Viral Vectors

Since the onset of the gene therapy era, viral vectors have been used as a primary vehicle for gene delivery to human cells. In 2019, over 3000 clinical trials related to gene therapy applications were conducted, proving that this field is gaining significant interest14. The clinical trials focused on cancer therapy, monogenic diseases, infectious diseases, and cardiovascular diseases. Interestingly, although more efficient non-viral vector-based methods have been developed recently, viral vectors were used in nearly 70% of the trials.

The recombinant viral vectors often used in gene therapy applications include Ad, RV, lentivirus (LV), HSV, and AAV, which range in their genomic size from ~5 to 150 kb. The suitability of each type of viral vector for different applications is decided based on various features. There are two major classes of recombinant viral vectors generally used to deliver genes, grouped according to whether their genomes exist as extrachromosomal episomes (AAVs, HSVs, and Ads) or integrate into the recipient’s chromosome (RVs and LVs). This feature is crucial for determining the suitability of each vector for certain applications. Although many targeted diseases have been responsive to gene therapy, no single vector is suitable for use in all applications. The essential features required of all vectors include assured safety, reproducibility, stability of propagation, the ability to be purified to high titers, and the ability to mediate specific targeted gene delivery, where only the therapeutic gene specific to the tissue or organ of interest is transferred without widespread vector dissemination. The features of these five commonly used viral vectors are discussed in detail in the following section. Table 1 contains an overview of the viral vectors and their comparison.

Adenoviral vectors

Ads are non-enveloped viruses with a double-stranded DNA genome and icosahedral nucleocapsid. Around 57 previously discovered human Ad serotypes are divided into 7 subgroups (A–G)15. The most extensively characterized Ads are the 36-kb serotypes 2 (Ad2) and 5 (Ad5), which belong to subgroup C. These Ads, especially those of serotype 5, are widely used in a broad range of gene therapies because they can transduce various types of dividing and non-dividing cells both and 16. Most recombinant adenoviral systems designed for gene transfer applications are replication-defective or, in other words, not capable of replicating themselves15, 17, 18. They are constructed into a vector expressing the therapeutic gene, while a helper construct encodes for the viral proteins and makes the virus incapable of replicating19. Recombinant Ad vectors are the most efficacious medium by which to deliver genes . In 2013, 476 human gene therapy studies were conducted using recombinant Ad vectors, representing 23.5% of all gene therapy trials; with this, Ad vectors were recognized as the viral vector most applied in clinical trials globally20. This is due to their broad cellular tropism for and applications. Despite having significant advantages for various gene therapy applications, Ads lack native integration machinery that would enable the attainment of permanent gene expression . Ad vectors do not integrate into the host genome and remain episomal. Studies have demonstrated that random integration of replication-defective Ad vectors into the host genome occurred at frequencies of 0.001% – 1% of infected cells, reducing the risk of insertional mutagenesis. Current advances have led to the emergence of a range of hybrid vectors combining the vastly efficient DNA delivery of Ads with the integration machinery of RVs, AAVs, and transposons to improve the integration frequencies of Ad vectors. These hybrid vectors have shown promise, at least in systems21.

Ad vectors are highly immunogenic and can induce strong innate and adaptive immune responses. Recombinant Ad vectors have been constructed by deleting numerous parts of their coding regions. The first-generation Ad vectors were made by substituting the early gene (E1) with a transgene. Although the deletion of E1 resulted in a replication-defective vector, the E2, E3, and E4 promoters remained active and led to the expression of viral genes at low levels15. This leaky viral protein expression induced strong cytotoxic immune reactions that rapidly eliminated transgene expression in transduced cells19. Additional attempts were made to delete several early genes (E2a, E2b, or E4) besides E1 to reduce the immune response to the viral antigens produced due to leaky expression. Although these second-and third-generation Ad vectors reduced toxicity, the gene sequences encoding the viral particles remained active, leading to the expression of viral proteins that induced secondary innate and adaptive immune responses against the vectors22, 23. Because of these factors, the sufficient expression period for Ad vectors in humans peaks at 1 week and is restricted to about 2 weeks, making these vectors ideal for use whenever a shorter duration of vector expression is required, such as for generating new biological structures20. Despite the safety of Ad vectors, considering the immune response against them, helper-dependent Ad (HDAd) vectors with large-scale cloning capacity were introduced. All viral genes except the inverted terminal repeats and packaging signal are deleted in these HDAd vectors24. HDAd vectors were found to reduce chronic toxicity in comparison to the first- and second-generation viruses in which only a part of the viral genome was deleted25. Although the safety profiles of HDAd vectors were improved, the dose of the vector was shown to trigger an acute immune response mediated by capsid proteins of the vector in a dose-dependent manner26.

