Viral vector

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Viral vectors
)
Flowchart showing a viral vector delivering genes into a neuron

Viral vectors are tools commonly used by molecular

Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage λ to infect monkey kidney cell maintained in culture.[1]

In addition to their use in molecular biology research, viral vectors are used for gene therapy and the development of vaccines. Vectors can either integrate into a cell's genome or transiently express a gene with non-integrative vectors.[2]: 50 

Key properties of a viral vector

Viral Vectors are tailored to their specific applications but generally share a few key properties.[citation needed]

  • Safety: Although viral vectors are occasionally created from
    virions
    .
  • Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects.
  • Stability: Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design.
  • Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral vector can be modified to target the virus to a specific kind of cell. Viruses modified in this manner are said to be pseudotyped.
  • Identification: Viral vectors are often given certain genes that help identify which cells took up the viral genes. These genes are called markers. A common marker is resistance to a certain antibiotic. The cells can then be isolated easily, as those that have not taken up the viral vector genes do not have antibiotic resistance, and so cannot grow in a culture with the relevant antibiotic present.

Applications

Basic research

Viral vectors were originally developed as an alternative to

integrate into the cell genome facilitating stable expression.[citation needed
]

Protein coding genes can be expressed using viral vectors, commonly to study the function of the particular protein. Viral vectors, especially retroviruses, stably expressing marker genes such as GFP are widely used to permanently label cells to track them and their progeny, for example in xenotransplantation experiments, when cells infected in vitro are implanted into a host animal.[citation needed]

Gene insertion, which can be done with viral vectors, is cheaper to carry out than gene knockout. But as gene silencing, an effect that may be intended with gene insertion, is sometimes non-specific and has off-target effects on other genes, it hence provides less reliable results. Animal host vectors also play an important role[clarification needed
].

Gene therapy

Main article: Gene therapy

Gene therapy is a technique for correcting defective genes responsible for disease development. In the future, gene therapy may provide a way to cure genetic disorders, such as severe combined immunodeficiency, cystic fibrosis or even haemophilia A. Because these diseases result from mutations in the DNA sequence for specific genes, gene therapy trials have used viruses to deliver unmutated copies of these genes to the cells of the patient's body. There have been a huge number of laboratory successes with gene therapy. However, several problems of viral gene therapy must be overcome before it gains widespread use. Immune response to viruses not only impedes the delivery of genes to target cells but can cause severe complications for the patient. In one of the early gene therapy trials in 1999 this led to the death of Jesse Gelsinger, who was treated using an adenoviral vector.[3]

Some viral vectors, for instance gamma-retroviruses, insert their genomes at a seemingly random location on one of the host chromosomes, which can disturb the function of cellular genes and lead to cancer. In a severe combined immunodeficiency retroviral gene therapy trial conducted in 2002, four of the patients developed leukemia as a consequence of the treatment;[4] three of the patients recovered after chemotherapy.[5] Adeno-associated virus-based vectors are much safer in this respect as they always integrate at the same site in the human genome, with applications in various disorders, such as Alzheimer's disease.[6]

Vaccines

Main article: Viral vector vaccine

A live vector vaccine is a

pathogenic organism. They are then inserted into the genome of a non-pathogenic organism, where they are expressed on the organism's surface and can elicit an immune response.[clarification needed
]

Unlike attenuated vaccines, viral vector vaccines lack other pathogen genes required for replication, so infection by the pathogen is impossible. Adenoviruses are being actively developed as vaccine vectors.

Medicine delivery

A strain of

interleukin-2 is used to treat cats with fibrosarcoma.[8]

Types

Retroviruses

Main article:
Retroviruses

SCID-X1 trial.[9]

Retroviral vectors can either be replication-competent or replication-defective. Replication-defective vectors are the most common choice in studies because the viruses have had the coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted. These virus are capable of infecting their target cells and delivering their viral payload, but then fail to continue the typical

lytic pathway
that leads to cell lysis and death.

Conversely, replication-competent viral vectors contain all necessary genes for virion synthesis, and continue to propagate themselves once infection occurs. Because the viral genome for these vectors is much lengthier, the length of the actual inserted gene of interest is limited compared to the possible length of the insert for replication-defective vectors. Depending on the viral vector, the typical maximum length of an allowable DNA insert in a replication-defective viral vector is usually about 8–10 kB.[verification needed][10] While this limits the introduction of many genomic sequences, most cDNA sequences can still be accommodated.

The primary drawback to use of retroviruses such as the Moloney retrovirus involves the requirement for cells to be actively dividing for

neurons
are very resistant to infection and transduction by retroviruses.

