Replicating-Competent VSV-Vectored Pseudotyped Viruses.

Vesicular stomatitis virus (VSV) is prototype virus in the family of Rhabdoviridae. Reverse genetic platform has enabled the genetic manipulation of VSV as a powerful live viral vector. Replicating-competent VSV is constructed by replacing the original VSV glycoprotein gene with heterologous envelope genes. The resulting recombinant viruses are able to replicate in permissive cells and incorporate the foreign envelope proteins on the surface of the viral particle without changing the bullet-shape morphology. Correspondingly, the cell tropism of replicating-competent VSV is determined by the foreign envelope proteins. Replicating-competent VSVs have been successfully used for selecting critical viral receptors or host factors, screening mutants that escape therapeutic antibodies, and developing VSV-based live viral vaccines.

Viral Vectors in Gene Therapy: Where Do We Stand in 2023?

Viral vectors have been used for a broad spectrum of gene therapy for both acute and chronic diseases. In the context of cancer gene therapy, viral vectors expressing anti-tumor, toxic, suicide and immunostimulatory genes, such as cytokines and chemokines, have been applied. Oncolytic viruses, which specifically replicate in and kill tumor cells, have provided tumor eradication, and even cure of cancers in animal models. In a broader meaning, vaccine development against infectious diseases and various cancers has been considered as a type of gene therapy. Especially in the case of COVID-19 vaccines, adenovirus-based vaccines such as ChAdOx1 nCoV-19 and Ad26.COV2.S have demonstrated excellent safety and vaccine efficacy in clinical trials, leading to Emergency Use Authorization in many countries. Viral vectors have shown great promise in the treatment of chronic diseases such as severe combined immunodeficiency (SCID), muscular dystrophy, hemophilia, ß-thalassemia, and sickle cell disease (SCD). Proof-of-concept has been established in preclinical studies in various animal models. Clinical gene therapy trials have confirmed good safety, tolerability, and therapeutic efficacy. Viral-based drugs have been approved for cancer, hematological, metabolic, neurological, and ophthalmological diseases as well as for vaccines. For example, the adenovirus-based drug Gendicine® for non-small-cell lung cancer, the reovirus-based drug Reolysin® for ovarian cancer, the oncolytic HSV T-VEC for melanoma, lentivirus-based treatment of ADA-SCID disease, and the rhabdovirus-based vaccine Ervebo against Ebola virus disease have been approved for human use.

Progress in Respiratory Gene Therapy.

The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.

The Adenovirus Vector Platform: Novel Insights into Rational Vector Design and Lessons Learned from the COVID-19 Vaccine.

The adenovirus vector platform remains one of the most efficient toolboxes for generation of transfer vehicles used in gene therapy and virotherapy to treat tumors, as well as vaccines to protect from infectious diseases. The adenovirus genome and capsids can be modified using highly efficient techniques, and vectors can be produced at high titers, which facilitates their rapid adaptation to current needs and disease applications. Over recent years, the adenovirus vector platform has been in the center of attention for vaccine development against the ongoing coronavirus SARS-CoV-2/COVID-19 pandemic. The worldwide deployment of these vaccines has greatly deepened the knowledge on virus-host interactions and highlighted the need to further improve the effectiveness and safety not only of adenovirus-based vaccines but also of gene therapy and oncolytic virotherapy vectors. Based on the current evidence, we discuss here how adenoviral vectors can be further improved by intelligent molecular design. This review covers the full spectrum of state-of-the-art strategies to avoid vector-induced side effects ranging from the vectorization of non-canonical adenovirus types to novel genome engineering techniques.

miRNA Pathway Alteration in Response to Non-Coding RNA Delivery in Viral Vector-Based Gene Therapy.

Gene therapy is widely used to treat incurable disorders and has become a routine procedure in clinical practice. Since viruses can exhibit specific tropisms, effectively penetrate the cell, and are easy to use, most gene therapy approaches are based on viral delivery of genetic material. However, viral vectors have some disadvantages, such as immune response and cytotoxicity induced by a disturbance of cell metabolism, including miRNA pathways that are an important part of transcription regulation. Therefore, any viral-based gene therapy approach involves the evaluation of side effects and safety. It is possible for such effects to be caused either by the viral vectors themselves or by the delivered genetic material. Many gene therapy techniques use non-coding RNA delivery as an effective agent for gene expression regulation, with the risk of cellular miRNA pathways being affected due to the nature of the non-coding RNAs. This review describes the effect of viral vector entry and non-coding RNA delivery by these vectors on miRNA signaling pathways.

Mechanisms and challenges of nanocarriers as non-viral vectors of therapeutic genes for enhanced pulmonary delivery.