To sum up, Ad has a high transduction capacity and is the most effective gene delivery method in cell and tissue types of varying complexity. The strong inflammatory response to the virus’s capsid proteins is the greatest flaw of the Ad system. It is anticipated that additional development will soon provide patients with efficient and safe Ad gene transfer.

Retroviral vectors

RVs, which are enveloped viruses with RNA genomes, can reverse transcribe their genetic material from RNA to cDNA. Most recombinant RV systems are replication-defective, and a transgene expression vector along with a helper virus that expresses viral proteins are used in gene therapy applications. However, the ability of the virus to replicate is limited. Packaging features are provided by the helper virus that carries co-transfected plasmids in a packaging cell (., the HEK293 cell line). The packaging cell lines encode sequences involved in viral packaging functions (gag, pro, pol, and env). These genes cannot be packaged into RV particles because they are supplied during transformation using molecular constructs. The transgene is delivered into a genetic vector that mimics the viral genome structure but contains fewer cis-acting sequences, permitting its effective incorporation into viral particles, reverse transcription, and expression in target cells.

The use of RV vectors is a classic approach in long-term gene therapy applications, and RVs have been used to deliver transgenes for the past three decades9. As the gold standard, RV vectors could accommodate up to 8 kb of foreign genes and were proven effective in therapies involving bone marrow stem cell transplants27. The most commonly used RV vector is derived from the murine leukemia virus (MLV). In 1990, the first human gene therapy using MLV-based gamma-retroviral (γ-RV) vectors carrying therapeutic genes was performed in two young girls suffering from severe combined immunodeficiency (SCID). The clinical trial involved the implantation of autologous bone marrow cells transduced with gamma RV vectors28, 29. Although the clinical trial restored T-cell immunity in the SCID patients, vector-induced insertional oncogenesis due to random incorporation of the vector into the host genome was identified as a significant issue30, 31, 32. Another limitation of RVs is their inability to infect non-dividing cells, which enhanced the interest in applying LV vectors for gene therapy. LVs are capable of infecting both dividing and non-dividing cells even though they belong to the RV family. Therefore, LVs exhibit low cytotoxicity33.

In sum, because of their persistent and long-term gene transfer in proliferating cells, RVs are a promising option for gene transfer. However, RVs only transduce diving cells and integration may lead to oncogenesis in certain cases.

Lentiviral vectors

LVs are a type of RVs. Scientists showed interest in LVs for the first time in the early 1980s when the AIDS pandemic introduced the world to the most well-known and feared LV: human immunodeficiency virus type 1 (HIV-1). Since replication-defective LVs derived from HIV-1 can incorporate into the genome of non-dividing cells such as neurons and hematopoietic stem cells (HSCs) with a sustained, long-term expression of therapeutic genes, they were adopted as vehicles for gene delivery33. This is a unique feature of LVs as other RVs require proliferating cells to integrate into the cellular genome34. These properties underscore the potential benefits of using LVs over other RV vectors in human gene therapy applications. Naldini (1996) were the first to show that LVs derived from HIV-1 could offer efficient preclinical gene delivery, stable incorporation into the cellular genome, and constitutive expression of the therapeutic genes in non-dividing cells such as neurons35. However, potent host immune responses against the LV capsid and/or their transgene products hinder the success of gene therapy using LV vectors. The transgene expression period and potency of immune reactions to LV-encoded transgene products have differed among studies on delivery. The efficacy of LV vector-mediated gene delivery in immunocompetent mice leads to effective transduction and transgene expression in various cell types, including the retina, muscle, and hematopoietic cells, but the transduction of hepatocytes is relatively inefficient.

LVs function like RVs in that the genomic RNA of LVs is reverse-transcribed into cDNA as the virus invades the cells. The cDNA produced is then integrated into the cellular genome with the aid of a viral integrase enzyme at a random site. The integrated viral genome, now recognized as a provirus, remains in the host genome and is transferred to cell progeny as the cells undergo division. However, the integration site cannot be predicted, which may lead to some issues. The function of cellular genes may be interrupted by the provirus, inducing oncogene activation and enhancing carcinogenesis, raising safety concerns around the use of LVs in gene therapy.

