Current Status of Gene Engineering Cell Therapeutics

Ex vivo manipulations of autologous patient’s cells or gene-engineered cell therapeutics have allowed the development of cell and gene therapy approaches to treat otherwise incurable diseases. These modalities of personalized medicine have already shown great promises including product commercialization for some rare diseases. The transfer of a chimeric antigen receptor or T cell receptor genes into autologous T cells has led to very promising outcomes for some cancers, and particularly for hematological malignancies. In addition, gene-engineered cell therapeutics are also being explored to induce tolerance and regulate inflammation. Here, we review the latest gene-engineered cell therapeutic approaches being currently explored to induce an efficient immune response against cancer cells or viruses by engineering T cells, natural killer cells, gamma delta T cells, or cytokine-induced killer cells and to modulate inflammation using regulatory T cells.

iNTRODUCTiON Cell and gene therapy is an emerging field with the high potential to offer a curative therapy. Gene therapy is defined as the use of genetic material such as DNA to manipulate a patient's cells, and cell therapy is defined as the administration of live whole cells or of a specific cell population to a patient. In many diseases, cell and gene therapies are combined as gene engineering cell therapeutics in the development of promising therapies for the treatment of an acquired or inherited disease. The number of applications for gene engineering cell therapeutics is increasing at a very rapid pace, with these applications being at different development stages from preclinical to clinical.
Autologous gene engineering cell therapeutics have the potential to correct the underlying genetic cause of some monogenic disorders and potentiate immune responses against cancers to provide sustained clinical responses (1)(2)(3)(4)(5). In addition, one of the main advantages of autologous therapies is their full major histocompatibility complex (MHC) compatibility leading to a better engraftment and persistence of the cells and a low risk of graft versus host disease (GvHD). Gene transfer into autologous hematopoietic stem cells (HSC) has shown potential especially in treating primary immunodeficiencies such as X-linked severe combined immunodeficiency (X-SCID) or adenosine deaminase deficiency-SCID. The transfer of a chimeric antigen receptor (CAR) or T cell receptor (TCR) genes into autologous T cells allows redirecting the genetically engineered T cells towards specific antigens expressed on cancer cells or presented as peptides on MHC molecules, respectively. In particular, the transfer of autologous CD19-CAR T cells in patients with hematological malignancies has been very successful, achieving impressive remission rates (6). Notably, the Food and Drug Administration (FDA) recently approved the first CAR T cell therapy, Kymriah ® (or tisagenlecleucel), for patients with B cell acute lymphoblastic leukemia (ALL). In addition, another CAR therapy was approved by the FDA, "Yescarta ® " (axicabtagene ciloleucel), for the treatment of adult patients with certain types of non-Hodgkin lymphoma.
However, some of the trials testing gene engineering cell therapeutics have not been without setbacks such as the incidence of insertional mutagenesis observed in the first clinical trials for X-SCID, which has led to the design of new vectors allowing reducing their potential for insertional mutagenesis. This also highlighted the clear need for long-term follow-up for the patients receiving these live gene engineering cell therapeutics. In addition, several deaths linked to neurotoxicity in patients treated with CD19-CAR T cells have been reported and the cytokines produced after infusion of the product can lead to adverse effects such as cytokine release syndrome (CRS) that many patients experienced, highlighting the fact that we still need to gain a better understanding of the effects of gene engineering cell therapeutics in patients so as to make these therapies safer. Here, we review the latest gene engineered cell therapeutic approaches being currently explored preclinically but emphasizing those that have been clinically tested (Figure 1), to induce an efficient immune response against cancer cells or viruses by engineering T cells, natural killer (NK) cells, gamma delta T cells or cytokineinduced killer (CIK) cells and to modulate inflammation by using regulatory T cells (Tregs).

CeLL AND GeNe THeRAPieS FOR ONCOLOGY
Different cell types can be used to develop gene engineering cell therapeutics to induce an immune response against tumor cells.
Here, we will review the use of TCR-or CAR-modified T cells (TCR-T or CAR-T), NK cells, CIK cells, and gamma delta T cells for oncology.

