Unlocking the potential: advancements and applications of gene therapy in severe disorders

Abstract

Introduction

Several severe disorders, such as inherited diseases (e.g. cystic fibrosis and beta thalassemia), genetic diseases (e.g. malignant tumors and diabetes), and infectious diseases (e.g. HIV) are pose significant challenges to human health.

Background

Over the past few decades, researchers have been working on gene therapies, and currently, terrible dreams have come true. To date, the Food and Drug Administration (FDA) has approved multiple gene therapies such as Kynamro for familial hypercholesterolaemia, Exondys51 for duchenne muscular dystrophy, Spinraza for spinal muscular atrophy, etc., rest for cancer, infectious diseases, and rare diseases.

Discussion

The authors have summarized recent advances in gene therapy, its background, molecular basis (e.g. viral and non-viral vectors), gene-editing techniques (e.g. CRISPR/Cas9, TALEN, ZFN), and its foremost applications in severe disorders, such as cancer, monogenic disorders (e.g. spinal muscular atrophy), polygenic disorders (e.g. autism), neurogenic disorders (e.g. Parkinson disease and Alzheimer’s disease), and infectious diseases (e.g. HIV).

Challenges

In addition, we explored the major challenges faced by gene therapies during targeted delivery, immunogenicity, efficacy, and safety.

Conclusion

To date, most of the promising approaches, such as different vectors, target cell populations, and both in vivo and ex vivo have paved the foundation for applications of gene therapies. Additionally, advances in enhancing the immune system that would certainly lower the healthcare costs. This review highlights the translatory potential of gene therapy in revolutionizing the treatment landscape for severe disorders.

1. Introduction

Current revolutionary trends in medical technologies ‘gene therapy’, which mimics the delivery of genetic material to change, regulate gene expression, or modify the characteristics of living cells for therapeutic purposes [1]. At present, various gene therapies such as miRNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), antisense oligonucleotides (ASOs), clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), and plasmid-DNA have shown limited potential in biomedical applications for malignant tumors, acquired immunodeficiency syndrome (AIDS), cardiovascular diseases, and hereditary diseases (e.g. cancer and diabetes). According to the European Medicines Agency (EMA), gene therapies generally consist of a vector or delivery system containing a genetically engineered gene to express a specific transgenic cascade, that is, a therapeutic sequence [2]. Transgenic cascade is a series of biological events that occur following the introduction of a transgene – an artificially introduced gene from one organism into the genome of another organism.

Gene therapy can restore defective proteins in hereditary disorders by directly repairing or replacing disease-causing genes at the molecular level. Gene therapy can fix disease mutations in three ways, as shown in Figure 1, to cure inherited disorders at their source. There are three main methods of gene therapy: (i) introduction or overexpression of a therapeutic gene or synthetic construct (a system that is artificially created), (ii) mutation of the mutant version of the gene, and (iii) replacement of the defective gene with a functional copy. Three ways to silence cells are through zinc finger silencing technology (e.g. zinc finger nucleases), shRNA introduction, and miRNA structure embedding [3–5].

Figure 1.

Illustration of gene therapy techniques.

Gene therapy, the experimental technique that uses genes to treat or prevent disease, brings forth a host of ethical considerations that must be carefully examined. Patients must be aware about the risks and benefits of gene therapy, including potential long-term effects. The long-term safety of gene therapies is still under scrutiny. As gene therapy develops, issues of accessibility and equity come to the forefront due to their high costs. Gene therapy also involves modifications to reproductive cells, meaning changes can be passed on to next generations. Ethical considerations demand rigorous preclinical and clinical testing, transparency about research findings.

The development of gene therapy has its roots in the 1970s. Alternatively, The first mention of gene therapy was from Wilhelm Johansen in 1909, who coined the term ‘Gene discovery’. In 1970, Rogers replaced defective deoxyribonucleic acid (DNA) with normal DNA. Subsequently, in 1972, Theodore Friedmann and Richard Roblin proposed the concept of gene therapy. Jesse Gelsinger has a rare ornithine transcarbamoylase (OTC) deficiency and willingly participated in gene therapy clinical trials. Unfortunately, in 1999, his death during the clinical trial was shocked the entire world, and gene therapy research absolutely collapsed due to safety issues [6]. However, till 2010 Food and Drug Administration (FDA) has been approved several gene therapies such as Virtanen for CMV retinitis, Gendicine for squamous cell carcinoma, Oncorine for cancer, and Rexin-G for metastatic cancer, respectively [7–11]. The development timeline of the current approved gene therapy is shown in Figure 2.

Figure 2.

FDA and EMA approved gene therapies.

Gene therapy has long been considered a potentially effective treatment for a number of serious illnesses, such as hemophilia [12], human severe combined immunodeficiency [13], spinal muscular atrophy (SMA) [14], cystic fibrosis [15], and acquired disorders (such as several malignancies) [16]. Recently, hematological cancers have been the most investigated, partly because of their promising development. CAR T-cell therapy is progressing rapidly to improve the cancer treatment, expanding its use to more cancers with excellent accessibility and broad surface interactions. The range of target disorders is still expanding due to numerous ongoing clinical investigations. As of 2023, 68.5% of gene therapy clinical trials have focused on cancer, making it the primary area of investigation. Additionally, 12.8% of cases involve inherited monogenic disorders, which have achieved remarkable success. Cardiovascular disorders (5%) and infectious diseases (5%) have also garnered attention. Furthermore, gene therapy is being explored for several other conditions, including rare disorders, demonstrating its versatility [17,18].

This review emphasizes the translational landscape of gene therapy, exploring its background, molecular underpinnings, diverse applications across various diseases, and challenges that lie ahead. Through a comprehensive analysis, the authors aimed to shed light on the current status of gene therapy development and its promising potential to revolutionize the future of biomedicine. The world is on the verge of entering a golden age of somatic gene therapies, with over 1,600 trials currently recruiting, despite disagreements among scientists regarding the future of heritable genome editing. As we explore how genome editing will supplement current gene therapy techniques, which still primarily rely on gene addition strategies, we provide examples from human clinical trials to contextualize these remarkable advancements within the framework of modern medicine [18,19].