Nevertheless, a few studies showed that the selection of LV insertion sites differs from that of RVs such as γ-RV vectors. This could be due to the differences in the structure of the integrase enzyme of these two types of viruses and the viral long terminal repeats (LTRs) in the γ-RV vectors. LVs incorporate randomly throughout the genome, although they only target active transcription units. On the other hand, the integrase and LTRs of γ-RV vectors were found to promote selected interaction with active host cell promoters and enhancers36. A previous study conducted by Aiuti et al. suggested that lentiviral gene therapy is safer than retroviral gene therapy since LVs have a lower risk of causing insertional mutagenesis. This lentiviral gene therapy did not promote the selection of integration sites near oncogenes and there was no aberrant clonal expansion detected37, 38.

The use of lentiviral vectors in gene therapy has emerged owing to the persistence of gene transfer in most tissues after the insertion of the vector. Its biggest flaw is integration possibly leading to oncogenesis in certain situations.

Herpes simplex virus vector

HSVs are large, enveloped viruses belonging to the subfamily of the family . Amongst the herpesviruses, HSV serotype 1 (HSV-1) has several natural properties that make it attractive for use as a viral vector, particularly for treatments related to diseases of the nervous system12, 39. In contrast to other viral systems, HSV-1-derived vectors can package and efficiently transfer up to 150 kb of transgenes to the nucleus of mammalian cells. HSV-1-based vectors can accommodate multiple transgene cassettes or single large genes, which is very practical for applications that require 97 expressions of one or a few gene products for an effective therapeutic outcome (., tumor-killing). HSV-1-based vectors can infect many cell types, including both dividing and non-dividing cells; however, they cannot incorporate into cellular DNA40, 41. These viruses lack lytic viral protein production and thus are episomal. Interestingly, latent, lifelong infections in the nervous system with the long-term expression of the transgene in brain neurons and without any leaky expression of the viral genes make HSV vectors a promising vehicle for gene delivery to the central nervous system (CNS)42. Several clinical studies using HSV vectors were performed to test therapies against recurrent breast cancer, head and neck cancer, unresectable pancreatic cancer, refractory superficial cancer, melanoma, and other conditions, mainly neurological and pain disorders43.

Amplicons are vectors harboring only a minimal number of virally derived sequences and are non-integrative, replication-defective, and helper-dependent HSV-1-derived vectors. They have the same properties as typical HSV-1 particles, including structure, tropism, and immunogenicity. However, they carry a concatemeric DNA plasmid containing the HSV-1 origin of replication (ori), HSV-1 packaging signal (pac), and transgene rather than a functional HSV genome44. These sequences enable the packaging of infectious HSV-1 particles. HSV-1 amplicon vector systems offer some advantages in gene therapy. They do not carry any viral genes, providing more space to accommodate larger foreign DNA molecules (up to almost 150 kb). These advantages make the HSV vector a powerful, interesting, and useful gene delivery tool.

Amplicons are completely safe for inoculation in animals, non-toxic to the infected cells, and only induce low levels of the adaptive immune response because they do not encode any viral proteins45. Like other HSV vectors, amplicons remain episomal, therefore posing no risk of insertional mutagenesis. Moreover, HSV-1 naturally spreads in the nervous system via neuronal transport, and it infects several species, which is useful for the assessment of vectors in various animal models. Transduction in the brain is generally more effective with HSV vectors than with other vectors (lower titers are required) since neurons are the natural target for HSV-1 infection.

To summarize, the benefits of HSV-1 vectors include their ability to infect both quiescent and proliferating cells effectively as well as their ability to encode for transgenes of significant size. On the other hand, when primary fibroblasts are used, there is a rapid reduction in the expression of the transgene.

Adeno-associated virus vector

AAV, a small, non-enveloped virus that infects human cells, is naturally replication-defective and does not cause disease in humans. So far, AAV appears to be the most promising means of gene delivery since the wild-type virus has never been reported to cause any disease in humans46, 47. The general principles of recombinant AAV (rAAV) vector construction involve the modification of molecular clones by replacing the AAV coding gene sequence with exogenous DNA to generate a plasmid vector. Recombinant vectors derived from these viruses are composed of two inverted terminal repeats flanking an expression cassette encoding a therapeutic transgene, deleting all viral open reading frames. To propagate rAAVs, co-infection with a helper virus, mainly Ad, is required47. This system facilitates rAAV production at relatively high titers. However, this preparation is contaminated with the helper virus and therefore needs further purification. For gene therapy applications in humans, this requirement for co-infection offers a natural safety feature that helps prevent the improper spread of rAAVs following clinical administration.

