Application Of Viral Vectors Research Articles

Adeno-associated viruses in gene therapy for spinal muscular atrophies: trend or triumph?

The development of gene therapy in the 21st century is largely based on application of viral vectors, which have shown their effectiveness along with a fairly high safety profile. Among the vector systems, one of the leading places was taken by adeno-associated viruses (AAV), on the basis of which drugs were created for the treatment of severe hereditary monogenic diseases, including spinal muscular atrophies (SMA). Their use, on the one hand, is justified by the flexibility of AAV as a platform for the creation of gene therapy drugs, and on the other hand, it is often perceived as a kind of trend that has significant limitations. In this review, the focus is on two main aspects: AAV as a vector for the treatment of diseases from the SMA group and possible directions of development in this area and in gene therapy in general.
 The review operates with recent data published after clinical trials and experimental studies during last decade, and also critically examines the possibilities of gene therapy using AAV, mentioning other existing approaches, incl. medical therapy for SMA.
 Attention is also paid to the situation in the field of using AAV for the treatment of other hereditary diseases and the most acute problems faced by the use of drugs created on the basis of this promising vector system.

Seroprevalence of binding and neutralizing antibodies against 18 adeno-associated virus types in patients with neuromuscular disorders.

High levels of pre-existing antibodies are a major challenge for the application of viral vectors since they can severely limit their efficacy. To identify promising candidates among adeno-associated virus (AAV) based vectors for future gene therapies for the treatment of hereditary neuromuscular disorders (NMDs), we investigated the antibody levels in sera from patients with NMDs against 18 AAV types, including 11 AAVs with wild-type capsids, 5 AAVs with peptide-modified capsids and 2 AAVs with shuffled capsids. With regard to the wild-type capsid AAVs, the lowest binding antibody levels were detected against AAV6, AAV5, AAV12 and AAV9, whereas the highest binding antibody levels were detected against AAV10, AAV8, AAV1, and AAV2. The lowest neutralizing antibody levels against wild-type AAVs were detected against AAV12, AAV5, AAV9, AAV7, AAV8 and AAV10, and the highest neutralizing antibody levels were detected against AAV13, AAV2 and AAV3. Interestingly, the influence of peptide modifications or shuffling of AAV capsids on antibody binding and AAV neutralization seemed to depend on the parental AAV. While the sex of the serum donors had no significant impact on binding or neutralizing antibody levels, we observed a trend to higher binding antibodies in older serum donors against some AAV types and a clear positive correlation of neutralizing antibody titers with the age of the serum donors. The disease status on the other hand did not have a meaningful impact on antibody levels, with no changes in AAV neutralization. Our data indicate that several wild-type or peptide-modified AAV may be good candidates for therapeutic application due to low pre-existing antibody levels, and that the age of potential recipients rather than their health status with regard to NMDs has the biggest impact on vector applicability.

Gene therapy for primary myopathies: literature review and prospects

Gene therapy has emerged as a promising frontier in the pursuit of effective treatments for primary myopathies. This scientific review explores the application of viral vectors and more specifically of recombinant adeno-associated virus (rAAV) vectors as a potent gene delivery tool in the context of primary myopathies, highlighting its transformative potential. Focusing on primary myopathies, including Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophies (LGMDs), X-linked myotubular myopathy (XLMTM), and Pompe disease, we review the ongoing pre-clinical and clinical trials that underscore the therapeutic promise of rAAV-based gene therapies. Recent developments in gene therapy have unveiled innovative gene transfer approaches, particularly with rAAV vectors. These vectors offer a well-tolerated and efficient means of delivering corrective genetic material to diseased muscles, thereby addressing the root causes of primary myopathies. Encouraging data from pre-clinical studies and early clinical trials have demonstrated the potential to ameliorate muscle function, reduce pathological manifestations, and enhance the quality of life for patients afflicted with these devastating diseases. However, the transition from bench to bedside is not without challenges. This review emphasizes the critical need for a comprehensive risk management strategy to better handle potential side effects and immune responses associated with gene therapy. As the field of gene therapy for primary myopathies is advancing, it is imperative to refine and optimize safety measures, ensuring that the transformative potential of these therapies is realized while the risks are minimized. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.

