Reverse genetics#Vaccine synthesis

In order to learn the influence a sequence has on phenotype, or to discover its biological function, researchers can engineer a change or disrupt the DNA. After this change has been made a researcher can look for the effect of such alterations in the whole organism. There are several different methods of reverse genetics:

Site-directed mutagenesis is a sophisticated technique that can either change regulatory regions in the promoter of a gene or make subtle codon changes in the open reading frame to identify important amino residues for protein function.{{citation needed|date=January 2020}}

Image:Physcomitrella knockout mutants.JPG and knockout mosses: Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row; scale bar corresponds to 1 mm) and a close-up (bottom row; scale bar equals 0.5 mm) are shown. A: Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B–E: Different mutants.{{cite journal | vauthors = Egener T, Granado J, Guitton MC, Hohe A, Holtorf H, Lucht JM, Rensing SA, Schlink K, Schulte J, Schween G, Zimmermann S, Duwenig E, Rak B, Reski R | display-authors = 6 | title = High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library | journal = BMC Plant Biology | volume = 2 | pages = 6 | date = July 2002 | pmid = 12123528 | pmc = 117800 | doi = 10.1186/1471-2229-2-6 | doi-access = free }}]]

Alternatively, the technique can be used to create null alleles so that the gene is not functional. For example, deletion of a gene by gene targeting (gene knockout) can be done in some organisms, such as yeast, mice and moss. Unique among plants, in Physcomitrella patens, gene knockout via homologous recombination to create knockout moss (see figure) is nearly as efficient as in yeast.{{cite journal | vauthors = Reski R |author-link=Ralf Reski |title=Physcomitrella and Arabidopsis: the David and Goliath of reverse genetics |journal=Trends Plant Sci |volume=3 |pages=209–210 |date=1998 |doi=10.1016/S1360-1385(98)01257-6 |issue=6}} In the case of the yeast model system directed deletions have been created in every non-essential gene in the yeast genome.{{cite journal | vauthors = Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M'Rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Véronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, Johnston M, Davis RW | display-authors = 6 | title = Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis | journal = Science | volume = 285 | issue = 5429 | pages = 901–906 | date = August 1999 | pmid = 10436161 | doi = 10.1126/science.285.5429.901 }} In the case of the plant model system huge mutant libraries have been created based on gene disruption constructs.{{cite journal | vauthors = Schween G, Egener T, Fritzowsky D, Granado J, Guitton MC, Hartmann N, Hohe A, Holtorf H, Lang D, Lucht JM, Reinhard C, Rensing SA, Schlink K, Schulte J, Reski R | display-authors = 6 | title = Large-scale analysis of 73 329 Physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes | journal = Plant Biology | volume = 7 | issue = 3 | pages = 228–237 | date = May 2005 | pmid = 15912442 | doi = 10.1055/s-2005-837692 | s2cid = 260251655 }} In gene knock-in, the endogenous exon is replaced by an altered sequence of interest.{{cite journal | vauthors = Manis JP | title = Knock out, knock in, knock down--genetically manipulated mice and the Nobel Prize | journal = The New England Journal of Medicine | volume = 357 | issue = 24 | pages = 2426–2429 | date = December 2007 | pmid = 18077807 | doi = 10.1056/NEJMp0707712 | publisher = Massachusetts Medical Society | oclc = 34945333 }}

In some cases conditional alleles can be used so that the gene has normal function until the conditional allele is activated. This might entail 'knocking in' recombinase sites (such as lox or frt sites) that will cause a deletion at the gene of interest when a specific recombinase (such as CRE, FLP) is induced. Cre or Flp recombinases can be induced with chemical treatments, heat shock treatments or be restricted to a specific subset of tissues.{{citation needed|date=January 2020}}

Another technique that can be used is TILLING. This is a method that combines a standard and efficient technique of mutagenesis with a chemical mutagen such as ethyl methanesulfonate (EMS) with a sensitive DNA-screening technique that identifies point mutations in a target gene.{{citation needed|date=January 2020}}

