melanoma most commonly arises from epidermal skin melanocytes ( cutaneous melanoma ) , but primary tumors can also be found lining the choroidal layer of the eye ( uveal melanoma ) or the mucosal surfaces of the respiratory , genitourinary , and gastrointestinal surfaces . similar to other tumors , the progression stage of melanoma is predictive for therapeutic success . early stage melanomas ( thin tumors ) result in a 97% 5-year survival rate of the patients , after surgical removal . conversely , advanced melanoma patients , comprising metastasis in regional lymph nodes or other organs , face 5-year survival rates of less than 10% . due to the intrinsic tendency of melanoma to early metastasis , even small primary tumors have already led to metastasis and a substantial portion of diagnosed melanoma cases are of late progression stages . treatment of advanced or metastatic melanoma has proven a challenge , as the conventional therapeutic approaches failed to translate into improved or significant survival rate in phase iii clinical trials . newer treatments were established in the last years that elicit unprecedented response rates in late stage melanoma , for example , up to 80% in the case of braf inhibitors . however , almost all tumors become resistant within months , and the treatment is available only for a subset of melanomas . altogether , despite substantial improvements in therapeutic options during the last years , there is still an urgent need for alternative approaches . based on clinical and histopathological features melanoma cancer cells undergo four sequential phases before reaching metastasis . these phases ensue from several genetic , epigenetic , and microenvironmental , modifications . in the last decade , a number of reports have brought significant insight into melanoma genetics and molecular markers , which are essential for the development of therapies , and in particular targeted regimens . this paper will focus on melanoma targeted gene delivery ; we aim at providing a general view on melanoma - targeting ligands , and other forms of specifically driving gene expression , reported in the literature , as well as review the most recent and/or relevant nucleic acid therapeutics employed in this field . the current paper will not dwell upon melanoma mutations or cancer transcriptional regulators ( for reviews , see [ 4 , 5 ] ) . instead , the following melanoma section serves rather as a comprehensive overview on the key players of the neoplasia , which is essential for the understanding of targeted therapies . presently , it is generally believed that melanomagenesis instigates from alterations in multiple molecules or pathways rather than a single high - risk melanoma loci . moreover , melanoma progression is a dynamic process involving several steps , each requiring the activation of different genes . first , normal melanocytes undergo genetic alterations that lead to their transformation into benign nevi . benign nevi differ from normal melanocytes in that they have initially proliferated in the basal layer of the epidermis ; however , they entered a long - term dormant status due to the lack of additional oncogenic alterations . for example , the most frequent activating mutation in the braf gene occurs in the same frequency in nevi , where it causes a dormant status called oncogene - induced senescence . this progression stage is characterized by noninvasive horizontal growth and spread through the epidermis and has been termed as radial growth phase ( rgp ) . this phase has been termed as vertical growth phase ( vgp ) . for invasion , alterations like loss of adhesive molecules together with an increase in extracellular matrix degrading enzymes are characteristic . for metastasis , cell populations have to migrate to distant locations . for this , cells have to acquire more alterations that enable the complex processes underlying metastasis . these processes involve tissue invasion , entering , and evasion of blood or lymphatic vessels to reach distant location but also survival and proliferation at distinct locations . whole genome sequencing revealed that melanoma is the tumor type with the most dna mutations  many being typical for uv - induced mutations . despite the plethora of dna alterations , two gene mutations were found to be rather common in melanoma . a general overview on these mutations and their key players are schematically represented in figure 1 . with respect to mutation frequency , the mitogen - activated protein kinase ( mapk ) pathway plays a central role in melanoma . activation of growth factor receptors leads to activation of ras molecules which activate in a downstream phosphorylation cascade raf , mek , and erk kinases . ras molecules , comprising hras , kras , and nras , are small gtpases or g proteins , and activating mutations in nras are found in 10%20% of melanomas . a single nucleotide mutation in braf at amino acid 600whereupon a valine ( v ) aminoacid is replaced by glutamic acid ( e)represents the most common mutation in braf . this mutant braf leads to an alternative protein structure and to a constitutive active protein . the outstanding importance of the ras / raf signaling pathway is documented by the observation that braf and nras mutations  exclusively nras or braf is mutated in a tumor  together are found in over 80% of melanomas and by inhibitors of mutated braf that are clearly effective in melanoma therapy . interestingly , braf has also been reported in melanocytic nevi [ 1012 ] , which rarely develop into melanoma . nevi are described to be senescent , and , similarly , expression of braf in melanocytes induces oncogene - induced senescence . these findings imply