Editing DNA vaccine (section)

=== Vector design ===

DNA vaccines elicit the best immune response when high-expression vectors are used. These are [[plasmids]] that usually consist of a strong viral [[Promoter (genetics)|promoter]] to drive the in vivo [[Transcription (genetics)|transcription]] and [[Translation (biology)|translation]] of the gene (or [[complementary DNA]]) of interest.<ref name=Mor1995>{{cite journal | vauthors = Mor G, Klinman DM, Shapiro S, Hagiwara E, Sedegah M, Norman JA, Hoffman SL, Steinberg AD | display-authors = 6 | title = Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA | journal = Journal of Immunology | volume = 155 | issue = 4 | pages = 2039–2046 | date = August 1995 | doi = 10.4049/jimmunol.155.4.2039 | pmid = 7636255 | s2cid = 37290980 | url = http://www.jimmunol.org/cgi/content/abstract/155/4/2039 | doi-access = free }}</ref> [[Interferon alfa-2b|Intron A]] may sometimes be included to improve [[mRNA]] stability and hence increase protein expression.<ref name=Leitner1997>{{cite journal | vauthors = Leitner WW, Seguin MC, Ballou WR, Seitz JP, Schultz AM, Sheehy MJ, Lyon JA | title = Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites | journal = Journal of Immunology | volume = 159 | issue = 12 | pages = 6112–6119 | date = December 1997 | doi = 10.4049/jimmunol.159.12.6112 | pmid = 9550412 | s2cid = 37685499 | url = http://www.jimmunol.org/cgi/content/abstract/159/12/6112 | doi-access = free }}</ref> Plasmids also include a strong [[polyadenylation]]/transcriptional [[termination signal]], such as bovine [[growth hormone]] or rabbit [[beta-globulin]] polyadenylation sequences.<ref name=Alarcon1999 /><ref name=Robinson2000 /><ref name="Böhm1996">{{cite journal | vauthors = Böhm W, Kuhröber A, Paier T, Mertens T, Reimann J, Schirmbeck R | title = DNA vector constructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibody responses in mice after intramuscular injection | journal = Journal of Immunological Methods | volume = 193 | issue = 1 | pages = 29–40 | date = June 1996 | pmid = 8690928 | doi = 10.1016/0022-1759(96)00035-X }}</ref> [[Cistron|Polycistronic]] vectors (with multiple genes of interest) are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.<ref name=Lewis1999>{{cite journal | vauthors = Lewis PJ, Babiuk LA | title = DNA vaccines: a review | journal = Advances in Virus Research | volume = 54 | pages = 129–88 | year = 1999 | pmid = 10547676 | doi = 10.1016/S0065-3527(08)60367-X | url = https://books.google.com/books?id=lrMc3G9nIpkC&q=lewis&pg=PA129 | publisher = Academic Press | isbn = 978-0-12-039854-6 }}</ref>

Because the plasmid{{snd}}carrying relatively small genetic code up to about 200 K[[Base pair|bp]]{{snd}}is the "vehicle" from which the immunogen is expressed, optimising vector design for maximal protein expression is essential.<ref name=Lewis1999 /> One way of enhancing protein expression is by optimising the [[codon]] usage of pathogenic mRNAs for [[eukaryotic]] cells. Pathogens often have different [[GC-content|AT-contents]] than the target species, so altering the [[gene sequence]] of the immunogen to reflect the [[genetic code|codon]]s more commonly used in the target species may improve its expression.<ref name=Andre1998>{{cite journal | vauthors = André S, Seed B, Eberle J, Schraut W, Bültmann A, Haas J | title = Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage | journal = Journal of Virology | volume = 72 | issue = 2 | pages = 1497–1503 | date = February 1998 | pmid = 9445053 | pmc = 124631 | doi = 10.1128/JVI.72.2.1497-1503.1998 }}</ref>

Another consideration is the choice of [[promoter (biology)|promoter]]. The [[SV40]] promoter was conventionally used until research showed that vectors driven by the [[Rous Sarcoma Virus]] (RSV) promoter had much higher expression rates.<ref name=Alarcon1999 /> More recently, expression and immunogenicity have been further increased in model systems by the use of the [[cytomegalovirus]] (CMV) immediate early promoter, and a retroviral [[Cis-regulatory element|cis-acting transcriptional element]].<ref name=Muthumani1998>{{cite journal | vauthors = Muthumani K, Zhang D, Dayes NS, Hwang DS, Calarota SA, Choo AY, Boyer JD, Weiner DB | display-authors = 6 | title = Novel engineered HIV-1 East African Clade-A gp160 plasmid construct induces strong humoral and cell-mediated immune responses in vivo | journal = Virology | volume = 314 | issue = 1 | pages = 134–146 | date = September 2003 | pmid = 14517067 | doi = 10.1016/S0042-6822(03)00459-8 | doi-access = free }}</ref> Additional modifications to improve expression rates include the insertion of enhancer sequences, synthetic [[introns]], [[adenovirus]] tripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences.<ref name=Alarcon1999 /> An example of DNA vaccine plasmid is pVAC, which uses SV40 [[promoter (biology)|promoter]].

Structural instability phenomena are of particular concern for plasmid manufacture, DNA vaccination and gene therapy.<ref>{{cite journal | vauthors = Oliveira PH, Prather KJ, Prazeres DM, Monteiro GA | title = Structural instability of plasmid biopharmaceuticals: challenges and implications | journal = Trends in Biotechnology | volume = 27 | issue = 9 | pages = 503–511 | date = September 2009 | pmid = 19656584 | doi = 10.1016/j.tibtech.2009.06.004 }}</ref> Accessory regions pertaining to the plasmid backbone may engage in a wide range of structural instability phenomena. Well-known catalysts of genetic instability include direct, inverted and tandem repeats, which are conspicuous in many commercially available cloning and expression vectors. Therefore, the reduction or complete elimination of extraneous noncoding backbone sequences would pointedly reduce the propensity for such events to take place and consequently the overall plasmid's recombinogenic potential.<ref>{{cite journal | vauthors = Oliveira PH, Mairhofer J | title = Marker-free plasmids for biotechnological applications - implications and perspectives | journal = Trends in Biotechnology | volume = 31 | issue = 9 | pages = 539–547 | date = September 2013 | pmid = 23830144 | doi = 10.1016/j.tibtech.2013.06.001 }}</ref>