Can CRISPR-Cas9 deliver on its promise to transform genome therapy?
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Gene genie: can CRISPR-Cas9 deliver on its promise to transform genome therapy?

By Sally Turner 11 Apr 2019

Experimental treatments based on CRISPR-Cas9 gene editing technology are at a crossroads, with the first European trial now enrolling patients and new clinical applications and delivery methods steadily emerging. It’s not all good news though; two studies in 2018 found that a majority of patients had T-cell immunity against a type of Cas9. Sally Turner investigates at this important juncture.

Gene genie: can CRISPR-Cas9 deliver on its promise to transform genome therapy?
CRISPR-Cas9 is now touted as a novel gene-editing technology with the ability to modify, delete or correct precise regions of our DNA. Credit: AstraZeneca.

CRISPR gene-editing therapy has been heralded as the highly sort after cure for multiple diseases including those affecting the liver and eyes, Sickle Cell disease and other blood disorders.

It also has potential as a diagnostic tool in a healthcare setting, and beyond the medical sphere as a transformative agent in the consumer food industry. But can this innovative technology live up to the hype?

‘CRISPR’ is an acronym for the Clustered Regularly Interspaced Short Palindromic Repeats of genetic information that some species of bacteria utilise as part of an antiviral response.

In 2012, scientists Jennifer Doudna and Emmanuelle Charpentier re-engineered the protein Cas9 to edit genes, vastly improving on previous techniques which were more costly and unwieldy, and which edited DNA strands piece by piece.

CRISPR-Cas9 is now touted as a novel gene-editing technology with the ability to modify, delete or correct precise regions of our DNA.

CRISPR/Cas9 edits genes by precisely cutting DNA and then enabling natural DNA repair mechanisms to kick in. The Cas9 enzyme acts as biological ‘scissors’ to cut DNA at a location specified by the guide ribonucleic acid (gRNA).

“We have evolved from simply adding new genes to human cells,” says Kamaljit Behera, healthcare industry analyst at Frost & Sullivan’s Transformational Healthcare Practice. “Now we can precisely manipulate DNA to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences.”

CRISPR-Cas9 technology exceeds what was possible with other classes of engineered nucleases employed for genome editing purposes, such as ZFN, TALEN and mega nucleases.

“With Cas9 the specificity is dictated by DNA complementarity, without the need for multistep protein engineering,” explains Behera. “The CRISPR/Cas9 technology is considered to be a more straightforward, affordable and accurate way for genome-editing in comparison to traditional ZFN and TALENs approaches.”

CRISPR is almost 150 times cheaper than the ZFN method and in terms of high volume requirements, CRISPR can cost as little as $30. Furthermore, it enables multiplexed mutations – multiple genes can be injected at the same time via multiple gRNAs. The low cost of CRISPR tools and enzymes has resulted in significant interest from researchers in gene-editing-based therapies. Furthermore, researchers are exploring CRISPR’s possible applications in developing low-cost diagnostic kits and tools.

The CRISPR/Cas9 system can also be used for multiplex genome editing, in which modification of multiple loci can be performed simultaneously by multiple or single target-specific gRNA(s). “JAX Assistant Professor Dr Haoyi Wang and his former colleagues in Rudolf Jaenisch’s group at the Whitehead Institute recently reported using the CRISPR/Cas system to successfully introduce mutations in five different genes in mouse ES cells simultaneously,” says Behera. “This also makes CRISPR a more viable option for drug compound discovery, the development of agriculture-focused tools, and innovation in animal and disease models for drug discovery.”

The trials ahead

Approximately 50 companies are competing in the US CRISPR/Cas9 gene-editing tools market (covering all applications) which means the evolving market space is highly fragmented with low barriers to entry and exit.

“There are at least 25-30 CRISPR/Cas9 clinical trials that are ongoing or recruiting – about 1,000 patients,” says Aarti Chitale, senior research analyst at Frost & Sullivan’s Transformational Health Practice. “China and the US are the most active countries and some of the leading companies include Dharmacon™ (a Horizon Discovery Group company), Thermo Fisher Scientific, MilliporeSigma, Agilent Technologies, Editas Medicine, CRISPR Therapeutics, DuPont, and Intellia Therapeutics Inc.”

The first European clinical trial is underway using CRISPR technology, which will treat 12 patients who live with the blood disorders sickle cell anaemia or beta-thalassemia, and a similar trial is running in the US. The results of the European trial are eagerly awaited and will be published in May 2022.