that braf mutations are involved in the first transition state of melanoma progression . hence , this mutation per se is insufficient to drive tumorigenesis , rather additional alterations are required to avoid dormancy . several pathways have been shown to cooperate with ras / raf signaling and to reduce ras / raf - mediated senescence . dna damage due to oncogene - induced dna replication stress has been proposed as an important mechanism of senescence . accordingly , molecules involved in dna damage signaling have been shown to promote oncogenesis together with braf , for example , the loss of p53 . pten is a tumor suppressor gene that negatively modulates signal transduction via phosphatidylinositol phosphatase ( pip3 , a cytosolic second messenger ) . allelic loss or altered expression of pten can be observed in tumors . in melanoma , this lost / modified expression is present in 20%/40% of melanoma tumors , respectively [ 15 , 16 ] . in a mouse model , it was shown that expression of braf in melanocytes leads to benign lesions that do not progress to melanoma . however , when pten was silenced , these mice developed metastatic tumors with high penetrance . regarding the family history of melanoma , a two - fold risk increase has been reported , and it was associated to the 9p12 chromosome . in 1994 , the cyclin - dependent kinase n2a ( cdkn2a ) gene was identified , and it is now hold as a high - risk melanoma locus . the cdkn2a gene encodes for two tumor suppressor proteins , p16 and p14 , involved in cell cycle and apoptosis , respectively . explicitly , p14 directly promotes the degradation of human double minute 2 ( mdm2 ) . accordingly , inactivation of p14 leads to increased mdm2 levels leading to increased degradation of p53 . the other product of the cdkn2a locus , p16 , prevents cell cycle progression by binding to cdk4/6 and through a series of events prevents the release of e2f1 ( a transcriptional inducer of s - phase genes ) . mutations of p16 , and similarly of cdk4 gene [ 23 , 24 ] , can therefore lead to increased cell cycle progression . however , despite the contribution of cdkn2a mutations for oncogenesis , the absolute risk of melanoma in mutation carriers is still highly shaped by environmental and pedigree factors . in close relation to pedigree structure is skin pigmentation ; the positive connection between light skin color and melanoma risks is well known . melanocortin-1 receptor ( mc1-r ) is responsible for the cutaneous pigmentation , and , interestingly , it has been reported as being overexpressed in both melanotic and amelanotic melanomas . there are two forms of epidermal melanin : eumelanin ( with a black - brown color ) and pheomelanin ( red - yellow color ) . the synthesis of eumelanin  in charge of uv attenuation  is stimulated by the activation of the mc1-r , through the binding of the tridecapeptide -msh , or -melanocortin stimulating hormone [ 2729 ] . the binding of -msh results in an increment of camp , which in turn upregulates the microphthalmia - associated transcription factor ( mitf ) inducing the transcription of pigment synthetic genes and the production of eumelanin . mitf , on the other hand , is also involved in the regulation of the cell cycle and proliferation , and few variants of the gene have been found in melanoma patients [ 31 , 32 ] . in particular , mitf(e318  k ) was reported to represent a gain - of - function allele for the gene , supporting mitfs role as an oncogene . however , mitfs expression in melanoma metastasis is yet to be clarified , as there are also studies showing that downregulation and ablation of this gene create a more invasive phenotype in vitro   and increase tumor growth in vivo , respectively . the transcription factor activator protein-2 ( ap2 ) has been suggested as a major key player in the transition from rgp to vgp . similar to several other mediators , ap2 also modulates a variety of cellular processes , including cell growth and apoptosis . in tumors , ap2 acts as a tumor suppressor , and high cytoplasmatic to nuclear expression ratio was shown to correlate with poor patients ' prognosis [ 35 , 36 ] . in particular , the promoters for the adhesion molecule mcam / muc18 , which is overexpressed in tumors , and tyrosinase kinase receptor , c - kit ( silenced in 70% of metastatic tumors ) , have ap2 binding sites . ap2 has been described to directly bind to mcam / muc18 promoter and to inhibit its transcription , whereas it promotes c - kit expression . therefore , the loss of this transcription factor during melanoma results in high mcam / muc18 levels and c - kit downregulation . in addition , the loss of ap2 was also appointed as a probable cause for the upregulation of the g - protein - coupled receptor protease activated receptor-1 , par-1 [ 10 , 39 ] . in par-1 promoter region , there are two binding complexes for ap2 and sp1 . in normal melanocytes , ap2 binds to par-1 inhibiting its transcription . however , upon melanoma progression , the levels of ap2 decrease , and sp1 binds to the par-1 promoter instead , driving its expression . ras , phosphoinositide-3 kinase ( pi3  k ) , and mapk pathways are all signaling events downstream par-1 , and hence closely related to tumor progression . during the metastatic process , following evasion into the blood circulation , tumor cells adhere to the endothelium at distant sites , and herein adhesion molecules are necessary . together with selectins , integrins have been found to play crucial roles in these steps . integrins are a family of transmembrane glycoproteins that mediate cell - cell and cell - matrix adhesion . it is therefore expected that their expression pattern changes during tumor growth , metastasis , and angiogenesis . in particular , v3 and 41 ( very late activation antigen-4 , vla-4 ) have been reported as overexpressed in numerous cancer types [ 41 , 42 ] and have served as therapeutic targets . vla-4 has been shown to be used by malignant melanoma cells to adhere to the endothelium ( binding to the ligand vcam-1 ) [ 43 , 44 ] , and to promote transmigration [ 42 , 45 ] and metastasis [ 46 , 47 ] . overall , melanoma incidence has been increasing over the years , reaching an annually increase of 3.1% during the past two decades . , unresectable advanced melanoma is characterized by an aggressive behaviour , fast spread and metastasis , and a strong resistance to chemotherapy . therefore , and in spite of the extensive research , the current prognosis for patients with advanced melanoma is limited . the earlier conventional chemotherapeutic treatment approved by us food and drug administration ( fda ) , dacarbazine , alternative chemotherapeutic agents include fotemustine , temozolomide , paclitaxel ( often in combination with carboplatin ) , and docetaxel all not yielding larger progression - free survival ( pfs ) or overall survival ( os ) than dacarbazine [ 50 , 51 ] . generally , chemotherapeutics suffer from a lack of targeting specificity ; their low molecular mass results in easy and fast body secretion , and thus the need of increased doses , which leads to inevitable toxicity . similarly , immunotherapy based on interleukine 2 ( il-2)also fda approved  has comparable response rates , and it is further restricted by the ensuing multiorgan toxicity , requiring management in specialized cancer centers . although combined therapies resulted in higher response rates , they still failed to translate into improved survival , with no impact on pfs or os compared to dacarbazine alone [ 1 , 52 ] . another alternative is the combined treatment with the cytokine tnf in combination with the alkylating drug melphalan . although highly successful , this treatment is limited to local treatment of melanoma in - transit metastases in limbs by isolated limb perfusion due to live threatening systemic toxicity of therapeutically active tnf doses . in the last decade , much progress was achieved due to the discovery of mutations in the braf gene . this led to the development of therapies interfering with ras / raf signaling and to specific braf inhibitors . in august 2011 , an alternative melanoma regimen , for patients positive for braf mutations , was brought into the market with the fda approval of vemurafenib ( zelboraf , plexxikon / roche ) . in phase ii and iii studies , vemurafenib showed a response rate up to 50% , yet the response duration varied between the phase studies [ 5456 ] . in addition , vemurafenib induces acanthopapillomas , keratoacanthomas , and cutaneous squamous cell carcinomas in the early treatment [ 57 , 58 ] . unfortunately , these unprecedented response rates are limited by the fact that almost all tumors become resistant to this therapy and the overall survival of patients was 6.7 months . in addition , the treatment is only available for 50%60% of patients with mutated tumors because it is not effective in tumors with wildtype braf . nevertheless , this success has led to the development of other ras / raf pathway inhibitors , for example , for mutated braf or downstream kinases like mek . alternative activation of ras / raf pathway has been proposed as a resistance mechanism . in line with this , the combination of braf inhibition with mek inhibition led to an improved survival of 9.4 months . other new therapies that add to the therapeutic options for melanoma patients are immunotherapies . an anti - ctla-4 antibody ( ipilimumab ) improved survival of stage ii and iv melanoma patients ( 10.1 versus 6.4 months ) . cytotoxic t - lymphocyte antigen 4 ( ctla-4 ) inhibits t - cell responses and respectively , ctla-4 blockade promotes immune responses and antitumor activity . in an early analysis of anti - pd - l1 antibody , similar to ctla4 , pd-1 reduces immune activation , and its inhibition can lead to reactivation of immune responses . altogether , even with respect to the recent advances in melanoma therapy , the high resistance rates and the restriction to certain patient subgroups demonstrate that there is still an urgent need to develop alternative therapies . as also observed for other tumor entities , melanoma treatment with low molecular weight chemotherapeutic drugs often results in the rise of resistant cancers cells , especially in case of relapsed disease . a well - known mechanism of resistance is the elevated expression of multidrug transporter proteins , like p - glycoprotein , which actively pump chemotherapeutics out of the cell . here as an example , the attachment of chemotherapeutics to polymers via reversible covalent bonds helps to overcome this type of resistance ( for a recent review see ) . also , biotherapeutics , such as antibodies , have been successfully applied in melanoma therapy ( see above ) , but also here resistance can occur , for example , when blocking of one cellular pathway responsible for cancer cell proliferation can be replaced by another . in this case , the application of therapeutically active nucleic acids comes into play . firstly , they exhibit a relatively high molecular weight , which prevents resistance mediated by p - glycoprotein upregulation . secondly , nucleic acids can be designed to affect only malignant cells , for example , by using promoter elements being only activated in tumors , or as rna oligonucleotides ( like sirna ) , which will enable the knockdown of a specific protein overexpressed in tumor tissue . furthermore , the delivery of