Autologous TCR-T and CAR-T
T cell engineering provides the possibility to generate antigenspecific T cells for many types of cancer. The identification of a graft versus leukemia (GvL) effect and subsequent recognition that T cells played a role provided an early indicator of the potential of T cells to mediate antitumor effects and led to the use of donor lymphocyte infusion (DLI). Subsequent studies of autologous tumor infiltrating lymphocytes (TIL) in melanoma patients for whom suitable TIL could be isolated and expanded successfully, allowing adoptive transfer, led to significant clinical responses (7,8). The difficulty to isolate TIL from some patients' tumors, especially with non-melanoma tumors, led to the concept of modifying autologous peripheral blood T cells to redirect them to recognize tumors offering an alternative approach to develop T cell therapies. Two principal strategies were developed, one using natural TCR that recognized human leukocyte antigen (HLA)-restricted tumor-associated antigen peptides (9)(10)(11)(12) and the other seeking to use antibody recognition domains linked to TCR signaling molecules in CAR (13)(14)(15).
Both strategies have been continually refined over the last 15 years, with promising early data culminating in a number of medicines in late-stage clinical development and two approved medicines (Tables 1 and 2). The first clinical study of TCR-T  (12). There were some initial signs of clinical activity with 2/17 melanoma patients undergoing a partial response, and no related toxicities were observed. Subsequent studies included using TCR of mouse origin (16)(17)(18)(19)(20) and affinity-enhanced TCR (16,(21)(22)(23). A variety of different tumor antigen targets have also been evaluated in different tumor types ( Table 1). The most advanced TCR-T clinical asset, NY-ESO-1-specific TCR-T cells, has shown particularly promising clinical responses in multiple myeloma (MM) and synovial sarcoma (19,23,24), with a 61% response rate and a 38% overall survival (OS) at 3 years (19,24,25) and a 91% response rate and a median OS of 3 years in MM in the context of autologous hematopoietic stem cell transplantation (HSCT) (23,26). As shown in Table 1, improved clinical response rates have been observed in multiple TCR-T studies compared to the original study of Morgan et al. including complete responses, but there have also been some serious, sometimes fatal adverse events.
Early CAR-T studies, using what is now termed a firstgeneration construct design with the antigen-binding domain scFV linked to CD3 zeta or FcR γ chain as a signaling domain, provided evidence of feasibility for CAR-T cell production but lacked clinical efficacy in HIV-infected patients or patients with solid tumors ( Table 2) (28-30, 32, 33). Indeed, activation through the CD3zeta chain or FcR γ chain was insufficient to produce productive immunity (43,44). Second-generation construct designs were developed by addition of a costimulatory domain (typically CD28 or 41BB) to CD3zeta to simultaneously provide both activation and co-stimulatory signals [several recent reviews have addressed CAR design in detail (45)(46)(47)(48)(49)]. The CAR-T field was truly energized when Carl June and colleagues at the University of Pennsylvania, using second generation CD19-specific CAR-T cells, achieved two complete responses in the treatment of three patients with refractory advanced chronic lymphocytic leukemia (CLL) using anti-CD19 CAR T cells (4,35). Subsequently, CD19 CAR-T cell therapy has generated complete and durable remissions in patients with refractory and relapsed B cell malignancies (5,6,37,50). As previously mentioned, the FDA has recently approved the CD19 CAR-T cell approach from Novartis, Kymriah ® , to treat patients with B cell ALL and the CD19 CAR-T cell approach from Kite Pharma, YESCARTA™, for the treatment of diffuse large B-cell lymphoma.
These autologous CAR-T and TCR-T cell therapies show great promise but there remain many aspects of these approaches that need to be further refined. The highly potent nature of these modified T cells produces dramatic tumor regressions; however, toxicities have also been observed. There have been a number of deaths on TCR-T and CAR-T trials. A detailed review of toxicity is beyond the scope of this review, but it is important to briefly underscore the risks. Some of these deaths have been due to apparent on target, off tumor toxicity, where expression of the target antigen on normal tissues occurs. For example, a patient died from rapid respiratory failure and multiorgan dysfunction in a CAR-T trial targeting ErbB2 in patients suffering from lung carcinoma, due to the recognition of ErbB2 on normal lung cells (51). Both Morgan et al. and Linette et al. reported instances where TCR-T targeting different MAGEA3 epitopes led to reactivity against proteins with amino acid sequence homolog to the target epitope in the brain (MAGEA12) and the heart (Titin), respectively (20, 21) ( Table 1). In various CD19 CAR-T trials, patient deaths have been reported due to neurotoxicity caused by cerebral edemas [reviewed in Ref. (52)]. Here, the exact mechanism is less clear. Other severe side effects, such as CRS and tumor lysis syndrome, can also mean that patients often require aggressive support in an intensive care unit setting (53).
Another area for improvement is to induce better efficacy in more indications. Indeed, clinical response rates and the durability of response in some hematologic malignancies and all solid tumors are currently lower than those seen with CD19 CAR-T in ALL. The tumor microenvironment in solid tumors is a hostile environment for T cells, and low persistence of gene-engineered T cells with low efficacy has been observed. Since there is a dearth of truly tumor-specific targets because most tumor-associated antigens are also naturally expressed on some normal tissues, there is a need for new targets and ways to better control "on target, off-tumor" toxicity. Additional strategies will likely be needed to improve the effectiveness of CAR-T and TCR-T in the face of these obstacles [reviewed in Ref. (49,54)]. In addition, the scale-up in manufacturing capability to supply thousands of patients has yet to be demonstrated and will be critical for the success of these autologous T cell approaches.