In the field of virology, reverse-genetics techniques can be used to recover full-length infectious viruses with desired mutations or insertions in the viral genomes or in specific virus genes. Technologies that allow these manipulations include circular polymerase extension reaction (CPER) which was first used to generate infectious cDNA for Kunjin virus a close relative of West Nile virus.{{cite journal | vauthors = Edmonds J, van Grinsven E, Prow N, Bosco-Lauth A, Brault AC, Bowen RA, Hall RA, Khromykh AA | display-authors = 6 | title = A novel bacterium-free method for generation of flavivirus infectious DNA by circular polymerase extension reaction allows accurate recapitulation of viral heterogeneity | journal = Journal of Virology | volume = 87 | issue = 4 | pages = 2367–2372 | date = February 2013 | pmid = 23236063 | pmc = 3571472 | doi = 10.1128/JVI.03162-12 }} CPER has also been successfully utilised to generate a range of positive-sense RNA viruses such as SARS-CoV-2,{{cite journal | vauthors = Amarilla AA, Sng JD, Parry R, Deerain JM, Potter JR, Setoh YX, Rawle DJ, Le TT, Modhiran N, Wang X, Peng NY, Torres FJ, Pyke A, Harrison JJ, Freney ME, Liang B, McMillan CL, Cheung ST, Guevara DJ, Hardy JM, Bettington M, Muller DA, Coulibaly F, Moore F, Hall RA, Young PR, Mackenzie JM, Hobson-Peters J, Suhrbier A, Watterson D, Khromykh AA | display-authors = 6 | title = A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses | journal = Nature Communications | volume = 12 | issue = 1 | pages = 3431 | date = June 2021 | pmid = 34103499 | pmc = 8187723 | doi = 10.1038/s41467-021-23779-5 | bibcode = 2021NatCo..12.3431A }} the causative agent of COVID-19.

The discovery of gene silencing using double stranded RNA, also known as RNA interference (RNAi), and the development of gene knockdown using Morpholino oligos, have made disrupting gene expression an accessible technique for many more investigators. This method is often referred to as a gene knockdown since the effects of these reagents are generally temporary, in contrast to gene knockouts which are permanent.{{citation needed|date=January 2020}}

RNAi creates a specific knockout effect without actually mutating the DNA of interest. In C. elegans, RNAi has been used to systematically interfere with the expression of most genes in the genome. RNAi acts by directing cellular systems to degrade target messenger RNA (mRNA).{{citation needed|date=January 2020}}

RNAi interference, specifically gene silencing, has become a useful tool to silence the expression of genes and identify and analyze their loss-of-function phenotype. When mutations occur in alleles, the function which it represents and encodes also is mutated and lost; this is generally called a loss-of-function mutation.{{cite web| vauthors = McClean P |title=Types of Mutations |url=http://www.ndsu.edu/pubweb/~mcclean/plsc431/mutation/mutation4.htm |website= Genes and Mutations |publisher=North Dakota State University |access-date=April 27, 2015 }} The ability to analyze the loss-of-function phenotype allows analysis of gene function when there is no access to mutant alleles.{{cite web| vauthors = Tierney MB, Lamour KH |title=An Introduction to Reverse Genetic Tools for Investigating Gene Function |url=http://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx |website=APSnet |publisher=The American Phytopathological Society |access-date=2015-04-28 |archive-date=2018-11-23 |archive-url=https://web.archive.org/web/20181123100354/http://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx |url-status=dead }}

While RNA interference relies on cellular components for efficacy (e.g. the Dicer proteins, the RISC complex) a simple alternative for gene knockdown is Morpholino antisense oligos. Morpholinos bind and block access to the target mRNA without requiring the activity of cellular proteins and without necessarily accelerating mRNA degradation. Morpholinos are effective in systems ranging in complexity from cell-free translation in a test tube to in vivo studies in large animal models.{{citation needed|date=January 2020}}

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A molecular genetic approach is the creation of transgenic organisms that overexpress a normal gene of interest. The resulting phenotype may reflect the normal function of the gene.

Alternatively it is possible to overexpress mutant forms of a gene that interfere with the normal (wildtype) gene's function. For example, over-expression of a mutant gene may result in high levels of a non-functional protein resulting in a dominant negative interaction with the wildtype protein. In this case the mutant version will out compete for the wildtype proteins partners resulting in a mutant phenotype.

Other mutant forms can result in a protein that is abnormally regulated and constitutively active ('on' all the time). This might be due to removing a regulatory domain or mutating a specific amino residue that is reversibly modified (by phosphorylation, methylation, or ubiquitination). Either change is critical for modulating protein function and often result in informative phenotypes.

Reverse genetics plays a large role in vaccine synthesis. Vaccines can be created by engineering novel genotypes of infectious viral strains which diminish their pathogenic potency enough to facilitate immunity in a host. The reverse genetics approach to vaccine synthesis utilizes known viral genetic sequences to create a desired phenotype: a virus with both a weakened pathological potency and a similarity to the current circulating virus strain. Reverse genetics provides a convenient alternative to the traditional method of creating inactivated vaccines, viruses which have been killed using heat or other chemical methods.

