Gene replacement therapy has emerged as a central strategy in the field of gene therapy and has shown great promise in the treatment of certain pediatric cancers (Pearl et al., 2023). The thrust of this approach is to identify a particular mutated gene in a tumor and use viral vectors to deliver a normal, “wild type” version of that gene to the cancer cells (Bhattacharjee et al., 2022). The goal of introducing a functional copy of the aberrantly mutated gene is to normalize the cancer’s genetics and limit its proliferation (Pscherer et al., 2006).

This therapy is particularly suitable for cancers driven by single-gene mutations, such as the BCR-ABL1 translocations characteristic of pediatric ALL (Sugapriya et al., 2012). However, there are challenges in selecting the optimal vector for gene delivery. While viruses could effectively infect human cells, they can induce immune responses against the introduced gene, potentially limiting efficacy (Aledo-Serrano et al., 2021). Another challenge is to ensure sustained expression of the delivered gene.

Nevertheless, gene replacement therapy has been successful in certain pediatric cancers. For example, in ALL, the introduction of CAR genes into patients’ T cells has resulted in high remission rates in refractory disease (Wu et al., 2021). Early studies have shown promising results in using gene replacement to correct mutated MERTK genes in neuroblastoma. Vollrath et al. found that administering recombinant adenovirus encoding MERTK subretinally reversed the defect in retinal pigment epithelium phagocytosis and rescued photoreceptors from degeneration in juvenile rats. As a result, the treated areas in animal models, which already exhibited significant pathology, appeared nearly normal (Vollrath et al., 2001).

The potential of gene replacement therapy to restore conventional function by targeting single genetic drivers underscores its relevance for precision treatment of pediatric malignancies (Senft et al., 2017). Expansion of this promising approach will require refinement of gene delivery methods to improve safety and efficacy, long-term outcome studies, and expansion of trials across the spectrum of pediatric cancers driven by targetable mutations (Magnani et al., 2013). Overcoming existing barriers could pave the way for a new generation of highly specific cancer therapies that could potentially eradicate mutation-driven pediatric tumors.

Gene suppression therapy, often referred to as gene silencing, symbolizes a breakthrough approach to gene therapy that aims to combat disease by silencing harmful genes responsible for disease development. This method of silencing deleterious genes appears promising in the treatment of cancers activated by gain-of-function mutations, particularly those associated with pediatric malignancies (He and Jia, 2020).

RNA interference (RNAi), a powerful tool for gene silencing, inhibits the translation of targeted mRNAs via complementary base pairing. This is generally accomplished by the incorporation of either synthetic siRNAs or vector-derived shRNAs (Li et al., 2018). This approach has potential advantages in the treatment of pediatric cancers. For example, neuroblastoma cells harboring anaplastic lymphoma kinase (ALK) mutations experienced growth inhibition in experimental models following ALK silencing by RNAi (Schulte and Eggert, 2021). Thus, the approach of silencing oncogenic ALK could develop beneficial therapies for patients with this particular abnormality (Ji et al., 2021). Furthermore, RNAi-mediated targeting of beta-catenin in hepatoblastoma models resulted in tumor growth suppression, suggesting another potential target (Indersie et al., 2017). However, the challenge is to deliver RNAi triggers specifically to the tumor tissue. MicroRNAs (miRNAs), another regulatory approach, are short non-coding RNAs that reduce protein production by binding to mRNAs (Arraiano, 2021). A positive correlation was observed between miRNAs expression and tumor grade and type, with the highest expression noted in medulloblastomas, followed by ependymomas, and the lowest in pilocytic astrocytomas. Upregulation was predominantly observed in most members of the miR-17–92, miR-106a-363, and miR-106b-25 clusters, with miR-18a and miR-18b exhibiting the highest expression. Other miRNAs such as miR-19a, miR-92a, miR-106a, miR-93, and miR-25 also demonstrated elevated values. Additionally, miR-17-5p and miR-20a displayed high expression levels in medulloblastomas and ependymomas while approaching control levels in pilocytic astrocytoma samples. Notably, miRNA expression was also influenced by tumor grade and histology in pediatric tumorigenesis, suggesting therapeutic potential for miRNA (Gruszka et al., 2021). Measures such as adjusting abnormal miRNA levels or using miRNAs to suppress fusion oncogenic transcripts could be employed (Gambari et al., 2016). For example, chemotherapy showed higher efficacy in neuroblastoma cells where let-7 miRNA levels were restored (J et al., 2015). Nevertheless, the development of safe and effective miRNA-based therapies has encountered many obstacles, such as stability, off-target effects, and efficient delivery to target tissues (Roy et al., 2022).