NK Cells
Natural killer cells are defined as CD56 + CD3 − cells and are innate immune lymphocytes able to exert cytotoxicity against tumor cells and virally infected cells without prior stimulation. Because of their antitumor activity, NK cells have been put forward as potential candidates for cancer immunotherapy. Cell therapy using autologous NK cells has proven to be safe and feasible when treating hematological malignancies such as CLL or different types of solid tumors but with however quite limited clinical responses (55)(56)(57)(58)(59).
The first evidence that allogeneic NK cells could be more cytolytic against tumor cells than autologous NK cells stems from the study by Ruggeri et al., who showed that allogeneic NK cells were very potent effectors of the GvL especially in the case of a killer-cell immunoglobulin-like receptors ligand incompatibility in the graft versus host direction in patients who received HLA-mismatched HSCT (60). In addition, the infusion of allogeneic NK cells together with interleukin (IL)-2 has shown some promising results in patients with different types of advanced cancers (61,62) and several groups reported similar results using allogeneic NK cells mainly in the context of haploidentical HSCT (63-69) ( Table 3). Although NK cells are short-lived, they may provide a potential off-the-shelf therapy with limited toxicity as they do not induce GvHD. A recent first-time-in-human study has also been reported exploring the use of umbilical cord blood (CB)-derived NK cells in MM (70).
A number of gene engineering cell therapy using human NK cell lines have been established. In particular, NK92 has been investigated as a therapy, either unmodified or with a variety of different genetic modifications. The first human clinical trial report using NK92 cells by Arai et al. showed some initial signs of clinical activity (73). Subsequently, a multicenter trial enrolled 15 patients, showing as well signs of clinical activity (74). Recently, clinical trials results with NK92 cells were reported by Keating and colleagues at the Princess Margaret Cancer Centre, Toronto (NCT00990717) (75). In that study, 5 of 12 patients exhibited a clinical response, including 2 complete responses, 1 of which is sustained and ongoing 10 years posttherapy. Boyiadzis et al. also recently reported results of a clinical trial of NK92 cells in 7 refractory/relapsed AML patients with 3/6 showing transient clinical responses (76).
Since NK92 cells do not express CD16 and therefore cannot mediate ADCC, a NK92 variant that has been engineered to express CD16 and intracellularly retained IL-2 (haNK) to be combined with monoclonal antibody therapies and clinical development is underway (e.g., NCT03027128). No clinical results with haNK cells have yet been reported.
Natural killer cells could also be used for the generation of CAR NK cell products for immunotherapy to enhance their effector function by providing antigen specificity. This could be especially effective in the case of cancers resistant to NK cell killing. Moreover, CAR-NK cells could be used to bridge the patient's immunity during HSCT or prior to administration of another therapy such as CAR-T cells. Preclinical studies using CAR-NK cells have shown promising results in vitro and in animal models against different types of solid tumors (77,78) and hematological malignancies (79)(80)(81). Thus, first-time-in human testing is underway. Notably, Shook and colleagues at St. Judes Children's Research Hospital, Memphis, USA, have completed enrollment in a trial of CD19 CAR-expressing NK cells for B-Lineage ALL (NCT00995137), and results are  Overall, all the ongoing clinical trials will provide key data on safety and efficacy of gene engineering NK cell therapeutics. It will be interesting to evaluate whether NK cell therapies can be as effective as their T cell counterparts for certain indications and whether CAR-NK cells lead to similar adverse effects such as CRS and neurotoxicity.

