Manual Viruses in Human Gene Therapy

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Transduction with MV-SCD showed replication and efficient lysis of human ovarian cancer cell lines and primary tumor cells. Moreover, precision-cut tumor slices from human ovarian cancer patients demonstrated efficient infection by MV-SCD. Furthermore, MV-SCD generated long-term virus replication in tumor tissue and induced apoptosis-like cell death independent of intact apoptosis pathways. Moreover, tumor reduction and significant survival benefits were observed in a HuCCT1 xenograft model [ 71 ]. Newcastle disease virus NDV vectors have been frequently used in preclinical cancer therapy studies due to their oncolytic activity [ 72 ].

However, the day survival rate was Likewise, the survival rate was The rescued virus showed tumor-selective replication and induced apoptosis in tumor cells in athymic mice with implanted lung tumors. It has also been demonstrated that expression of IL-2 and tumor necrosis factor-related apoptosis inducing ligand TRAIL enhanced inherent antineoplasticity by inducing apoptosis [ 74 ]. Coxsackieviruses have been used for several gene therapy applications [ 23 ]. For instance, the coxsackievirus B3 CVB3 expressing the human fibroblast growth factor 2 FGF2 was injected into ischemic hindlimbs of mice showing protection from ischemic necrosis [ 76 ].

The treatment improved the blood flow in ischemic limbs for more than 3 weeks. A single administration of CAV21 was sufficient to provide efficient oncolysis and the systemic spread of CAV21 showed efficient regression in tumors distantly located from the site of viral injection. A single intravenous injection generated significant regression of pre-established tumors and also targeting and elimination of metastases. Furthermore, intravenous injection of CVA21 expressing ICAM-1 and DAF in combination with intraperitoneal injection of doxorubicin hydrochloride provided significantly enhanced tumor regression in comparison to either virus or drug alone in mice with implanted MDA-MB tumors [ 79 ].

Systemic delivery induced regression of tumor xenografts and a therapeutic dose-response was obtained for escalating doses of EV1 in the LNCaP mouse model. Finally, poxviruses have found several applications as gene therapy vectors. For instance, vaccinia virus vectors have demonstrated potential for treatment of pancreatic cancer [ 81 ]. In this context, the PANVAC system comprising of recombinant vaccinia and fowlpox viruses, carrying the tumor-associated antigens epithelial MUC-1 and carcinomebryonic antigen CEA as well as T cell stimulatory molecules, have been applied [ 82 ]. In the case of HCC, the light-emitting recombinant GLV-2b vaccinia virus was injected into HCC xenografts in the flank of athymic nude mice for assessment of tumor growth and inhibition of viral biodistribution [ 83 ].

Combination therapy was superior to individual treatments both in xenograft and immunocompetent transgenic adenocarcinoma of the mouse prostate TRAMP mouse models, demonstrating restricted tumor growth and improved survival rates. A vaccinia virus was engineered by mutating the F4L gene, the viral homologue of the cell-cycle-regulated small subunit of ribonucleotide reductase 2 RRM2 , which provided tumor-selective replication and cell killing [ 85 ].

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It was confirmed that the F4L-mutated vector selectively replicated in immune-competent rat AY and xenografted human RTluc orthotopic bladder cancer models, resulting in substantial tumor regression or complete ablation without causing any cytotoxicity. Moreover, antitumor immunity was established in rats cured of AY tumors. Systemic administration of the modified CPXV vector showed accumulation in tumor cells and low infection and toxicity of normal cells.

Moreover, intratumoral administration in UMG glioblastoma and LoVo colon cancer models, induced relevant tumor growth inhibition. A substantial number of clinical trials have been conducted or are currently in progress applying viral vectors Table 3. For instance, the tumor-selective chimeric Enadenotucirev adenovirus vector was subjected to intravenous delivery in 17 patients with resectable colorectal cancer, non-small-cell lung cancer, urothelial cancer and renal cancer [ 87 ].

Tumor-specific delivery was observed in most tumor samples with no treatment-related serious adverse events.

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Related to hemophilia, gene therapy has been employed already for three decades, mainly focusing on AAV-based vectors [ 88 ]. In addition to discovery of pre-existing neutralizing antibodies in animal models, clinical trials have revealed that liver transaminase levels are elevated and immune-related loss of transgene expression.