more than one sirna targeting different pathways can prevent tumor resistance by blocking different resistance or escape strands . last but not least , nucleic acid delivery permits systemic delivery of toxic agents , such as diphtheria toxin a   or tumor necrosis factor ( tnf ) , as they only become toxic after transcription in the target cell . solid tumors exceeding a certain size rely on a functional blood supply for access to nutrients and oxygen . in contrast to nonmalignant tissues , tumor vasculature often exhibits a leaky appearance , which in principle also allows nanosized particles to reach tumor cells . being packed into nanoparticles or polyplexes , nucleic acids can be protected from nucleases which are present in the bloodstream . nevertheless , systemic delivery of nanopharmaceutics offers several pitfalls and obstacles , such as aggregation with blood cells , undesired adherence to the vessel wall , or opsonization with plasma proteins followed by clearance through tissue macrophages ( a key component of the reticulo - endothelial system ) . blood proteins interact both with negatively and positively charged nanosystems , whereas a neutral surface charge enables , in principle , blood circulation , as it has been shown for small nanocrystals , so called quantum dots . alternatively , nanosystems can be decorated with hydrophilic polymers , which , owing to their excessive hydration , shield the particles ' surface charge , hereby preventing the aggregation with protein components . from the group of hydrophilic polymers , like n-(2-hydroxypropyl)methacrylamide ( hpma ) , hydroxyethyl starch ( he s ) , or polyethyleneglycol ( peg ) , peg is the most commonly used one . in addition , targeting entities can be used to direct the nanocarrier to specific cells . commonly , these are ligands that bind to receptors , or other cell surface molecules , that are overexpressed in tumor cells . macromolecular drugs , which exceed the renal excretion limit and are able to circulate in the blood stream , can benefit from the so - called enhanced permeability and retention ( epr ) effect : nanopharmaceutics accumulate in tumor tissue as they can penetrate the leaky vasculature but are retained within the tumor tissue due to incomplete lymphatic drainage . this tumor deposition is a prerequisite for all steps that follow : binding to and internalization of the particles into target cells . the latter can be promoted by the incorporation of the earlier mentioned cell - binding ligands into the carrier system . figure 2 summarizes the limitations in nucleic acids delivery , the solutions for such limitations , and the therapeutic advantages of nucleic acid nanosystems . directed approaches are of special interest as they have the potential to specifically distress malignant cells causing increased local concentrations of the active agent and avoiding undesired side effects . tracking down melanoma - associated molecular targets involves identifying signaling pathways ' key players , earlier described , as much as cancer cell surface markers . in particular , for gene therapy , cell surface markers are important , and these abide with the conception of a treatment addressing multiple melanoma subgroups  as cells with different mutations can still exhibit common surface markers . ergo , it is crucial to identify critical and idiosyncratic targets for these cells . already reported in the early seventies , one of the largely explored targets is the melanocortin-1 receptor ( mc1-r ) , which is also overexpressed in numerous melanoma cases . mc1-r belongs to a class of g - coupled protein receptors ( mc1-r  mc5-r ) , where the different receptors allocate in different tissues , reflecting their functions . while mc1-r is found in hair and skin , mc2-r is localized in adrenal glands , whereas mc3-r and mc4-r are in hypothalamus   and mc5-r in kidneys . however , owing to their similarity their binding domains may share common affinities , and certain peptide motifs can bind to several receptors . for targeting purposes , the most well - known and used mcr-1 the substitution of methionine in position four by norleucine ( nle ) and of phenylalanine for its d - counterpart in position seven ( d - phe ) renders this peptide with higher affinity and resistance to enzyme degradation than its native form . however , ndp--msh was shown to have a strong nanomolar binding affinity towards mc3-r , mc4-r , and mc5-r , and , for gene delivery , it is crucial to decrease off - target effects . aiming at the design of ligands suitable for micelle conjugation , and with an adequate selectivity to mc1-r , barkey et al . this paper allowed the following conclusions : ( 1 ) free rotation of carbons that compose the peptide 's biding motif seems to be required for mc1-r avidity ; ( 2 ) alkyl modifications , for the attachment of triblock polymer micelle , at the n - terminal of the peptide , did not affect binding affinity in the short four amino acid peptide ; ( 3 ) for peptides twice as long , c - terminal modifications for micelles ' attachment did not altered binding affinities . in addition , the authors have synthesized micelles conjugated to the short peptide version [ 4-phenylbutyril - hist - dphe - arg - trp - gly - lys(hex-5ynoyl)-nh2 ] , through a peg linker . and importantly , in vitro cell - uptake studies showed the ability of conjugated micelles to selectively bind to mc1-r receptor , and , whether due to multivalent interactions or other factors , the micelles had higher avidity for the receptor than the ligand alone . nevertheless , further studies ( i.e. , by flow cytometry or confocal laser microscopy ) to quantify the uptake of these conjugated