Lipid nanoparticle-based mRNA vaccines: a new frontier in precision oncology

Abstract The delivery of lipid nanoparticle (LNP)-based mRNA therapeutics has captured the attention of the vaccine research community as an innovative and versatile tool for treating a variety of human malignancies. mRNA vaccines are now in the limelight as an alternative to conventional vaccines owing to their high precision, low-cost, rapid manufacture, and superior safety profile. Multiple mRNA vaccine platforms have been developed to target several types of cancer, and many have demonstrated encouraging results in animal models and human trials. The effectiveness of these new mRNA vaccines depends on the efficacy and stability of the antigen(s) of interest generated and the reliability of their delivery to antigen-presenting cells (APCs), especially dendritic cells (DCs). In this review, we provide a detailed overview of mRNA vaccines and their delivery strategies and consider future directions and challenges in advancing and expanding this promising vaccine platform to widespread therapeutic use against cancer.


Introduction
Cancer imm unother a pies r esha pe the tumor imm une micr oenvir onment and driv e the activ ation of host anti-tumor imm unity to suppress tumorigenesis and growth.Cancer vaccines thus repr esent a pr omising str ategy for ac hie ving this specialized antitumor imm unother a py as they can mobilize T cell r esponses against tumor-associated antigens ( TAAs ) or tumor-specific antigens ( TSAs ) to specifically attack and destroy malignant tumor cells ( Fig. 1 ) [ 1 ].The high expression of antigens induced by vaccines can sustain organismal tumor-killing ability through immune memory.T hus , cancer vaccines could theoretically provide ther a py that is mor e pr ecise, safer , better -tolerated, and longerlasting than other a ppr oac hes ( Fig. 1 ) .
After its discovery in 1961 [ 2 ], mRN A w as soon recognized as a potential ther a peutic deliv ery system and in the 1970s nucleic acid-based vaccine solutions became the subject of intense drug r esearc h [ 3 ].After years of effort, and as medical technology progr essed, pr eclinical studies of mRNA-based vaccines and cancer imm unother a pies began to show impr ov ed r ates of success [ 4 ].In 1990, the first successful expression of in vitro transcription ( IVT ) of mRNA in mouse skeletal muscle cells through direct injection into animals demonstrated the feasibility of mRNA vaccine development [ 5 ].Since then, mRNA has been widely credited with the potential to r e volutionize v accination, cancer imm unothera pies, cellular r epr ogr amming, and genome editing [ 6 ].On the other hand, although manipulating protein expression may ultimatel y pr ov e a po w erful w ea pon in conquering diseases, pr oteins have been limited as therapeutic agents by their large size and high cost of production, and research has therefore focused on exogenousl y pr oduced nucleic acids and their introduction into cells.Plasmid DN A w as initially pursued as a ther a peutic v ector [ 7 ], ho w e v er, IVT of mRNA had v arious adv anta ges in ther a peu-tic applications, including low toxicity and high translatability in both dividing and non-dividing cells, as RNA only needs to be internalized into the cytoplasm ( rather than the nucleus ) , where a one-step translation can produce the antigen ( s ) of interest and initiate imm unostim ulatory activity for cancer imm unother a py [ 7 ].mRNA v accines hav e higher r ates and gr eater ma gnitude of pr otein expr ession than DNA v accines due to the r elativ el y high transfection efficacy of mRNA.Additionally, mRNA cannot integrate into the genome sequence, precluding any risk of insertional m uta genesis.Mor eov er, synthesizing mRNA in a cell-free system in suitable standardized and controlled conditions makes production of good manufacturing pr actice-gr ade mRNA compar ativ el y simple, r a pid, and inexpensiv e at a r ange of scales [ 8 ].These attributes gave mRNA vaccines critical advantages in response to the coronavirus pandemic, and US Food and Drug Administration ( FDA ) a ppr ov al of tw o mRN A-based vaccines from Pfizer -BioNT ech and Moderna for emergency use against COVID-19 led to a dramatic rise in the mRNA-based pharmaceutical market and de v elopment of applications of mRNA therapies against both cancer and infectious disease [ 9 ].