The mechanism of the decrease in expression levels is not fully understood, but the use of different serotypes for consecutive administration of AAV has provided improved transgene expression [ 9 ], which has resulted in long-term expression of factors VIII FVIII and IX FIX and furthermore allows a cure of severe bleedings and joint damage associated with hemophilia. Furthermore, stem cell-based lentiviral vector delivery has proven successful in establishing sustained high level FIX expression after differentiation of adipogenic, chondrogenic, and osteoblastic cells [ 90 ], which potentially can be applied for treatment of hemophilia B.

Likewise, stem cell-based lentiviral gene therapy can provide life-long production of FVIII and the potential cure of hemophilia A [ 89 ]. The oncolytic HSV HF10 vector has been subjected to clinical trials in recurrent breast cancer, head and neck cancer, unresectable pancreatic cancer, refractory superficial cancer, and melanoma [ 42 ]. The studies demonstrated high safety and a low frequency of adverse effects in treated patients. Moreover, HF10 antigens were detected days after immunization in pancreatic cancer patients.

Related to retroviruses, a clinical trial in patients with recurrent high-grade glioma HGG is currently in progress with the Toca retrovirus [ 47 ]. Moreover, Toca was subjected to an open-label, ascending dose, multicenter phase I trial in patients with recurrent or progressive HGG [ 91 ]. The overall survival was Moreover, tumor samples from patients surviving more than a year demonstrated survival-related RNA expression in correlation with treatment-related survival.

In another approach, a gammaretroviral vector was employed for the treatment of chronic granulomatous disease CGD , which relates to primary immunodeficiency, resulting in an impaired antimicrobial activity in phagocytic cells [ 48 ]. In another cancer-related approach MV-NIS has been approved by the FDA for human clinical trials in myeloma patients, which could provide a potential strategy for targeting iodine-resistant ATC [ 66 ].

Oncolytic vaccinia viruses have also been subjected in a phase I clinical trial in 11 patients with refractory advanced colorectal or other solid cancers [ 93 ]. The study showed neither dose-related toxicity nor any treatment-related severe adverse events. However, a strong induction of inflammatory and Th1 cytokines indicated a potent mediation of potential immunity against cancer, which supports further trials with intravenously administered vaccinia virus in combination with expression of therapeutic genes, immune checkpoint blockade, or complement inhibitors.

However, a phase III trial targeting patients with metastatic pancreatic cancer failed to meet the therapeutic targets and was terminated [ 95 ]. In the context of HSV-based clinical trials, the oncolytic HSV M vector expressing IL has been subjected to a phase I dose-escalating study in patients with recurrent or progressive malignant glioma [ 97 ]. Moreover, the HSV strain G lacking genes essential for replication in normal cells were evaluated in patients with recurrent glioblastoma multiforme [ 98 ].

After two doses of HSV G totaling 1. Furthermore, the study demonstrated safe multiple dose delivery including direct injections into the brain. Furthermore, preclinical studies with HSV G have generated highly sensitive tumor killing, which support the initiation of the first-in-children study of intratumoral administration in children with recurrent or progressive supratentorial malignant tumors [ ].

Alphaviruses have so far been subjected to only a limited amount of clinical trials. In this context, recombinant VEE replicon particles expressing the prostate specific membrane antigen PSMA were administered to patients with castration resistant metastatic prostate cancer in a phase I dose-escalation study [ ]. Similar results occurred when immunizations were performed with 3. Despite the lack of clinical benefit and robust immune responses, immunizations elicited neutralizing antibodies, which encourages further dose optimization studies. In another approach, liposome-enveloped SFV vectors expressing IL were subjected to systemic administration in a phase I study in melanoma and kidney carcinoma patients [ ].

Intravenous injections provided a transient 5-fold increase of IL in the plasma. Due to the encapsulation procedure, tumor targeting and protection against recognition by the host immune system was obtained, which also allowed repeated vector administration. NDV has been used in a number of clinical trials [ ].

For instance, NDV expressing multiple tumor-associated antigens TAAs has been demonstrated to provide long-term survival in phase II trials in patients with ovarian, stomach, and pancreatic cancer [ ]. However, the study results suggested that there were no remarkable differences between the vaccinated individuals and those in the placebo group.

It was demonstrated that vaccination with NDV provided prolonged survival and short-term improved quality of life. Moreover, a self-activating lentiviral vector has been engineered to express a combination of the sh5 anti-HIV gene and the C46 antiviral fusion inhibitor peptide, which provided a synergistic effect on HIV-1 inhibition [ ]. The latter could be further enhanced by combination therapy with immune checkpoint blockade. In another phase II trial, CVA21 demonstrated induced immune cell infiltration in the tumor microenvironment of patients with melanoma [ ].