Vaccines created through reverse genetics methods are known as attenuated vaccines, named because they contain weakened (attenuated) live viruses. Attenuated vaccines are created by combining genes from a novel or current virus strain with previously attenuated viruses of the same species.{{cite journal | vauthors = Hoffmann E, Krauss S, Perez D, Webby R, Webster RG | title = Eight-plasmid system for rapid generation of influenza virus vaccines | journal = Vaccine | volume = 20 | issue = 25–26 | pages = 3165–3170 | date = August 2002 | pmid = 12163268 | doi = 10.1016/s0264-410x(02)00268-2 | url = https://pdfs.semanticscholar.org/bf1e/0eb494d3867e1fbd2f4dcd6240a130e57176.pdf | url-status = dead | s2cid = 17514938 | archive-url = https://web.archive.org/web/20170402165400/https://pdfs.semanticscholar.org/bf1e/0eb494d3867e1fbd2f4dcd6240a130e57176.pdf | archive-date = 2017-04-02 }} Attenuated viruses are created by propagating a live virus under novel conditions, such as a chicken's egg. This produces a viral strain that is still live, but not pathogenic to humans,{{cite journal | vauthors = Badgett MR, Auer A, Carmichael LE, Parrish CR, Bull JJ | title = Evolutionary dynamics of viral attenuation | journal = Journal of Virology | volume = 76 | issue = 20 | pages = 10524–10529 | date = October 2002 | pmid = 12239331 | pmc = 136581 | doi = 10.1128/JVI.76.20.10524-10529.2002 }} as these viruses are rendered defective in that they cannot replicate their genome enough to propagate and sufficiently infect a host. However, the viral genes are still expressed in the host's cell through a single replication cycle, allowing for the development of an immunity.{{cite journal | vauthors = Lauring AS, Jones JO, Andino R | title = Rationalizing the development of live attenuated virus vaccines | journal = Nature Biotechnology | volume = 28 | issue = 6 | pages = 573–579 | date = June 2010 | pmid = 20531338 | pmc = 2883798 | doi = 10.1038/nbt.1635 }}

A common way to create a vaccine using reverse genetic techniques is to utilize plasmids to synthesize attenuated viruses. This technique is most commonly used in the yearly production of influenza vaccines, where an eight plasmid system can rapidly produce an effective vaccine. The entire genome of the influenza A virus consists of eight RNA segments, so the combination of six attenuated viral cDNA plasmids with two wild-type plasmids allow for an attenuated vaccine strain to be constructed. For the development of influenza vaccines, the fourth and sixth RNA segments, encoding for the hemagglutinin and neuraminidase proteins respectively, are taken from the circulating virus, while the other six segments are derived from a previously attenuated master strain. The HA and NA proteins exhibit high antigen variety, and therefore are taken from the current strain for which the vaccine is being produced to create a well matching vaccine.

The plasmid used in this eight-plasmid system contains three major components that allow for vaccine development. Firstly, the plasmid contains restriction sites that will enable the incorporation of influenza genes into the plasmid. Secondly, the plasmid contains an antibiotic resistance gene, allowing the selection of merely plasmids containing the correct gene. Lastly, the plasmid contains two promotors, human pol 1 and pol 2 promotor that transcribe genes in opposite directions.{{cite journal | vauthors = Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG | title = A DNA transfection system for generation of influenza A virus from eight plasmids | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 97 | issue = 11 | pages = 6108–6113 | date = May 2000 | pmid = 10801978 | pmc = 18566 | doi = 10.1073/pnas.100133697 | bibcode = 2000PNAS...97.6108H | doi-access = free }}

cDNA sequences of viral RNA are synthesized from attenuated master strains by using RT-PCR. This cDNA can then be inserted between an RNA polymerase I (Pol I) promoter and terminator sequence through restriction enzyme digestion. The cDNA and pol I sequence is then, in turn, surrounded by an RNA polymerase II (Pol II) promoter and a polyadenylation site.{{cite journal | vauthors = Mostafa A, Kanrai P, Petersen H, Ibrahim S, Rautenschlein S, Pleschka S | title = Efficient generation of recombinant influenza A viruses employing a new approach to overcome the genetic instability of HA segments | journal = PLOS ONE | volume = 10 | issue = 1 | pages = e0116917 | date = 2015-01-23 | pmid = 25615576 | pmc = 4304806 | doi = 10.1371/journal.pone.0116917 | bibcode = 2015PLoSO..1016917M | doi-access = free }} This entire sequence is then inserted into a plasmid. Six plasmids derived from attenuated master strain cDNA are cotransfected into a target cell, often a chicken egg, alongside two plasmids of the currently circulating wild-type influenza strain. Inside the target cell, the two "stacked" Pol I and Pol II enzymes transcribe the viral cDNA to synthesize both negative-sense viral RNA and positive-sense mRNA, effectively creating an attenuated virus. The result is a defective vaccine strain that is similar to the current virus strain, allowing a host to build immunity. This synthesized vaccine strain can then be used as a seed virus to create further vaccines.

