Viral vectors are modified viruses designed to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture. They have widespread applications for use in basic research, therapies, and vaccines.
Viruses have evolved specialized molecular mechanisms to efficiently target and transport their genomes into infected cells, a process termed transduction. Viral vectors may integrate the genetic material—the transgene—into the host genome, although non-integrative vectors have also been designed. Viral vectors are widely used in gene therapy due to their specific tropism and efficient transgene expression: as of 2022, all approved gene therapies were viral vector-based. Compared to traditional vaccines, the intracellular antigen expression enabled by viral vector vaccines offers robust activation of both the innate and adaptive immune system.
Molecular biologists first harnessed this machinery in the 1970s. In 1972, Paul Berg demonstarted the first viral vector, a modified SV40 virus. However, recombinant DNA research was temporarily suspended following the Asilomar Conference. Following the creation of formal policies by the National Institutes of Health, work proceeded. The 1980s saw both the first recombinant viral vector gene therapy and the first viral vector vaccine. Although the 1990s saw significant advances in viral vector design and genetics, viral vector clinical trials had a number of setbacks, culminating in the death of patient Jesse Gelsinger.
However, the 2000s saw a clinical resurgence of viral vector vaccines, partly due to the development of ex vivo viral vector gene therapy. Viral vectors have been globally approved for the treatment of various diseases. During the COVID-19 pandemic, billions of people received viral vector vaccines.
This ability to transfer foreign genetic material has been exploited by genetic engineers to create viral vectors, which can transduce the desired transgene into a target cell.[2] Viral vectors consists of three components:[5][6]
A protein capsid and sometimes an envelope that encapsidates the genetic payload. This determines the range of cell types that the vector infects, termed its tropism.
A genetic payload: the transgene that results in the desired effect when expressed.
Mice transduced by a lentiviral vector fluoresce under UV-illumination
Basic research
Viral vectors are routinely used in a basic research setting, and can introduce genes encoding, for instance, complementary DNA, short hairpin RNA, and CRISPR/Cas9 for gene editing.[8] Viral vectors can be used for cellular reprogramming, for instance, inducing pluripotent stem cells or differentiating adult somatic cells into different cell types.[9] They are also frequently used to create transgenic mice and rats for experiments.[10] Viral vectors can be used for in vivo imaging via the introduction of a reporter gene. Further, transduction of stem cells can allow for the tracking of cell lineage during development.[9]
Viral vector transduction of a cell for gene therapy
Gene therapy is a therapeutic effect that seeks to modulate or otherwise affect gene expression via the introduction of a therapeutic transgene. Gene therapy by viral vectors can be performed by in vivo delivery by directly administering the vector to the patient, or ex vivo by extracting cell from the patient, transducing them, and then reintroducing the modified cells into the patient.[11]
There are four broad categories of gene therapy: gene replacement, gene silencing, gene addition, or gene editing.[11][12] Relative to other non-integrative gene therapy approaches, transgenes introduced by viral vectors offer multi-year long expression.[13]
Shipments of the Russian-made Sputnik V COVID-19 vaccine, an adenoviral vector, are seen in Guatemala in 2021.