CiK Cells
Cytokine-induced killer cells are a heterogeneous effector CD3 + CD8 + cell population that exhibit non-MHC-restricted cytotoxicity [reviewed in Ref. (85)]. There has been a long history of clinical studies testing CIK cells, with evidence of antitumor effects of CIK cells against hematologic malignancies and solid tumors, along with studies exploring their antivirus potential and anti-GVHD potential. The first human clinical trial of autologous peripheral blood-derived CIK transfected to express human IL-2 by electroporation demonstrated the safety in patients with metastatic renal cell carcinoma, colorectal carcinoma, and lymphoma (86) ( Table 4). In one patient with follicular lymphoma grade I, a pre-existing bone marrow involvement as the only sign of disease resolved after CIK cell therapy and was scored as a complete clinical response.
Various CIK clinical trials have combined CIK cells with other therapies, including mAb therapies, chemotherapies, radiotherapy, and dendritic cell (DC) therapy. An in-depth review of all CIK clinical studies is beyond the scope of this article because more than 100 clinical trials of CIK have been reported, and readers are referred to Gao et al (85), for a review (91). Some examples of key clinical studies are listed in Table 4. In 2012, a randomized phase II clinical study showed that CIK therapy could enhance the efficacy of conventional chemotherapy in patients with NSCLC (87). The study was  The next few years will see clinical results from CAR-modified NK cell and CAR-modified CIK cell trials and will determine if these approaches are efficacious and can complement or perhaps replace CAR-T/TCR-T approaches.

Gamma Delta T Cells
Gamma-delta (γδ) T cells represent less than 5% of circulating T cells and unlike conventional αβ T cells, the TCR repertoire of γδ T cells is very restricted. They can be cytotoxic and secrete cytokines like conventional αβ T cells, but γδ T cells do not require MHC antigen presentation for antigen recognition. γδ T cells are also more widely distributed in tissues throughout the body, in addition to also being present in the typical T cell compartments, the lymph nodes, and spleen, where most αβ T-cells reside. Thus, γδ T cells could potentially offer differentiated activity from αβ T cells, and some investigators are beginning to explore their utility as CAR carriers. Indeed, one specific subset of γδ T cells identified by their expression of a Vg9Vd2 TCR can be expanded using bisphosphonates and are known to recognize in an MHC-independent manner phosphoantigens, and these tend to be enriched in tumors (97). The MHCindependent nature of the Vg9Vd2 antigen recognition also means that they are not alloreactive and therefore should not cause GvHD. Deniger et al. recently reported on a methodology to expand polyclonal γδ T cells in vitro and electroporation with Sleeping Beauty transposon and transposase to enforce expression of a CD19 CAR (98). The demonstrated killing of CD19 + tumor cell lines and adoptive transfer of CAR γδ T cells reduced growth of CD19 + leukemia xenografts in mice. Clinical translation of CAR γδ T cell approaches will be needed to determine if these cell carriers provide advantages over CAR αβ T cells. In that regard, Chen and colleagues at the Fuda Cancer Hospital, Guangzhou, China have recently opened four studies for enrollment to assess safety and efficiency of autologous peripheral blood mononuclear cell-derived γδ T cells against lung cancer (NCT03183232) and liver cancer (NCT03183219), breast cancer (NCT03183206), and pancreatic cancer (NCT03180437) respectively ( Table 5).