Vaccines engineered from reverse genetics carry several advantages over traditional vaccine designs. Most notable is speed of production. Due to the high antigenic variation in the HA and NA glycoproteins, a reverse-genetic approach allows for the necessary genotype (i.e. one containing HA and NA proteins taken from currently circulating virus strains) to be formulated rapidly. Additionally, since the final product of a reverse genetics attenuated vaccine production is a live virus, a higher immunogenicity is exhibited than in traditional inactivated vaccines,{{cite journal | vauthors = Stobart CC, Moore ML | title = RNA virus reverse genetics and vaccine design | journal = Viruses | volume = 6 | issue = 7 | pages = 2531–2550 | date = June 2014 | pmid = 24967693 | pmc = 4113782 | doi = 10.3390/v6072531 | doi-access = free }} which must be killed using chemical procedures before being transferred as a vaccine. However, due to the live nature of attenuated viruses, complications may arise in immunodeficient patients.{{Cite web | vauthors = Kroger AT, Atkinson WL, Sumaya CV, Pickering LK | collaboration = Advisory Committee on Immunization Practices (ACIP) |url= https://www.cdc.gov/mmwr/preview/mmwrhtml/rr6002a1.htm?s_cid=rr6002a1_e |title=General Recommendations on Immunization| date = 28 January 2011 | id = 60(RR02);1-60 | work = Morbidity and Mortality Weekly Report (MMWR) | publisher = U.S. Centers for Disease Control and Prevention |language=en |access-date=2017-04-01}} There is also the possibility that a mutation in the virus could result the vaccine to turning back into a live unattenuated virus.{{cite journal | vauthors = Shimizu H, Thorley B, Paladin FJ, Brussen KA, Stambos V, Yuen L, Utama A, Tano Y, Arita M, Yoshida H, Yoneyama T, Benegas A, Roesel S, Pallansch M, Kew O, Miyamura T | display-authors = 6 | title = Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001 | journal = Journal of Virology | volume = 78 | issue = 24 | pages = 13512–13521 | date = December 2004 | pmid = 15564462 | pmc = 533948 | doi = 10.1128/JVI.78.24.13512-13521.2004 }}

• Forward genetics

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• {{cite journal | vauthors = Neumann G, Hatta M, Kawaoka Y | title = Reverse genetics for the control of avian influenza | journal = Avian Diseases | volume = 47 | issue = 3 Suppl | pages = 882–887 | date = 2003 | pmid = 14575081 | doi = 10.1637/0005-2086-47.s3.882 | s2cid = 30492604 }}
• {{cite journal | vauthors = Neumann G, Fujii K, Kino Y, Kawaoka Y | title = An improved reverse genetics system for influenza A virus generation and its implications for vaccine production | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 102 | issue = 46 | pages = 16825–16829 | date = November 2005 | pmid = 16267134 | pmc = 1283806 | doi = 10.1073/pnas.0505587102 | bibcode = 2005PNAS..10216825N | doi-access = free }}
• {{cite journal | vauthors = Ozaki H, Govorkova EA, Li C, Xiong X, Webster RG, Webby RJ | title = Generation of high-yielding influenza A viruses in African green monkey kidney (Vero) cells by reverse genetics | journal = Journal of Virology | volume = 78 | issue = 4 | pages = 1851–1857 | date = February 2004 | pmid = 14747549 | pmc = 369478 | doi = 10.1128/JVI.78.4.1851-1857.2004 }}

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{{Library resources box

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• [http://www3.niaid.nih.gov/healthscience/healthtopics/Flu/Research/ongoingResearch/FluVirusChanges/ReassortmentReverseGenetics.htm Reassortment vs. Reverse Genetics]
• [https://www.niaid.nih.gov/topics/Flu/Research/vaccineResearch/Pages/ReverseGenetics.aspx Reverse Genetics: Building Flu Vaccines Piece by Piece]

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Category:Genetic engineering

Category:Molecular genetics