Neither dose-limiting toxicity nor grade 3 or higher treatment-related adverse events were observed. The first viral-based gene therapy drugs were approved some time ago in China [ ]. In this context, oncolytic adenoviruses expressing the p53 gene Gendicine TM [ ] and AdH containing the E1bK deletion [ ] are used for treatment of cancers with mutated p53 and head and neck cancer, respectively. Gendicine TM has been used for 12 years in more than 30, patients with an exemplary safety record and has provided significantly better responses compared to standard therapies when combined with chemotherapy and radiotherapy [ ].

Moreover, the progression-free survival times were significantly extended. Unfortunately, although the AAV-based Glybera drug was approved for treatment of the rare inherited disorder lipoprotein lipase deficiency, the high costs and limited demand forced the withdrawal from the market [ ]. Additionally, several other viral-based drugs will most likely be on the market in the near future. In summary, the field of gene therapy has seen some significant progress with nearly clinical trials conducted by [ ].

Not surprisingly, Furthermore, Although recent developments in gene manipulation methods, such as CRISPR, and more efficient delivery methods for nonviral vectors, viral vectors still remain attractive. In attempts to further improve the applicability of viral vectors, a number of modifications have been introduced. For instance, the issue of insertional oncogenesis of retroviruses leading to activation of LMO2 or other oncogenes has been a major concern in treatment of SCID [ 2 , 3 ].

For this reason, a new assay has been developed for assessing vector safety related to insertions into the LMO2 locus and other T-cell proto-oncogenes [ ]. It was revealed that gamma-retrovirus vectors with full viral long-terminal repeats were most prone to LMO2 pathway activation. On the other hand, lentiviral vectors showed a significantly lower tendency of proto-oncogene activation. The third generation lentiviruses have also contributed to improved delivery and safety [ ].

For instance, deletion of the viral tat gene makes the vector replication-incompetent [ ]. Furthermore, lentiviral vector packaging functions have been placed on three plasmids instead of two to reduce the risk of recombination. Additionally, VSV-G pseudotyped lentiviral vectors allow transduction of a much wider range of cell type and the enhanced vector stability, which facilitates high titer vector concentration by ultracentrifugation. Another issue relates to host genetic variations affecting transgene expression from lentivirus vectors, which was studied in 12 collaborative cross mouse strains [ ].

Total body and hepatic luciferase expression was monitored in female mice after administration of a lentivirus vector with a liver-specific promoter. The study revealed major strain-specific transduction, vector biodistribution, and maximum luciferase expression and kinetics, highlighting the importance of genetic variation and the need for redesigning preclinical studies.

The future of viral-based gene therapy, even taking a cautious approach, sounds bright. The approval of several drugs for the treatment of different conditions by applying various viral vectors provides substantial flexibility and alternative strategy options. The progress made in vector engineering and safety development has also put viral vectors in a favorable position. However, both preclinical and clinical studies have confirmed that there is no single universal viral vector suitable for the treatment of all indications.

As personalized medicines have become an essential part of modern drug development, viral vectors should also find potential opportunities in this area. In this context, improved adenovirus vectors could be engineered for personalized drug delivery [ ]. In another approach, a library of tumor antigen-specific T-cell receptor TCR genes from frozen tumor biopsies were introduced into a retroviral vector, which allowed rapid generation of therapeutic personalized antitumor T-cell products [ ]. It is therefore not useful to try to make suggestions of which viral vector system to use, but rather encourage parallel development of several systems and then make appropriate decisions based on publications and previous experience.

Anyway, viral-based gene therapy has developed substantially and is on the way to becoming a key treatment in modern medicine. National Center for Biotechnology Information , U. Journal List Diseases v. Published online May Kenneth Lundstrom. Author information Article notes Copyright and License information Disclaimer.

Received Apr 30; Accepted May This article has been cited by other articles in PMC. Abstract Applications of viral vectors have found an encouraging new beginning in gene therapy in recent years. Keywords: prevention, therapy, immunotherapy, gene silencing, clinical trials, approved drugs. Introduction After genuine excitement in the s of the potential of gene therapy providing more or less unlimited opportunities to cure the majority of human diseases, a severe setback was encountered when adenovirus vectors were employed for the treatment of the non-life threatening disease ornithine transcarbomylase resulting in the death of a young patient [ 1 ].