Research Status and Prospect of Non-Viral Vectors Based on siRNA: A Review

Gene therapy has attracted much attention because of its unique mechanism of action, non-toxicity, and good tolerance, which can kill cancer cells without damaging healthy tissues. siRNA-based gene therapy can downregulate, enhance, or correct gene expression by introducing some nucleic acid into patient tissues. Routine treatment of hemophilia requires frequent intravenous injections of missing clotting protein. The high cost of combined therapy causes most patients to lack the best treatment resources. siRNA therapy has the potential of lasting treatment and even curing diseases. Compared with traditional surgery and chemotherapy, siRNA has fewer side effects and less damage to normal cells. The available therapies for degenerative diseases can only alleviate the symptoms of patients, while siRNA therapy drugs can upregulate gene expression, modify epigenetic changes, and stop the disease. In addition, siRNA also plays an important role in cardiovascular diseases, gastrointestinal diseases, and hepatitis B. However, free siRNA is easily degraded by nuclease and has a short half-life in the blood. Research has found that siRNA can be delivered to specific cells through appropriate vector selection and design to improve the therapeutic effect. The application of viral vectors is limited because of their high immunogenicity and low capacity, while non-viral vectors are widely used because of their low immunogenicity, low production cost, and high safety. This paper reviews the common non-viral vectors in recent years and introduces their advantages and disadvantages, as well as the latest application examples.

Design and Gene Delivery Application of Polymeric Materials in Cancer Immunotherapy

Immunotherapy has offered an alternative therapy method for cancer patients with metastatic tumors or who are not suitable for surgical resection. Different from traditional surgery, radiotherapy and chemotherapy, immunotherapy mainly restores the activity of the body’s own immune cells silenced in the tumor microenvironment to achieve anticancer therapy. Gene therapy which corrects abnormal expression of immune cells in tumor microenvironment by delivering exogenous genes to specific immune cells, is the most widely studied immunotherapy. Although most available gene delivery vectors are still viral vectors, the further application of viral vectors is still limited by the immunogenicity and mutagenesis. Based on this, cationic polymeric gene vectors with high flexibility, high feasibility, low cost and high safety have been widely used in gene delivery. The structural variability of polymers allows specific chemical modifications to be incorporated into polymer scaffolds to improve their physicochemical properties for more stable loading of genes or more targeted delivery to specific cells. In this review, we have summarized the structural characteristics and application potential in cancer immunotherapy of these polymeric gene vectors based on poly(L-lysine), poly(lactic-co-glycolic acid), polyethyleneimine, poly(amidoamine) and hydrogel system.

Tackling scalability challenges in AAV manufacture

Kyle Grant is a trained PhD chemical engineer with a concentration in molecular virology. Kyle has extensive experience in the molecular analysis of virus host cell interactions, as well as innovation around commercial grade unit operations to support CMC activities for the application of viral vectors in gene therapy. This multi-scale approach allows for comprehensive insight into the development of methods for provision and release of novel drug candidates to address the unmet need for patients where there is no available cure.

Application of Viral Vectors for Vaccine Development with a Special Emphasis on COVID-19.

Viral vectors can generate high levels of recombinant protein expression providing the basis for modern vaccine development. A large number of different viral vector expression systems have been utilized for targeting viral surface proteins and tumor-associated antigens. Immunization studies in preclinical animal models have evaluated the elicited humoral and cellular responses and the possible protection against challenges with lethal doses of infectious pathogens or tumor cells. Several vaccine candidates for both infectious diseases and various cancers have been subjected to a number of clinical trials. Human immunization trials have confirmed safe application of viral vectors, generation of neutralizing antibodies and protection against challenges with lethal doses. A special emphasis is placed on COVID-19 vaccines based on viral vectors. Likewise, the flexibility and advantages of applying viral particles, RNA replicons and DNA replicon vectors of self-replicating RNA viruses for vaccine development are presented.