There is concern that

SCID-X1 patients using Moloney murine leukemia virus[11] resulted in two cases of leukemia caused by activation of the LMO2 oncogene due to nearby integration of the vector.[12]

Lentiviruses

Main article: Lentivirus
Packaging and transduction by a lentiviral vector.

reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position (although recent findings suggest that the insertion of viral DNA is not random but directed to specific active genes and related to genome organisation[13]) by the viral integrase enzyme. The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. There are, as yet, no techniques for determining the site of integration, which can pose a problem. The provirus can disturb the function of cellular genes and lead to activation of oncogenes promoting the development of cancer, which raises concerns for possible applications of lentiviruses in gene therapy. However, studies have shown that lentivirus vectors have a lower tendency to integrate in places that potentially cause cancer than gamma-retroviral vectors.[14] More specifically, one study found that lentiviral vectors did not cause either an increase in tumor incidence or an earlier onset of tumors in a mouse strain prone to tumors.[15] Moreover, clinical trials that utilized lentiviral vectors to deliver gene therapy for the treatment of HIV experienced no increase in mutagenic or oncologic events.[16] Versions of the lentiviral vector have been developed that do not integrate their genetic material into the cell's genome but only transiently express genes.[17]
: 50 

For safety reasons, lentiviral vectors never carry the genes required for their replication. To produce a lentivirus, several

transcribed
to produce the single-stranded RNA viral genome and is marked by the presence of the ψ (psi) sequence. This sequence is used to package the genome into the virion.

Adenoviruses

Main article:
Adenovirus

As opposed to lentiviruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division.

SARS-CoV-2 spike gene in Oxford AstraZeneca COVID vaccine. PEGylation of adenoviruses for gene therapy can help prevent adverse reactions due to pre-existing adenovirus immunity.[20]

Adeno-associated viruses

Main article: Adeno-associated virus
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 virus (AAV) is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease, and causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. Moreover, AAV mostly stays as episomal (replicating without incorporation into the chromosome); performing long and stable expression.[21] These features make AAV a very attractive candidate for creating viral vectors for gene therapy.[1] However, AAV can only bring up to 5kb which is considerably small compared to AAV's original capacity.[21]

Adeno-associated viral vectors have been engineered to evade virus recognition by

TLR9 receptors by incorporating TLR9-inhibiting genes into the vector.[22]

Furthermore, because of its potential use as a gene therapy vector, researchers have created an altered AAV called self-complementary adeno-associated virus (scAAV). Whereas AAV packages a single strand of DNA and requires the process of second-strand synthesis, scAAV packages both strands which anneal together to form double stranded DNA. By skipping second strand synthesis scAAV allows for rapid expression in the cell.[23] Otherwise, scAAV carries many characteristics of its AAV counterpart.

Plant viruses

Main article: Plant virus

genetic material into plant cells; they are also sources of biomaterials and nanotechnology devices.[24][25] Tobacco mosaic virus (TMV) is the first virus to be discovered. Viral vectors based on tobacco mosaic virus include those of the magnICON[26] and TRBO plant expression technologies.[24]

Hybrids

Hybrid vectors are

genetically engineered to have qualities of more than one vector. Viruses are altered to avoid the shortcomings of typical viral vectors, which may have limited loading capacity, immunogenicity, genotoxicity, and fail to support long-term adequate transgenic expression. Through the replacement of undesirable elements with desired abilities, hybrid vectors may in the future outperform standard transfection vectors in terms of safety and therapeutic efficiency.[27]

Challenges in application

The choice of a

clinical trials
, the virus can't be used again in the patient for a different vaccine or gene therapy in the future.

Pre-existing

immunity against the viral vector could also be present in the patient, rendering the therapy ineffective for that patient.[28][30] Because priming with a naked DNA vaccine and boosting with a viral vector results in a robust immune response via yet indefinite mechanism(s), despite pre-existing viral vector immunity, this vaccination strategy can counteract this problem.[31]
However, this method may present another expense and obstacle in the vaccine distribution process. Pre-existing immunity may also be challenged by increasing vaccine dose or changing the vaccination route.[32]

Some shortcomings of viral vectors (such as genotoxicity and low transgenic expression) can be overcome through the use of

hybrid vectors
.

See also

References

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  3. ^ Beardsley T (February 2000). "A tragic death clouds the future of an innovative treatment method". Scientific American.
  4. ^ McDowell N (15 January 2003). "New cancer case halts US gene therapy trials". New Scientist.
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  7. ^ "Live-Vector Vaccine". American Institute of Chemical Engineers. 17 December 2014. Archived from the original on 2021-02-03. Retrieved 2021-02-03.
  8. ^ "EPAR summary for the public: Oncept IL-2 (Feline interleukin-2 recombinant canary pox virus) [EMA/151380/2013 EMEA/V/C/002562]" (PDF). European Medical Agency. 2013.
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    S2CID 219588089
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  26. ^ magnICON
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Further reading
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