Viral Vectors The spectrum of viral vectors is very broad including both delivery vehicles developed for transient short-term and permanent long-term expression. Table 1 Examples of viral vectors applied for gene therapy. Open in a separate window. Table 2 Examples of gene therapy applications in animal models. Table 3 Examples of clinical trials using viral vectors. Types of Vectors The most applied viral vectors are certainly based on adenoviruses [ 4 ]. Preclinical Studies 2. Adenoviruses Due to the many gene therapy applications of a number of viral vectors evaluated in preclinical animal models, only some examples can be presented here Table 2.

Herpes Simplex Viruses Due to the long-term effect, HSV vectors have found many applications in various disease areas. Retroviruses Retroviruses present the classic approach for long-term gene therapy applications and the first human gene therapy trial involved implantation of autologous bone marrow cells transduced ex vivo with gamma retrovirus vectors [ 44 ].

Alphaviruses Alphaviruses have been mainly applied in preclinical gene therapy studies for cancer treatment [ 57 ]. Flaviviruses In the context of flaviviruses, the granulocyte macrophage colony-stimulating factor GM-CSF expressed from a Kunjin virus vector was subjected to intratumoral administration in mice with subcutaneous CT26 colon carcinoma [ 62 ]. Rhabdoviruses Among rhabdoviruses, recombinant vesicular stomatitis virus VSV has been applied for preclinical gene therapy studies. Measles Viruses Measles viruses have found a number of gene therapy applications, which have been evaluated in preclinical animal models.

Newcastle Disease Viruses Newcastle disease virus NDV vectors have been frequently used in preclinical cancer therapy studies due to their oncolytic activity [ 72 ]. Coxsackieviruses Coxsackieviruses have been used for several gene therapy applications [ 23 ]. Poxviruses Finally, poxviruses have found several applications as gene therapy vectors. Clinical Trials A substantial number of clinical trials have been conducted or are currently in progress applying viral vectors Table 3.

Approved Drugs The first viral-based gene therapy drugs were approved some time ago in China [ ]. Conclusions In summary, the field of gene therapy has seen some significant progress with nearly clinical trials conducted by [ ]. Conflicts of Interest The authors declare no conflict of interest. Funding This research received no external funding. References 1. Raper S. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer.

McCormack M. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. Hacein-Bey-Abina S. Schiedner G. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Wang F.

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Biodistribution and safety assessment of bladder cancer specific oncolytic adenovirus in subcutaneous xenografts tumor model in nude mice. Gene Ther. Wei Q.

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Engineering the rapid adenovirus production and amplification RAPA cell line to expedite the generation of recombinant adenoviruses. Samulski R. AAV-mediated gene therapy for research and therapeutic purposes. Park K. Cancer gene therapy using adeno-associated virus vectors. Mingozzi F. Immune responses to AAV vectors: Overcoming barriers to successful gene therapy. Grieger C. Packaging capacity of adeno-associated virus serotypes: Impact of larger genomes on infectivity and postentry steps. McClements M. Adeno-associated virus AAV dual vector strategies for gene therapy encoding large transgenes.

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Schambach A. Retroviral vectors for cancer gene therapy. Recent Results Cancer Res. Design of retroviral vectors and helper cells for gene therapy. Vigna E. Lentiviral vectors: Excellent tools for experimental gene transfer and promising candidates for gene therapy. Kay M.

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1. Introduction

Oncolytic viruses: Adenoviruses. Virus Genes. Nagasato M. A tumor-targeting adenovirus with high gene-transduction efficiency for primary pancreatic cancer and ascites cells. Anticancer Res. Yamamoto Y. Strong antitumor efficacy of a pancreatic tumor-targeting oncolytic adenovirus for neuroendocrine tumors. Cancer Med. Emdad L. Ehrke-Schulz E. Recent advances in preclinical developments using adenovirus hybrid vectors. Panek W. Hitting the nail on the head: Combining oncolytic adenovirus-mediated virotherapy and immodulation for the treatment of glioma.

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Yue Y. Prospect of gene therapy for cardiomyopathy in hereditary muscular dystrophy. Expert Opin. Orphan Drugs. Kodippili K. Greig J. Thankur V. Viral vector mediated continuous expression of interleukin in DRG alleviates pain in type 1 diabetic animals. Chattopadhyay M. Targeted delivery of growth factors by HSV-mediated gene transfer for peripheral neuropathy. Eissa I. Genomic signature of the natural oncolytic herpes simplex virus HF10 and its therapeutic role in preclinical and clinical trials.