2. The molecular basis of gene therapy techniques

The molecular basis of gene therapy relies on the modification and introduction of genetic materials into target cells to introduce therapeutic genes, modify gene expression, and replace or repair damaged genes. The key elements are as follows:

2.1. Gene transfer technique

The key step in gene therapy is efficient gene delivery to target tissue/cells via vectors [20]. Vectors offer efficient gene delivery to treat genetic diseases [21–23]. Vectors are generally classified as viral or non-viral depicted in Figure 3.

Figure 3.

Illustration of gene transfer techniques.

2.1.1. Viral vectors

Generally, studies have demonstrated the effectiveness of viral vectors in delivering genes to target cells or tissues, which is a crucial step towards achieving therapeutic efficacy [21]. The benefits of viral vectors, such as improved transduction efficiency, greater engineering versatility, and highly specific gene delivery, have made a broader range of applications possible. Adenovirus (Ad) [24], adeno-associated viruses (AAV) [25,26], alphaviruses [27], flaviviruses, herpes simplex viruses (HSV), measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), poxviruses, and picornaviruses are among the viruses used in viral vector-based gene therapy. The increased use of lentiviral vectors in vivo delivery in gene therapy, including the development of in vivo CAR-Ts as well as their applications in vivo vectors for rare diseases, oncology, and infectious diseases. The latest advancements in the delivery of in vivo lentiviral vectors are the use of different pseudotypes (integrating and non-integrating), optimised packaging plasmids, and modified cell lines to increase targeting of lentiviral vectors. The development of good manufacturing practice (GMP)-compliant instruments, technologies, and protocols will play a crucial role in the development of lentiviral T cell therapies [26,28].

So far, 22 gene therapies have been approved. Of these, 45% use the Lentiviral (LV) vector, 36% use the Adeno-Associated Virus (AAV) vector, 9% use the retroviral vector, and both the Adenoviral (AV) vector and the Herpes Simplex Virus (HSV) vector each account for 5% as shown in Figure 4. According to the FDA, viral vectors have been utilized in several clinical trials, primarily for gene therapy. In some of these experiments, undesired insertions or the activation of proto-oncogenes have been observed, which has led to cancer in certain individuals [29].

Figure 4.

Types of vectors with approved gene therapies.

An alternative delivery method for therapeutic transgenes is ex vivo, which involves removing patient cells and cultivating them outside of the body. The patient’s cells are genetically modified by introducing a therapeutic transgene, which is then reintroduced into the patient’s body following the injection. One of the most well-known of these is the delivery of RPE65 using an AAV2 vector to treat Laber congenital amaurosis (LCA), which has resulted in the restoration of visual function in multiple patients with long-lasting benefits [28].

Although several viral vectors are safe, some have the potential to cause oncogenesis (e.g. gamma retroviral vectors) [29]. Furthermore, few viral vectors (e.g. adenovirus vector and herpes viral vector) are toxic and can be immunogenic [8].

2.1.2. Non-viral vectors

Lipid or polymeric particles, which encase genetic material to facilitate its entry into cells, constitute the majority of non-viral vectors. Non-viral vectors have a number of benefits over viral vectors, including easier scalability of production, longer shelf life, potentially boundless payload size of genetic material, and an improved safety profile [30]. In recent years, with the continuous development of nanotechnology, nanomaterials (NMs) have provided more options for delivering nucleic acids (NA) into target cells. Various NMs of interest include liposomes [31,32], polymer nanostructures [33], nanogolds [34], silicon NMs [35], quantum dots [36], magnetic NMs [37], and carbon nanotubes [38]. Despite the fact that these vectors exhibit biocompatibility and stability, there are still significant questions regarding the host response mechanism, low transfection effectiveness, and gene transfection methods. However, vector modifications can potentially achieve high transfection efficiency. We could learn more about host response mechanisms, gene transfection, and non-viral vectors for targeted NA delivery, severe genetic illnesses, and new ways to treat cancer through research in this area [39]. Construction of a safe and high transfection efficiency non-viral gene delivery vector to improve the effectiveness of gene delivery is vital for cancer treatment. Li et al. demonstrated 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol to prepare cationic liposomes in combination with poly(aspartate-graft-PEI423) (PAE) and plasmid DNA to prepare liposomes-poly(aspartate-graft-PEI423)/DNA (LP-PAE/DNA) to improve gene transfection efficiency. Therefore, development of non-viral vectors with synergy could be beneficial in near future for betterment of gene transfection efficiency [40].

2.2. Gene editing technologies

Certainly, the development of higher-precision methods like CRISPR-Cas has been driven by several key motivations, especially when compared to more traditional types of genetic modification such as random mutagenesis or vector-based approaches. It features improvements such as precision, efficiency, flexibility, and multiplexing capabilities, among others. On the other hand Savulescu et al. has demonstrated due to unpredictable effects of genome editing in human embryos makes it ethically unacceptable [41]. Other controversies include eugenics; patentability including the void of research on human germ cell biology [42]. This suggests that pursuing GGE is morally permissible and indeed morally desirable [43].

2.2.1. CRISPR/Cas9

By altering DNA sequences, CRISPR-associated nuclease 9 (Cas9) is a potentially effective genome editing technique for hereditary diseases that are incurable. Compared with other genome-editing methods, CRISPR-Cas9 is an easy, effective, and highly targeted method. The immune system of naturally occurring bacteria has developed a new and effective RNA-guided endonuclease-based genome-editing method called CRISPR Cas9 [32,44]. CRISPR/Cas9 technology uses Cas9 endonuclease and single-guide RNA (sgRNA) sequences. Combining trans-activating crRNA (tracrRNA) and CRISPR RNA (crRNA), sgRNA recognizes and binds the sgRNA-Cas9 ribonucleoprotein complex to the target DNA [45]. After targeting DNA, Cas9 produces double-stranded breaks (DSBs) at the target DNA location [46]. Figure 5 illustrates how insertions, deletions, additions, or inversions further repair this DSBs. PAM, protospacer adjacent motif, it serves as a critical recognition element for the Cas protein to bind and initiate DNA cleavage. The host’s own natural repair mechanisms or tailored DNA sequences can carry out DNA repair [47].