Direct injection of naked mRNA into the body, ho w e v er, faces se v er al hurdles in clinical application, including its quick degradation via endonucleases, the obstruction of cell internalization due to dense negativ e c har ges of nucleic acids, and the triggering of nonspecific interferon responses [ 10 ].Remedies such as ad ding effecti v e shielding fr om degr adation and enhancing gene translation in the cell became necessary to ensure the efficacy of v accine deliv ery.In addition, a nonvir al v ector deliv ery system was de v eloped that utilized lipid nanoparticles ( LNPs ) as carriers due to their nanoscopic size ( diameter < 200 nm ) , biocompatibility , safety , and ease of scalability [ 11 ].These nano-delivery systems have now become widely favored for the delivery of anti-inflammatory, antioxidant, and anti-cancer agents due to their many advantages over conventional medicines [ 8 ].Specificall y, the enca psulation of mRNA within LNPs impr ov es stability and mediates contr olled, tar geted deliv ery of the ther a peutics to diseased cells [ 12 ].
In this article we will provide an ov ervie w of curr ent adv ances and challenges in different LNP-based mRNA vaccines for cancer imm unother a pies, including v accines that encode ( i ) monoclonal antibodies ( mAbs ) , ( ii ) interleukins ( ILs ) and cytokines, ( iii ) TAAs, TSAs , and neoantigens , or that impro ve ( iv ) chimeric antigen receptor ( CAR ) T-cell ther a py ( Fig. 2 ) .

LNP aiding mRNA delivery
Curr entl y the most well-de v eloped tools for mRNA delivery depend on lipid-based systems.A cationic lipid not only aids in encapsulating the polyanionic mRNA but also interacts with negativ el y c har ged phospholipids in the plasma membrane to stimulate internalization by endocytosis [ 8 ].The typical constituents of an LNP ( Fig. 3 ) are a combination of four k e y elements: first, for the encapsulation of the polyanionic mRNA, a pHr esponsiv e or cationic lipid bearing tertiary or quaternary amine is essential; second, a neutral helper lipid, such as 1,2-dioleoylsn -gl ycer o-3-phosphoethanolamine ( DOPE ) or 1,2-distear oyl-sngl ycer o-3-phosphoc holine ( DSPC ) , maintains the bilayer structure and aids cellular uptake; third, a sterol lipid-like c holester ol stabilizes the lipid bilayer of LNPs and pr omotes membr ane fusion, thereby boosting the efficiency of mRNA delivery; and finally, the addition of polyethylene glycol ( PEG ) -lipid can reduce aggregation and establish a hydration layer over the LNPs that decreases non-specific uptake and pr e v ents the absor ption of plasma pr oteins and avoids reticuloendothelial clearance, thus enhancing colloidal stability [ 13 , 14 ].
Kranz et al .[ 15 ] demonstrated that by optimizing the mRNA/cationic lipid ratio and using N -[1-( 2,3-diole ylo xy ) propyl]-N , N , N -trimethylammonium chloride ( DOTMA ) /DOPE or 1,2dioleoyl-3-trimethylammonium-pr opane ( DOTAP ) /DOPE form ulations, dendritic cells ( DCs ) could be passiv el y tar geted by solel y adjusting the negative net c har ge of the nanoparticles; no molecular ligand functionalization was r equir ed.Deliv ery of the mRNA w as safeguar ded via a lipoplex nanostructure that minimized the degradation inflicted by extracellular ribonucleases .T he internalization of the LNPs in various lymphoid organs induced tr anslation fr om the enca psulated mRNA in a range of DC subsets and macr opha ges .T he mRNA-lipoplex vaccine also stimulated both an innate type-I interferon ( IFN ) -mediated immune response and a potent ada ptiv e r esponse, whic h together led to a strong rejection of aggressive tumors [ 15 ].One substantial obstacle to the delivery of mRNA vaccines for cancer immunotherapy, i.e. inadequate accumulation in antigen-presenting cells ( APCs ) , was successfull y addr essed by a r ecent study.By exploiting mannose receptor-mediated endocytosis, Lei et al .