CATALYSTS for identifying and developing an independent and proprietary CRISPR IP position .
The prime selection parameter for Cas nuclease development was to achieve freedom to operate ( FTO ). In addition to legal hurdles , there are also limits on what can be done with CRISPR , for which some of our technologies can provide gamechanging competitive advantages . Here we focus on G-dase E *, previously known as BEC . This has a novel MoA and is significantly different to classical CRISPR variants like Cas9 or Cpf1 . In terms of G-dase E the European Patent Office granted a new patent in September 2023 covering Brain Biotech ’ s G-dase E nucleases as a composition of matter1 and still pending in other jurisdictions .
Advanced metagenomics approach
From a scientific point of view , Cas nucleases are incredibly powerful tools . When the first publications on CRISPR Cas nucleases appeared , Brain immediately began testing and adapting the system for its own basic research .
The functionality was amazing and allowed for new technological applications in the field of biotechnology . However , as explained above , the IP and licensing situation was not acceptable . Therefore , the company initiated a programme using
Figure 2 - Homology modelling of G-dase E nuclease an advanced metagenomics approach to identify new Cas nucleases with the aim of gaining FTO .
Figure 1 shows the workflow . A Nagoya-compliant collection of appropriate samples from carefully chosen habitats , followed by the enrichment of specific microorganisms with the highest potential for novel CRISPR systems , then complete DNA extraction and deep next-generation sequencing . Together with a partner , we screened approximately 100 million genes in a bioinformatics pipeline .
Finally , two enzyme families with different activities were identified . The sequences of the first originate directly from the metagenome ( G-dase M ), while the enzymes of the second , G-dase E , have been heavily engineered using advanced methods ( Figure 2 ). G-dase E in particular stands out from the crowd as its unusual capabilities open up a whole new range of applications .
Novel enzymatic mechanism
The Akribion Genomics team has since developed these novel Cas nucleases , known as G-dases , with strong activity in various organisms , including bacteria and mammalian cells . The enzymatic mechanism of G-dase E differs significantly from that of previously known CRISPR systems .
Classical Cas nucleases like Cas9 or Cpf1 target DNA and usually create a double strand break in a genomic DNA sequence that is defined and bound by the so-called spacer sequence of the gRNA . G-dase E , like Cas13 , instead targets RNA by binding to a sequence of a RNA biomarker in the cell .
However , upon binding it gets activated and shreds down all RNAand DNA-type nucleic acids through a constitutive collateral activity , whereas Cas13 only depletes RNA . This mechanism has been described in our patent application and a similar MoA has been published for Cas12a2 nucleases in prokaroytes . 1 , 2
The fundamental difference in the MoA enables G-dase E to kill mammalian cells when activated by a targeted RNA biomarker , since these cells cannot cope with the complete destruction of RNA and DNA . This particular ability has not been demonstrated for other Cas nucleases .
Attempts by Kwon et al . to test and qualify Cas9 as a programmable cell killing tool revealed that multiple genomic double strand breaks are required for such an application , disqualifying them as tools for cell depletion . Hence the complexity of providing multiple guide RNAs ( for extreme multiplexing ) and identifying multiple DNA biomarkers is preventive for therapeutics . 3
However , since G-dase E is a Cas nuclease , its activation is programmable and should be adaptable to the genomic make-up of a cell by employing one designated gRNA . Thus , one RNA biomarker should be sufficient to kill a cell . Based on this concept , G-dase E , which uses guided toxicity in a cellular context based on a known cellular RNA biomarker , could become a novel class of drug that could even become a personalised drug based on a patient ' s genetic makeup in the future .
Potential in cancer
In general , G-dase E ’ s novel enzymatic mechanism opens up applications in therapeutics and diagnostics . It could also be highly synergistic with other genome editing applications by depleting unedited cells , which could be beneficial , for example in ex vivo cell therapies . However , there is a lot of evidence that the ‘ guided toxicity ’ mechanism described above or the targeted elimination of cells based on an RNA biomarker has the greatest potential in cancer therapies .
The vast majority of cancer indications are characterised by genetic alterations , such as oncogenic fusions or mutations , when compared to healthy cells . These changes lead to the appearance of RNA molecules with unique sequence motifs that can be targeted and addressed by G-dase
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