For use as vaccine platforms, viral vectors can be engineered to carry a specific antigen associated with an infectious disease or a tumor antigen.[14][15] Conventional vaccines are not suitable for protection against some pathogens due to unique immune evasion strategies and differences in pathogenesis.[16] Viral vector-based vaccines, for instance, could eventually offer immunity against HIV-1 and malaria.[17]
While traditional subunit vaccines elicit a humoral response,[18] viral vectors allow for intracellular antigen expression that activates MHC pathways via both direct and crosspresentation pathways. This induces a robust adaptive immune response.[19][20] Viral vector vaccines also have intrinsic adjuvant properties via innate immune system activation and the expression of pathogen-associated molecular patterns (PAMPs), negating the need for any additional adjuvant.[21][14] In addition to a more robust immune response in comparison to other vaccine types, viral vectors offer efficient gene transduction and can target specific cell types.[18] Pre-existing immunity to the virus used as the vector, however, can be a significant issue.[17]
Prior to 2020, viral vectors were widely administered in but confined to veterinary medicine.[21] In the global response to the COVID-19 pandemic, viral vector vaccines played a fundamental role and were administered to billions of people, particularly in low and middle-income nations.[22]
Retroviruses—enveloped RNA viruses—are popular viral vector platforms due to their ability to integrate genetic material into the host genome.[2] Retroviral vectors comprise two general classes: gamma retroviral and lentiviral vectors. The fundamental difference between the two are that gamma retroviral vectors can only infect dividing cells, while lentiviral vectors can infect both dividing and resting cells.[23] Notably, retroviral genomes are composed of single-stranded RNA and must be converted to proviral double-stranded DNA, a process known as reverse transcription—before it is integrated into the host genome via viral proteins like integrase.[24]
The most commonly used gammaretroviral vector is a modified Moloney murine leukemia virus (MMLV), able to transduce various mammalian cell types. MMLV vectors have been associated with some cases of carcinogenesis.[25] Gammaretroviral vectors have been successfully applied to ex vivo hematopoietic stem cell to treat multiple genetic diseases.[26]
Lentiviral vectors are able to carry up to 10 kb of foreign genetic material, although 3-4 kb was reported as optimal as of 2023.[23][27] Relative to other viral vectors, lentiviral vectors possess the greatest transduction capacity, due to the formation of a three-stranded "DNA flap" during retro-transcription of the single-strand lentiviral RNA to DNA within the host.[27]
Although largely largely non-inflammatory,[28] lentiviral vectors can induce robust adaptive immune responses by memory-type cytotoxic T cells and T helper cells.[29] This is largely due to lentiviral vectors' high tropism for dendritic cells, which activate T cells.[29] However, they can infect all types of antigen-presenting cells.[30] Moreover, as they are the only retroviral vectors able to efficiently transduce both dividing and non-dividing cells, make them the most promising vaccine platforms.[30] They have also been trialed as vaccines against cancer.[31]
Adenoviruses (visualized via electron micrograph at left and right and depicted graphically at center) are commonly used as viral vector platforms. Note the icosahedron capsid structure.
Adenoviruses are double-stranded DNA viruses belonging to the family Adenoviridae.[33][34] Their relatively large genomes, of approximately 30-45 kb, make them ideal candidates for genetic delivery;[33] newer adenoviral vectors can carry up to 37 kb of foreign genetic material.[35] Adenoviral vectors display high transduction efficiency and transgene expression, and can infect both dividing and non-dividing cells.[36]
The adenoviral capsid, an icosahedron, features a fibre "knob" at each of its 12 vertices. These fibre proteins mediate cell entry—greatly affecting efficacy and contribute to its broad tropism—notably via coxsackie–adenovirus receptors (CARs).[33][36] Adenoviral vectors can induce robust innate and adaptive immune responses.[37] Its strong immunogenicity is particularly due to the transduction of dendritic cells (DC), upregulating the expression of both MHC I and II molecules and activating the DCs.[38] They have a strong adjuvant effect, as they display several pathogen-associated molecular patterns.[37] One disadvantage is that pre-existing immunity to adenovirus serotypes is common, reducing efficacy.[36][39] The use of chimpanzee adenoviruses may circumvent this issue.[40]
While the activation of both innate and adaptive immune responses is an obstacle for many therapeutic applications, it makes adenenoviral vectors an ideal vaccine platform.[34] The global response to the COVID-19 pandemic saw the development and use of multiple adenoviral vector vaccines, including Sputnik V, the Oxford–AstraZeneca vaccine and the Janssen vaccine.[41]
Lentivirus (upper panel) – To produce lentiviruses with the gene of interest as the lentiviral DNA construct, first transfect cells with a packaging plasmid and the envelope vector (VSVG). Adeno Associated Virus (AAV) (lower panel) – To produce AAV, package a gene of interest into the AAV-ITR vector and transfect cells with a Helper vector and the Rep/Cap DNA integration vector.