[ 16 ] designed DC-targeting LNPs ( STLNPs-Man ) for mRNA delivery in vitro and in vivo .Compared to vaccines based on commercially available  LNPs, the mRNA vaccine ( STLNPs-Man@mRNA O VA ) showed a 4-fold increase in DC uptake when administered intramuscularly.STLNPs-Man@mRNA O VA necessitated only one-fifth the dose of commercial LNPs, thus reducing the likelihood of adverse effects.Additionally, STLNPs-Man@mRNA O VA inhibited the CD206/CD45 axis, r esulting in downr egulation of cytotoxic T-l ymphocyteassociated protein 4 ( CTLA-4 ) on T cells .T his implies substantial potential for impr ov ed efficacy when used in conjunction with imm une c hec kpoint inhibitors ( CPIs ) , and this method is ther efor e a promising advance in the design of mRNA vaccines for cancer treatment, as it offers significantly reduced toxicity as well as impr ov ed efficacy [ 16 ].Additionall y, the incor por ation of imm une adjuv ant α-galactosylcer amide along with the mRNA enca psulated in lipopolyplex nanoparticles enabled passive targeting of DCs and induced a substantial ther a peutic effect against a highly malignant B16-F10 melanoma tumor [ 17 ].Mor eov er, mRNA lipopolyplexes with mannose receptor-targeting moieties did not depend on type-I IFN for effective T-cell immunity.This differential anti-tumor T-cell immunity of mRNA lipopolyplexes enabled the incor por ation of N1 methyl pseudouridine nucleoside-modified mRNA to generate mRNA vaccines with potent immunogenicity yet low and safe inflammatory responses [ 18 ], which could provide a po w erful therapeutic alternative to mRNA-lipoplex v accines curr entl y being e v aluated in earl y phase clinical trials.

Application of LNP-based mRNA cancer vaccines
The r esurr ection of mRNA cancer v accines as a gener al ther a peutic application can be attributed to the swift de v elopment of RNAbased vaccines during the COVID-19 pandemic and their success against SARS-COV-2 [ 19 ]. mRNA has a brief half-life in the blood and is hamper ed fr om entering tar get cells [ 20 ].A wide variety of LNPs, including lipids, lipid deri vati ves, and hybrid particles , ha ve been through extensive testing and have been effectiv el y implemented for clinical trials in the delivery of mRNA [ 8 ] ( Table 1 ) , and these nanomedicine techniques have transformed the management of many diseases , including cancer.T he small size ( 1-200 nm ) and lar ge surface-to-volume r atio of LNPs, along with their tunable surface functionalization properties, offer even bio-distribution, enhanced encapsulation of drugs and nucleic acid, contr olled drug r elease, and r educed systemic toxicities [ 21 ].Mor eov er, these LNPs boast favorable pharmacokinetics and have shown potent pharmacological effects against numerous diseases [ 22 ].Curr entl y, v arious imm unother a pies based on mRNAs have been applied in clinical trials, and results have confirmed the efficacy of a range of treatments against solid tumors [ 23 ].The following section describes LNP-based mRNA cancer vaccines that ar e curr entl y in pr eclinical or clinical trials ( Table 1 ) .