Targeted delivery of gene silencing therapies to malignant cells is a challenging task (Safarzadeh Kozani et al., 2021a). Systemic administration may induce off-target gene silencing and consequent toxicity. Therefore, innovative delivery systems are being explored to increase specificity and avoid activation of immune responses (Zhao et al., 2020a). Studies are considering the use of bioengineered viruses, liposomes, and nanoparticles loaded with ligands for receptors overexpressed on tumor cells (He et al., 2021). However, other challenges remain, such as transient gene silencing and variable silencing efficacy due to accessibility of the target site (Safari et al., 2017).

In summary, despite the obstacles, gene silencing methods could play a critical role in accurately targeting and silencing genes that promote pediatric cancer growth (Verreault et al., 2006). In summary, the advent of paradigm-shifting gene editing technologies such as CRISPR and transcription activator-like effector nucleases (TALENs) has invigorated genetic research by providing customizable, targeted genome editing capabilities. However, with the tremendous opportunity comes the responsibility to guide the appropriate use of these rapidly advancing tools that hold the power to permanently recode life. Realizing their potential while mitigating the risks will require ongoing ethical discussions and flexible regulations based on scientific evidence. The coming decade promises exciting advances in gene editing, but it also demands caution.

Viral vectors have gained significant attention in gene therapy, as they have been transformed into useful gene delivery mechanisms due to their inherent ability to deliver genetic material into host cells. The main variants used in this context are retroviruses, lentiviruses, and adenoviruses (Giacca and Zacchigna, 2012).

Retroviruses, being RNA viruses, undergo a reverse transcription process in which their RNA genome is converted to DNA and further integrated into the host genome, facilitating persistent and stable gene expression within the host cell. The potential tumorigenesis induced by insertional mutagenesis is a notable risk (Steckbeck et al., 2014; Sabatino et al., 2022). Indeed, retroviruses exhibit efficient infection properties toward dividing cells—an aspect that is critical for their application in ex vivo treatments, as in the case of CAR T-cell therapy for leukemia (Heyman et al., 2022).

Similar to retroviruses, lentiviruses can affect both dividing and non-dividing cells, expanding their potential therapeutic applications, including but not limited to neuroscience and human immunodeficiency virus (HIV) treatment (Fassati, 2006). One of their notable advantages is their broad cell tropism, which allows them to transduce multiple cell type (Rainey and Coffin, 2006; Poletti and Mavilio, 2018). However, the risk of insertional mutagenesis associated with lentiviruses remains significant (Woods et al., 2003). The class of adenoviruses, which are double-stranded DNA viruses that do not integrate into the host genome, greatly reduces the risk of mutagenesis (Charman et al., 2019). Their efficacy in delivering transgenes to a variety of tissues in vivo and ex vivo is complemented by their large transgene capacity (Morrissey et al., 2012). They have shown impressive results in cancer gene therapy, enabling localized delivery of tumor suppressor genes (Wang et al., 2010). Their efficacy may be limited in certain therapeutic settings due to anti-adenovirus immune responses (Zhou et al., 2020).

Significant progress has been made in refining viral vector design and production methods, which have greatly improved safety and delivery efficiency (Li et al., 2021). An example is the advent of self-inactivating vectors that reduce the oncogenic risks associated with retroviruses and lentiviruses. Similarly, third-generation adenoviral vectors are designed to minimize unwanted immune activation (Troyanovsky et al., 2015; Shaw and Suzuki, 2019).

