Viral vector‐based gene therapies in the clinic

Abstract Gene therapies are currently one of the most investigated therapeutic modalities in both the preclinical and clinical settings and have shown promise in treating a diverse spectrum of diseases. Gene therapies aim at introducing a gene material in target cells and represent a promising approach to cure diseases that were thought to be incurable by conventional modalities. In many cases, a gene therapy requires a vector to deliver gene therapeutics into target cells; viral vectors are among the most widely studied vectors owing to their distinguished advantages such as outstanding transduction efficiency. With decades of development, viral vector‐based gene therapies have achieved promising clinical outcomes with many products approved for treating a range of diseases including cancer, infectious diseases and monogenic diseases. In addition, a number of active clinical trials are underway to further expand their therapeutic potential. In this review, we highlight the diversity of viral vectors, review approved products, and discuss the current clinical landscape of in vivo viral vector‐based gene therapies. We have reviewed 13 approved products and their clinical applications. We have also analyzed more than 200 active trials based on various viral vectors and discussed their respective therapeutic applications. Moreover, we provide a critical analysis of the major translational challenges for in vivo viral vector‐based gene therapies and discuss possible strategies to address the same.

cells and represent a promising approach to cure diseases that were thought to be incurable by conventional modalities. In many cases, a gene therapy requires a vector to deliver gene therapeutics into target cells; viral vectors are among the most widely studied vectors owing to their distinguished advantages such as outstanding transduction efficiency. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes with many products approved for treating a range of diseases including cancer, infectious diseases and monogenic diseases. In addition, a number of active clinical trials are underway to further expand their therapeutic potential. In this review, we highlight the diversity of viral vectors, review approved products, and discuss the current clinical landscape of in vivo viral vectorbased gene therapies. We have reviewed 13 approved products and their clinical applications. We have also analyzed more than 200 active trials based on various viral vectors and discussed their respective therapeutic applications. Moreover, we provide a critical analysis of the major translational challenges for in vivo viral vectorbased gene therapies and discuss possible strategies to address the same.

| INTRODUCTION
Gene therapy, modifying expression of genes or correcting dysfunctional genes, offers great potential as a therapeutic modality for treating a plethora of diseases. [1][2][3] Unlike traditional drugs, gene therapy genetically modifies cells and thus opens possibilities of curing diseases that were once thought to be incurable. 4,5 The concept of gene therapy can date back to the 1960s when early studies demonstrated that DNA sequences could be introduced into mammalian cells for gene repair. 6 Decades of scientific efforts led to the first human gene therapy clinical trial in 1990 using a retrovirus vector technology for treating severe combined immunodeficiency. 7,8 However, gene therapy experienced a major setback in late 1990s following the death of a patient due to immune responses caused by the viral vector in a trial in 1999 and the development of viral vectorinduced leukemia in four patients after receiving a retrovirus-based gene therapy in another trial in 2000. 9 These two events brought gene therapy to a temporary halt in the clinic, raised concerns about its safety, and highlighted the critical need for safer viral vectors. The following decade focused on better understanding of the biology of viral vectors and advancing the engineering of safe and effective vectors; this led to the first clinical approvals of gene therapy products.
China approved the first gene therapy in the world for treating head and neck cancer in 2003 (Gendicine). EMA approved its first gene therapy product in 2012 (Glybera), and the United States approved its first in 2017 (Kymriah). 10 With the development of gene editing technologies (e.g., CRISPR/Cas9) that could precisely modify genes at a base level, [11][12][13] gene therapy is stepping into a new era and rapidly expanding its therapeutic horizon in treating a broader spectrum of diseases.
Current gene therapies fall into two broad categories, that is ex vivo and in vivo gene therapies. Particularly, in vivo gene therapies involve the direct infusion of gene therapeutics into patients' bloodstream or injection into target organs. 12,14 A vector is generally required for in vivo gene therapies, with an aim to pack and deliver gene therapeutics into target cells. Engineered viruses are the dominant vectors in current gene therapy clinical studies. 15,16 To date, a plethora of viral vectors including adenovirus (Ad), adeno-associated virus (AAV), and herpes simplex virus (HSV) have proven their potential in safely and efficiently delivering gene therapies. 17,18 Excitingly, over a dozen viral gene therapy products have been approved for treating cancer, infectious diseases, and monogenic diseases and their clinical investigation is still expanding. 5 Here, we provide an overview of clinical advances of viral vector-based gene therapies. Particularly, we restrict our discussion to in vivo gene therapies. Clinical advances of ex vivo gene therapies, often related to a subclass of cell therapies, have been recently discussed in other reviews. 19 We identified more than 10 approved products and more than 200 active clinical trials ( Figure 1) and provided a snapchat of the current clinical landscape by discussing the diversity of clinically relevant viral vectors, reviewing the clinical applications of approved products, and discussing active clinical trials based on their corresponding vectors and disease indications. We also discuss critical challenges associated with the clinical translation of in vivo viral vector-based gene therapies, with an aim to provide a framework for better product design.