mRNA cancer vaccines encoding mAbs
mAbs have been exploited for clinical use since they were first successfull y pr oduced in the labor atory thr ough immortalizing B cells mimicking the naturally produced antibodies within the body that target specific antigens [ 38 ]. mAbs have revolutionized clinical a ppr oac hes to ther a py as they are precise, effectiv e, and r esult in r educed side effects, especiall y in tr eating certain types of cancer [ 38 ].Imm une c hec kpoint mAbs such as antipr ogr ammed cell death 1 ( PD-1 ) /pr ogr ammed cell death-ligand 1 ( PD-L1 ) and CTLA-4 have shown durable responses in the treat-ment of melanoma, non-small cell lung cancer ( NSCLC ) , and renal cancer carcinoma [ 39 ].Although r ecombinant tec hnologies hav e expanded the utilization of this quic kl y gr owing class of ther apeutics, the utilization of these recombinant antibodies r equir ed impr ov ements in suc h aspects as the form ulation featur es that suppr ess a ggr egation during long-term stor a ge, br oader biodistribution, the necessity for large-scale manufacturing, the need for complex protein characterization, and the high costs of repeated administration during treatment [ 38 , 40 ].Since mRNAs encode proteins, full-size mAbs, antibody fr a gments, or an y antibody variants can theoretically be coded and delivered to cells, whic h subsequentl y pr oduce these pr oteins.P ardi et al .[ 41 ] first demonstrated the feasibility of using mRNA to encode the light and heavy chains of VRC01, an antibody a gainst HIV-1.Thr an et al .[ 42 ] independently validated the feasibility of using mRNA for passiv e v accination a gainst infectious a gents , toxins , and tumors .Their findings demonstrated that single injections of mRNA-LNPs were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo , and that ther a peutic mRNA-mediated antibody expression allo w ed mice to survive an otherwise lethal tumor challenge.An optimized IVT-mRNA system for in vivo delivery of a humanized anti-Her2 antibody, Trastuzumab, was developed by Rybak ov a et al. [ 43 ].Systemic delivery of optimized IVT-mRNA loaded into LNPs impr ov ed the pharmacokinetic profile for in vivo pr oduced Tr astuzumab compar ed to injected Tr astuzumab pr otein, and resulted in selective anti-tumor activity in HER2-positive tumors while improving animal survival.Wu et al. [ 44 ]. designed the IVT-mRNA encoding Pembrolizumab, a commercially available anti-PD-1 mAb.Maximized Pembr olizumab expr ession le vels from IVT-mRN A w ere achieved by optimizing the signal peptide and the molar ratio of the heavy/light chain.A single dose of IVT-mRNA loaded into LNPs in mice resulted in serum Pembr olizumab le v els that endur ed for > 35 days and displayed a significantly enhanced pharmacokinetic profile compared to the same dose of dir ectl y injected Pembr olizumab.Chr onic tr eatment of tumor-bearing mice with LNP-encapsulated Pembrolizumab mRNA effectiv el y suppr essed the growth of intestinal tumors and impr ov ed animal surviv al.These studies indicate that mRNAencoding antibodies appear to represent a viable therapeutic str ategy a gainst v arious biological thr eats, including ( vir al ) infections, intoxication, and cancer [41][42][43][44].
Furthermor e, other r esearc h has yielded an innov ativ e a ppr oac h to mRNA delivery with bispecific T-cell engaging ( BiTE ) antibodies .T he ther a peutic potential of BiTE antibodies has been hindered by manufacturing challenges and their short serum half-life [ 45 ].Stadler et al. [ 46 ]. hypothesized that these limitations could be cir cumvented b y generating BiTE antibodies using mRNA.RiboMABs, BiTE antibodies directed against the T cell receptor-associated molecule CD3 and one of three TAAs ( CLDN6, CLDN18.2, or EpCAM ) , wer e gener ated in vivo using 1methylpseudouridine-containing mRNAs.RiboMAB le v els wer e sustained over several days in vivo and their ther a peutic efficacy a gainst adv anced xenogr aft tumors was equiv alent to that of r ecombinant antibodies.Additionally, mRNA encoding B7H3 × CD3 BiTE encapsulated in LNPs have achieved high transfection efficiency and targeted delivery to hepatosplenic tissues .T his therapy also resulted in high BiTE concentr ations, pr olonged half-life, and robust antitumor efficacy against hematologic malignancies and melanoma [ 47 ].This mRNA-based a ppr oac h offers a flexible and cost-effecti ve alternati ve for conventional recombinant antibody manufacturing and has ther efor e pr ompted a sur ge of clinical a pplications in cancer treatment [ 47 ].