Adeno-associated viruses (AAVs) are relatively small single-stranded DNA viruses belonging to Parvoviridae and, like lentiviral vectors, AAVs can infect both dividing and non-dividing cells.[42] AAVs, however, require the presence of a "helper virus" such as an adenovirus or herpes simplex virus to replicate within the host, although it can do so independently if cellular stress is induced or the helper virus genes are carried by the vector.[43]
AAVs insert themselves into a specific site in the host genome, particularly AAVS1 on chromosome 19 in humans. However, recombinant AAVs have been designed that do not integrate. These are instead stored as episomes that, in non-dividing cells, can last for years.[44] One disadvantage is that they are not able to carry large amounts of foreign genetic materials. Furthermore, the need to express the complementary strand for its single-stranded genome may delay transgene expression.[44]
As of 2020, 11 different AAV serotypes—differing by capsid structure and consequently by tropism—had been identified.[42] The tropism of adeno-associated viral vectors can be tailored by creating recombinant versions from multiple serotypes, termed pseudotyping.[42] Due to their ability to infect and induce longlasting effects within nondividing cells, AAVs are commonly used in basic neuroscience research.[45] Following the approval of the AAV Alipogene tiparvovec in Europe in 2012,[46] in 2017, the FDA approved the first AAV-based in vivo gene therapy—voretigene neparvovec—which treated RPE65-associated Leber congenital amaurosis.[32] As of 2020, 230 clinical trials using AAV-based treatments were either underway or had been completed.[46]
Vaccinia virus, a poxvirus, is another promising candidate for viral vector development.[47] Its use as the smallpox vaccine—first reported by Edward Jenner in 1798—led to the eradication of smallpox and demonstrated vaccinia as safe and effective in humans.[48][47][note 1] Moreover, manufacturing procedures developed to mass-produce smallpox vaccine stockpiles may expedite vaccinia viral vector production.[50]
Vaccinia possesses a large DNA genome and can consequently carry up to 40 kb of foreign DNA.[48][51][52][51] Further, vaccinia are unlikely to integrate into the host genome, decreasing the chance of carcinogenesis.[51] Attenuated strains—replicating and non-replicating—have been developed.[48] Although widely characterized due to its use against smallpox, as of 2019 the function of 50 percent of the vaccinia genome was unknown. This may lead to unpredictable effects.[52]
As a vaccine platform, vaccinia vectors display highly effective transgene expression and create a robust immune response.[50] The virus fast-acting: its life cycle produces mature progeny vaccinia within 6 hours, and has three viral spread mechanisms.[52] Vaccinia also has an adjuvant effect, activating a strong innate response via toll-like receptors.[50] A significant disadvantage that can reduce its efficacy, however, is pre-existing immunity against vaccinia in those who received the smallpox vaccine.[50]
Of the nine herpesviruses that infect humans, herpes simplex virus 1 (HSV-1) is the most well characterized and most commonly used as a viral vector.[53] HSV-1 offers several advantages: it had broad tropism and can deliver therapeutics via specialized expression systems.[54] Moreover, HSV-1 can cross the blood brain barrier if medically-disrupted, enabling it to target neurological diseases. Also, HSV-1 does not integrate into the host genome and can carry large amounts of foreign DNA. The former feature prevents harmful mutagenesis, as can occur with retroviral and adeno-associated vectors. Replication-deficient strains have been developed.[55]
Cytomegalovirus (CMV), a herpesvirus, has also been developed for use as a viral vector.[58] CMV can infect most cell types and can thus proliferate throughout the body. Although a CMV-based vaccine provided significant immunity against SIV—closely related to HIV—in macaques, development of CMV as a reliable vector was reported to still be in early stages as of 2020.[59][60]
Plant viruses are also engineered viral vectors for use in agriculture, horticulture, and biologic production.[61] These vectors have been employed for a range of applications, from increasing the aesthetic quality of ornamental plants to pest biocontrol, rapid expression of recombinant proteins and peptides, and to accelerate crop breeding.[62]
Replicating virus-based vectors are typically used.[63] RNA viruses used for monocots include wheat streak mosaic virus (WSMV) and barley stripe mosaic virus (BSMV) and, for dicots, tobacco rattle virus (TRV). Single-stranded DNA viruses like geminiviruses have also been utilized.[63] Viral vectors can be administered to plants via several pathways termed "agro-inoculation", including via rubbing, a biolistic delivery system, agrospray, agroinjection, and even via insect vectors.[64][62] However, Agrobacterium-mediated delivery of viral vectors—in which bacteria are transformed with plasmid DNA encoding the viral vector construct—is the most common approach.[65]