Promising Cytomegalovirus-Based Vaccine Vector Induces Robust CD8+ T-Cell Response.

Vaccination has had great success in combating diseases, especially infectious diseases. However, traditional vaccination strategies are ineffective for several life-threatening diseases, including acquired immunodeficiency syndrome (AIDS), tuberculosis, malaria, and cancer. Viral vaccine vectors represent a promising strategy because they can efficiently deliver foreign genes and enhance antigen presentation in vivo. However, several limitations, including pre-existing immunity and packaging capacity, block the application of viral vectors. Cytomegalovirus (CMV) has been demonstrated as a new type of viral vector with additional advantages. CMV could systematically elicit and maintain high frequencies of effector memory T cells through the “memory inflation” mechanism. Studies have shown that CMV can be genetically modified to induce distinct patterns of CD8+ T-cell responses, while some unconventional CD8+ T-cell responses are rarely induced through conventional vaccine strategies. CMV has been used as a vaccine vector to deliver many disease-specific antigens, and the efficacy of these vaccines was tested in different animal models. Promising results demonstrated that the robust and unconventional T-cell responses elicited by the CMV-based vaccine vector are essential to control these diseases. These accumulated data and evidence strongly suggest that a CMV-based vaccine vector represents a promising approach to develop novel prophylactic and therapeutic vaccines against some epidemic pathogens and tumors.

Generation of a caged lentiviral vector through an unnatural amino acid for photo-switchable transduction.

Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging–uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.

Viral vectors as a novel tool for clinical and neuropsychiatric research applications

BackgroundA viral vector is a genetically modified vector produced by genetic engineering. As pathogenic genes in the virus are completely or largely eliminated, it is safe to be widely used...

Viral Vectors in Gene Therapy

Applications of viral vectors have found an encouraging new beginning in gene therapy in recent years. Significant improvements in vector engineering, delivery, and safety have placed viral vector-based therapy at the forefront of modern medicine. Viral vectors have been employed for the treatment of various diseases such as metabolic, cardiovascular, muscular, hematologic, ophthalmologic, and infectious diseases and different types of cancer. Recent development in the area of immunotherapy has provided both preventive and therapeutic approaches. Furthermore, gene silencing generating a reversible effect has become an interesting alternative, and is well-suited for delivery by viral vectors. A number of preclinical studies have demonstrated therapeutic and prophylactic efficacy in animal models and furthermore in clinical trials. Several viral vector-based drugs have also been globally approved.

Development of Methods for Safe Application of Viral Vectors for Production of Gene-Engineering Vaccines

Development of Methods for Safe Application of Viral Vectors for Production of Gene-Engineering Vaccines

Stimulation and Inhibition of Lymphangiogenesis Via Adeno-Associated Viral Gene Delivery.

The lymphatic vessels can be selectively stimulated to grow in adult mice, rats and pigs by application of viral vectors expressing the lymphangiogenic factors VEGF-C or VEGF-D. Vice versa, lymphangiogenesis in various pathological settings can be inhibited by the blocking of the VEGF-C/VEGFR3 interaction using a ligand-binding soluble form of VEGFR3. Furthermore, the recently discovered plasticity of meningeal and lacteal lymphatic vessels provides novel opportunities for their manipulation in disease. Adenoviral and adeno-associated viral vectors (AAVs) provide suitable tools for establishing short- and long-term gene expression, respectively and adenoviral vectors have already been used in clinical trials. As an example, we describe here ways to manipulate the meningeal lymphatic vasculature in the adult mice via AAV-mediated gene delivery. The possibility of stimulation and inhibition of lymphangiogenesis in adult mice has enabled the analysis of the role and function of lymphatic vessels in mouse models of disease.