Figure 5.

The gene editing with CRISPR-Cas9.

In their study, Koo et al. discovered that using an adenovirus vector to deliver EGFR mutation-specific CRISPR/Cas9 effectively cut and disrupted mutant EGFR alleles in tumors with EGFR mutations. Disruption of the EGFR mutation (L858R) in H195 tumors leads to cancer cell death and a significant decrease in tumor size in vivo; however, CRISPR/Cas9 is still an emerging technology that could be effective in severe disorders. To date, clinical trials have focused on the safety and efficacy of genome editing in humans to improve the molecular processes involved in genome editing [48]. However, the journey of CRISPR/Cas9 could be interesting, and it may offer a significant contribution in the treatment of severe diseases [47,49]. Additionally, the latest Cas9 research findings, such as Cytosine Base Editors (CBEs), A3A/Y130F-iSpyMacCas9-CBE_V01, smaller Cas9 variants, and R-loop assay. CBEs are a type of base editor developed based on the CRISPR/Cas9 system, which can perform C > T (G > A) transitions without generating DNA double-stranded breaks. To date, improved fourth-generation CBE4 was generated by fusing an additional copy of UGI to the N terminus of nCas9 with an optimized 27 bp linker. Other CBE systems such as BE4max and ancBE4max are developed by adding two nuclear localization signals (NLS) to nCas9. Target-AID (activation-induced cytidine deaminase) is developed by nCas9, Petromyzon marinus cytidine deaminase 1 (pmCDA1), respectively [48].

These base editors have been widely used in gene therapy, animal model construction, precision animal breeding, and gene function analysis, providing a powerful tool for both basic and applied research. CBE like A3A/Y130F-iSpyMacCas9-CBE_V01 outperformed other iSpyMacCas9 CBEs in rice protoplasts, with a base editing efficiency of 68.8%.

2.2.2. Zinc finger nuclease (ZFN)

The traditional in vivo gene targeting approach uses normal homologous recombination, which is a tedious, time-consuming, and inefficient process. The development of transcription activator-like effector nucleases ZFN and TALEN (transcription-activator-like effector nucleases (TALEN) as genome editing tools was overwhelming. It contains the catalytic domains of the restriction endonuclease FokI, which is essential for producing double-strand breaks (DSB) with cohesive overhangs during targeted genome editing.

Synthetic DNA-binding proteins (ZFNs) are composed of two domains joined by a linker sequence. The first is a modified zinc-finger DNA-binding domain that provides a 24 bp DNA stretch with sequence-specificity. On the other hand, the second is a DNA cleaving domain mediated by the restriction endonuclease FokI, which cleaves the sequence in the 5–7 bp spacer regions [50].

2.2.3. TALEN

The foundation of TALENs is the in vivo homologous recombination method, which encourages extremely repetitive sequences. The N-terminal transcription activator-like effector (TALE) DNA-binding domain and the C-terminal catalytic domain of restriction endonuclease FokI are the two domains of TALENs that are similar to ZFNs. Figure 6 shows these domains. A linker region links the nuclease domain to DNA binding, whereas a spacer segment, which spans 12–25 base pairs, divides the two binding sites. Furthermore, the binding sites of TALENs are found on opposing strands and they function in a dimeric form to create a double-strand break. Tandem repetitions in the monomers that make up the DNA-binding domains are two highly variable nucleotides that recognize and bind to a single nucleotide in the target sequence [51].

Figure 6.

Illustration of zinc-finger nucleases (ZFN) and transcription activator- like effector nucleases (TALEN) - the gene editing technology.

3. Forefront applications of gene therapy

3.1. Cancer therapy

To date, global cancer prevalence is at its utmost peak [52]. According to the World Health Organization (WHO), cancer is the second leading cause of death globally [53]. As conventional cancer therapy, surgical excision followed by chemo-radiotherapy can improve the overall survival rate. However, long-term chemotherapy could be responsible for several adverse effects and impact the quality of life of patients [54]. Currently, there is an urgent need to develop effective and sustainable therapies (e.g. gene therapy and stem cell therapy) to treat cancer [55].

The current scope of cancer gene therapy includes tumor suppressor gene therapy [56], gene silencing therapy [57], suicide gene therapy [58], immunological gene therapy [59], anti-tumor angiogenesis therapy [60], oncolytic therapy [61], and other subtypes. Few gene therapies have been used in clinical trials to treat different types of cancer [62]. Examples include liver cancer [63], pancreatic cancer [64], and head and neck tumors [65]. Researchers have administered therapeutic NAs ex vivo and/or in vivo to demonstrate gene transfer into tumor cells [53,66].

There are several ways to target cancer with gene therapy: (a) expressing a gene to enhance tumor sensitivity to radiation therapy or induce apoptosis; (b) inserting a wild-type tumor suppressor gene to compensate for its loss or deregulation; (c) using an antisense (RNA/DNA) approach to block the expression of an oncogene; and (d) increasing the tumor’s immunogenicity to stimulate immune cell recognition [67]. To date, approved gene therapies are depicted in Table 1.

Table 1.

Approved gene therapies for cancer and monogenetic disorder [159].