| CLINICALLY RELEVANT VIRAL VECTORS
The primary reason of using a virus as a vector for gene delivery is to employ their natural capability of infecting cells and to efficiently shuttle gene materials of interest into host cells. 17 Major viral vector types used for in vivo gene therapies in the clinic include Ad, AAV, HSV, retrovirus, and lentivirus ( Figure 1). 24,25 Here we briefly introduce these vectors with a focus on their primary applications and distinguishing characteristics. Comprehensive reviews of different viral vectors can be found elsewhere. 18,24 Adenovirus (Ad) is one of the earliest viral vectors investigated in the clinic for in vivo gene therapies. [26][27][28] Ads are a class of DNA viruses with a 34-43 kb genome enclosed in a nonenveloped icosahedral viral particle. 29 There are more than 50 Ad serotypes, with Ad5 and Ad26 being most widely used for gene therapy. 16 Three generations of Ad vectors have been engineered to fit different therapeutic applications. The first generation involves the removal of the E1 and E3 units that makes the vector capable of carrying up to 7.5 kb foreign DNA. 30,31 The E1 and E4 units are deleted in the second generation of Ad vectors that significantly reduces immunogenicity. 32 All viral genes are deleted in the third generation that allows the vector to carry more than 30 kb foreign DNA. 33  Herpes simplex virus (HSV) is an enveloped virus with a doublestranded NDA genome of over 150 kb. 44 The virus genome encodes approximately 90 genes; half of the genes are nonessential that can be removed/replaced in recombinant vectors and thus affords a high capacity for foreign DNA. 16 Eight human HSV serotypes have been identified and each serotype exhibits distinct tropism. 18,45,46 Currently, three major types of HSV vectors have been engineered for gene therapy applications, including amplicon HSV, replicationdefective HSV, and replication-competent HSV. 16,44 Amplicon HSV is an engineered vector to carry a large foreign DNA of over 100 kb.
Replication-defective HSV vector is typically created by depletion of necessary genes for the lytic cycle of HSV and is less toxic and immunogenic. Replication-competent HSV vector is an engineered HSV that keeps the genes for replication in vitro but deletes the genes for replication in vivo. 16,44 Major characteristics of HSV vectors include (1) their ability to evade the immune system, (2) capability to deliver large DNA cargos and multiple genes, and (3) intrinsic or engineered cell-specific lytic property. 44 Clinical application of HSV vector-based gene therapies has been primarily focused on cancer treatment, mostly attributed to their intrinsic oncolytic capability. 47 Retrovirus is the first viral vector that was studied in clinical trials for in vivo gene therapy. Retrovirus is an enveloped spherical virus that carries their genetic materials in the form of RNA. Retrovirus vectors can reverse transcribe their genetic materials (single-stranded RNA) into double-stranded DNA and integrate it into host cells' genome. 48,49 Major advantages of retrovirus vectors for gene therapy is that they can carry a large gene of interest (9-12 kb) and result in long-term gene expression due to their integration into host genome. 50 However, several major drawbacks limit their applications. 51 First, retrovirus vectors require cell division to integrate its DNA into host genome, and thus they can only transduce dividing cells. In addition, retrovirus vectors have the risk to randomly insert its DNA into host chromosome and leads to insertional mutagenesis. 51 Self-inactivating vectors that have the promotor or enhancer of the long terminal repeat deleted have been developed to reduce the risk of insertional mutagenesis. 16 Due to these limitations, retrovirus vectors are currently not often used in clinical studies any more.
Lentivirus is another important viral vector for gene therapy. Its major application is for ex vivo gene therapies. 52 However, it is also currently investigated in the clinic for in vivo gene therapy applications. Lentivirus is a subtype of retrovirus and carries the genetic materials in the form of RNA. However, unlike retrovirus, lentivirus can integrate its genome into and transduce nondividing cells. 53 The first generation of lentivirus vectors is originally derived from (human immunodeficiency virus 1) HIV-1 and has proven its capability of efficiently transducing CNS organs in vivo. 53 (Table 1). Their clinical use covers therapies of cancer, infectious diseases, and monogenic diseases. In this section, we discuss these approved products and their respective clinical indications.