Development of Methods for Safe Application of Viral Vectors for GeneEngineering Manipulations

Additional oncogene copy was transferred in normal cells, and additional tumor-suppression gene copy-in malignant cells, respectively, by transfection with the appropriate recombinant DNA-constructs, containing each one the respective gene. The results obtained were compared with the data from insertion of copy from the respective gene in prokaryotic bacteria strains E. coli, by analogical transfection with appropriate gene constructs. In application of low initial infections (high initial dilutions of viral suspensions, respectively) of eventually attenuated by many passages vaccine strains from the heterologous for mammals and mammalian cells avian viral species, only slight ultra-structural changes and signs of partially-formed immature viral particles in the cells, were established, but no cytopathic effect (CPE was observed). In freezing of the so inoculated mammalian cells after addition of cryoprotector Dimethylsulfoxide (DMSO), thawing and re-incubation in fresh cultivation medium, signs of activated cell proliferation were noted. One of the possible explanations could be eventual transfer of nucleotide sequences from viral particles to separate cells because of activated fusion processes on the influence of DMSO in drastic temperature changes. The results obtained proposed a possibility about application of the described methods as available alternatives for both vaccine production and gene-engineering manipulations with cells, compared with the procedures of preparation and application of designed quite expensive recombinant DNA-constructs. Future studies are necessary in this direction.

Strategies and methods for the detection and identification of viral vectors.

The production and application of viral vectors are frequently performed genetic engineering operations. HIV-1-based lentiviral vectors, AAV2-based, and adenoviral vectors are amongst the most abundant viral vectors utilized for gene delivery. They are generally classified into risk group 1 or 2 (according to EU directive 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work).

Application of viral vectors to the study of neural connectivities and neural circuits in the marmoset brain.

ABSTRACTIt is important to study the neural connectivities and functions in primates. For this purpose, it is critical to be able to transfer genes to certain neurons in the primate brain so that we can image the neuronal signals and analyze the function of the transferred gene. Toward this end, our team has been developing gene transfer systems using viral vectors. In this review, we summarize our current achievements as follows. 1) We compared the features of gene transfer using five different AAV serotypes in combination with three different promoters, namely, CMV, mouse CaMKII (CaMKII), and human synapsin 1 (hSyn1), in the marmoset cortex with those in the mouse and macaque cortices. 2) We used target‐specific double‐infection techniques in combination with TET‐ON and TET‐OFF using lentiviral retrograde vectors for enhanced visualization of neural connections. 3) We used an AAV‐mediated gene transfer method to study the transcriptional control for amplifying fluorescent signals using the TET/TRE system in the primate neocortex. We also established systems for shRNA mediated gene targeting in a neocortical region where a gene is significantly expressed and for expressing the gene using the CMV promoter for an unexpressed neocortical area in the primate cortex using AAV vectors to understand the regulation of downstream genes. Our findings have demonstrated the feasibility of using viral vector mediated gene transfer systems for the study of primate cortical circuits using the marmoset as an animal model. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 354–372, 2017

214. Specific Targeting of IL-13 Receptor alpha 2 Expressing Breast Cancer Cells by Paramyxovirus-Pseudotyped Lentiviral Vectors Displaying IL-13

The ability to selectively target specific cell types is highly desirable for the in vivo application of viral vectors for the treatment of multiple diseases including cancer. Lentiviral vectors (LV) have the advantage of being easily pseudotyped allowing their tropism to be altered. LV can also be engineered to deliver therapeutic or cytotoxic genes directly to cancer cells. IL-13 receptor alpha 2 (IL-13Rα2) is overexpressed in many different tumor types, including glioma, sarcoma, kidney, breast and ovarian cancer, making it an attractive target for tumor therapy. We have previously shown that firefly luciferase-expressing LV pseudotyped with a truncated fusion (F) protein derived from measles virus (MV) and a tail-truncated, receptor-blind MV hemagglutinin (H) protein bearing IL-13 at the C-terminus were capable of transducing IL-13Rα2-positive U251 glioma cells when administered intratumorally. In addition, we have shown the ability of LV to be pseudotyped using the F and H proteins from the Tupaia paramyxovirus (TPMV). TPMV does not infect human cells, potentially making it a safer choice for pseudotyping LV for in vivo delivery. Similar to the MV H protein, the TPMV H protein can also display IL-13 and bind specifically to IL-13Rα2-positive tumor cells. We plan to confirm and extend these findings by examining the targeting of LV-MV-IL-13 and LV-TPMV-IL-13 in metastatic orthotopic models of breast cancer by intratumoral and intravenous administration. In addition, LV expressing HSV1 thymidine kinase (TK) were constructed and are being evaluated for antitumor efficacy.

