Messenger RNA (mRNA) is a promising new class of therapeutics that has potential for treatment of diseases in fields such as immunology, oncology, vaccines, and inborn errors of metabolism. mRNA therapy has several advantages over DNA-based gene therapy, including the lack of the need for nuclear import and transcription, as well as limited possibility of genomic integration. One drawback of mRNA therapy, especially in cases such as metabolic disorders where repeated dosing will be necessary, is the relatively short in vivo half-life of mRNA (∼6–12 h). We hypothesize that protein engineering designed to improve translation, yielding longer-lasting protein, or modifications that would increase enzymatic activity would be helpful in alleviating this issue. In this study, we present two examples where sequence engineering improved the expression and duration, as well as enzymatic activity of target proteins in vitro. We then confirmed these findings in wild-type mice. This work shows that rational engineering of proteins can lead to improved therapies in vivo.
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References
1.
SahinU, KarikoK and TureciO. (2014). mRNA-based therapeutics—developing a new class of drugs. Nat Rev Drug Discov, 13:759–780.
2.
SchwanhäusserB, BusseD, LiN, DittmarG, SchuchhardtJ, WolfJ, ChenW and SelbachM. (2011). Global quantification of mammalian gene expression control. Nature, 473:337–342.
3.
YinH, KanastyRL, EltoukhyAA, VegasAJ, DorkinJR and AndersonDG. (2014). Non-viral vectors for gene-based therapy. Nat Publ Gr, 15:541–555.
4.
BaleJB, GonenS, LiuY, ShefflerW, EllisD, ThomasC, CasicoD, YeatesTO, GonenT, KingNP and BakerD. (2016). Accurate design of megadalton-scale two-component icosahedral protein complexes. Science, 353:389–393.
5.
SchottJW, GallaM, GodinhoT, BaumC and SchambachA. (2011). Viral and non-viral approaches for transient delivery of mRNA and proteins. Curr Gene Ther, 11:382–398.
6.
KoreAR, ShanmugasundaramM, CharlesI, VlassovAV and BartaTJ. (2009). Locked nucleic acid (LNA)-Modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization. J Am Chem Soc, 131:6364–6365.
7.
FeriziM, AnejaMK, BalmayorER, BadieyanZS, MykhaylykO, RudolphC and Plank.C (2016). Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts. Sci Rep, 6:39149.
8.
BellP, WangL, ChenS-J, YuH, ZhuY, NayalM, HeZ, WhiteJ, Lebel-HaganD and WilsonJM. (2016). Effects of self-complementarity, codon optimization, transgene, and dose on liver transduction with AAV8. Hum Gene Ther Methods, 27:228–237.
9.
LiB, LuoX and DongY. (2016). Effects of chemically modified messenger RNA on protein expression. Bioconjug Chem, 27:849–853.
10.
Grudzien-NogalskaE, JemielityJ, KowalskaJ, DarzynkiewiczE and RhoadsRE. (2007). Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells. RNA, 13:1745–1755.
11.
LiJ, WangW, HeY, LiY, YanEZ, ZhangK, IrvineDJ and HammondPT. (2017). Structurally programmed assembly of translation initiation nanoplex for superior mRNA delivery. ACS Nano, 11:2531–2544.
12.
LagasséHAD, AlexakiA, SimhadriVL, KatagiriNH, JankowskiW, SaunaZE and Kimchi-SarfatyC. (2017). Recent advances in (therapeutic protein) drug development. F1000Res, 6:113.
13.
CasiG and NeriD. (2012). Antibody-drug conjugates: basic concepts, examples and future perspectives. J Control Release, 161:422–428.
14.
JefferisR. (2009). Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov, 8:226–234.
15.
GrabowskiGA, GolemboM and ShaaltielY. (2014). Taliglucerase alfa: an enzyme replacement therapy using plant cell expression technology. Mol Genet Metab, 112:1–8.
16.
FoxJL. (2012). First plant-made biologic approved. Nat Biotechnol, 30:472.
17.
MossnerE, BrunkerP, MoserS and PuntenerU. (2010). Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II antibody with enhanced direct and immune effector cell mediated B-cell cytotoxity. Blood, 115:4393–4402.