mRNA cancer vaccines encoding ILs and cytokines
Cytokines are among the most important agents in the imm unother a peutic tr eatment of cancer.In 1986 the FDA first appr ov ed the delivery of IFN-α for treatment of hairy cell leukemia [ 48 ], which w as follo w ed b y a ppr ov al of IL-2 in 1992 and 1998 for metastatic renal cancer and advanced melanoma, respectively [ 49 , 50 ].Curr entl y, clinical studies ar e being conducted on a v ariety of cytokines for the treatment of various malignancies.Howe v er, these efforts faced tight ther a peutic mar gins owing to their paracrine or autocrine effect and gener all y short half-life.In vivo studies of IL-12, IL-15, and IL-27 have frequently shown anticancer effectiveness [ 51 ], but to achieve the desired therapeutic window through systemic administration in the tumor microenvironment, higher concentrations of cytokines are required, which often results in dose-limiting toxicities [ 52 , 53 ].Alternative approaches with more efficient encapsulation, more proficient delivery of imm unostim ulatory cytokines to tumors, and lower systemic toxicity are urgently needed for clinical applications [ 54 ].Encapsulated mRN A-encoding c ytokines in LNPs have been shown to induce robust tumor infiltration of immune effectors and have inhibited tumor growth with reduced toxicity [54][55][56].In comparison to carrier-free IL-12 or layer-free liposomal NPs, IL-12 ther a pies utilizing systemicall y deliv er ed lay er-b y-lay er NPs have exhibited diminished toxicity and sustained anti-tumor efficacy, resulting in a 30% complete survival rate in ovarian cancer [ 57 ]. mRNA-2416 LNPs by Moderna, which encapsulate mRNAencoding wild type human OX40L ( a ligand of OX40 ) , are under clinical e v aluation as a monother a py and in combination with administered fixed doses of Durvalumab for the treatment of patients with ovarian cancer ( NCT03323398 ) .The intr atumor al administration of mRNA 2416 in this trial was designed for up to 12 doses e v ery 2 weeks, with four dose le v els fr om 1 to 8 mg.mRN A-2416 w as gener all y found to be well-toler ated at the different dose le v els .T he analysis of paired biopsies from injected lesions r e v ealed an ele v ation in OX40L expr ession and PD-L1, as well as heightened T cell le v els and pr o-inflammatory activity.Howe v er, this trial was pr ematur el y ended in July 2022 since the effecti veness objecti ves of neither the monotherapy nor combination tr eatment wer e fulfilled [ 24 ].
Similarly, mRNA 2752 LNPs, which encapsulate mRNA encoding human OX40L, IL-23, and IL-36 γ , are now in clinical trials ( NCTO3739931 ) for intr atumor al injection alone and in combination with immune CPIs.mRNA-2752 was intr atumor all y administer ed e v ery 2 weeks for up to se v en doses, alone or in combination with the infusion of Durvalumab.A total of 23 solid tumor patients ( monother a py: n = 14; combination: n = 9 ) tolerated the treatment well with no dose-limiting toxicities.Six patients had stable disease, 10 had partial disease and 1 had partial responses ( 52% tumor reduction ) .A total of 5 individuals had tumor r eduction in tr eated or untr eated ar eas thr oughout tr eatment [ 25 ].In recent years, cancer immunotherapy research has focused on IL-2 as a target to reduce regulatory T cell development, despite the challenge of potentially severe systemic toxicity.The peritumoral injection of 'BALLkine-2', recombinant human IL-2 ( rIL-2 ) loaded in mesoporous silica NPs, elicited beneficial immunotherapeutic effects while mitigating concerns and adverse effects associated with rIL-2-related systemic and vascular toxicity.The impr ov ed ther a peutic outcomes associated with B ALLkine-2 w ere ascribed to two factors: the establishment of an rIL-2 depot facilitating the continuous release of rIL-2 and the heightened exposure of tumors to rIL-2, as well as the instantaneous localiza-tion of BALLkine-2 to secondary lymph nodes by DCs from the depot.These c har acteristics of locall y injected BALLkine-2 make it attr activ e as an innov ativ e cytokine ther a p y that offers lo w er medical cost and impr ov ed patient compliance [ 58 ].