Trade name	Diseases/disorders	Vectors and modified gene	Sponsor(s)	Year/approval agency
Recombinant human p53 adenovirus (GENIDICINE)	Head and neck squamous cell carcinoma	Adenoviral vector p53	Biotech Company, Anges MG	2012/2003 (EMA/FDA) [82]
(ONCORINE)	Head and neck and esophagus cancer, nasopharyngeal cancer	Adenovirus Type 5	Fujian Cancer Hospital	2015 (FDA) [83]
Tisagenlecleucel (KYMRIAH)	Acute lymphoblastic leukemia	Lentiviral	Novartis Gene Therapies Inc.	2018/2017 (EMA/FDA) [84]
Axicabtagene ciloleucel (YESCARTA)	Non-Hodgkin lymphoma	Retroviral	Kite Pharma	2018/2017 (EMA/FDA) [85]
Talimogene laherparepvec (IMLYGIC)	Melanoma	HSV-1	Amgen	2015/2015 (EMA/FDA) [86]
Brexucabtagene autoleucel (TECARTUS)	Mantel cell lymphoma or B-cell acute lymphoblastic Leukemia	Retroviral	Kite Pharma	2020/2020 (EMA/FDA) [87]
Lisocabtagene maraleucel (BREYANZI)	B-cell lymphoma	Lentiviral	Juno Therapeutics Inc.	2022/2021 (EMA/FDA) [88]
Idecabtagene vicleucel (ABECMA)	Multiple myeloma	Lentiviral	Celgene Corporation	2021/2021 (EMA/FDA) [89]
Ciltacabtagene autoleucel (CARVYKTI)	Multiple myeloma	Lentiviral	Janssen Biotech Inc.	2022/2022 (EMA/FDA) [90]
Onasemnogeneabeparvovec (ZOLGENSMA)	Spinal muscular atrophy	Adeno-associated virus 9	Novartis	2019/FDA [91]
Voretigene neparvovec (LUXTURNA)	Leber congenital amaurosis	Adeno-associated virus	Spark Therapeutics	2017/FDA [92]
Valoctocogeneroxaparvovec (ROCTAVIAN)	Hemophillia A	Adeno-associated virus	BioMarin Pharmaceutical	2023/FDA [93]
Etranacogenedezaparvovec (HEMGENIX)	Hemophillia B	Adeno-associated virus	CSL/ UniQure	2022/FDA [94]
Fidanacogeneelaparvovec (BEQVEZ)	Hemophillia B	Adeno-associated virus	Pfizer/ Spark	2024/FDA [95]

3.1.1. Apoptosis inducers gene therapy

Mutations or abnormalities in the expression of pro- and anti-apoptotic genes cause ineffective or inefficient apoptotic signalling, rendering malignant cells resistant to apoptosis. This explains why targeting the apoptotic mechanism using gene therapy makes sense. To induce apoptosis, the most common method of cancer gene therapy involves inserting genes that encode the inducer, mediator, or executor of apoptosis. For instance, ONYX-015 (Delta-24-RGD) is an engineered adenovirus designed to selectively replicate in and kill cancer cells [68]. Gene therapy approaches to induce apoptosis typically involve oncogene targeting, pro-apoptotic gene delivery, inhibition of anti-apoptotic proteins, and cytokine-induced apoptosis. Approaches incorporating apoptosis induction with other therapeutic modalities, such as immunotherapy, chemotherapy, and targeted drug delivery, are showing promise.

3.1.2. Suicide gene therapy

Suicide gene therapy is a therapeutic strategy in which suicide-inducing transgenes are introduced into cancer cells. It involves the introduction of a gene into target cancer cells that encodes an enzyme capable of converting a non-toxic prodrug into a toxic compound. When the prodrug is administered, only the cells expressing the suicide gene can convert the drug into a lethal form, leading to selective cell death.

The most dominant suicide gene therapy is the herpes simplex virus (HSV thymidine kinase/ganciclovir) system. Recent studies have revealed that HSV thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy and radiofrequency hyperthermia combination is a promising technique. It has shown promising anticancer effects in rats and mice. Various gene therapy systems, excluding HSV-TK/GCV, are under investigation, such as cytosine deaminase/5-fluorocytosine, cytochrome P450/cyclophos-phamideand carboxypeptidase/4-[2-chloroethyl-2-mesyloxyetel-0-amino] benzoyl-l-glutamic acid. The use of suicide gene therapy has been explored in various clinical trials, particularly for treating conditions like glioblastoma multiforme and other aggressive cancers [69].

The transition from suicide gene therapy to apoptosis inducers gene therapy represents a enhancement in the approach to targeted therapy for cancer and other several diseases. By focusing on the pathways that govern cell death directly, apoptosis inducers offer potentially greater efficacy and flexibility in the treatment of various cancers, paving the way for more effective and less toxic therapeutic options. Apoptosis inducers target a broader array of cancer types, and avoid the potential risk of resistance caused by suicidal gene therapy as the apoptosis induction mechanisms can be more universally applicable.

3.1.3. Cancer suppressor gene therapy

Based on familial, epidemiological, and cytogenetic studies, cancer is a genetic disease. The major contribution to the development of cancer is a multistage process, involving inherited and somatic mutations, that is, proto-oncogenes and tumor suppressor genes. A large number of tumor suppressor genes, including p53, retinoblastoma gene Rb, p16INK/CDKN2, PTEN, have been identified, and tremendous efforts have been made to deliver targeted genes to cancer cells for normal functioning [67]. An intriguing strategy in cancer treatment is the high mutation frequency of the tumor suppressor p53 in human cancers and restoration of wild-type p53 function. One of the most notable applications of p53 gene therapy occurred in clinical trials involving patients with head and neck squamous cell carcinoma. Researchers developed a therapeutic approach known as ‘Gendicine’, which involves the delivery of a functional copy of the p53 gene to tumor cells. Gendicine is a genetically modified adenovirus that carries the normal p53 gene.