18.
CostaAR, RodriguesME, HenriquesM, OliveiraR and AzeredoJ. (2013). Glycosylation: impact, control and improvement during therapeutic protein production. Crit Rev Biotechnol, 8551:1–19.
19.
Di CostanzoL, SabioG, MoraA, RodriguezPC, OchoaAC, CentenoF and ChristiansonDW. (2005). Crystal structure of human arginase I at 1.29-A resolution and exploration of inhibition in the immune response. Proc Natl Acad Sci U S A, 102:13058–13063.
HäberleJ, BoddaertN, BurlinaA, ChakrapaniA, DixonM, HuemerM, KarallD, MartinelliD, CrespoPS, et al. (2012). Suggested guidelines for the diagnosis and management of urea cycle disorders. Orphanet J Rare Dis, 7:32.
22.
KeoughDT, BreretonIM, De JerseyJ and GuddatLW. (2005). The crystal structure of free human hypoxanthine-guanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle. J Mol Biol, 351: 170–181.
23.
TewariN, MathurVP, SardanaD and BansalK. (2017). Lesch-Nyhan syndrome: the saga of metabolic abnormalities and self-injurious behavior. Intractable Rare Dis Res, 6:65–68.
24.
BellS, KolobovaI, CrapperL and ErnstC. (2016). Lesch-Nyhan syndrome: models, theories, and therapies. Mol Syndromol, 7:302–311.
25.
ThompsonJD, HigginsDG and GibsonTJ. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res, 22:4673–4680.
26.
LehmannM, PasamontesL, LassenSF and WyssM. (2000). The consensus concept for thermostability engineering of proteins. Biochim Biophys Acta, 1543:408–415.
27.
Mesa-TorresN, YuntaC, Fabelo-RosaI, María Gonzalez-RubioJ, Anchez-RuizJM, SalidoE, AlbertA and PeyAL. (2014). The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine–glyoxylate aminotransferase. Biochem J, 462:453–463.
ZhaoS, XuW, JiangW, YuW, LinY, ZhangT, YaoJ, ZhouL, ZengY, et al. (2010). Regulation of cellular metabolism by protein lysine acetylation. Science, 327:1000–1004.
30.
RichnerJM, HimansuS, DowdKA, PiersonTC, CiaramellaG, DiamondMS, RichnerJM, HimansuS, DowdKA, et al. (2017). Modified mRNA vaccines protect against Zika virus article modified mRNA vaccines protect against Zika virus infection. Cell, 168:1–12.
31.
AnD, SchnellerJL, FrassettoA, LiangS, ZhuX, ParkJ-S, TheisenM, HongS-J, ZhouJ, et al. (2017). Systemic messenger RNA therapy as a treatment for methylmalonic acidemia. Cell Rep, 21:3548–3558.
32.
DegorceF, CardA, SohS, TrinquetE, KnapikGP and XieB. (2009). HTRF: a technology tailored for drug discovery—a review of theoretical aspects and recent applications. Curr Chem Genomics, 3:22–32.
33.
SenisterraGA and FinertyPJ. (2009). High throughput methods of assessing protein stability and aggregation. Mol Biosyst, 5:217–223.
34.
Jeremy JohnsonR, SavasCJ, KartjeZ and HoopsGC. (2014). Rapid and adaptable measurement of protein thermal stability by differential scanning fluorimetry: updating a common biochemical laboratory experiment. J Chem Educ, 91:1077–1080.
35.
Rodriguez-LarreaD, Perez-JimenezR, Sanchez-RomeroI, Delgado-DelgadoA, FernandezJM and Sanchez-RuizJM. (2010). Role of conservative mutations in protein multi-property adaptation. Biochem J, 429:243–249.
36.
KarikóK, MuramatsuH, KellerJM and WeissmanD. (2012). Increased erythropoiesis in mice ynjected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol Ther, 20:948–953.
37.
KanastyR, DorkinJR, VegasA and AndersonD. (2013). Delivery materials for siRNA therapeutics. Nat Mater, 12:967–977.
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