The challenges for CRISPR-Cas9

Although CRISPR tools show great promise, the technology is not without risk and controversy.

DNA damage and the cancer risk

A British study published in September 2018, demonstrated that the CRISPR process is not always entirely accurate and may in some instances inadvertently edit untargeted DNA sequences, potentially causing unintended damage to the genome.

Swedish scientists have also published research indicating that CRISPR technology may, in certain circumstances, increase the risk of cancer.

“It is important to clarify that these reports did not claim that use of CRISPR-Cas9 technology can directly cause cancer if used as a gene therapy,” states Behera. “The studies focused on a gene called Tumour Protein 53 (TP53), which encodes a protein known as p53 that functions to repair DNA damage and prevent tumour formation. Mutations in this gene are found in the majority of human cancers, meaning that it plays a crucial role in preventing cancer.”

Behera believes that eventually CRISPR technology will be fine-tuned to the degree that precision targeting will be greater, off-setting this issue.

“CRISPR-Cas9 editing of stem cells, including pluripotent stem cells and hematopoietic stem and progenitor cells, have been shown in other studies to be more efficient than what was reported in this work, suggesting that other factors may be involved in successful genome editing of these types of cells,” he adds.

T-cell immunity

Another study in 2018, made headlines because it revealed that 96% of participants had T-cell based immunity against Cas9, and 85% had antibodies against it. If our immune systems are already primed to attack the bacterial component of CRISPR-Cas9, this a huge potential blow for the technology.

“As well as this finding, the researchers also claim that they have developed a test that will allow us to ensure that cell products can be used with confidence,” counters Behera. “It can be used to reliably determine whether the risk of an immune reaction is low.”

Some forms of gene-editing can be done outside the body in vitro, which may reduce the immune response, but Behera concedes that some genetic diseases produce tissue defects that cannot be modified outside the human body: “New solutions must be found to prevent dangerous immune responses to the CRISPR-Cas9 gene editing scissors and tighter regulations around genome editing will improve CRISPR safety.”

Gene-editing – the next ten years

“The overall investment in the gene-editing sector is worth more than $1 billion and is expected to increase over the next three to four years,” predicts Chitale. “Pharmaceutical companies have financially supported private enterprise and venture capital firms by signing product development-focused partnerships, and biopharma and manufacturing are finding huge areas of application for this technology.”

Along with the scientific fine-tuning of CRISPR-Cas9 for current and potential applications, both analysts forecast high growth potential for automated CRISPR identification software and CRISPR informatics platforms.

“Companies should develop machine learning-based tools to automate sgRNA’s identification process in databases and integrate the overall process with the laboratory information management system,” comments Behera.

Researchers are exploring different ways of using CRISPR in both diagnostic kits and therapeutics development and the successful commercialisation of such developments is likely to drive the market’s revenue growth.

In terms of therapeutics, as the CRISPR research tools mature, the applications will diversify.

“It will find application in rare and hereditary diseases, blood disorders and infectious diseases such as severe combined immunodeficiency (SCID) and HIV,” continues Behera, “and even be applicable in treating some chronic health conditions such as diabetes and cardiac issues which have a high correlation with genetics and family history.”

Additionally, a large number of agricultural companies are using CRISPR to develop gene-edited crops and food products.

“A company called Dupont Pioneer has developed an advanced plant breeding platform, which is capable of producing seeds and seed products with high sustainability, resilience and productivity,” says Chitale. “They successfully developed waxy corn hybrids, which produce a high amount of amino starch content and this can be used for processed foods and even in the production of high gloss paper and adhesives. Efforts are also ongoing in imparting resistance to viruses in plants.”

CRISPR – the ethical question

Limits to CRISPR’s broad applications may well be imposed by ethics and cultural perceptions of the technology, rather than the science itself.

“Questions around ethics are potential challenges that will govern how CRISPR technology is developed and used,” concludes Chitale. “For example, finding a cure for a genetic disorder versus choosing the external characteristics for your baby highlights a critical aspect of exploiting the very boundaries of this technology and we must be prepared for that debate.”

It is also worth noting that almost all of the current efforts to use CRISPR-Cas9 in humans involve somatic cell editing – changes to DNA that affect just an individual and are not heritable. But, cautions Chitale, “a major issue is if CRISPR technology is used for germline editing, any changes to DNA in embryos, sperm or eggs can easily pass down to future generations.”