| Cancer therapy products
Four viral gene therapy products have been approved globally for the treatment of cancer, solid tumors in particular ( is based on: (1) the engineered viral vectors have intrinsic oncolytic properties and/or (2) the carried gene leads to expression of tumor suppressors or immunomodulators that enhance the anti-tumor efficacy. 18 One notion is that following their approval for a specific cancer type, all of these products have been or are being studied in the clinic for treating various additional types of cancers.
IMLYGIC ® is the first and only US FDA-approved viral gene therapy for cancer. IMLYGIC ® was approved for the treatment of local recurrent unresectable cutaneous, subcutaneous, and nodal melanoma after initial surgery via direct intralesional administration. 62 IMLYGIC ® is based on a modified HSV1 in which two viral genes, Of note, apart from melanoma, IMLYGIC ® has been clinically investigated for treating other solid tumors. 64

| Vaccination products against infectious diseases
Six viral gene therapy products have been approved or authorized for emergency use for vaccination against infectious diseases, specifically Ebola virus infections and COVID-19 ( Table 1) Two viral gene therapy products have been approved for vaccina- vaccine and has now received authorization for use in more than 30 countries. However, it was recently suspended in several countries due to increased risk of blood clots.

| Products for monogenetic diseases
Another important category of approved viral gene therapies is for treating monogenic diseases. In this category, three products have been approved for the therapy of metabolic diseases, ophthalmological diseases, and neurological diseases (Table 1). Although they are indicated for different diseases, their fundamental mechanism of action is similar, that is to deliver a corrected gene to replace the mutated/dysfunctional copy in target cells to alleviate or even cure diseases. 5 Notably, all three approved products use AAV as the viral vector. This is mostly attributed to the distinct feature of AAVs that they lack pathogenicity, exhibit low immunogenicity, and can typically lead to long-lasting gene expression. clinical trials to study its long-term safety and efficacy or to explore its therapeutic potential using other administration routes.

| CURRENT CLINICAL TRIALS FOR VIRAL VECTOR-BASED IN VIVO GENE THERAPIES
We performed the search for active clinical trials on clinicaltrials.gov using the following methods/criteria. We used "gene therapy" as the keyword in the "Other terms" category and the system also automatically searched for "gene transfer" and "DNA therapy". Under the and musculoskeletal disorders such as Duchenne muscular dystrophy.
We analyzed the serotypes/variants of AAV vectors used in active trials. Historically, AAV2 was the first AAV serotype that was studied in the clinic for in vivo gene therapy. 43   Nearly one third (29.4%) of the active trials are investigated for treating ophthalmological diseases (Figure 3c and Table 2). Of note, in all of these trials, AAV gene therapies are administered directly into the eye using a localized method such as subretinal and intravitreal injections. This localized administration enables efficient delivery of viral-vectors to target retinal compartments and also mitigates offtarget effects. More than nine types of ophthalmological diseases are currently studied (Table 2) and a detailed analysis of our search results, by disease indication, is shown in Figure S1c. Notably, retinitis pigmentosa is the most studied ophthalmological indication in the clinic. In these retinitis pigmentosa-related trials, a functional gene was delivered to retinal cells to replace the mutated or lost copy to restore vision. Depending on the specific type of retinitis pigmentosa, various genes were used in these trials, such as genes encoding channelrhodopsin, Phosphodiesterase 6A, and retinitis pigmentosa GTPase regulator. It should be noted that the mechanism involved in the age-related macular degeneration trials is different from that in other indications. Instead of replacing/restoring a gene, these trials for treating age-related macular degeneration use AAV vectors to express a therapeutic protein such as anti-VEGF Fab.
Other than ophthalmological diseases, neurological, metabolic, hematological, and musculoskeletal diseases are four additional major indications that the current AAV trials are focused on (Figure 3c and Table 2). AAVs are used to treat many diseases within these catego-   to name a few (Figure 4b and Table 3). Notably, ChAd is emerging as an important variant in the ongoing clinical trials, most likely attributed to the factor that ChAd is not infectious to humans and thus has a good safety profile. 92,93 Infectious diseases and cancer are two major indications that the current active trials are focused on ( Figure 4c). New areas of indications such as cardiovascular diseases and degenerative diseases are emerging; however, the number of ongoing clinical trials in these areas are only a few (Table 3).