AAVs have a good safety profile for use as a vector in gene therapy. Because of the absence of viral genes, AAVs do not cause a deleterious host immune response47. A clinical trial in hemophilia B patients using an AAV-2 vector resulted in long-term expression in skeletal muscles and deficient inflammatory responses48, 49. However, the effectiveness of AAVs relies on the serotype, administration route, effective multiplicity of infection of the vector employed, transgene and expression cassette, and dosing schedule of injection50. Although AAV vectors have low immunogenicity, a primary host immune response (., a neutralizing antibody response) may be elicited against the vector capsid protein or transgene product, which might impact their use. For example, the decline in expression of the factor IX transgene 6 weeks after hepatic gene transfer and destruction of hepatocytes have been observed in patients in the highest dose cohort. Other significant positive features of AAV vectors include their efficacy in transducing proliferating cells as well as non-proliferating tissues, such as neurons, photoreceptor cells, hepatocytes, and muscle cells and their promotion of the long-term expression of transgenes . This is due to their integration into the host chromosome. Although AAV vector genomes can persist within cells as episomes, vector integration has been seen in numerous experimental settings51. About 20 AAV vectors were used in clinical settings for various therapeutic and prophylactic applications (., hemophilia B and CF)52. As of 2020, AAV vectors have been used in more than 12 clinical studies on hemophilia alone53.

Although AAV has a good safety profile compared to other viruses and may mediate long-term gene expression in non-proliferating or slowly proliferating cells, it is limited by several factors. For example, the immune response was shown to be triggered by the repeated administration of AAV vectors54. To overcome this problem, a different AAV serotype is applied for each re-administration. Another related issue is the very small packaging size of AAV (~4.7 kb), which limits the size of transgenes that can be packaged into the vector55. AAV vectors are unable to efficiently infect certain types of cells, such as HSCs, which are considered potential targets for numerous gene therapy applications. Furthermore, the safety of AAVs as a vector in clinical applications is limited because of concerns about AAV integration-mediated tumorigenicity. A previous study reported that the development of hepatocellular carcinoma was associated with AAV gene delivery in mice56.

AAVs offer similar benefits to Adv vectors, such as high transduction efficiency and long-lasting transgenic expression. They have a high biosafety rating and low immunogenicity, making them a good choice for gene therapy. However, AAVs also have several drawbacks, including a reduction in transgene expression over time due to episomal integration and a modest packaging capacity.

Oral administration

Oral gene therapy remains a promising and attractive strategy despite its challenges and obstacles. Oral administration holds great promise as it allows the daily intake of genetic medicine, easing the administration route and allowing transient gene expression. The idea of oral gene therapy is exciting because the body is not subjected to invasive surgery or deep wounds or exposed to local drug effects upon administration. Gene therapy clinical trials using oral administration that are actively recruiting participants include those testing treatments for typical Canavan disease in children57 and high-risk prostate cancer58 using AAV and Ad, respectively. The main obstacles to effective oral administration of gene therapy include withstanding harsh and everchanging conditions, such as the acidic pH and degradative enzymes in the gastrointestinal tract, without compromising the transgene while simultaneously maintaining efficiency.

IV administration

IV administration, also known as systemic delivery, is one of the most preferred viral gene delivery methods. IV administration is well tolerated at higher viral doses and provides a high level of gene expression. It has been reported as an effective and safe route for cancer gene therapy using HSV59. One of the system’s limitations is its broad distribution of vectors throughout the body that can easily lead to side effects. For instance, liver tropism is often observed after Ad administration through the IV route.

Topical administration

One of the major challenges in gene delivery is to deliver the transgene specifically to the target tissue; in this regard, local gene delivery holds great potential compared to systemic gene delivery. In the last 5 years, 40% of ongoing Phase III clinical trials on cancer gene therapy have utilized local gene delivery systems. A study on bevacizumab delivery using an AAV gene system showed sustained gene expression with less off-target effects and toxicity in lung cancer60. Another study showed that regional IV application using suitable AAV vectors has the most promise as a gene therapy approach for ischemic heath disease61. Local delivery, like pleural delivery, was also shown to have little risk of gene- or virus-induced inflammation62.