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CRISPR/Cas9 gene editing in hematology
Efficiency
Suboptimal DNA repair outcomes or insufficient target conversion might prevent an intervention from reaching a critical gene-editing threshold necessary to rescue the genetic defect. Strategies to enhance the frequency of HDR in CRISPR/Cas9-mediated transgenesis have been reported and need to be tested in the clinical context.74 Moreover, it should be considered that efficacy could be reduced if the CRISPR/Cas9-induced mutation is detri- mental to the cells, having a negative, non-reversible effect.
Immunogenicity
The immune system reaction to in vivo administration of gene editing reagents or ex-vivo genetically modified cells is also a cause for concern.75 The presence of antibodies against Cas9, mainly isolated from Staphylococcus aureus or Streptococcus pyogenes, is common in neonates and adults. Similarly, T lymphocytes against Staphylococcus aureus Cas9 constitute an obstacle to CRISPR/Cas9 therapeutic gene editing.76 Accordingly, the possible immune response must be examined in depth to ascertain whether it could compromise the efficacy of CRISPR-based treatments. Strategies to minimize/eliminate immunogenicity include the use of nucleases other than Cas9 that have not been exposed to the human immune system, or novel nucleases that do not activate an immune response. Other strategies could be: (i) to design an in silico prediction tool for immunogenic predisposition; (ii) to understand the innate immune mechanism against CRISPR/Cas9 in order to help in vector choice and engineering; (iii) to identify antigenic regions on CRISPR/Cas9 to enable deimmunization and epitope masking; and (iv) to employ immunosuppression by using drugs and/or regulatory T cells to reduce unde- sired immune reactivity.77
p53-mediated DNA damage response
CRISPR/Cas9 genome editing has recently been shown to induce a p53-mediated DNA damage response in some human cell types,78,79 which is in part responsible for the low targeting efficiencies observed in these cells. Consequently, p53 inhibition may improve the efficiency of genome editing in wild type cells; however, a caveat to this approach would be the increased likelihood of cancer- ous transformation of cells in which the “guardian” activ- ity of p53 is inhibited. p53 gene sequence and function should, therefore, be monitored closely in cells destined for therapy when developing CRISPR/Cas9 cell-based therapies.
Bioethical regulation
CRISPR/Cas9 gene editing is associated with several ethical issues; for example, its application to humans, embryos or germline cells. While the clinical application in human somatic cells to treat hematologic diseases is gen- erally accepted, there is consensus among geneticists that its application in human embryos and germline cells (except for research purposes), in which genetic changes would be inherited by future generations, should be impermissible. That being said, some alarming news was recently reported about the use of CRISPR/Cas9 in human embryos to inactivate the CCR5 receptor and provoke resistance to HIV infection. The biophysicist He Jiankui presented limited (and non-peer-reviewed) data on the birth of twin girls genetically edited with CRISPR/Cas9.
This claim, whether true or not, urgently imposes the establishment of strict regulations on human CRISPR/Cas9 genome editing such that it should only be considered for therapeutic uses, but not for human enhancement or eugenics, although it could be used as a research tool to understand early human development or disease pathogenesis. Thus far, no patients have been clin- ically treated with in vivo CRISPR-based therapy, whereas patients have been given infusions of ex vivo modified T cells (Table 6).
The ethical and regulatory aspects of therapeutic CRISPR/Cas9 genome editing are very complex.80 Given the proven potential of CRISPR/Cas9 to modify the human genome, there are naturally great expectations for future applications. To discuss all these concerns, a multi- disciplinary regulatory committee, composed of geneti- cists, lawyers, society representatives and clinicians, should be created to define a legislative framework to reg- ulate permission or prohibition of CRISPR applications and any genome engineering technique in the future. Global scientific and biological ethics communities must take the lead and establish standards and procedures that reduce the dangers of these powerful new technologies without forgetting the benefits.
What is necessary to move the hematology field forward
As with any new treatment, safety and efficacy are very important. Efforts should be made to develop novel vector systems to maximize delivery to target cells with minimal side effects. In this sense, platforms for systemic and local administration of CRISPR/Cas9 would be highly desir- able, including non-integrative vectors for gene therapy or systems based on nanoparticle technology, ribonucleopro- teins or other novel approaches.
In the last decade, next-generation sequencing has led to enormous advances in the molecular diagnostics of hema- tologic malignancies. Somatic mutations have been revealed in many diseases and some may have important prognostic value. The functional impact of these muta- tions on tumor initiation and/or maintenance needs to be addressed in the next years, and CRISPR/Cas9-based screens in patient-derived cells will be powerful tools to undertake this endeavor. In addition, some hematologic malignancies are characterized by mutations in epigenetic modifiers, proteins that modify DNA at cytosine residues or cause post-translational histone modifications. Some therapies already exploit epigenetic targets, such as DNA methyltransferase 3A (DNMT3A) or histone deacetylase (HDAC), and hypomethylating agents, including the DNA methyltransferase inhibitors azacytidine and decitabine, are used to treat myelodysplastic syndrome and AML. Accordingly, targeted epigenome editing,81 which is the modification of the epigenome at specific sites as opposed to whole-genome modification, could be an area for research development in hematology – for example, for fine-tuning gene expression by locally modulating DNA methylation or determining the function of specific methylation sites. Because epigenome editing does not involve genetic changes, it may also be less hazardous with respect to off-target effects. The challenges will be how to administer epigenome-editing tools in vivo, to achieve reversible epigenetic modifications at precise sites
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