micelles are needed to better evaluate the delivery efficiency of this platform . more recently , -msh peptide has been conjugated to a nanoplatform based on the heavy chain of the human protein ferritin ( hft ) . ferritin can be used to build a hollow nanocage that can transport materials such as fe3o4 , co3o4 , mn3o4 , pt , and au and hence be used for imaging and therapeutic purposes . unfortunately , the authors have not analyzed the in vivo distribution of their nanoparticles , and the targeting efficiency was evaluated by immunohistochemistry in the tumor tissue in relation to normal skin . in a similar approach to that of hft nanocages , lu and collaborators have used hollow gold nanospheres , conjugated to ndp--msh , aiming at cancer photothermal ablation . in this study , nude mice were subcutaneously inoculated with b16/f10 murine melanoma cells , and the nanoparticles were administered intravenously . the authors have collected different organs and were able to show the targeting effect by the ndp--msh - gold nanospheres . interestingly , targeting of mc1-r by -msh peptide has been mostly used in radionuclide therapy studies and for diagnostic purposes . currently , 2-[f]fluoro-2-deoxy - d - glucose ( f - fdg ) is the only radioactive probe used in the clinic to detect melanoma . be that as it may , f - fdg is an unspecific positron emission tomography ( pet ) imaging agent with poor sensitivity towards micrometastatic sites [ 78 , 79 ] , a fact that underlines the general insufficiency in melanoma targeting . regarding mc1-r targeting , yubin miao and thomas p. quinn 's extensive work is of particular interest , reporting on two generations of an ndp--msh - based peptide used for melanoma imaging by single - photon emission - computed tomography ( spect ) and more recently by pet . what distinguishes the two -msh peptide generations is mostly the peptide 's length , being twelve aminoacid - long in the first generation ( cycmsh ) [ 8082 ] and six in the second ( cycmshhex ) [ 83 , 84 ] . in both generations , the peptide is cyclized ( cyc ) , and the mc1-r binding motif ( his - dphe - arg - trp ) is conserved . the peptides have also undergone structural modifications concerning the aminoacid linkers , which are used to support the peptide cyclization and bridge the targeting ligand and the radiometal chelator . interestingly , the authors have observed that the exchange of single aminoacids in these linkers , and the introduction of  glygly  linker between the chelator and the peptide   resulted in improved melanoma targeting , with decreased renal excretion and liver uptake of the radiolabelled peptide in b16/f1 melanoma - bearing c57 mice . these studies underscore the structural role of the targeting moiety but also of the integral component being delivered . in other words , the addition of a targeting entity to a carrier does not necessarily suffice for efficient deliver ; the number of peptides conjugated to the delivery platform , the site of conjugation and the size and type of the linker play an important role . after the discovery of adhesion molecules as mediators of tumor metastasis , the identification of their binding motifs opened the possibilities for targeted therapies . several peptide fragments have been employed to target these mediators , either as antagonists or as ligands for drug delivery purposes . the formation of new vessels and , by serving as receptor for extracellular matrix proteins , it mediates migration of endothelial cells into the basement membrane , and regulates their growth , survival , and differentiation . it is therefore no surprise that such integrin is found upregulated in different tumor cells , where it is involved in processes that govern metastasis . the integrin 's binding peptide motif has been identified in 1990 arginine - glutamine - aspartate or rgd  but studies that followed have shown that the cyclic version of rgd ( crgd ) has higher binding affinities towards the integrin [ 86 , 87 ] . either alone or in combination with other ligands , crgd has been conjugated to several nanocarriers for both diagnostic and therapeutic purposes [ 8890 ] . another integrin reported to have a dominant function in the metastatic spread is 41 or vla-4 . okumura , and more recently schlesinger , have shown , in different settings , that inhibition of vla-4 by natalizumab ( an antibody against 4 integrin ) significantly decreased melanoma lung metastases in murine models [ 42 , 44 , 122 ] . in 1991 , makarem and humphries have identified the leucine - aspartate - valine ( ldv ) sequence as the integrin 's motif , and a few years later , vanderslice et al . have reported on a series of cyclized peptides based on ldv that were assayed for the inhibition of the integrin . however , and despite the numerous reports relating this agent to tumor metastasis , and to melanoma in particular , most of the literature relies on the ldv sequence as an antagonist , rather than for deliver purposes , where , to our knowledge , there is only one paper reporting on in vitro studies . indeed , vla-4 is found in multiple leukocyte populations ; vla-4 is a vital receptor of leukocytes , and it is involved in the immune response . hence , a systemic application of vla-4 inhibitors , or binding peptides , could induce undesired partially immunosuppressive effects . in this context , the application of transcriptional - targeting strategies could potentially prevent off - target effects and prove this ligand a promising tool . in fact , tissue - specific elements as components of the dna vector can provide a tight control over gene expression and complement and strengthen targeted - delivery . commonly , tumor cells ' surface markers entail receptors that are also present in nontumor cells but are rather overexpressed in their malignant form . this is the case for both the integrins here described , but also the transferrin receptor all used as melanoma targets . therefore , off - target effects can occur , and for gene delivery purposes , tissue - specific control elements are an elegant way to bypass undesired side effects . these control elements consist of nucleic acid sequences that are recognized by proteins or other nucleic acids , which hereby regulate gene expression . for the case of melanoma , tissue specific promoters have been described , and these include tyrosinase [ 9395 ] and melanoma inhibitory activity ( mia ) [ 96 , 97 ] . micrornas are a class of short ( 2022 nucleotides long ) regulatory rnas , which are believed to regulate as many as 30% of all genes . several micrornas are tissue - specific and fine - tune genetic circuits , some of which are critical for normal development , cellular differentiation , and normal cellular homeostasis . if the target sequence and microrna have perfect complementarity , the mrna is eliminated by a rna degradation pathway . in the context of transcriptional control , this means that a dna vector that contains specific mir - binding sites is only translated in cells where the mir in question is absent [ 125 , 126 ] . in tumor cells , several micrornas are deregulated , while mirs enrolled in cell homeostasis are downregulated those involved in cell proliferation and differentiation are upregulated . for the case of melanoma , mir let-7b , mir-193b , mir-34a , mir-155 , mir-205 , mir148 , mir-137 , and mir-152 have been found downregulated ( for a review on melanoma micrornas , see ) and can therefore be suitable targets for transcriptional regulation when expressed in normal tissue . as opposed to conventional therapy , traditionally , that is , in the case of loss of function , gene therapy aims at permanent correction of a defected or missing gene by replacing with or providing , respectively , the corrected version  for example , by the introduction of plasmid dna ( pdna ) . ideally , this approach translates into a single treatment , or few initial treatments , rather than several ( or life long ) required to provide the patients with the functional form of the protein . however , this permanent correction treatment has proven very challenging . in the last twenty years , new nucleic acids with attractive therapeutic properties were discovered , notably , sirna and micrornas . small interference rna ( sirna ) has the ability to specifically silence protein expression  an asset particularly valuable for antiviral and cancer regimens . in general , also mirna negatively regulates gene expression , although via two different mechanism depending on the degree of complementarity towards its mrna target . nucleic acid - based approaches offer several advantages when compared to treatment with small molecules or proteins . they can be seen as mostly inactive prodrugs , which are activated at the tumor site producing a therapeutically active protein or knocking down a specific target gene . importantly , nucleic acid targeted delivery systems , preferably also relying in transcriptional targeting , decreasing off - target effects and toxicity , and permitting a systemic administration otherwise not feasible with a therapeutic agent with toxic properties . in parallel with new therapeutic nucleic acid tools , the last two decades brought insight into tumorgenesis in general and unveiled a plethora of therapeutic concepts against cancer ( figure 3 ) . the following paragraphs will deal with different antimelanoma approaches based on nucleic acids . despite the apparent tumor tolerance , humoral and cellular immune responses hence , whether the tumor grows as a result of stealth and nonrecognition or as the result of escape and immunological shaping , its recognition by the immune system can still be prompted . indeed , at a later stage , during the progressive growth phase , tumors may become more immune - activating for varies reasons : damage or disruption of surrounding tissue , generation of reactive oxygen species , upregulation of stress protective factors , or death by necrosis or apoptosis . however , at this stage , it is not known whether the tumor still needs to escape immune recognition , as it is unclear that these immune responses can cause tumor destruction . therefore , a number of studies have focused in eliciting earlier and suitable tumor recognition by the immune system . in a nucleic acid therapy context , this transliterates into genetic immunization or dna vaccination : the delivery and transcription of a gene encoding antigens or immunestimulatory molecules that elicit an immune response . as an example , interleukine-12 ( il-12 ) has been used and studied in different animal models [ 104 , 105 ] . il-12 is originally produced by mononuclear phagocytes and dendritic cells and is responsible for activating nk and cd4 t cells and inducing the production of high levels of interferon gamma ( inf- ) . interestingly , il-12 has been described to increase antitumor immune responses [ 129 , 130 ] , and later studies investigated its suitability for a dna vaccine approach against melanoma . il-12 effects appeared to be long lasting and efficient against tumor metastases , although not mainly mediated by inf- . the murine studies also revealed moderate toxicity caused by il-12 , and while lower il-12-encoding pdna doses can be administered , ideally the gene expression should be controlled , regarding the tissue and the durability of the expression . although dna vaccination against a strong