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Risk Factors and Biosafety Issues of Gene Therapy Viral Vectors

Singer O. Piedrahita D. Ringpis G. We have previously addressed these scaling-up limitations in Cell Gene Therapy Insights [ 4 ]. With viral vectors dominating cell and gene therapy, when should one turn to a non-viral delivery method? Which methods can be used, and how does one choose a method while keeping the end-game in mind: commercialization of an efficient and affordable drug? Non-viral vectors sidestep the main concerns that come with using viruses: safety, immunogenicity and manufacturing limits yield, scaling-up and costs.

Several non-viral methods have been developed and are worth mentioning: naked nucleic acid, physical-mediated methods gene gun, electroporation, hydrodynamic and chemical-based nanoparticles. While these techniques are all used in gene delivery studies performed in small animal models e. Naked nucleic acid delivery without any carrier is by far the simplest approach for gene delivery. The range of action of naked nucleic acid is often limited due to low dissemination and low cellular internalization, attributed respectively to a lack of protection from endonuclease degradation and its uncondensed shape and polyanionic charge.

Typically, the order of magnitude of the half-life of plasmid DNA is estimated to 10 minutes following systemic injection in mice [ 5 ]. To overcome these challenges, chemical-based nanoparticles have been developed to interact with nucleic acids in order to protect them from degradation and condense them into nanosized complexes that can be more easily internalized by cells. There are two main types of nanoparticles that are under clinical evaluation: lipid and cationic polymer-based. Lipid-based nanoparticles LNPs are synthesized taking into account the chemical properties of lipids and identification of conjugates to increase delivery specificity to a cell type.

While LNPs are the most clinically advanced non-viral delivery system e. Conversely, synthetic cationic polymers, and especially polyethyleimine PEI bring flexibility with the delivery of different nucleic acids and compatibility with the use of both systemic and local injection routes. It is a cationic polymer composed of PEI that efficiently encapsulates and protects negatively charged nucleic acids by forming nanopolyplexes with an overall net cationic charge. These positively charged complexes enhance interaction with negatively charged extracellular membrane and favor intracellular uptake.

As pointed out by Rouanet et al. This versatility is essential when the aim is to target organs other than the liver, which is usually where accumulation is observed with LNPs. Figure 1. As a positive control, LPS was injected intraperitoneally. Although non-viral vectors represent a small percentage of ongoing human gene therapy clinical trials, PEI-based non-viral delivery is increasingly used in treatments of pathologies such as cancer e.

Figure 2. As a positive control, CCl4 was subcutaneously administered. In cancer treatment, it was shown to be efficient in selectively delivering suicide genes to actively dividing cancer cells, and in regulating gene expression of tumor suppressor gene and oncogenes: a perfect example of turning a weakness into a strength.

Anchiano Therapeutics has been working on such an approach to treat Human bladder cancer using PEI-based non-viral delivery of a recombinant plasmid DNA construct BC that encodes for lethal diphteria toxin specifically in H19 overexpressing cancer cells [ 12 , 13 ]. Promisingly, at the early development phase, there is an increase in the number of studies in which PEI-based delivery is chosen for nucleic-acid based therapies, as well as in treatment of medical conditions such as brain injury [ 15 ] or sepsis [ 16 ].

Figure 3. Type of nucleic acid delivered, administration route and therapeutic application are very diverse. As transfection experts, we are convinced that there is sufficient space for both viral and non-viral vectors to cover the unaddressed needs of drug-based treatments with gene therapy. This is why we are continuously at the forefront with the development of transfection reagents to overcome inherent limitations of viral vector and non-viral vector systems.

To conclude, we would like to recommend investigators to carefully take into account advantages and disadvantages of each method, and to keep in mind that viral vectors unleash their full potential for treatment of rare diseases, while non-viral delivery vectors are optimal for treatment of common diseases such as cancer, infectious diseases, and other chronic diseases. The authors are all employees of Polyplus Transfection.

No writing assistance was utilized in the production of this manuscript. Non-viral vectors for gene-based therapy. Hidai C, Kitano H. Nonviral gene therapy for cancer: Review. Diseases ; 6 3. Genes ; 8 2 : E