With the rapid development of biopharmaceuticals and the outbreak of COVID-19, the world has ushered in a frenzy to develop gene therapy. Therefore, therapeutic genes have received enormous attention. However, due to the extreme instability and low intracellular gene expression of naked genes, specific vectors are required. Viral vectors are widely used attributed to their high transfection efficiency. However, due to the safety concerns of viral vectors, nanotechnology-based non-viral vectors have attracted extensive investigation. Still, issues of low transfection efficiency and poor tissue targeting of non-viral vectors need to be addressed. Especially, pulmonary gene delivery has obvious advantages for the treatment of inherited lung diseases, lung cancer, and viral pneumonia, which can not only enhance lung targeting and but also reduce enzymatic degradation. For systemic diseases therapy, pulmonary gene delivery can enhance vaccine efficacy via inducing not only cellular, humoral immunity but also mucosal immunity. This review provides a comprehensive overview of nanocarriers as non-viral vectors of therapeutic genes for enhanced pulmonary delivery. First of all, the characteristics and therapeutic mechanism of DNA, mRNA, and siRNA are provided. Thereafter, the advantages and challenges of pulmonary gene delivery in exerting local and systemic effects are discussed. Then, the inhalation dosage forms for nanoparticle-based drug delivery systems are introduced. Moreover, a series of materials used as nanocarriers for pulmonary gene delivery are presented, and the endosomal escape mechanisms of nanocarriers based on different materials are explored. The application of various non-viral vectors for pulmonary gene delivery are summarized in detail, with the perspectives of nano-vectors for pulmonary gene delivery.

Adenoviral Vectors: Potential as Anti-HBV Vaccines and Therapeutics.

Adenoviral vaccines have been at the front line in the fight against pandemics caused by viral infections such as Ebola and the coronavirus disease 2019. This has revived an interest in developing these vectors as vaccines and therapies against other viruses of health importance such as hepatitis B virus (HBV). Current hepatitis B therapies are not curative; hence, chronic hepatitis B remains the major risk factor for development of liver disease and death in HBV-infected individuals. The ability to induce a robust immune response and high liver transduction efficiency makes adenoviral vectors attractive tools for anti-HBV vaccine and therapy development, respectively. This review describes recent developments in designing adenoviral-vector-based therapeutics and vaccines against HBV infection.

A Novel Piggyback Strategy for mRNA Delivery Exploiting Adenovirus Entry Biology.

Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utility of this technology for the context of the COVID-19 pandemic. Nonetheless, broad implementation of these promising strategies has been restricted by the limited repertoires of delivery vehicles capable of mRNA transport. On this basis, we explored a strategy based on exploiting the well characterized entry biology of adenovirus. To this end, we studied an adenovirus-polylysine (AdpL) that embodied "piggyback" transport of the mRNA on the capsid exterior of adenovirus. We hypothesized that the efficient steps of Ad binding, receptor-mediated entry, and capsid-mediated endosome escape could provide an effective pathway for transport of mRNA to the cellular cytosol for transgene expression. Our studies confirmed that AdpL could mediate effective gene transfer of mRNA vectors in vitro and in vivo. Facets of this method may offer key utilities to actualize the promise of mRNA-based therapeutics.

An Adenoviral Vector as a Versatile Tool for Delivery and Expression of miRNAs.

Only two decades after discovering miRNAs, our understanding of the functional effects of deregulated miRNAs in the development of diseases, particularly cancer, has been rapidly evolving. These observations and functional studies provide the basis for developing miRNA-based diagnostic markers or new therapeutic strategies. Adenoviral (Ad) vectors belong to the most frequently used vector types in gene therapy and are suitable for strong short-term transgene expression in a variety of cells. Here, we report the set-up and functionality of an Ad-based miRNA vector platform that can be employed to deliver and express a high level of miRNAs efficiently. This vector platform allows fast and efficient vector production to high titers and the expression of pri-miRNA precursors under the control of a polymerase II promoter. In contrast to non-viral miRNA delivery systems, this Ad-based miRNA vector platform allows accurate dosing of the delivered miRNAs. Using a two-vector model, we showed that Ad-driven miRNA expression was sufficient in down-regulating the expression of an overexpressed and highly stable protein. Additional data corroborated the downregulation of multiple endogenous target RNAs using the system presented here. Additionally, we report some unanticipated synergistic effects on the transduction efficiencies in vitro when cells were consecutively transduced with two different Ad-vectors. This effect might be taken into consideration for protocols using two or more different Ad vectors simultaneously.

Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production.

Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.

Viral vector vaccines.

Over the past two years, the SARS-CoV-2 pandemic has highlighted the impact that emerging pathogens can have on global health. The development of new and effective vaccine technologies is vital in the fight against such threats. Viral vectors are a relatively new vaccine platform that relies on recombinant viruses to deliver selected immunogens into the host. In response to the SARS-CoV-2 pandemic, the development and subsequent rollout of adenoviral vector vaccines has shown the utility, impact, scalability and efficacy of this platform. Shown to elicit strong cellular and humoral immune responses in diverse populations, these vaccine vectors will be an important approach against infectious diseases in the future.

