How can we make CRISPR therapies safer?

• NHEJ is the most common DSB repair pathway in higher eukaryotes, being active in all phases of the cell cycle, but it is most active during the G1 phase . In short, this system involves several DNA repair factors that, after recognition of the DSB, lead to filling of the gap using random nucleotides. This system is very efficient at quickly repairing the DSB but lacks precision, usually resulting in insertion or deletion (INDELs) of random bases around the cleavage site. This feature of NHEJ is exploited in some gene editing approaches in order to alter the reading frame and thus knock out expression of targeted genes.

• HDR is mostly active in dividing cells, particularly in the G2 and S phases of the cell cycle. It is associated to DNA damage during chromosomal replication. After recognition of the DSB, HDR factors employ the sister chromatid as a template for strand invasion and homologous recombination followed by polymerase-mediated extension of the single-stranded DNA. This leads to precise repair of the DSB. This mechanism can be exploited in dividing cells by delivering a homologous recombination template flanked by homology arms. This allows correction of mutations as well as integration of exogenous sequences in targeted loci. This approach has been used extensively in vivo but can only be used in dividing cells and thus is very inefficient in most differentiated tissues of the organism.

• MMEJ is considered to be a mix between NHEJ and HDR. It involves proteins from both pathways but is active through all cell cycle phases, which implies it can be independent of both NHEJ and HDR. In short, the DNA repair machinery can detect microhomologous sequences flanking the site of the DSB and facilitate recombination between those sequences, generating a small and precise deletion between the two microhomologous regions. If possible, this mechanism is quite efficient and leads to predictable INDELs.

1. They can be tailored for delivering different forms of the CRISPR–Cas9 system.
2. Viral vectors are smaller and the gene-editing system needs to be divided and separately packed in two vectors.
3. Cells may have a preexisting immunity against viral vectors. The immunocompatibility of synthetic materials can be improved by optimizing the size, shape, coating, and surface chemistry and charge.
4. Retroviral and lentiviral vectors have a large genome of size. However, these vectors may integrate the transgene into the host genome, disrupting functional genes, and increasing off-target gene editing. Synthetic-material-mediated delivery can avoid such problems although random integration has been observed.
5. Synthesis/ production of the synthetic material and its conversion to nanoparticles is more cost-effective and suitable for large-scale production than viral vectors.

• it allows controlling edited cells without the need to integrate bulky construct in the cell. Like a safety-switch as you would do for CART cells for example. So you can envision to perform along with the editing at the desired target also the KO of the UMPS gene. This means that if after re-infusing the edited cells in the patient if some side effects are observed ( maybe as a consequence of undetected off-targets ), withdrawing the organic compound will be enough to get rid of the cells

• if you infuse such cells in the patient and the patient benefits from them, you will need to keep these cells alive. How you do it? By supplying for example Uridine. Unfortunately, even if Uridine has been used in humans for the treatment of hereditary orotic aciduria and fluoropyrimidine toxicity, it is poorly absorbed and broken down in the liver. This can be circumvented by the administration as the prodrug uridine triacetate (UTA; also known as PN401), which has approval from the US Food and Drug Administration (FDA). So, you are maybe solving one problem but you are creating a dependency on the patients on constant Uridine supplement. At least until the edited cells have completed their job
• to create cells holding both the UMPS gene KO and the modification in the gene of interest you will need to introduce two DNA double-stranded break (DSB) at the same time. This increases the probability to introduce gross DNA rearrangements like translocations or head-to-head chromosomic fusions .

1. They inhibit the guide RNA and Cas complex.
2. They prevent Cas binding to the DNA.
3. They prevent DNA cleavage by Cas.

1. why CRISPR is already safe enough
2. strategies used to refine CRISPR precision
3. why although precise, CRISPR has an inner “ defect “
4. alternative to CRISPR to address the previous point.

Background

The point of this

Ethical questions