In summary, despite their unique profiles, retroviral, lentiviral, and adenoviral vectors have been instrumental in the advancement of gene therapy. With continued improvements in viral vector biology and engineering, their medical utility is expected to expand. A better understanding of cell-specific targeting may lead to vectors with superior therapeutic precision in the future.

Due to the various challenges associated with viral vectors, such as immunogenicity risk, limited payload capacity, and high production costs (Luis, 2020), alternative non-viral vectors for gene delivery have come to the forefront. These alternatives, which include liposomes, nanoparticles, and others, have their own unique advantages and specific issues that need to be addressed (Musale and Giram, 2021).

Liposomes, spherical vesicles composed of lipid bilayers, have been identified as safer alternatives capable of encapsulating and delivering genes, proteins, or drugs with the added benefit of minimizing immunogenic responses or infections (Liu et al., 2022; van der Koog et al., 2022). One of the identified problems with liposomes is that they often exhibit encapsulation efficiencies below 20% (Costa et al., 2021), and there is a constant risk of rapid degradation of the cargo before it reaches its intended target (Eloy et al., 2014).

To address these limitations, cationic liposomes have been developed to enhance encapsulation by facilitating the formation of complexes with negatively charged nucleic acids (Ewert et al., 2021). In addition, surface modifications, such as the attachment of antibodies, peptides, or saccharides, can ensure that target specificity is enabled (Lestini et al., 2002). For example, anti-EGFR nanobodies have been attached to liposomes to facilitate targeted drug delivery to colon cancer cells (Narbona et al., 2023). Further refinement of the size, composition, and surface charge of these liposomes aims to improve stability, efficiency, and cellular uptake (Ma et al., 2013). Nanoparticles represent a diverse range of non-viral vectors incorporating polymers, lipids, metals, or other elements (Botto et al., 2018). For example, gold nanoparticles provide controlled, sustained release of genes, making them invaluable in the treatment of solid tumors (Zhao and Liu, 2014). Other nanoparticle vectors include polymeric nanoparticles, quantum dots, and liposomal nanoparticles (Ishihara et al., 2011). Polyplexes, a type of nanoparticle vector, are widely used due to their ease of production, safety, customization potential, and ability to encapsulate various nucleic acids (Bai et al., 2014). Despite these advantages, they still present challenges such as short circulation time, low transfection efficiency, and cytotoxicity (Ita, 2020).

LNPs have shown potential for mRNA delivery, as demonstrated by the use of mRNA COVID-19 vaccines (Melamed et al., 2022). With high encapsulation efficiency, biocompatibility, and stability, LNPs can be functionalized for targeting (Alipour et al., 2017). To realize their full potential, new surface modifications are being explored to increase stability, reduce off-target effects, improve specificity, and decrease toxicity (Kulkarni and Feng, 2011).

In conclusion, replacement non-viral vectors such as liposomes and nanoparticles offer versatility that is stimulating research aimed at overcoming current limitations. As safer alternatives to viral vectors with the potential for significant optimization, they could greatly improve gene therapy and drug delivery methods. Looking ahead, the future seems bright for non-viral vectors as newer designs, modifications, and combinations continue to emerge.

CAR T-cell therapy is a revolutionary approach to cancer treatment that differs significantly from previous immunotherapy techniques. This innovative therapy uses a patient’s own lymphocytes, commonly referred to as T-cells, to identify and eradicate cancer cells with extreme precision (Xu et al., 2021). The key steps in CAR T-cell therapy involve the isolation of a patient’s T cells, followed by genetic modification using viral vectors to create CARs, their subsequent expansion in vitro, and finally their reintroduction into the patient’s body (Ke et al., 2021). These engineered CARs can bind to target antigens on cancer cells with an affinity in the nanomolar range, 100 to 1,000 times stronger than natural antibodies (Harris and Kranz, 2016). This binding induces cytotoxicity, resulting in the elimination of the cancer (Wang et al., 2019). The structure of CARs consists of three main components—an external moiety that binds to the tumor antigen, a membrane spanning region, and an internal domain that induces T-cell activation (Muller et al., 2021). The outer segment typically uses a single-chain variable fragment (scFv) derived from an antibody that confers specificity (Krokhotin et al., 2019). The internal segment carries a CD3ζ domain required for T-cell activation and costimulatory domains such as CD28 or 4-1BB to enhance the activation signal (Boucher et al., 2021).