This could be achieved by introducing an intact p53 gene using a viral vector (for example, an adenoviral vector (Adp53) [70]. Additionally, preclinical studies have reported that Adp53 induces tumor regression in various cancers, including head and neck cancer [71], colorectal cancer [72], lung cancer [73], ovarian cancer [74], bladder cancer [75], and prostate cancer [76]. The world’s first Adp53-based gene therapy (e.g. Gendicine) has been approved by the State FDA of China and later on by US-FDA for the treatment of head and neck squamous cell carcinoma [77]. The primary function of the p53 protein, which is encoded by the TP53 gene located on human chromosome 17, is tumor suppression. Through complex interactions with various signaling pathways that are essential for vital cellular functions such as cell division, maintenance of genomic stability, apoptosis, autophagy, immune response, and regulation of the tumor microenvironment (TME), the p53 protein is thought to prevent the phenotypic and genomic changes associated with the development of cancer. The inactivation of the TP53 gene, by promoting invasion, proliferation, and cell survival, facilitates the spread and metastasis of cancer. In nearly 75% of TP53 gene mutations, the functions of wild-type p53 are lost. Mutated p53 proteins may display distinct tumorigenic characteristics that undermine the protective role of wild-type p53 or may act as a dominant negative, inhibiting the function of wild-type p53 [78].

3.1.4. Immunization gene therapy

Immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T-cells, immune checkpoint inhibitors (ICIs), mRNA vaccines, ZyCoV-D, and oncolytic viruses, have recently helped cancer patients live longer on average. Increased synergistic efficacy has been observed when immunotherapy drugs are used with or without cytotoxic therapies. Talimogene laherparepvec (T-VEC), the first gene therapy approved by the FDA, has sparked research on gene therapy treatments intended to elicit tumor-specific immune responses. T-VEC is an oncolytic HSV that contains the GM-CSF gene and has changed so that it can only multiply in tumor cells. GM-CSF draws dendritic cells to the tumor microenvironment, where they secrete tumor antigens and induce tumor-specific immunity [79].

3.1.5. Gene editing technique

Gene editing is a novel technology for this purpose. It specifically modifies target genes using nucleases to achieve DNA knock-out, knock-in, and/or mutation, subsequently downregulating or upregulating gene expression to attain an altered or new cell phenotype. Recently, the most useful nucleases were ZFN, TALEN, and CRISPR/Cas9. For example, urologic neoplasms, gynecological oncology, respiratory tumors, and digestive system tumors have been the subject of CRISPR/Cas9 technical trials. Lu Yu et al. at Huaxi Hospital deleted the PD-1 gene from T cells extracted from a patient with non-small cell lung cancer in 2016. It was the world’s first CRISPR gene therapy technology tested in a patient with a bright future for clinical treatment [80]. A well-publicized case involved the use of CRISPR to attempt to treat a patient with sickle cell disease. Future gene editing techniques could be focus on increasing the personalized therapy. Techniques like CRISPR-Cas9 have already demonstrated tremendous potential and other advance techniques under pipeline like CRISPR-Cas12 and CRISPR-Cas13. The combination of gene editing techniques with artificial intelligence and machine learning could enhance the design of more effective and targeted editing strategies. To date, the CRISPR Therapeutics and Vertex Pharmaceuticals conducting notable trials of CRISPR-Cas9 (e.g. STARR) to treat sickle cell disease and beta-thalassemia, respectively. Researchers are also trying to correct mutations in the CFTR gene responsible for cystic fibrosis with the help of CRISPR technology.

3.1.6. Oncolytic gene therapy

Introduction of genetically altered viruses into the body to eradicate malignant cells, either by producing cytotoxic proteins or by inducing cytolysis as a result of the spread of the virus, is known as oncolytic gene therapy (e.g. Talimogene Laherparepvec). Viruses such as vaccinia, adenovirus, HSV-type I, retrovirus, and Newcastle disease virus were chosen because of their inherent ability to infect cancer cells or because of their easy genetic manipulation [81].

Compared with the control survival rate of 28%, the oncolytic adenovirus (OAd) Delta-24-RGDOX significantly increased the survival rate to 85% in glioma-bearing mice by expressing OX40L, an immunological co-stimulator, in conjunction with an anti-PD-L1 antibody. A recent study featuring rAd.sT (telomerase reverse transcriptase promoter-regulated OAd) combined with a soluble transforming growth factor receptor II and human IgG Fc fragment (sTGFβRIIFc) gene demonstrated dose-dependent cytotoxicity in breast and kidney cancer patients [82].

3.2. Applications of gene therapy for genetic disorders

3.2.1. Monogenetic disorder (MD)

MDs are a range of human diseases that are caused by mutations in specific genes. Gene therapy has emerged as a promising therapeutic strategy for the treatment of genetic disorders. Gene therapy has shown promising results in treating MD, such as hemophilia [96–99], LCA [100–104], and SMA [105,106]. SMA is a genetic disease that causes weakness and wasting in the voluntary muscles. The SMA phenotype is categorized into four grades of severity (SMA I, SMA II, SMA III, SMA IV) based on the age of onset and motor function achieved. Type 1 is the most severe, where the patient is unable to sit; Type 2 is unable to walk unaided; Type 3 is able to achieve some walking abilities; and Type 4 is adult-onset SMA. For instance, onasemnogene abeparvovec (Zolgensma), is a one-time intravenous infusion to treat SMA in pediatric patients. Additionally AAV-9 mediated gene transfer could significantly improve lifespan and motor symptoms in SMA patients [14].

It paves the way for its broader application in other genetic disorders, such as Duchenne muscular dystrophy (DMD), Huntington’s disease (HD), transthyretin amyloidosis (ATTR), familial hypercholesterolemia (FH), and familial amyotrophic lateral sclerosis. Moreover, gene therapy is often used in combination with hematopoietic stem cell transplantation (HSCT) to treat diseases such as hemoglobin disorders, metabolic disorders, and storage disorders such as beta-thalassemia and leukodystrophies [106]. Few MDs and their respective approved gene therapies are listed in Table 1.