Characterization of a dynamic self-replicating mammalian expression vector based on the circular ssDNA genome of beak and feather disease virus.

In vivo nucleic expression technologies using DNA or mRNA offer several advantages for recombinant gene expression. Their inherent ability to generate natively expressed recombinant proteins and antigens allows these technologies to mimic foreign gene expression without infection. Furthermore, foreign nucleic acid fragments have an inherent ability to act as natural immune adjuvants and stimulate innate pathogen- and DNA damage-associated receptors that are responsible for activating pathogen-associated molecular pattern (PAMP) and DNA damage-associated molecular pattern (DAMP) signalling pathways. This makes nucleic-acid-based expression technologies attractive for a wide range of vaccine and oncolytic immunotherapeutic uses. Recently, RNA vaccines have demonstrated their efficacy in generating strong humoral and cellular immune responses for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). DNA vaccines, which are more stable and easier to manufacture, generate similar immune responses to RNA, but typically exhibit lower immunogenicity. Here we report on a novel method of constructing self-amplifying DNA expression vectors that have the potential to amplify and enhance gene/antigen expression at a cellular level by increasing per cell gene copy numbers, boost genomic adjuvating effects and mitigate through replication many of the problems faced by non-replicating vectors such as degradation, methylation and gene silencing. These vectors employ a viral origin rolling circle replication cycle in mammalian host cells that amplifies the vector and gene of interest (GOI) copy number, maintaining themselves as nuclear episomes. We show that these vectors maintain persistently elevated GOI expression levels at the cellular level and induce morphological cellular alterations synonymous with increased cellular stress.

Adenoviral vectors for cardiovascular gene therapy applications: a clinical and industry perspective.

Despite the development of novel pharmacological treatments, cardiovascular disease morbidity and mortality remain high indicating an unmet clinical need. Viral gene therapy enables targeted delivery of therapeutic transgenes and represents an attractive platform for tackling acquired and inherited cardiovascular diseases in the future. Current cardiovascular gene therapy trials in humans mainly focus on improving cardiac angiogenesis and function. Encouragingly, local delivery of therapeutic transgenes utilising first-generation human adenovirus serotype (HAd)-5 is safe in the short term and has shown some efficacy in drug refractory angina pectoris and heart failure with reduced ejection fraction. Despite this success, systemic delivery of therapeutic HAd-5 vectors targeting cardiovascular tissues and internal organs is limited by negligible gene transfer to target cells, elimination by the immune system, liver sequestration, off-target effects, and episomal degradation. To circumvent these barriers, cardiovascular gene therapy research has focused on determining the safety and efficacy of rare alternative serotypes and/or genetically engineered adenoviral capsid protein-modified vectors following local or systemic delivery. Pre-clinical studies have identified several vectors including HAd-11, HAd-35, and HAd-20-42-42 as promising platforms for local and systemic targeting of vascular endothelial and smooth muscle cells. In the past, clinical gene therapy trials were often restricted by limited scale-up capabilities of gene therapy medicinal products (GTMPs) and lack of regulatory guidance. However, significant improvement of industrial GTMP scale-up and purification, development of novel producer cell lines, and issuing of GTMP regulatory guidance by national regulatory health agencies have addressed many of these challenges, creating a more robust framework for future adenoviral-based cardiovascular gene therapy. In addition, this has enabled the mass roll out of adenovirus vector-based COVID-19 vaccines. KEY MESSAGES: First-generation HAd-5 vectors are widely used in cardiovascular gene therapy. HAd-5-based gene therapy was shown to lead to cardiac angiogenesis and improved function. Novel HAd vectors may represent promising transgene carriers for systemic delivery. Novel methods allow industrial scale-up of rare/genetically altered Ad serotypes. National regulatory health agencies have issued guidance on GMP for GTMPs.

Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector.

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.

Human adenovirus type 26 basic biology and its usage as vaccine vector.

Due to their nature, adenoviruses have been recognised as promising candidates for vaccine vector development. Since they mimic natural infection, recombinant adenovirus vectors have been proven as ideal shuttles to deliver foreign transgenes aiming at inducing both humoral and cellular immune response. In addition, a potent adjuvant effect can be exerted due to the adenovirus inherent stimulation of various elements of innate and adaptive immunity. Due to its low seroprevalence in humans as well as induction of favourable immune response to inserted transgene, human adenovirus type 26 (HAdV-D26) has been recognised as a promising platform for vaccine vector development and is studied in number of completed or ongoing clinical studies. Very recently HAdV-D26 based Ebola and Covid-19 vaccines were approved for medical use. In this review, current state of the art regarding HAdV-D26 basic biology and its usage as vaccine vector will be discussed.

Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.