Building on the market approvals and sales of recombinant virus-based biopharmaceuticals for veterinary and human medicine, the use of engineered plant viruses has been proposed to enhance crop performance and promote sustainable production.[66]
In 1972, Stanford University biochemist Paul Berg developed the first viral vector, incorporating DNA from the lambda phage into the polyomavirus SV40 to infect monkey kidney cells maintained in culture.[67][68][69] The implications of this research troubled scientists like Robert Pollack, who convinced Berg not to insert DNA from SV40 into E. coli via a bacteriophage vector. They feared that introducing the purportedly cancer-causing genes of SV40 may have created carcinogenic bacterial strains.[70][71] These concerns and others in the emerging field of recombinant DNA led to the Asilomar Conference of 1975, where attendees agreed to a voluntary moratorium on cloning DNA.[72]
In 1977, the National Institutes of Health (NIH) issued formal guidelines confining viral DNA cloning to rigid BSL-4 conditions, practically preventing such research. However, the NIH loosened these rules in 1979, permitting Bernard Moss to develop a viral vector utilizing vaccinia.[72] In 1982, Moss reported the first use of a viral vector for transient gene expression.[17] The following year, Moss used the vaccinia vector to express a hepatitis B antigen, creating the first viral vector vaccine.[21]
Although a failed gene therapy attempt utilizing wild-type Shope papilloma virus had been made as early as 1972, Martin Cline attempted the first gene therapy utilizing recombinant DNA in 1980. It proved unsuccessful.[74][11] In the 1990s, as genetic diseases were further characterized and viral vector technology improved, there was overoptimism about the capabilities the technology. Many clinical trials proved failures.[75] There were minors successes, such as the first effective gene therapy for severe combined immunodeficiency (SCID) in patient Ashanthi DeSilva. It employed a recombinant retrovirus.[11]
However, during a 1999 clinical trial at the University of Pennsylvania, Jesse Gelsinger died from a fatal reaction to an adenoviral vector-based gene therapy.[73][75] It was the first death related to any form of gene therapy.[76] Consequently, the FDA suspended all gene therapy trials at the University of Pennsylvania and investigated 60 others across the US.[76] An anonymous editorial in Nature Medicine noted that it represented a "loss of innocence" for viral vectors.[75] Shortly therafter, the field's reputation was further damaged when 5 children treated with a SCID gene therapy developed leukemia due to an issue with the retroviral vector.[75][note 2]
Viral vectors experienced a resurgence when they were employed for ex vivo hematopoietic gene delivery.[77] In 2003, China approved the first gene therapy for clinical use: Gendicine, an adenoviral vector encoding p53.[78][79] In 2012, the European Union issued its first approval of a gene therapy, an adeno-associated viral vector.[80] During the COVID-19 pandemic, viral vector vaccines were used to an unprecedented extent.[81] As of 2022, all approved gene therapies were viral vector-based and over 1000 viral vector clinical trials targeting cancer were underway.[77]
In popular culture
The film I Am Legend (set pictured) depicts a viral vector-created apocalypse.
↑ Edward Jenner's use of material from cow blisters containing vaccinia was the basis for vaccinology and ultimately led to the eradication of smallpox from Earth.[49]
↑ Ultimately, one child died. According to Cormac Sheridan, the backlash was unfair as the overall mortality rate for the viral vector therapy was lower than equivalent approaches.[75]
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Carmen I (1985). Cloning and the Constitution: An Inquiry into Governmental Policymaking and Genetic Experimentation. University of Wisconsin Press. ISBN9780299103408.
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Gene therapy is a medical technology that aims to produce a therapeutic effect through the manipulation of gene expression or through altering the biological properties of living cells.
A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.
Vaccinia virus is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, which encodes approximately 250 genes. The dimensions of the virion are roughly 360 × 270 × 250 nm, with a mass of approximately 5–10 fg. The vaccinia virus is the source of the modern smallpox vaccine, which the World Health Organization (WHO) used to eradicate smallpox in a global vaccination campaign in 1958–1977. Although smallpox no longer exists in the wild, vaccinia virus is still studied widely by scientists as a tool for gene therapy and genetic engineering.