3.2.2. Polygenetic disorder

Independently acting or interacting polymorphic genes are responsible for polygenetic disorders. Due to the social impact of polygenetic disorders, it could be a major concern to find makers for it. Experimental approaches have suggested that analysis of genomic linkage or association with the disease could be a major breakthrough [107]. Analysis of genomic linkage in polygenic disorders is a complex yet essential aspect of genetic research that aims to identify the contributory alleles and their interactions involved in these multifactorial diseases. There are several methods like genome-wide association studies, linkage disequilibrium mapping, and family-based studies are useful in genome linkage analysis. Polygenetic disorders occur because of various factors, such as genetic variations and environmental factors (e.g. nutrition, exercise, and environmental exposure). Major psychiatric disorders such as attention deficit hyperactivity disorder (ADHD), autism (AUT), bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SZ) are highly heritable and polygenic [108]. The polygenic risk score (PRS) has been used in several disorders to predict disease status in case-control studies, shared genetic correlation between comorbid diseases, and shared genetic correlation between a disease and specific endophenotypes [109]. Clinical implementation of PRS may be useful in cohorts where there is a higher prior probability of disease; for example, in the early stages of diseases to assist in diagnosis or to inform treatment choices [110].

In developed countries, the primary cause of visual impairment is diabetic retinopathy (DR), which is characterized by vascular lesions and neuronal damage to the retina. The advent of therapy targeting VEGF has demonstrated significant benefits to patients with DR. However, this treatment is limited by its short half-life and requirement for frequent invasive intravitreal injections. Gene therapy could be a better alternative to treat DR with distinct advantages, such as a longer therapeutic effect, lower injection frequency, ability to intervene at disease onset, and potentially fewer side effects [111,112].

3.3. Infectious diseases

Owing to the massive success in MDs [113] and cancer [114], gene therapy has been considered a promising method for treating severe infectious diseases, such as human immunodeficiency virus (HIV) [115]. Instead of antiretroviral therapy (ART) or latency-reversing agents (LRAs), gene therapy could be promising for the treatment of infectious diseases [116].

3.4. Gene therapy approaches

Recently, three primary gene editing methods that involve nucleases, namely TALENs, ZFNs, and CRISPR/CRISPR-associated nuclease 9 (Cas9), have gained significant popularity in the field of HIV-1/AIDS treatment [117,118]. A successful clinical trial was done using ZFN-mediated editing of the C-C chemokine receptor type five (CCR5) in autologous CD4 T cells of HIV-1 infected individuals. The CCR5 gene is significant in HIV due to its role in facilitating viral entry into T cells. During HIV entry CCR5 could be act as a co-receptor which lead to replication of HIV. Gene editing leads to mutated CCR5-Δ32 which is resistant for HIV and prevent its entry into CD4 cells. This trial revealed that modifying ZFNs-CCR5 may be an effective and safe approach in the treatment of AIDS in humans.

Due to their high cost and time requirements [119], the latest gene-editing method, CRISPR/Cas9, has been swiftly developed to offer the advantages of accurately inserting, deleting, and replacing specific DNA sequences [120]. In 2013, the first use of CRISPR/Cas9 to prevent HIV-1 infection was destroying the latent HIV-1 provirus [121]. So far, there have been many research on the use of CRISPR/Cas9 technology for gene therapy in HIV-1/AIDS. These studies indicate that this technology has great potential for treating HIV-1/AIDS [122].

In 2013, the first experimentation with a CRISPR/Cas9-based method was conducted to assess its efficacy in treating HIV1/AIDS. The CRISPR/Cas9 system has effectively suppressed the expression of HIV-1 genes in Jurkat cell lines by specifically targeting the HIV-1 LTR [121]. The specific locations targeted were the NF-κB binding cassettes found in the U3 region of the LTR and TAR sequences in the R region. This led to a highly effective suppression of HIV-1 provirus transcription and replication [121]. CRISPR/Cas9 has the ability to remove the viral genes that have become part of the infected host cell’s chromosome. This indicates that CRISPR/Cas9 could be a promising technique for treating HIV-1/AIDS [121].

Recent research has indicated that CRISPR/Cas9 has the ability to cut non-integrated HIV-1, leading to a 3–4 times decrease in the integrated HIV-1 provirus. Remarkably, the NHEJ-mediated DNA repair process also reacts to non-integrated HIV-1 provirus [123]. Therefore, CRISPR/Cas9 operates in both HIV-1 and proviral DNA within dormant cells, which makes it a highly promising contender for the treatment of HIV-1/AIDS [122]. CCR5 acts as a co-receptor that aids in the cellular entrance of CCR5-tropic HIV-1 viruses. This makes it a desirable target for developing therapeutic strategies against HIV, as depicted in Figure 7. Not surprisingly, CCR5 has been focused on using various CRISPR-based methods in diverse animal models, human cell lines, and primary human cells [125–127]. A number of researchers and academics have devised novel strategies to combat HIV-mediated gene transfer or genome editing. To initiate autologous gene therapy, the initial procedure involves the extraction of CD4 + T cells or CD34 + HSCs from persons who are HIV-positive. Afterwards, these cells are made resistant to HIV through genetic manipulation, either by transferring genes using retroviral/lentiviral methods or by altering the genome with targeted techniques. Alternatively, cells can be enhanced with supplementary characteristics to eradicate cells infected with HIV [123,127–129].

Figure 7.

Life cycle of HIV and its possible targets [124]. CD4: cluster of differentiation 4; CCR5: C–C chemokine receptor type 5; CXCR4: C–X–C chemokine receptor type 4.

3.5. Neurodegenerative disorders

Millions of patients with neurodegenerative disorders may benefit from gene therapy using a variety of strategies, such as direct pathogenic mechanism rectification, neuronal protection, neuronal repair, and symptom management. For instance, brain-derived neurotrophic factor, CRISPER/Sas9, RNA interference and glial-derived neurotrophic factor, RPE65 gene are the prime examples in a neuronal protection and neuronal repair, respectively. Therefore, understanding the pathophysiology of the disease and the necessary temporal and spatial specificity of gene expression are critical for effective therapy as well as achieving maximal transduction of the target structure while preventing leakage into adjacent regions or perivascular spaces. The field of gene therapy has just transitioned into a new technical era, in which interventional MRI-guided convection-enhanced delivery (iMRI-CED) is the gold standard for confirming precise vector administration in real-time [130].

The availability of this advanced neurosurgical technique may accelerate the translation of the promising preclinical therapeutics under development for neurodegenerative disorders, including Parkinson’s, Huntington’s, and Alzheimer’s diseases (AD), etc. To date, several gene therapies in the clinical pipeline are depicted in Table 2.

