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promise for the clinical translation of CRISPR/Cas9 gene editing therapies for primary immunodeficiencies.
Acquired immunodeficiencies
Human immunodeficiency virus type-1 (HIV-1) infec- tion gives rise to acquired immune deficiency syndrome (AIDS). Several different CRISPR/Cas9 gene-editing strategies have been used to target HIV-1 along its replica- tion cycle.61 For example, disruption of the CCR5 receptor gene, which is present in the host cell and is a co-factor for entry of HIV into the cell, represents a promising strategy to combat the disease, and several studies have reported NHEJ-mediated CRISPR/Cas9 inactivation of CCR5 and other co-factors in lymphocytes. In one study, generation of iPSC homozygous for the naturally occurring CCR5Δ32 mutation through CRISPR/Cas9 genome editing conferred resistance to HIV infection.56 Antiretroviral therapy fails to cure HIV-1 infection because of the persistence of HIV reservoirs harboring integrated HIV DNA. CRISPR/Cas9- mediated deletion or inactivation of proviral DNA could eliminate this source of HIV persistence, thereby being a potentially curative treatment. In preclinical studies, CRISPR/Cas9 efficiently mutated and deactivated HIV proviral DNA in latently infected Jurkat cells.62 However, complete eradication of HIV latent infection is challenging because of the development of mutations resistant to attack by DNA-shearing enzymes.51 Clinically, the safety of transplantation of CRISPR CCR5-modified CD34+ cells in HIV-infected patients with hematologic malignancies is under evaluation in clinical trials (Table 6).
Cancer immunotherapy using chimeric antigen receptor T cells
Cancer immunotherapy can be defined as the induction or enhancement of cancer-specific immune responses against malignant tumors. One approach to this is the ex vivo manipulation of patients’ T cells to express a chimeric antigen receptor (CAR) including an intracellular chimeric signaling domain capable of activating T cells and an extracellular binding domain that recognizes an antigen specific for and strongly expressed on tumor cells. CAR-T cells are re-infused into patients to attack cancer cells in vivo. Currently, CAR-T cells expressing CD19, CD20, CD22, or dual B targeting CAR-T cells are available to treat acute lymphoblastic leukemia, non-Hodgkin lym- phoma and chronic lymphocytic leukemia.63 Unfortunately, CAR-T-cell administration can have adverse effects, such as neurotoxicity, cytopenia and cytokine release syndrome, which can be life-threatening. CRISPR/Cas9 can be utilized to complement CAR-T-cell therapy, for example, via disruption of the endogenous T- cell receptor (TCR). Upon its interaction with engineered, transgenic TCR in patients’ cells, endogenous TCR can alter the antigen specificity of CAR-T cells. In a study using CRISPR/Cas9 to knock out endogenous TCR-β, with simultaneous introduction of CAR-T cells, the authors found that this replacement strategy resulted in more robust transgenic anticancer T cells.64 The CRISPR/Cas9 system has also be applied to eliminate other genes that encode inhibitory T-cell surface receptors, such as programmed cell death protein 1 (PD1), to improve the efficiency of T-cell-based immunotherapy.65 To exploit CAR-T-cell therapy beyond the autologous set- ting, allogeneic universal T cells derived from healthy donors could be engineered by CRISPR/Cas9 upon disrup-
tion of TCR to prevent graft-versus-host disease, or beta-2- microglobulin, to eliminate major histocompatibility com- plex I (MHC I) expression, or by integrating a CAR pre- cisely at the disrupted T-cell receptor a constant (TRAC) locus to improve safety and potency.66-68 Thus, CRISPR/Cas9 technology holds enormous promise to advance the field of cancer immunotherapy and several clinical trials are running to assess the efficacy of CRISPR/Cas9-edited CAR-T cells (Table 6).
Challenges and opportunities for CRISPR/Cas9 therapeutic applications
Delivery of editing tools
Delivery platforms that ensure the access of editing components into a large number of target cells are critical for the clinical development of this technique. Ribonucleoprotein is the cargo format preferred over other transient delivery methods such as mRNA and non- integrating viral vectors because of its hit-and-run mecha- nism, which reduces the risk of undesired off-target effects, and also because of its ability to efficiently modify cells with low translation rates.47 Nevertheless, the bene- fit-to-harm ratio of the CRISPR/Cas9 system must be maximized. Possible solutions include the development of novel approaches to integrate ribonucleoprotein and donor template DNA for gene correction in a unique sys- tem.
Safety
Off-target DSB can result from non-specific Cas9 cleavage at unwanted genome sites, which is perhaps the major concern regarding therapeutic CRISPR/Cas9 edit- ing. Accordingly, genome-wide sequencing approaches should be employed to thoroughly examine for modifi- cations at unexpected genome locations, or at anticipated off-target sites indicated by in silico prediction tools. The issue of off-target activity is, nevertheless, controversial since studies have yielded contrasting results.69,70 Along this line, several methods have been developed in the last years to detect CRISPR off-target mutations;71 however, there is a lack of consensus on how to predict which putative off-target sites should be examined via deep tar- geted sequencing. Additionally, the possibility of Cas9- induced on-target mutagenesis, including large deletions and rearrangements that may have pathogenic conse- quences, has been highlighted as another safety con- cern.72 Accordingly, more research is needed for a defini- tive understanding of the in vivo genomic effects of CRISPR/Cas9. Indeed, the possibility of producing unde- sired gene modifications raises concerns about the use of the CRISPR/Cas9 system for therapy in humans. For instance, infused gene-edited HSC could have the poten- tial to expand clonally and induce leukemia, and so clin- ical gene editing might cause panic. Possible solutions include the substitution of Cas9 with a different nucle- ase, for example, Cas12a (also known as Cpf1), which prohibits mismatches between the 18 nucleotides next to the protospacer adjacent motif.73 Other alternatives include the use of paired nickases, guided by two differ- ent gRNA targeting the same locus but on opposite DNA strands, or “base editors” editing nucleotides without inducing a DNA break.17
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