| Adenovirus vector-based clinical trials
Ongoing clinical trials using Ad vector-based gene therapies for treating infectious diseases are abundant (Table 3)  Cancer is the second most investigated area for the identified Ad vector-based gene therapy clinical trials (Table 3)

| Other viral vector-based clinical trials
We identified 18 ongoing clinical trials using newer viral vectors other than AAV, Ad, and HSV for in vivo gene therapies ( With better understanding of the biology of these newer viral vectors, it is expected that they will be more extensively examined in clinical trials for various indications.

| CLINICAL CHALLENGES
Viral vector-based gene therapies face many challenges that can affect their implementation and adoption into standard clinical care; indeed, these challenges have been extensively reviewed in previous articles. [94][95][96][97][98][99][100][101][102][103] A main challenge is the reliance on animal models that inaccurately predict transduction efficiency in humans; one effort in this area is the development of humanized mouse models or animal models that use tissue grafts from humans to better predict potential efficacy and success of viral vector-based gene therapies in the clinic. 42 Closely connected to efficacy in humans, immune response from viral vectors presents a significant hurdle to the translation of viral vector-based gene therapies. Briefly, both innate and adaptive immune responses against viral vector-based gene therapies can occur in human patients, which can limit: (1) repeated use of the same viral vector due to host-generation of neutralizing antibodies, which can alter biodistribution and thus prevent the successful long-term use of viral vectors, and (2) transduction efficiency due to immune system response to the introduced viral structures. 94 Importantly, understanding how to navigate and interact with the immune system, and possibly control immune response, could lead to the expansion of applications that viral vector-based gene therapies could be clinically used for. Approaches to mitigate immune responses against viral vectors include introduction of novel vectors with tissue-specific tropism, 104 genetically engineering the capsid through directed evolution, 42 and altering the route of administration, 105 to name a few currently emerging approaches. [106][107][108] There also exists considerable manufacturing challenges, which have been previously and extensively reviewed [95][96][97][101][102][103] ; importantly, these limitations with manufacturing and productions can contribute to the high costs of many viral vector-based gene therapies, especially those for rare diseases. 98 To address these production and manufacturing challenges, emerging efforts include transition from transfected producer cells to stable producer cell lines, 95 development of processes that enable suspension culture (as opposed to adherent-culture) of producer cell lines, 96 development of serotype-independent purification processes for viral vectors, 109 and new methodologies to identify contaminating proteins or bacteria of vector stocks, 102 to name a few. Through our search, we identified greater than 20 recent viral vector-based clinical trials that had been withdrawn or terminated; however, reasons for the withdrawal or termination for the overwhelming majority of these trials were either not stated or provided generic reasons (e.g., sponsor decision, low accrual). Two studies provided specific reasons for trial termination. NCT02387125, a clinical trial studying the lentiviral vector CMB305 to target dendritic cells as a primeboost vaccine against tumors that express NY-ESO-1, was terminated on July 1, 2020 due to the clinical trial not meeting the efficacy objective. Likewise, NCT02609984, which also studied CMB305 was terminated on July 7, 2020, also due to lack of meeting the efficacy objective. While specific reasons for lack of efficacy were not listed, the results for the latter study have been posted on clinicaltrials.gov; overall, these studies highlight the difficulty in achieving efficacy for viral vectors, potentially due to the aforementioned clinical challenges.

| CONCLUSIONS
Gene therapy directly corrects/modifies genes and represents a revolutionary modality to treat diseases. Gene therapy holds a unique position in the therapeutics spectrum in a sense that it is the only approach that can potentially cure some diseases such as genetic