Table 2.

List of gene therapies for neurodegenerative disorders.

Disorders	Clinical trial code	Delivery route	Gene therapy	Phase	Sponsor(s)
Alzheimer’s disease	NCT00876863	Direct basal forebrain injection	AAV2-NGF	II	Sangamo Therapeutics
Alzheimer’s disease	NCT05400330	–	AAVrh.10hAPOE2	I	Lexeo Therapeutics
Alzheimer’s disease	NCT03634007	Intrathecal injection	AAVrh.10hAPOE2	I/II	Lexeo Therapeutics
Huntington’s disease	NCT02519036	Intrathecal injection	ASOs to HTT messenger RNA	II	Ionis Pharmaceuticals, Inc.
Huntington’s disease	NCT03225833	Intrathecal injection	ASOs to HTT	II	Wave Life Sciences Ltd.
Huntington’s disease	NCT06444217	In-Vitro	Trans-splicing	NA	University Hospital, Angers
Pompe’s disease	NCT00976352	Intramuscular injection	rAAV1-CMV-hGAA	II	University of Florida
Parkinson’s disease	NCT02418598	Intraputaminal injection	AAV-hAADC-2	II	Jichi Medical University
Parkinson’s disease	NCT00643890	Injection into the subthalamic nucleus	AAV2-GAD	II	Neurologix, Inc.
Parkinson’s disease	NCT04167540	Infusion into Putamen	AAV2-GDNF	I	Brain Neurotherapy Bio, Inc.
Parkinson’s disease	NCT01973543	injected into the striatum	AAV2-hAADC	I	NeurocrineBiosciences
Parkinson’s disease	NCT05822739	intracranial injection	AAV	Early Phase I	Xiangya Hospital of Central South University
Parkinson’s disease	NCT03065192	injected into the striatum	VY-AADC01	I	Neurocrine Biosciences
Spinal Muscular Atrophy	NCT02122952	Intravenous injection	AAV9-SMN	I	Novartis Gene Therapies
Spinal Muscular Atrophy	NCT02292537	Intrathecal injection	ASOs targeting SMN2 splicing	III	Biogen
Mucopolysaccharidosis type III A	NCT02053064	Intracerebral injection	AAVrh10-SUMF1	II	LYSOGENE
Mucopolysaccharidosis type II	NCT03041324	Intravenous injection	AAV6-IDS	II	Sangamo Therapeutics

3.5.1. Alzheimer’s diseases (AD)

AD is a progressive and fatal neurodegenerative disorder characterized by cognitive and memory deterioration, progressive impairment of daily activities, and various neuropsychiatric symptoms and behavioral disturbances [131]. Currently, there are more than 26.6 million patients with AD worldwide. As the world population ages, the number of AD patients is expected to exceed 106.2 million by 2050; one in every 85 people will live with AD [130,132]. The marketed drugs are donepezil, rivastigmine, and galantamine, which enhance cholinergic neurotransmission and compensate for cholinergic neurodegeneration [133], while limiting excitotoxicity-induced neurodegeneration [134,135]. The current treatments primarily focus on managing symptoms and slowing disease progression, rather than providing a cure. Gene therapy offers several potential advantages over current treatments such as proper management of symptoms by targeting root cause, effect durability, tailored and diverse treatment approaches.

3.5.1.1. Gene therapy for AD

3.5.1.1.1. Reducing the generation of Aβ-peptides

AD is characterized by the accumulation of β-amyloid peptide (Aβ) within the brain, along with hyperphosphorylated and cleaved forms of the microtubule-associated protein tau [136,137]. Hanseul and co-workers. (2019) investigated the potential of Cas9 nanocomplexes targeting Bace1 to reduce Aβ levels and improve memory impairment in an AD model. Four weeks after injecting Cas9-Bace1 nanocomplexes into the CA3 hippocampal area, the researchers tested the behaviour and biochemistry of 5XFAD transgenic AD mice that were 6 months old. Using Sanger sequencing, they were able to effectively target Bace1 in the brains of mice injected with nanocomplexes. Mice that received nanocomplexes showed an almost 70% decrease in Bace1 expression in the CA3 region compared to the control group. Additionally, the brains of 5XFAD mice treated with Cas9 nanocomplex showed significantly fewer APP cleavage products (CTF, C99) [137].

3.5.1.1.2. Gene therapies that modulate neuroinflammation

The release of pro-inflammatory cytokines by activated microglia and astrocytes is a crucial aspect of neuroinflammation in the brains of AD patients. However, the role of anti-inflammatory cytokines in AD pathology remains unclear [138]. For example, short-term AAV-mediated expression of the anti-inflammatory cytokine IL-4 in the TgCRND8 transgenic mouse model exacerbated Aβ peptide deposition [139], but long-term expression in APP/PS1 mice significantly reduced microglial accumulation, astrogliosis, and Aβ peptide loading. The link between anti-inflammatory cytokine levels and AD-relevant pathology appears to be complex, and it may not be influenced by their effects on Aβ peptide. Further research is required to characterize the mechanisms underlying cytokines as potential therapies for AD [137].

3.5.1.1.3. Enhancing proteins that regulate oxidative stress

In recent study, it was found that lentivirus-mediated gene transfer of Nrf2 to the hippocampus of APP/PS1 mice reduced astrocytosis and improved spatial learning. Nrf2 has also been implicated as a negative regulator of BACE1 transcription in mouse embryonic fibroblasts, suggesting that its upregulation could provide a multi-mechanistic treatment for AD involving both reduced oxidation and modified amyloid processing [135,140,141].