mRNA cancer vaccines encoding TAAs or TSAs
Cancer vaccines that target TAAs or TSAs can specifically attack and destroy malignant cells overexpressing the antigens of interest and can induce long-term ther a peutic r esponse thr ough immunologic memory [ 1 ].TAAs are attractive targets but are more suitable for certain solid tumors, such as melanoma and NSCLC.A typical example of the application of TAA targeting is the BNT111 cancer vaccine, the first candidate from the BioNTech FixVac platform to be tested in humans.BNT111 was FDA a ppr ov ed in 2021 for a Phase I trial ( Lipo-MERIT, NCT02410733 ) to e v aluate the safety and tolerability of vaccinating patients with stage III B, C, and stage IV melanoma.BNT111 is a nanoparticulate liposomal RN A ( RN A-LPX ) v accine deliv er ed intr av enousl y that targets DCs in lymphoid compartments throughout the body.During the clinical study, the following TAAs for melanoma were utilized: New York esophageal squamous cell carcinoma 1 ( NY-ESO-1 ) , melanoma-associated antigen A3 ( MAGE-A3 ) , tyrosinase, and tr ansmembr ane phosphatase with tensin homology ( TPTE ) .The study sho w ed that T cells induced by FixVac were fully functional, r ecognized their tar get epitopes on melanoma cells, and exhibited strong cytotoxic activity.Although the vaccination proved effective as a single agent, the immunotherap y w as markedly mor e effectiv e in combination with anti-PD1 antibodies in CPIpositive tumor patients.Vaccine-induced T cells were of the PD1 + effector memory phenotype and ther efor e could be activ ated by the anti-PD1 antibodies.In pr etr eated, CPI-experienced patients with melanoma, the FixVac/anti-PD1 combo induced > 35% tumor shrinkage.In individuals with CPI-naive metastatic melanoma, the objectiv e r esponse r ates wer e compar able to PD1 bloc king alone, as PD1 blockade works through the expansion of preexisting antigen-specific T cells, many of which target mutationderived neoantigens.Anti-PD1 treatment alone imposes a higher risk of disease r ecurr ence because most patients have moderate to low mutational burden, which has been linked to a lower c hance of pr e-formed neoantigen-specific T cells .T he four TAAs tar geted ar e highl y pr e v alent in human melanoma but ar e also found in normal cells.As vaccinations may induce both central and peripher al imm une toler ance r esponses, their ther a peutic effectiveness may be reduced.Consequently, most vaccines expr essing TAAs ar e administer ed in conjunction with imm unological CPIs .T his Phase I study was completed in 2023 and Lipo-MERIT vaccine was found to display several anti-tumor activities that contributed to its ther a peutic effect [ 27 ].

mRNA cancer vaccines encoding neoantigens and personalized vaccines
Mutated, non-self-peptides generated in cancer cells due to nonsynon ymous m utations ar e pr ocessed and pr esented by MHC on the cell surface, pr oficientl y stim ulating T-cell r esponses .T hese antigens, known as neoantigens, are capable of potent immunogenicity, arise from somatic mutations typically not exhibited in normal cells, and can be classified into two categories: private and public neoantigens.Private neoantigens differ from patient to patient and are designed as custom-made therapies based on the specifications of each patient [ 59 ].One pr e vious clinical study demonstrated that 62 of 75 ( 83% ) patients with common gastrointestinal cancers released tumor-infiltrating lymphocytes ( TIL ) that recognized neoantigens, and that the majority of the neoantigen determinants were unique and not shared among patients [ 60 ].This type of cancer vaccination permits individualized diagnosis and therapy for each patient and has become the focus of most current clinical studies.A Phase I ( NCT03313778 ) open-label, m ulticenter r esearc h trial examining the safety, tolerability, and immunogenicity of the personalized vaccine mRNA-4157 ( V940 ) alone and in combination with a CPI ( Pembrolizumab ) in patients with resected solid tumors found that mRNA-4157 ( V940 ) was safe and well-tolerated at all dose levels tested.Clinical responses have been observed in combination with Pembrolizumab and neoantigen-specific T cells have been induced, supporting the advancement of mRNA-4157 to Phase II [ 32 ].So far, 3-year data from a Phase II ( NCT03897881 ) trial of mRNA-4157 ( V940 ) in combination with Pembrolizumab has demonstrated sustained impr ov ement in r ecurr ence-fr ee surviv al [ 33 ] and distant metastasis-free survival [ 34 ] versus Pembrolizumab alone in patients with high-risk stage III/IV melanoma following complete resection.Multiple Phase III trials of mRNA-4157 ( V940 ) plus Pembr olizumab v ersus Pembr olizumab ( NCT06077760, NCT06295809, NCT05933577 ) wer e subsequentl y initiated.Similarl y, the BioN-Tec h v accine RO7198457 ( BNT122 ) is curr entl y being e v aluated in a multi-step, open-label, phase II ( NCT04486378 ) randomized trial v ersus watc hful waiting in patients with circulating tumor DNA ( ctDNA ) -positiv e, sur gicall y r esected Sta ge II/III r ectal cancer or Stage II ( high risk ) /Stage III colon cancer [ 37 ].