Developments in gene delivery vectors for ocular gene therapy

Gene therapy is quickly becoming a reality applicable in the clinic for inherited retinal diseases. Its remarkable success in safety and efficacy, in clinical trials for Leber's congenital amaurosis (LCA) type II generated significant interest and opened up possibilities for a new era of retinal gene therapies. Success in these clinical trials was mainly due to the favorable characteristics of the retina as a target organ. The eye offers several advantages as it is readily accessible and has some degree of immune privilege making it suitable for application of viral vectors. The viral vectors most frequently used for retinal gene delivery are lentivirus, adenovirus and adeno-associated virus (AAV). Here we will discuss the use of these viral vectors in retinal gene delivery with a strong focus on favorable properties of AAV. Thanks to its small size, AAV diffuses well in the inter-neural matrix making it suitable for applications in neural retina. Building on this initial clinical success with LCA II, we have now many opportunities to extend this proof-of-concept to other retinal diseases using AAV as a vector. This article will discuss what are some of the most imminent cellular targets for such therapies and the AAV toolkit that has been built to target these cells successfully. We will also discuss some of the challenges that we face in translating AAV-based gene therapies to the clinic.

Induced Pluripotent Stem Cells: Challenges and Opportunities

Regenerative capacity of mammals is limited and can rarely regenerate a specific organ or tissue fully. Due to these limitations, regenerative medicine seeks efficient and safe cell sources for regeneration of damaged tissues and organs or treatment for incurable diseases. Human embryonic stem cells (HESCs) hold two important properties called self renewal and pluripotency. However, the use of embryonic pluripotent stem cells in cell therapy faces two major obstacles. First, immunological incompatibility of ES cells with the recipient, and the second, ethical concerns about the destruction of human embryos during the ES cells. Thus, induction of somatic cells of individuals can be a proper way to overcome these problems. So far, several methods have been utilized to induce Pluripotency in Somatic cells. One of these methods is the technology of induced pluripotent stem cells (iPS) in contribution with Pluripotency factors. Yet, the use of these cells in the clinic, owing to application of viral vectors to transfer Pluripotency inducing factors, is quite limited. Therefore, recognition of a combination of small molecules to be replaced with exogenous factors is the ultimate goal of the study for the purpose of generating iPS cells. Recent progresses in development of iPS cells will be discussed here. Keywords: IPS, Plupotency, Repair, Regeneration, Stem Cell.

Gene therapy for inherited retinal degenerations

Gene therapy is quickly becoming a reality applicable in the clinic for inherited retinal diseases. Progress over the past decade has moved proof-of-concept gene therapies from bench to bedside. The remarkable success in safety and efficacy, in the phase I/II clinical trials for the form of the severe childhood-onset blindness, Leber's Congenital Amaurosis (LCA) type II (due to mutations in the RPE65 gene) generated significant interest and opened up possibilities for a new era of retinal gene therapies. Success in these clinical trials was due to combining the favorable features of both the retina as a target organ and adeno-associated virus (AAV) as a vector. The retina offers several advantages for gene therapy approaches. It is an anatomically defined structure that is readily accessible for therapy and has some degree of immune privilege, making it suitable for application of viral vectors. AAV, on the other hand, is a non-pathogenic helper dependent virus that has little immunogenicity. This viral vector transduces quiescent cells efficiently and thanks to its small size diffuses well in the interneural matrix, making it suitable for applications in neural tissue. Building on this initial clinical success with LCA II, we have now many opportunities to extend this proof-of-concept to other retinal diseases. This article will discuss what are some of the most imminent targets for such therapies and what are the challenges that we face in moving these therapies to the clinic.