Adenoviruses are medium-sized, nonenveloped viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from their initial isolation from human adenoids in 1953.
Virotherapy is a treatment using biotechnology to convert viruses into therapeutic agents by reprogramming viruses to treat diseases. There are three main branches of virotherapy: anti-cancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy. These branches use three different types of treatment methods: gene overexpression, gene knockout, and suicide gene delivery. Gene overexpression adds genetic sequences that compensate for low to zero levels of needed gene expression. Gene knockout uses RNA methods to silence or reduce expression of disease-causing genes. Suicide gene delivery introduces genetic sequences that induce an apoptotic response in cells, usually to kill cancerous growths. In a slightly different context, virotherapy can also refer more broadly to the use of viruses to treat certain medical conditions by killing pathogens.
Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. They are small replication-defective, nonenveloped viruses and have linear single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb).
Gammaretrovirus is a genus in the Retroviridae family. Example species are the murine leukemia virus and the feline leukemia virus. They cause various sarcomas, leukemias and immune deficiencies in mammals, reptiles and birds.
Modified vaccinia Ankara (MVA) is an attenuated (weakened) strain of the vaccinia virus. It is being used as a vaccine against smallpox and mpox, having fewer side effects than smallpox vaccines derived from other poxviruses.
Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.
Dependoparvovirus is a genus in the subfamily Parvovirinae of the virus family Parvoviridae; they are Group II viruses according to the Baltimore classification. Some dependoparvoviruses are also known as adeno-associated viruses because they cannot replicate productively in their host cell without the cell being coinfected by a helper virus such as an adenovirus, a herpesvirus, or a vaccinia virus.
Gene therapy using lentiviral vectors was being explored in early stage trials as of 2009.
A genetically modified virus is a virus that has been altered or generated using biotechnology methods, and remains capable of infection. Genetic modification involves the directed insertion, deletion, artificial synthesis or change of nucleotide bases in viral genomes. Genetically modified viruses are mostly generated by the insertion of foreign genes intro viral genomes for the purposes of biomedical, agricultural, bio-control, or technological objectives. The terms genetically modified virus and genetically engineered virus are used synonymously.
Retinal gene therapy holds a promise in treating different forms of non-inherited and inherited blindness.
Self-complementary adeno-associated virus (scAAV) is a viral vector engineered from the naturally occurring adeno-associated virus (AAV) to be used as a tool for gene therapy. Use of recombinant AAV (rAAV) has been successful in clinical trials addressing a variety of diseases. This lab-made progeny of rAAV is termed "self-complementary" because the coding region has been designed to form an intra-molecular double-stranded DNA template. A rate-limiting step for the standard AAV genome involves the second-strand synthesis since the typical AAV genome is a single-stranded DNA template. However, this is not the case for scAAV genomes. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. The caveat of this construct is that instead of the full coding capacity found in rAAV (4.7–6kb) scAAV can only hold about half of that amount (≈2.4kb).
Adenovirus varieties have been explored extensively as a viral vector for gene therapy and also as an oncolytic virus.
Lentiviral vectors in gene therapy is a method by which genes can be inserted, modified, or deleted in organisms using lentiviruses.
DNA-directed RNA interference (ddRNAi) is a gene-silencing technique that utilizes DNA constructs to activate an animal cell's endogenous RNA interference (RNAi) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs, that bring about the silencing of a target gene or genes once processed. Any RNA, including endogenous messenger RNA (mRNAs) or viral RNAs, can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target.
Adeno-associated virus (AAV) has been researched as a viral vector in gene therapy for cancer treatment as an oncolytic virus. Currently there are not any FDA approved AAV cancer treatments, as the first FDA approved AAV treatment was approved December 2017. However, there are many Oncolytic AAV applications that are in development and have been researched.
A viral vector vaccine is a vaccine that uses a viral vector to deliver genetic material (DNA) that can be transcribed by the recipient's host cells as mRNA coding for a desired protein, or antigen, to elicit an immune response. As of April 2021, six viral vector vaccines, four COVID-19 vaccines and two Ebola vaccines, have been authorized for use in humans.
Transgene S.A. is a French biotechnology company founded in 1979. It is based in Illkirch-Graffenstaden, near Strasbourg, and develops and manufactures immunotherapies for the treatment of cancer.
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