3.5.2. Parkinson’s disease

Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by both motor and non-motor features. Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), which projects to the striatum (the nigrostriatal pathway), results in the loss of dopaminergic function in individuals with PD [142]. Currently, gene therapy for PD is characterized by several approaches. Compared to conventional treatments, gene therapy offers the advantage of eradicating major symptoms of PD with few to no side effects. Identifying potential targets is the first step in genetic therapy. Targets for PD can be classified as either disease-modifying or non-disease-modifying. The difference between the two is that non-disease-modifying treatments involve the production of dopaminergic enzymes, while disease-modifying treatments aim to prevent the degeneration of neurons. Tyrosine hydroxylase (TH), GTP cyclohydrolase 1 (GCH), and aromatic L-amino acid decarboxylase (AADC) therapy are presented as a tricistronic system, as AADC therapy is utilized in non-modifying targets but not as a stand-alone treatment. A drug called ProSavin is currently undergoing a Phase 1 clinical trial as a result of these advancements. Glial cell-derived neurotrophic factor (GDNF) is one of the disease-modifying targets, and direct administration of GDNF has been shown to alleviate symptoms of PD [143].

3.5.2.1. Restoration of dopamine level

Aromatic l-amino acid decarboxylase (AADC) gene therapy: Toni et al. (2021) studied the safety and effectiveness of delivering a virus (AAV2-hAADC) that causes AADC to the midbrain of children who do not have enough of it (ClinicalTrials.gov Identifier NCT02852213). They found that administering AAV2-hAADC directly to both sides of the brain was safe, well-tolerated, and increased the metabolism of dopamine in all participants. It also increases the absorption of FDOPA in the striatum and midbrain. Furthermore, midbrain gene administration proved safe and feasible, and improved clinical motor function and symptoms in children with AADC deficiency [144].

3.5.2.2. Neuroprotection

Derek et al. discovered that injecting a replication-defective Ad vector encoding human GDNF close to the rat substantia nigra prevented the neurotoxic 6-hydroxydopamine (6-OHDA) injection into the striatum from gradually degenerating dopaminergic (DA) neurons. Comparing Ad GDNF gene therapy to neither treatment nor injection of Ad lacZ or Ad mGDNF (producing a physiologically inactive deletion mutant GDNF), the reduction in DA neuron loss was almost three times greater six weeks after the 6-OHDA injury.These results suggested that Ad vector-mediated GDNF gene therapy may slow the DA neuronal cell loss in humans with PD [145].

3.5.2.3. Alpha-synuclein gene therapy

Targeted gene silencing of α-syn has been performed using various approaches, including ASOs, siRNAs, shRNA, and ZFNs, with both liposomal and viral vectors as delivery modalities. More recently, CRISPR-based technologies have also been utilized to modulate α-syn expression via transcriptional regulation through the endonuclease-deficient dCas9-based system [146].

4. Challenges and limitations

Gene therapy using viral vectors faces the major challenge of immunogenicity [147]. One of the biggest obstacles to the application of viral gene therapy is immune-mediated rejection. For instance, hepatic, systemic, and ocular AAV gene therapy commonly uses steroid medications to suppress antiviral CD8+ T cell responses and inflammation [148]. Additionally, unregulated gene expression could be a major concern in limiting human applications owing to potential side effects. While low gene expression is of concern in a clinical setting, these levels may not be sufficient to elicit a response [149]. Stringent regulation of therapeutic gene expression by means of cell-type-specific and/or targeted vector systems will significantly decrease potential safety risks [150].

Another major challenge could be the high cost of gene therapy. For example, Glybera, the first gene therapy approved in Europe, was priced at €1 million and was later withdrawn because no country provided coverage owing to its high cost [151]. Gene therapies charge high-cost manufacturing, and several biopharmaceutical firms and many academicians are working together to decrease manufacturing costs [152]. However, the main obstacle to current gene therapies is their lack of specificity. Hence, achieving a defined target remains a major challenge in gene therapy. Many promoters exhibit specific activity only in certain cell types, making them potential candidates for transcriptional targeting. Ideally, such a promoter should provide maximum specific expression of the therapeutic gene in the target tissue and must be strong enough to ensure the safety and efficiency of the system [153,154].

5. Conclusions and future perspective

Gene therapy could be a breakthrough for several severe disorders, such as AD, HD, and PD. Gene editing technology has helped gene therapy to become a more precise and effective treatment owing to the development of CRISPR and other modern gene-editing tools. Gene therapies are also helpful beyond the scope of rare genetic disorders, cardiovascular disorders, neurodegenerative conditions, and even infectious diseases, such as HIV/AIDS, by boosting host resistance. Future policy and societal acceptance will be shaped by ethical issues, particularly with regard to germline editing, while regulatory frameworks will change to guarantee the safety and effectiveness of these treatments. With enormous market growth and potential savings in healthcare costs, as curative treatments take the place of long-term symptom management, the economic impact will be substantial. To avoid inequities in healthcare availability, it is crucial to guarantee equitable access and minimize expenses. The economic aspects and global scalability of gene therapy present both challenges and opportunities. While the high costs of development and treatment pricing pose barriers to accessibility, the potential for long-term cost savings and improved health outcomes offers a compelling argument for investment in this transformative field. Overcoming regulatory, infrastructural, and cultural hurdles will be crucial for the widespread adoption of gene therapy worldwide. As the field continues to evolve, collaborations among stakeholders will be key to navigating these complex but promising landscapes. In addition, advances in gene therapy hold transformative potential for treating genetic conditions; ethical considerations such as safety, germ line modification, regulation, and public perception must accompany these innovations to ensure responsible development and application. Balancing innovation with ethical standards will be critical as the field evolves. Gene therapy has the potential to become a better alternative to conventional therapies. This would have a profound impact on global healthcare and the quality of life of patients.

Funding Statement

The funding has been provided by Datta Meghe College of Pharmacy, Datta Meghe Institute of Higher Education and Research, Wardha, India and Gehan M. Elossaily would like to thank AlMaarefa University, Riyadh, Saudi Arabia.

Author contributions

R.G.I.: Conceptualization, Visualization, Methodology, Review, and Editing; G.M.E.: Supervision, Review, Software; M.N.A.: Formal analysis, Review & Editing, Supervision, S.M.: Formal analysis, investigation, writing–original draft, and data curation.

All the authors have read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.