melanoma tumor antigen should be possible , the authors have not seen an effect on lung metastases when using melanoma - associated glycoprotein 100 ( gp 100)/pmel17 pdna alone . adjuvants appear to be necessary for a successful dna vaccination : the authors have seen an effect when the gp 100-pdna was administered together with il-12 , similar to other murine study where granulocyte - macrophage colony - stimulating factor was used . alternatively , in a canine study , the developed vaccine was based on the human ( rather than canine ) gp 100 protein , where the human form of the antigen acted as adjuvant . together with gp 100 , and for the case of melanoma , two more tumor genes have been described for dna vaccination : mart-1 and tyrosinase [ 108 , 109 ] . also , the expression of chemokines , such as monocyte chemoattractant protein-1 ( mcp-1 ) and interferon - inducible protein-10 ( ip-10 ) , can mediate an immune response . in particular , ip-10 as been described by sgadari et al . as an antitumor agent and found to promote damage in established tumor vasculature as well as tissue necrosis in a murine model for the human burkitt lymphomas . based on this , and after their studies with il-12 , keyser and collaborators have investigated the efficiency of ip-10-encoding pdna therapy in murine melanoma models . the authors have used two murine tumor models , whereupon cells have been injected subcutaneously ( originating a solid tumor ) or intravenously , inducing lung metastases . when administered alone , and intramuscularly ( resulting in systemic circulation ) , ip-10-encoding pdna showed an antimetastatic effect , reducing the number of lung metastases as compared to the control - pdna treated group . when administered with il-12-encoding pdna , ip-10 pdna enhanced the il-12 effect , and decreased its earlier observed toxicity . this anti - neoplastic effect of ip-10 has been attributed to the engagement of nk cells and the inhibition of angiogenesis and cell proliferation . alternative antitumor strategies aim at a direct destruction of cancer cells , through the delivery of pdna encoding for a toxic protein  dna - based strategies . this is referred to as a suicide gene therapy or gene - directed enzyme prodrug therapy ( gdept ) , when the nucleic acid sequence encodes for an enzyme , which is not directly toxic but instead converts a nontoxic prodrug into a cytotoxic metabolite . the first proof of principle of gdept was presented in the mid - eighties and involved the herpes simplex thymidine kinase ( hsv - tk ) and the prodrug ganciclovir ( gcv ) . presently , hsv - tk as well as other approaches , such as diptheria toxin a chain ( dta ) , have been employed in the clinics , the most successful cases being reported in ovarian and prostate cancers [ 67 , 133 ] . as for melanoma treatments , hsv - tk has been the most commonly used [ 111113 ] , although there is no human clinical trial yet . suicide gene therapy has also been proven effective when used in combined approaches , such as with cytokine - enhanced vaccine in a clinical trial involving canine melanoma patients . despite promising , this strategy is currently restrained by a poor delivery ; most nanocarriers are not as target - specific and efficient as required , and the toxic gene does not reach the tumor cells in efficacious concentrations . a number of studies have instead focused on mediators of cell proliferation and differentiation , which are upregulated during tumorgenesis , aiming at their downregulation by means of sirna delivery [ 114 , 135137]these are rna - based approaches . as an example , based on the fact that in epithelial cells , n - cadherin induces changes in morphology of a fibroblastic phenotype ( rendering the cells more motile and invasive ) , the laboratory of laidler has investigated the outcome of n - cadherin silencing in human melanoma cell lines . although the results suggest that n - cadherin positively affects the regulation of the cell cycle and proliferation through activation of the akt kinase pathway , further investigations are needed to describe the mechanism . , upon the observation that thrombin receptor ( or protease - activated receptor-1 , par-1 ) is overexpressed in highly metastatic melanoma cell lines , has evaluated the therapeutic potential of sirna against par-1 . the authors have observed a significant reduction of in vivo tumor growth as well as in the number of metastatic lung colonies . this report showed that downregulation of par-1 decreased the expression of matrix metallopeptidase-2 ( mmp-2 ) , interleukin 8 ( il-8 ) , and vascular endothelial growth factor ( vegf ) , resulting in an overall decrease in angiogenesis and blood vessels . in 2010 , davis et al . reported on the first human clinical trial ( including three melanoma patients ) on sirna therapy against melanoma . the sirna targeted the m2 subunit of ribonucleotide reductase ( rrm2 ) , and the protein knock down was confirmed at the mrna level but not corroborated to the same extend by the protein analysis . nevertheless , the fact that the authors used a delivery vector targeting the transferrin receptor without showing analysis of such receptor expression in melanoma cells was left to be explained . of special interests are combinatorial strategies involving sirna delivery as these , similar to other combinatorial therapies , cause the most significant outcomes . the authors targeted bcl2 ( an apoptosis regulator protein ) , which was reported to play a central role in the resistance of melanoma cells to chemotherapy [ 7 , 116 , 139 , 140 ] . by adding 5-triphosphate ends to their sirna , the authors also activated innate immune cells , induced the