CAR T-cell therapy has shown impressive results in blood cancers, particularly B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL), with treatments such as Kymriah and Yescarta gaining FDA approval (Ma et al., 2019; Safarzadeh Kozani et al., 2021b). However, the efficacy of therapy in solid tumors is hampered by factors such as metastasis, tumor diversity, and the immunosuppressive tumor environment (Zhao et al., 2020b). Research is currently exploring potential solid tumor targets such as GD2 in neuroblastoma and human epidermal growth factor receptor 2 (HER2) in sarcoma (Straathof et al., 2020). To enhance the efficacy of CAR T-cell therapy in solid tumors, strategies such as regional administration and CARs that are immune to suppressive signaling are being considered (Huang et al., 2020).

Despite its great potential, CAR T-cell therapy has been observed to induce cytokine release syndrome (CRS) in more than 90% of patients and neurotoxicity due to overactivation in about half of the cases (Napolitano et al., 2022). CRS is often treated with drugs such as tocilizumab (Kauer et al., 2020). Ongoing efforts are directed at integrating suicide genes into CARs to improve their safety (Shi et al., 2020).

The future looks promising with the development of off-the-shelf universal CAR T-cells, multi-antigen targeting CARs, and the use of engineered Notch receptors to provide spatial control of CAR T-cell activation (Qu et al., 2019). Advances in CAR T-cell therapy could change the way cancer is treated. The ability to target solid tumors and improve the safety of treatments is a particularly exciting prospect in the evolution of this therapy.

Tumor-targeting antibodies are an integral part of immune-based cancer therapies. Their function depends on their ability to distinguish antigens that are differentially expressed on cancer cells, thus distinguishing these malignancies from healthy tissues. This leads to selective targeting and a reduction in untargeted or “off-target” effects (Hintz et al., 2021).

Over the past few decades, a number of these tumor-targeting antibodies have been approved. These developments have greatly improved the health outcomes of cancer patients (Sun et al., 2018). Rituximab, for example, targets CD20 on B cells, which are used to cure leukemia and lymphoma (Jain et al., 2021). Trastuzumab, on the other hand, reacts with HER2, which is overexpressed in about one-quarter to one-third of gastric and breast cancers and shows increased survival rates (Upton et al., 2021). Another example, cetuximab, blocks the epidermal growth factor receptor (EGFR), which plays a role in metastasis and growth, and has shown efficacy in colorectal and head and neck cancers (Dougherty et al., 2020; Trivedi and Ferris, 2021).

The most common mechanisms of anti-tumor action include blocking signaling pathways essential for cancer survival and proliferation. These mechanisms also recruit natural killer cells to enable antibody-dependent cellular cytotoxicity (ADCC). In addition, these mechanisms activate the complement system to enable complement-dependent cytotoxicity (CDC) (Murray et al., 2014; Zhu et al., 2020). Another effect of this mechanism is that the binding of the Fc region to cells of the immune system results in the stimulation of the immune defense against the cancerous tumor (Sasaki et al., 2020).

Nevertheless, resistance is observed in about half of all patients, often allowing relapse after an initial response to treatment (Licciardone and Aryal, 2014). This resistance can result from several mechanisms, including loss of expression of the target antigen, blockade of the antibody’s access to the cancer, and activation of other survival pathways (Fernandes et al., 2021). Another mechanism of resistance is through mutations in targets that allow evasion of antibodies (da Silva, 2012). Notably, even targeted therapies may have adverse effects due to non-specific distribution (Swati, 2021).