CeLL AND GeNe THeRAPieS USiNG GeNOMe eNGiNeeRiNG
A further step in simplifying supply and broadening access to donor cells is to engineer partially matched allogeneic cells. This approach is attractive for situations where dramatic clinical efficacy can be mediated rapidly and where perhaps sustained persistence of the cell product is not a requirement. One such example was recently reported whereby unmatched allogeneic donor T cells were lentivirally transduced to express an anti-CD19 CAR, followed by gene editing with TALENs to remove expression of the endogenous TCR alpha chain to prevent GvHD together with removal of CD52 to render them insensitive to the lymphodepleting agent Alemtuzumab (99). These TCR-ablated T cells were then administered to two relapsed refractory B-acute lymphoblastic leukemia patients as temporary "bridge to transplant" designed to eradicate their leukemia before a subsequent planned allogeneic HSCT. Recently, it has also been shown that gene editing could be used to generate high numbers of redirected NY-ESO-1 T cells with a predominant stem and memory T cell phenotype that were tested preclinically in vitro and in a mouse model of MM without inducing GvHD (100).
The "holy grail" would be an entirely "universal donor, " long-lived cell product providing an "off-the-shelf " cell therapy acceptable to any patient irrespective of their HLA and TCR specificity. Technically, genome editing technology offers the possibility to engineer such universal donor cells in a controlled manner. Cells could be modified ex vivo using gene editing to knockout expression of HLA class I and potentially HLA class II molecules to prevent GvHD that could otherwise lead to graft rejection. It has been shown that elimination of HLA class I molecules using zinc finger nucleases was possible in primary T cells as well as in embryonic stem cells (101,102). Furthermore, the removal of HLA-A molecules in HSC had no impact on their function as the cells could engraft in immuno-compromised mice (103). Depending upon the donor cell type, the removal of other molecules such as the TCR that could induce GvHD would be required. Additionally, other cell engineering may be necessary such as the introduction of a β-2 microglobulin (β-2M) gene fusion to a non-polymorphic HLA-E, F, or G gene in order to avoid NK cell-mediated lysis of the cell product that would otherwise occur if the engineered cells are completely devoid of class I molecules (102,104).
Zhao, June and colleagues have reported on preclinical studies combining lentiviral delivery of a CAR with electroporation of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2M, and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule, and PD1 by multiplex genome editing (105,106  in lung cancer patients that had been disrupted for PD1 using CRISPR/Cas9 (NCT02793856). These and other early studies will evaluate the safety of genome-edited T cells and pave the way for the more extensive multiplex genome editing required to create universal cell products. Once clinically validated, the genome editing solutions could equally be applied to different cell types such as T cells, DCs, or HSC. However, it remains to be demonstrated that such "universal" cells could readily engraft and provide long-term protection without inducing toxicity. In addition, the less than 100% efficiency achieved for each edit will require purification of "fully edited" from "partially edited" cells, which is a significant obstacle that is yet to be resolved. There may also be unidentified molecules that may need to be edited for a cell to be truly "invisible" to the host immune system and persist for the life of the individual.