Mutated antigens that ar e conserv ed among cancer patients ar e consider ed public neoantigens for groups with analogous genetic alterations .T he high specificity of neoantigens to cancer cells due to underlying mutations, while exerting minimal toxicity to non-cancerous cells, propels different screening techniques in cancer imm unother a py.Se v er al str ategies to scr een for candidate neoantigens, such as next-generation sequencing technology, whole-exome sequencing, computer algorithms, and imm unological effects e v aluation, ar e av ailable to facilitate more pr ecisel y identification and destruction of cancer cells by the immune system [ 61 ].Ho w ever, the current algorithm utilized for predicting neoantigens has a few dra wbacks , since it is largely confined to in vitro binding affinity data and is computationally constrained.As a result, it generates unavoidably large levels of false positives.Hao et al .[ 62 ] have therefore proposed a deep convolutional neural network titled APPM ( antigen presentation prediction model ) to predict antigen presentation in the context of HLA class I alleles .T he APPM pr ediction, combined with the imm une epitope database, can impr ov e the accur ate pr ediction of neoantigens.Another model, dubbed EDGE, was pr e viousl y de v eloped by Bulik-Sullivan et al .[ 63 ] based on a large HLA peptide and genomic dataset from various human tumors and improved the accuracy of HLA antigen prediction by as much as 9-fold.

mRNA cancer vaccines improving CAR T-cell therapy
Adoptive T-cell therapy is an umbrella term for therapeutic treatments involving the administration of enhanced or altered autologous cancer-specific T cells .T his in volves the ex vivo modification of isolated autologous cells to enhance patients' T-cells to identify and attack treatment-resistant cancer cells [ 64 ].There are two main types of T-cell ther a py: TIL ther a py and CAR-modified T-cell ther a py.The l ymphocyte tr eatment was deemed ineffective due to its inability to eradicate the tumor or counteract the signals that the tumor emits to inhibit the immune system [ 65 ], and therefore recent attention has focused on development of CAR T-cell therapy as an advanced personalized cancer imm unother a py.This engineer ed T-cell ther a py has r eceiv ed a ppr ov al fr om the FDA and the European Medicine Agency for clinical application in hematological cancers, including acute lymphoblastic leukemia and diffuse large B-cell lymphoma [ 66 , 67 ].CAR sequencing represents a major modification to T cells, and transduction is ac hie v ed by the use of r etr o-or lenti-vir al tr ansduction, whic h has r aised concerns regarding risks of insertional m uta genesis, imm unogenicity, and limited payload capacity [ 68 ].In response to these concerns, mRN A technology no w enables tr ansient expr ession of CAR, since mRNA sequencing can be customized, and the molecules are subject to decay after tr anslation, av erting an y risk of genomic v ector integration [ 69 ].As an alternative to integrating viral vectors, electr opor ation ( EP ) of CAS9 mRNA into human T cells allows directed integration of a CD19-specific CAR to the T cell receptor α constant ( TRAC ) locus, resulting in constant CAR expression as well as impr ov ed T cell potency [ 70 ].The adv anta ges of TRAC include promoting optimal baseline expression that can eradicate CAR tonic signaling in the absence of antigen and permit effective CAR internalization upon single or multiple contacts with antigen.TRAC also produces a balanced transcriptional response, resulting in kineticall y optimal r ecov ery of baseline CAR expr ession after antigen engagement.In contrast to T-cells with higher CAR expression, the TRAC -CAR profile has exhibited superior tumor eradication via decreased T-cell differentiation and exhaustion [ 71 , 72 ].Ho w e v er, EP uses pulsed electric fields, which entail a risk of cytotoxicity and irr e v ersible loss of cytoplasmic content, resulting in failure to guarantee consistent membrane penetration across cells for delivery [ 73 ].T hus , further in vestigation is necessary to analyze the potential risks associated with the long-term expression of transgenes and their behavior in cells [ 74 ].A recent investigation into the use of LNP to deliver CAR-mRNA to T-cells ex vivo also r e v ealed that its efficacy was prolonged in comparison to EP.Additional findings indicated that EP-CAR T cells exhibited decreased viability and efficiency against target cells .T his also increased fatigue and heightened off-target toxicity compared to LNP-CAR T cells 1 day post-transfection [ 75 ].Since the end of the last decade , nanoparticles , especially LNPs , ha ve come to dominate non-viral delivery systems for lymphocyte transfection.The exploitation of LNPs has been shown to facilitate the delivery of CAR-encoding mRNA into primary human T-cells, resulting in functional CAR T-cells that engage in high le v els of tumor-killing activity [76][77][78].

