Stem cells with shared genetic information aid in the study of human disease. Image: Kyoto University/Knut WoltjenSingle nucleotide polymorphisms (SNPs) are the most common form of genetic mutation, with more than ten million currently identified, and are often found in hereditary diseases – from Alzheimer’s to diabetes.
Due to the precise nature of SNPs, researchers need to compare genetic differences with isogenic twins – two cells which differ in their makeup by only a single gene.
To do this, scientists in Japan have used induced pluripotent stem (iPS) cells to create a novel gene editing technique that can modify DNA to a single gene.
iPS cells are unique in that they retain the genetic makeup of a donor and can be converted into any cell type. These characteristics mean the cells are perfect for testing new treatments in a laboratory setting.
The team – led by Dr Knut Woltjen and based at the Centre for iPS cell Research and Application at Kyoto University, Japan – use the method to insert an SNP modification along with a fluorescent report gene as a marker for the modified cells.
As adding the reporter gene is another modification to the genome, the researchers created a duplicated DNA sequence that flanks the gene in order to remove it.
These strands hang over the sequence of the reporter gene so that once the latter is removed, the two resulting strands can join together – a method known as microhomology-mediated end joining.
In the Alzheimer’s affected brain, abnormal levels of the beta-amyloid protein clump together to form plaques (seen in brown) that collect between neurons and disrupt cell function. Image:NIH Image Gallery
Unique target sites were also added to remove the gene using the enzyme CRISPR, which cuts DNA. As a result, only the modified SNP is left in the genome of the cell.
One of the isogenic twins receives the mutant SNP and the other receives the normal SNP, allowing for a comparison to be made.
Dr Woltjen calls the new technique Microhology-Assisted eXcision, or MhAX. ‘To make MhAX work, we duplicate DNA sequences which are already present in the genome. We then let the cell resolve this duplication. At the same time, the cells decide which SNPs will remain after repair,’ said Woltjen. ‘One experiment results in the full spectrum of possible SNP genotypes.’
The team have already collaborated with other Japanese universities on the application of their novel method, using the HPRT gene – a mutation that can lead to gout – as the first example of its potential use in therapy.
Their work shows that cells with the HPRT mutant SNP had similar issues with metabolism associated with gout patients, while the isogenic control cells had no problems.
Following on from this success, Woltjen and his team are now applying the technique to different diseases associated with SNPs, including diabetes.
The next five years will be the most promising in the fight against cancer with immunotherapies – such as CAR-T and moderating T-Cell approaches, and innate immunity therapies – delivering far better patient outcomes.
In the last five years, the industry has rapidly advanced its understanding of the body’s immune response and genetic markers. As a result, combination therapies – chemotherapies will continue to play an important role – are forecast to become an increasingly standardised treatment, with pharma keen to invest.
These newer options are bringing in transformative remission rates, and check-point inhibitors have already been seen to elicit long-term cures in patients, with success rates two-to-three times higher than standard chemotherapy approaches.
Over the next ten years, we will see significant breakthroughs as the industry’s understanding of the immune system improves. There are currently more than 130 biotechs – in addition to 20 big pharma companies – working on new therapies and it is believed the smaller companies are more aggressively bringing newer innovations to market. In the long run, pharma will undoubtedly absorb the most promising players in an effort to become leaders in combination therapy approaches, which many argue will deliver the best outcomes.
The current investor frenzy is comparable to that of the genomics industry at the turn of the century. Experts argue that a more complete understanding of the genome and promise of clinical data of these transformative modalities will create a golden age for cancer therapy over the next few years.
There are, however, a number of immediate challenges. For example, CAR-T, although demonstrating good efficacy in blood cancers, has yet to show enough efficacy in solid tumours. Another challenge is how far towards cures for all patients we can get, particularly for patients with late stage metastatic cancer.
Immunotherapies are moving cancer from treatment options that simply extend life or improve experience to more effective cures. The cost of newer therapies is also coming into focus; however, this is a positive pressure on companies to produce significant, not just incremental, outcomes for patients.