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Review
. 2020 Sep 29:11:570672.
doi: 10.3389/fimmu.2020.570672. eCollection 2020.

Using Gene Editing Approaches to Fine-Tune the Immune System

Kristina Pavlovic  1   2 , María Tristán-Manzano  1 , Noelia Maldonado-Pérez  1 , Marina Cortijo-Gutierrez  1 , Sabina Sánchez-Hernández  1 , Pedro Justicia-Lirio  1   3 , M Dolores Carmona  2 , Concha Herrera  2   4 , Francisco Martin  1 , Karim Benabdellah  1
Affiliations
Review

Using Gene Editing Approaches to Fine-Tune the Immune System

Kristina Pavlovic et al. Front Immunol. .

Abstract

Genome editing technologies not only provide unprecedented opportunities to study basic cellular system functionality but also improve the outcomes of several clinical applications. In this review, we analyze various gene editing techniques used to fine-tune immune systems from a basic research and clinical perspective. We discuss recent advances in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We also discuss the use of programmable nucleases and their derivative reagents such as base editing tools to engineer immune cells via gene disruption, insertion, and rewriting of T cells and other immune components, such natural killers (NKs) and hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric antigen receptors (CARs), we describe how different gene editing tools enable healthy donor cells to be used in CAR T therapy instead of autologous cells without risking graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs and instant treatment availability for patients. We pay particular attention to the delivery of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral damage and increases therapeutic effectiveness. Finally, we review creative innovations, including immune system repurposing, that facilitate safe and efficient genome surgery within the framework of clinical cancer immunotherapies.

Keywords: CARs; base editors; gene editing; graft-vs-host disease; immunotherapy.

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Figures

FIGURE 1
FIGURE 1
Representative scheme of the different genome editing tools used to improve the immune system. There are two variants of genome editing technologies: those that introduce double-stranded breaks (DSBs) into DNA and those that enable genome editing without DSBs. The first variant is mainly composed of the components ZFNs, TALEN, and CRISPR/CAS which are used to enhance immune system capacity. ZFNs are chimeric proteins containing a DNA binding domain (3–5 zinc-finger domains) and a Fok1 endonuclease domain (114). Each zinc-finger domain is specifically designed to bind to virtually any DNA sequence. ZFN cleavage activity needs to be dimerized given that Fok1 acts as a dimer. Two ZFNs therefore need to be designed, each targeting a DNA sequence separated by a short sequence from the recognition site of the other ZFNs in a head-to-head fashion. As ZFNs, TALENs contain two different domains: the DNA binding domain of the TALE protein designed to bind the desired sequence (10) and the Fok1 endonuclease domain (11). As TALENs only act as dimers, two TALENs must be designed to bind to the target locus in a face-to-face fashion to cleave the target sequence (110). CRISPR/Cas, the last described SENs, is the easiest to design and the most versatile gene editing tool. It is derived from the adaptive immune system of prokaryotes, which provides a defense mechanism against certain viral infections and plasmids. The CRISPR/Cas protein forms a complex with the RNA molecules crRNA (CRISPR RNA) and crRNA (tracrRNA) which guides the Cas protein to the target DNA and produces the cleavage (12). The only part that needs to be changed to specifically cut a new target site in the genome is 20 crRNA nucleotides (13, 14). Various modifications to the gRNA design and to the Cas9 protein have been made to reduce off-target activity. A mutated Cas9 nickase has been generated to expand the CRISPR genome editing system (111). The second group is mainly composed of CRISPR-CAS nickases variants and of two base editor (BE) variants, the cytosine base editor (CBE) and the adenine base editor (ABE). The first variant is based on simple APOBEC deaminase system named BE1, which fuses APOBEC1 and dead Cas9 from Streptococcus pyogenes with D10A and H840A mutations (28). Its lower efficiency is attributable to uracil DNA glycosylase (UDG) which catalyzes the removal of U from DNA in cells and initiates base-excision repair (BER), thus converting the U:G pair to the C:G pair (115). The uracil-DNA glycosylase inhibitor (UGI) was fused to the C terminus of BE1 to create the second-generation BE2 system, with an improved base editing yield of 50%. A further improvement was implemented for the third-generation BE3 system. The improved BE4 base editor contains a rAPOBEC cas9 linker expanded to 32 amino acids, a Cas9n-UGI linker expanded to 9 amino acids, as well as the addition of a second copy of UGI to the C terminus of the constructs (116); BE3 and BE4 have been validated for use as base editors of human primary T cells (55). The replacement of the APOBEC1 component in BE3 with natural adenine deaminase Escherichia coli TedA led to the creation of the first adenine base editor ABE which was followed by ABE1.2 After several target mutations and optimizations, the ABE7.1 base editor was released. This was followed by the latest version ABE8 with its base editing facility particularly for HSPCs and human primary T cells (33). Figures were created by BioRender.com.
FIGURE 2
FIGURE 2
Recent advances in engineering different immune cell types for immunotherapy applications. Engineered T cells in the B2M gene have lowered HLA class I antigen expression on the cell surface and have reduced the possibility of graft rejection. TCR/CD3 cells have been knocked out to reduce GVHD and to enable physiological CAR expression, thus enhancing CAR T potency. Tumor-suppressive microenvironments have been overcome by downregulating CTLA-4, PD1, and LAG-3. T cells have also been engineered to ignore suppressive signals by expressing dominant negative TGF beta receptors (TGBR2). On the other hand, to engraft T cells under lymphodepleting preparative conditions, the elimination of CD52 is required to enable T cells to resist alemtuzumab-mediated lymphodepletion. Targeting CD7 prevents fratricide and enables the expansion of CD7 CAR T cells without compromising their cytotoxic function. IL-2R has also been engineered to facilitate IL12P70 expression in a controlled manner. Cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, whose inhibition confers antitumor immunity, has been identified to regulate the PD-1/PD-L1 pathway. HSPCs are also gene edited to enhance adoptive immunotherapy. CD33-deficient human HSPCs resistant to CD33-targeted approaches have been produced to mitigate CART33 toxicity, to maintain myelopoiesis, and to prevent on-target off-tumor toxicity. Immune NK cells play an important role in host immunity against cancer and viral infections. Despite the low efficiency of viral and non-viral delivery methods, several NK cells can be edited to enhance their persistence, cytotoxicity, and tumor targeting (117). Dendritic cells (DCs) play a critical role in T-cell response instructions, with triple knockout established as proof of concept (118). A similar approach is used to target the costimulatory molecule CD40, whose disruption significantly inhibits T-cell activation, thus reducing graft damage and prolonging graft survival (88). Macrophages can also be edited using CRISPR-CAS9 by targeting USP7 and USP47, two genes that regulate inflammasome activation (119). The Ntn1 gene, thought to be involved in cell migration disruption, can also be targeted in vivo using nanoparticles encapsulating CRISPR/Cas9 RNPs under the control of the CD68 promoter (89). To elucidate its role in inflammation, the NEU1 gene can be targeted in macrophages using CRISPR-CAS9, thus demonstrating the role of NEU1 macrophages as inflammation enhancers (120). It is also possible to engineer B cells to express mature broadly neutralizing bNAb antibodies targeting IgH loci or safe harbor CCR 5 in the case of FIX. Figures were created by BioRender.com.
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