Current research is focused on a few areas to address these issues and further expand clinical utility. These areas include antibodies that are bispecific or multispecific, which can bind to more than one antigen as a method of not only improving specificity but also overcoming resistance (Ahamadi-Fesharaki et al., 2019). Another similar area of research is the study of drug–antibody conjugates, which use antibodies to selectively deliver chemotherapeutic agents or toxins (Pettinato, 2021). Another area of research is to increase the activation of the immune system through Fc engineering (van Tetering et al., 2020).

In sum, antibodies have had a significant impact on cancer care, leading to improved outcomes in certain malignancies. Therefore, it is highly likely that the future will see an expansion of antibodies as strategic, selective tools in therapy that harnesses the immune system to fight cancer.

Checkpoint inhibitors have significantly changed the landscape of cancer immunotherapy through their ability to interfere with the immune evasion mechanisms employed by tumors (Zhang et al., 2021a). Tumor cells have developed sophisticated strategies to evade the initial defenses of the immune system or to actively suppress anti-cancer immunity mechanisms that have received considerable attention in cancer research (Mahata et al., 2020).

Critical regulatory roles in modulating normal immune responses and maintaining immune self-tolerance are played by checkpoint proteins such as programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). PD-1, which is typically expressed on T-cells, binds to PD-L1 on healthy cells, and plays a regulatory role in preventing excessive T-cell activity (Schutz et al., 2017; Chun et al., 2022). Paradoxically, the same mechanism is used by tumor cells to evade T-cell attack by presenting PD-L1 on their surface (Hinterleitner et al., 2021). The introduction of PD-1 inhibitors interrupts this interaction, reviving the suppressed T cells and enabling them to identify and destroy tumor cells. This phenomenon has been demonstrated in the treatment of several cancers, including but not limited to melanoma and lung cancer (Curnock et al., 2021).

Also worthy of mention in the catalog of immune checkpoints is CTLA-4, which acts to inhibit T-cell activation during later stages of the immune response (Arra et al., 2021). Drugs such as ipilimumab are designed to target CTLA-4 and enhance anti-tumor immunity by removing this block to T-cell activation (Soldevilla et al., 2019). Clinical trials have demonstrated the efficacy of CTLA-4 inhibitors in the treatment of cancers, including melanoma (Hirshoren et al., 2020).

However, checkpoint inhibitors are not without limitations. More than half of treated patients experience immune-related adverse events (irAEs), the severity of which can range from mild to severe and in extreme cases can be life-threatening (Usui et al., 2022). Diarrhea, dermatitis, and hepatitis are among the most common irAEs (Spain et al., 2016). It is also noteworthy that some tumors are “cold” or non-inflamed, characterized by sparse immune cell infiltration into the tumor microenvironment. Patients with such tumors are less likely to respond to checkpoint inhibitor therapy (Gerard et al., 2021).

The scientific community is actively investigating strategies to overcome these challenges. Combination therapies using checkpoint inhibitors alongside other therapies such as chemotherapy, radiation, or targeted therapy are of great interest (Andersson and Ostheimer, 2019). Approaches aimed at making tumors more susceptible (or “hotter”) to immunotherapy are also under investigation, including the use of tumor antigen vaccines and optimized drug delivery approaches (Liu et al., 2020). Research is also underway to identify biomarkers that predict therapeutic response and risk of side effects (Bartsch et al., 2007).

In summary, although checkpoint inhibitors are revolutionizing cancer treatment through their ability to harness anti-tumor immunity, which is typically limited by immune evasion mechanisms employed by tumor cells, their efficacy is not universal, and side effects remain a notable concern. Current research efforts in combinatorial therapeutic strategies and predictive biomarker identification are aimed at improving clinical outcomes while minimizing toxic effects. Despite their limitations, checkpoint inhibitors show significant potential to improve cancer treatment. The challenge for the future is to extend the durability and safety of therapeutic responses, which will require continued research and innovation in the field.