CeLL THeRAPY TO CONTROL iNFeCTiON
Infections are still one of the main complications post-HSCT contributing to mortality and morbidity of the procedure.
Therefore, a lot of efforts have been dedicated in developing cell therapy approaches to control infection such as DLI as an alternative to more conventional therapies. Notably, it has been shown by several groups that the infusion of matched donorderived virus-specific T cells specific for cytomegalovirus, Epstein-Barr virus, or adenovirus can restore virus-specific immunity and control infection or could be used prophylactically in transplanted patients (107)(108)(109)(110)(111)(112)(113)(114) (Table 6). In addition, the use of multivirus-specific T cells has also been reported to be effective in either preventing or controlling viral infection (115)(116)(117)(118) (Table 6). However, this approach is not applicable to seronegative donors, patients undergoing immunosuppressive regimens, or for urgent use because of the time required to generate the cell product. In these specific conditions, the use of partially matched allogeneic virus-specific T cells has shown great promise and efficacy in controlling infection (116,119,120) ( Table 6), highlighting the possibility to develop banks of allogenic virus-specific T cells (113,121) whereby the virus-specific T cells of one donor could be expanded and banked for immediate use, which could allow treating several patients with the cells from the same donor thus decreasing the cost of the therapy.  (122)(123)(124). Moreover, double negative (DN) T cells have regulatory functions and play key roles in tolerance induction post-transplantation and in autoimmune diseases (125,126). DN T cells have been reported to suppress immune responses, in particular T cell responses and can improve allograft survival in experimental mouse models of cardiac transplantation (127). Mesenchymal stem cells (MSC) are multipotent stem cells that also have anti-inflammatory and regenerative properties. Their regulatory functions have been evaluated using in vitro and in vivo disease models (128). In addition, the infusion of autologous and allogeneic MSCs has been tested in numerous phase I studies as immunosuppressive regimen in the context of organ transplant as well as treatment for GvHD and different autoimmune diseases (129,130). Regulatory T cells are key players in inducing tolerance and maintaining immune homeostasis. They constitute 5-10% of CD4 T cells in peripheral blood and are defined as CD4 + CD25 + CD127lowFoxP3 + (131). They have been shown to be able to regulate the functions of numerous immune cells such as CD4/ CD8 T cells, DCs, NK cells, and B cells (132). Consequently, they have been evaluated when considering a cell therapy approach to induce tolerance especially for autoimmune diseases. Treg therapies have been shown to be safe and feasible with some promising results in clinical trials using autologous Tregs for GvHD (133)(134)(135)(136) and autoimmunity such as diabetes (137,138) and Crohn's disease (139) as well as third-party allogeneic Tregs (136) ( Table 7). All clinical studies have been done using polyclonal expanded Tregs; however, it has been shown that antigen-specific Tregs are more potent at suppressing undesired immune responses as compared to polyclonal Tregs. Some groups have studied the possibility to generate antigen-specific T cells by transferring a CAR into polyclonal expanded Tregs and showed their potency in vitro as well as in animal models (140)(141)(142)(143). However, this approach is still to be tested clinically.
Another approach to induce tolerance could be the transfer of a chimeric autoantibody receptor (CAAR) into T cells to induce cytotoxicity against autoreactive T or B cells. Such an approach has demonstrated exciting results in vitro and in vivo in the case of CAAR T cells redirected against desmogelin-3 (DSG3) as a therapeutic approach for pemphigus vulgaris where ReFeReNCeS B cells secrete autoantibodies against DSG3, which is an antigen expressed in the skin and mucosa (146). This study showed effective killing of autoreactive cells by DSG3-CAAR T cells in vitro and in vivo while sparing keratinocytes with no off-target effects. This approach showed promising results to potentially treat autoantibody mediated immune diseases. Numerous cell types with regulatory functions have now been tested in preclinical studies showing promising results. Further evaluation in clinical studies is warranted but these data will be key to evaluate whether gene engineering cell therapeutics could be as successful in inducing tolerance in chronic diseases as these types of approaches have been for rare diseases and oncology.

CONCLUDiNG ReMARKS
After many years of endeavor, the field of cell and gene therapy is starting to deliver remarkable clinical benefits in some patients, particularly in certain hematological malignancies. While this is a very exciting moment for the field, more work remains to be done to further develop and optimize therapies. More relevant disease models that can help predict not only efficacy but also adverse effects are much needed. In addition, making gene engineering cell therapeutics safer is key by adding, for example, safety or kill switch to the therapy or expression control elements. As this review shows, many cell types are being explored in early clinical trials and the results of these studies will provide important insights. A major factor in realizing the potential of cell and gene therapies in large numbers of patients and in more common diseases is the efficient and cost-effective manufacturing of gene engineering cell therapeutics. Advances in automation are a key component that needs to be addressed (147). The pace of clinical development is accelerating and this means that the next few years will provide many important learnings that will hopefully translate into new therapeutic options for patients.

AUTHOR CONTRiBUTiONS
AS, LJ, and TC all contributed to write the manuscript.

FUNDiNG
All the authors are supported by funding from GlaxoSmithKline.