimed at SMN2 and other specific genetic variants. Antisense oligonucleotides have been around for many years, but Singh and his colleagues helped popularize the approach by showing it could be used at very low concentrations, thereby avoiding many unwanted side effects, he says.  

Truncated Gene

In the years following the development of Spinraza, Singh’s group continued working on splicing applications in the SMN2 gene. Their focus turned to building a better model for seeing the outcomes from transcription to protein synthesis and steps in between that led to creation of the super minigene.  

Since mutations occur throughout the gene, and their effects are unknown, traditional minigenes would sometimes reveal the effect of a mutation locally but completely miss changes playing out across the gene expression process from transcription to splicing to RNA stability to protein production, says Singh. The super minigene system devised for SMN2 captures everything. 

This was accomplished by deleting intronic sequences in the middle that left splicing unaffected, he explains. The remaining sequences then had to be painstakingly stitched together to reconstruct the whole so it recapitulates the properties of the endogenous gene. The truncated version ends up being five to 10 times shorter than the original gene containing roughly 28,000 base pairs. 

Researchers individually looked at about 30 different steps in building the model, each separately optimized before being combined and checked for fit. Happily, Singh says, the super minigene produced the same results as the whole SMN2 gene in about 90% of the cases tested.  

“For every gene you can make a super minigene,” says Singh, which can then be introduced into cells within days to see its effects. The process of genetically altering model animals is not only difficult but can take months if not years. 

Super minigenes provide a simplified testing ground that enables research to be more holistic and thereby better capture interactions between splicing and the connected mechanisms of transcription, splicing, transcript stability, and translation (protein synthesis) all at the same time. “Instead of five experiments, you do one experiment, and it makes for more accurate results,” he says. 

The benefits of the system are enormous, Singh adds. A single gene can have potentially thousands of mutations, and, without the super minigene, there is no efficient way to know which are harmful and which are harmless. In terms of drug screening, it can be used to see what compounds make more SMN protein and which mechanism it works on in doing that. 

Development of the super minigene has set a precedent for quickly testing the effect of multiple mutations in a test tube to learn what they do, he says, which might logically extend to research on diseases such as amyotrophic lateral sclerosis or Parkinson’s with known associated mutations. For Singh and his team, the next goal is to use the super minigene system to test the effect of all known mutations of SMA.