expression of interferons , and caused specific cell tumor apoptosis . these actions are a consequence of the recognition of 5-triphosphate ends by the cytosolic retinoic acid - induced protein-1 ( rig-1 ) and synergized with the silencing effects originated from sirna resulting in massive tumor destruction in the murine lung metastases . first reported on a promising double stranded rna ( dsrna ) mimic polyinisine - polycytidylic acid ( pic ) . importantly , the therapeutic effect of the dsrna was significantly increased when delivered in the form of a complex , together with polyethyleneimine ( pei)-[pic ] . initially , the dsrna mimic was thought to engage toll - like receptors ( tlr ) , hereby mediating cellular tumor immunity . in turn , further investigation studies showed that it mobilizes the endo / lysosomal machinery of melanoma cells , and through melanoma differentiation associated gene-5 ( mda-5 ) induces self - degradation by ( macro ) autophagy and apoptosis , following the mda-5-mediated activation of proapoptotic factor noxa . interestingly , at the exact same time , mda-5 and noxa were also reported to play a role in interferon - independent apoptosis in human melanoma cells by besch and collaborators . not only were these findings meaningful , opening new windows for cancer therapy , but also , in particular in the dama tormo studies , was the murine model used very suited , whereupon mice overexpressing hepatocyte growth factor ( hgf ) and carrying an oncogenic mutation in the cyclin - dependent kinase-4 [ ( cdk4 ) ] developed invasive melanomas in the skin following neonatal exposure to carcinogenics . while a number of microrna has been described to play relevant roles in melanoma progression , only few in vitro studies have reported on the mirna potential for antimelanoma therapy [ 119 , 120 ] . however , pertinent therapeutic approaches targeting mirnas described for other tumor types [ 142 , 143 ] foretell the potential and the therapeutic window opportunities entailing these nucleic acids in metastatic melanoma . as an overview of this section , table 2 presents the therapeutic nucleic acids herein described , and figure 3 schematically summarizes the different strategies in nucleic acid therapies . it is of general consensus that the last decade of cancer research significantly expanded our knowledge in tumor development and progression . unfortunately  similar to the tumor escape shaped by the immune surveillance in an early growth phase  as new therapeutic strategies are applied , tumor cells undergo another round of selection , giving rise to therapy - resistant cells . it is therefore necessary to combine several approaches to attack different paths of tumor escape  a fact that is confirmed by the most significant results reported in studies where such strategies have been used . on this note , nucleic acids deliveries are truly advantageous tools as they allow the systemic delivery of potentially toxic molecules that can be combined with chemotherapy aiming at terminating possible resistant - tumor cells . as an example , recently , su and collaborators have reported on an antitumor strategy combining tnf - encoding pdna and chemotherapy . while systemically administered tnf is extremely toxic , in its genetic form , and when reaching specific target cells , tnf revealed to be a powerful antitumor agent . specific and efficient are indeed key words in this type of targeted approaches , as in suicide gene delivery . it is thus of extreme importance to thoroughly evaluate the target options and to verify the levels of the target molecule in the cells of interest . the activation of possible target - receptors may be desired , such as in the case reported by poeck et al . , but only when not hampering the therapeutic effect by activation of pathways that can lead to cell proliferation / differentiation , enhanced cell migration , or inhibition of apoptosis . , this can be the case when targeting the epidermal growth factor receptor ( egfr ) , and it is then desirable to design a ligand that targets the receptor circumventing its activation . on the other hand , the relevance of analyzing the targeted receptor has been well exposed in the short letter of perris in response to the work published by davis et al . . to avoid other pitfalls in nanovector development , also the in vivo distribution needs to be assessed , preferably by several approaches ( e.g. , bioluminescence imaging , positron emission tomography ( pet ) , and magnetic resonance imaging ( mri ) ) . to this end , immunohistochemistry studies may be suitable and very convenient to corroborate and support data collected by different means , but also microscopy ( mostly in vitro but also histochemistry analysis ) has had its traps . in summary , already a number of promising nucleic acid strategies exist , and these certainly present less hurdles for delivery than their protein counterpart , as they are smaller , less antigenic , and can bypass certain resistance mechanisms . nevertheless , further improvements in nonviral targeted delivery appear required to increase the efficacy of such therapies . a small final note regarding the potential of mirna approaches : microrna therapies can aim at ( 1 ) mirna upregulation , when the target nucleic acid is enrolled in cell homeostasis and is found silenced in tumor cells ; ( 2 ) mirna downregulation by antimirs , when it is upregulated in tumor cells due to its play in cell proliferation ; ( 3 ) alternatively , mirna can also have a role in cell - specific transcription in pdna vectors containing mirna binding - sites , allowing the expression of the gene of interest in cells , where the mirna is silenced .