Conclusions and future perspectives
mRNA-based ther a peutics ar e now the foundation of man y ne w treatments for various human malignancies and have transformed gene editing, protein replacement therapies, cell reprogr amming, and imm unother a pies via their immense a pplications.
As mRNA is synthesized thr ough cell-fr ee methods, the quick and cost-effectiv e pr oduction of mRNA-based medicines is feasible and efficient.Ho w e v er, the a pplication of these ther a pies is complicated by the inherent chemical instability of mRNA and its susceptibility to hydr ol ysis catal yzed by nucleases.Although mRNA v accinations pr ov ed critical in combating the COVID-19 pandemic, se v er al significant obstacles must be overcome for mRNA technology to succeed in treating diseases such as cancer.
A k e y obstacle is the lack of practical and scalable mRNA synthesis techniques that are compatible with current pharmaceutical tec hnology.Clinicall y tr anslatable mRNA tr ansporters with both a ppr opriate stability in the systemic circulation and r esolution of current immunogenicity issues are required for the development of potent mRNA ther a pies.Recent adv ances in non-vir al delivery systems and the development of no vel, effective , transfecting nanomaterials have provided solutions to some of these c hallenges.Se v er al anti-cancer imm unother a peutic tec hniques, including ther a py v accines , monoclonal antibodies , and C AR cells combined with mRNA-based tec hnologies thr ough LNPs , ha ve likewise enhanced the efficacy of various treatments.More studies are needed to determine the causes of the low le v els of mRNA tr ansfection effectiv eness sho wn b y non-vir al v ehicles, particularly in lymphoc ytes, monoc ytes, and other cells that have pr ov ed difficult to transfect.Combining mRNA-based therapies with other cancer treatments, including CPIs, c hemother a py, and r adiation, may r epr esent a viable str ategy for enhancing the effectiveness of these therapies.To increase delivery, the gaps in efficiency and usefulness found between small animals ( e .g. mice , r ats ) , lar ger animals ( non-human primates ) , and humans must be bridged by expediting the nanoparticle discovery pathway.It will be exciting to see the results of the current wave of clinical vaccination studies .T hese outcomes will have a significant impact on the whole arena of mRNA-based therapeutics as well as the futur e landsca pe of biomedicine.

Figure 1 .
Figure 1.Dia gr am illustr ating the in vivo administr ation of lipid nanoparticle-enca psulated mRNA v accine into tumor models, its internalization by dendritic cells, downstream immune activation, effector T cells homing from lymphoid tissues to the tumor sites through lymphatic recirculation, and subsequent tumor cell-killing.

Figure 3 .
Figure 3. Illustration of synthesis of LNP-encapsulated mRNA vaccine material for cancer treatment.

Table 1 .
Re presentati ve LNP-based mRNA vaccines in clinical trials

Vaccine/administr a tion Encoded protein Condition Adjuv ant ther apy NCT number
completed on 29 October 2021.The results sho w ed that at the eighth week after treatment, the PFS rate was 47.8 and 32.4% in the arm A and arm B cohorts, r espectiv el y.At the 24th week after treatment, the PFS rate was 43.5% in arm A and 8.8% in arm B.